Every year as spring approaches and day length increases, many birds begin to reproduce, an annual expression of seasonal phenology that requires physiological preparation. In species distributed over a broad geographic range, populations that breed at higher latitudes are often migratory and delay reproduction until later in the year as compared to those breeding at lower latitudes. Dark-eyed Juncos serve as an excellent model for understanding the timing mechanisms regulating population-level variation in seasonal reproductive responses. We compared two seasonally sympatric dark-eyed junco populations in early spring. One migrates (Junco hyemalis hyemalis) and breeds in Alaska and Canada, while the other remains resident (Junco hyemalis carolinensis) and breeds in the Appalachian Mountains of Virginia USA. These populations exhibit different photoperiodic responses to the same environment with respect to activation of the HPG axis, leading to earlier gonadal recrudescence in the resident population. We caught co-wintering sympatric male migrant (n = 6) and resident (n = 7) juncos from the field in March and collected the hypothalamic tissues. We also collected blood samples to determine circulating testosterone and a wing feather to determine stable isotope ratios (δ2H) as estimate of breeding latitude. We found three differentially expressed genes, among which gonadotropin releasing hormone 1 (GnRH1) showed significantly higher expression in early breeding residents as compared to migrant juncos. The δ2H showed a positive linear correlation with testosterone levels and GnRH1 mRNA, providing strong evidence for latitudinal variation in breeding phenology. This study provides insight into the underlying neuroendocrine response giving rise to a population-level difference in the timing of reproduction observed in a seasonally sympatric (co-wintering) population of resident and migrant juncos.

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species.

The ways in which animals sense the world around them change throughout development. Young of many species have absent or limited visual capabilities, but still make complex decisions about individuals with whom they interact. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems. Here, we examined visual system development in tadpoles of the Mimetic Poison Frog (Ranitomeya imitator) that use begging displays to stimulate egg feeding from their mothers. Neural activation in the retina increased in begging metamorphic tadpoles, but not in begging pre-metamorphic tadpoles. Molecular profiling of active eye neurons during begging identified numerous differentially expressed development-related transcripts, suggesting that developmental stage, not begging, was driving gene expression profiles. Using the neural tracer neurobiotin, we found that connections between the eye and brain proliferate during metamorphosis, with little retinotectal connections in recently-hatched tadpoles. To assess visual capabilities of tadpoles, we used a light/dark preference assay in early, middle, and late stages. All tadpoles showed a preference for the dark side, but the strength of preference increased with developmental stage and eyes were not required for this behavior. Taken together, these data indicate visual ontology of poison frog tadpoles is similar to that of other frogs, with poor visual capabilities at hatching and immense morphological and physiological changes occurring during metamorphosis. More broadly, this highlights the importance of multimodal cues, including photodetection via the pineal structure, in tadpole social interactions.

In contrast to mammals, birds have a higher basal metabolic rate and undertake wide range of energy-demanding activities. As a consequence, food deprivation for birds, even for a short period, poses major energy challenge. The energy-regulating hypothalamic homeostatic mechanisms, although extensively studied in mammals, are far from clear in the case of birds. We focus on the interplay between neuropeptide Y (NPY) and thyrotropin-releasing hormone (TRH), 2 of the most important hypothalamic signaling agents, in modulating the energy balance in a bird model, the zebra finch, Taeniopygia guttata. TRH neurons were confined to a few nuclei in the preoptic area and hypothalamus, and fibers widely distributed. The majority of TRH neurons in the hypothalamic paraventricular nucleus (PVN) whose axons terminate in median eminence were contacted by NPY-containing axons. Compared to fed animals, fasting significantly reduced body weight, PVN pro-TRH messenger RNA (mRNA) and TRH immunoreactivity, but increased NPY mRNA and NPY immunoreactivity in the infundibular nucleus (IN, avian homologue of mammalian arcuate nucleus) and PVN. Refeeding for a short duration restored PVN pro-TRH and IN NPY mRNA, and PVN NPY innervation to fed levels. Compared to control tissues, treatment of the hypothalamic superfused slices with NPY or an NPY-Y1 receptor agonist significantly reduced TRH immunoreactivity, a response blocked by treatment with a Y1-receptor antagonist. We describe a detailed neuroanatomical map of TRH-equipped elements, identify new TRH-producing neuronal groups in the avian brain, and demonstrate rapid restoration of the fasting-induced suppression of PVN TRH following refeeding. We further show that NPY via Y1 receptors may regulate PVN TRH neurons to control energy balance in T. guttata.

Altricial young of many species beg parents for the nutrients required for healthy development. From human crying to begging chicks, young expend precious energy reserves to communicate their hunger. Despite repeated independent evolutionary origins, the neural basis of parent-directed communication by infants is unknown. Here, we examined the sensory and neural basis of begging behavior in tadpoles of the monogamous and biparental Mimetic poison frog ( Ranitomeya imitator ). In this species, tadpoles beg parents for egg meals by dancing. We used this robust motor display to determine that tadpoles use multimodal cues for caregiver recognition, where olfactory cues are necessary for caregiver recognition while visual cues provide an orientation goal. We found that dopamine related brain regions have higher neural activity in begging tadpoles and that dopamine signaling had opposing modulatory effects through D1 and D2 family receptors, similar to swimming behavior in other tadpole species. We then identified caudal posterior tuberculum dopamine neurons as more active during begging behavior and sensitive to caregiver olfactory cues. Projections of these dopaminergic neurons to the spinal accessory motor nucleus are required for begging displays. These findings support the idea that dopamine regulates olfactory-guided parental recognition in young and opens many avenues for studying how new communication behaviors can evolve from ancestral motor circuits.

Many seasonally breeding animals exhibit a threshold day length (critical photoperiod; CPP) for gonadal growth, and populations breeding at higher latitudes typically have a higher CPP. Much less is known about latitudinal variation in CPP in migratory population that winter away from their breeding range and must time their reproduction to match favorable conditions at their destination. To address the relationship between migration, breeding latitude, and CPP, we held two closely related songbird populations in a common environment. One population is resident (Junco hyemalis carolinensis), the other winters in sympatry with the residents but migrates north to breed (Junco hyemalis hyemalis). We gradually increased photoperiod and measured indices of readiness to migrate (fat score, body mass) and breed (cloacal protuberance volume, baseline testosterone, and gonadotropin releasing hormone challenged testosterone). To estimate breeding latitude, we measured hydrogen isotopes in feathers grown the preceding year. As we predicted, we found a higher CPP in migrants than residents, and a higher CPP among migrants deriving from higher as opposed to lower latitudes. Migrants also terminated breeding earlier than residents, indicating a shorter breeding season. To our knowledge, this is a first demonstration of latitudinal variation in CPP-dependent reproductive timing in bird populations that co-exist in the non-breeding season but breed at different latitudes. We conclude that bird populations appear to exhibit local adaptation in reproductive timing by relying on differential CPP response that is predictive of future conditions on the breeding ground.

Daylength regulates the development of spring migratory and subsequent reproductive phenotypes in avian migrants. This study used molecular approaches, and compared mRNA and proteome-wide expressions in captive redheaded buntings that were photostimulated under long days (LD) for 4 days (early stimulated, LD-eS) or for ∼3 weeks until each bird had shown successive 4 nights of Zugunruhe (stimulated, LD-S); controls were maintained under short-days. After ∼3 weeks of LD, photostimulated indices of the migratory preparedness (fattening, weight gain and Zugunruhe) were paralleled with upregulated expression of acc, dgat2 and apoa1 genes in the liver, and of cd36, fabp3 and cpt1 genes in the flight muscle, suggesting an enhanced fatty acids (FAs) synthesis and transport in the LD-S state. Concurrently elevated expression of genes involved in the calcium-ion signaling and transport (camk1 and atp2a2; camk2a in LD-eS instead), cellular stress (hspa8 and sod1, not nos2) and metabolic pathways (apoa1 and sirt1), but not of the genes associated with migratory behaviour (adcyap1 and vps13a), were found in the mediobasal hypothalamus (MBH). Further, the MBH-specific quantitative proteomics revealed that out of 503 annotated proteins, 28 were differentially expressed (LD-eS vs. LD-S: 21 up- and 7 down-regulated) and they enriched five physiological pathways that are associated with fatty acids transport and metabolism. These first comprehensive results on gene and protein expressions suggest that changes in molecular correlates of fatty acids transport and metabolism may aid the decision for migratory departure from wintering areas in obligate songbird migrants.

We hypothesized that there were diurnal differences in metabolism, with major transcriptional changes occurring early during the day and/or night between migratory and non-migratory states of avian migrants. Present study investigated this in captive Black-headed Buntings (Emberiza melanocephala) exhibiting long-day stimulated vernal migratory and post-breeding non-migratory states, by using RNA-Seq and qPCR assays of liver samples collected at 1 h into day (hour 1) and 1 h into night (hour 17) of the 16-h photoperiod (16L:8D). There were differentially expressed genes (DEGs) both within (day vs. night) and between (vernal migratory vs. non-migratory) states. Within the state, 358 and 52 DEGs were identified in migratory and non-migratory states, respectively. In the migratory state, genes associated with oxidative phosphorylation (sdh) had higher expression, and genes associated with fat metabolism (acaca and elovl6) and ABC transporters (abcg5, abcg8) had lower expression at night, compared to the daytime. In the non-migratory state, among genes associated with fat metabolism and gluconeogenesis, ppara and hmgcl had higher while aacs had lower expression at night. Between states, 35 and 180 DEGs were identified in day and night, respectively, with highly expressed genes associated with fat metabolism (acsbg2, cetp) found in migratory, and those associated with cell death (casp7), gluconeogenesis (stat3) and circadian rhythm pathway (cry1) in the non-migratory state. These results demonstrate differentially activated hepatic molecular pathways during photostimulated vernal migratory and post-breeding non-migratory states of buntings and provide molecular insights into differential metabolic support to physiologically contrasting seasonal life-history states in migratory songbirds.

In the modern era of industrialization, illuminated nights have become a common defining feature of human-occupied environments, particularly cities. Artificial light at night (ALAN) imposes several known negative impacts on the neuroendocrine system, metabolism, and seasonal reproduction of species living in the wild. However, we know little about the impact of ALAN on populations of birds that either live year-round in the same location or move to different latitudes across seasons. To test whether ALAN has a differing impact on the reproductive timing of bird populations that winter in sympatry but breed at different latitudes, we monitored sedentary and migratory male dark-eyed juncos that were or were not exposed to low intensity (∼2.5 ± 0.5 lux) ALAN. All groups were held in common conditions and day length was gradually increased to mimic natural day length changes (NDL). We assessed seasonal reproductive response from initiation to termination of the breeding cycle. As expected based on earlier research, the sedentary birds exhibited earlier gonadal recrudescence and terminated breeding later than the migratory birds. In addition, resident and migrant birds exposed to ALAN initiated gonadal recrudescence earlier and terminated reproductive events sooner as compared to their conspecifics experiencing NDL. Importantly, the difference in the reproductive timing of sedentary and migratory populations was maintained even when exposed to ALAN. This variation in the seasonal reproductive timing may likely have a genetic basis or be the result of early developmental effects imposed due to different light regimes related to the latitude of origin. This study reveals first that ALAN accelerated reproductive development across both migrants and residents and second that latitude-dependent variation in reproductive timing is maintained despite exposure to ALAN. These results corroborate a relationship between latitude, population, and reproductive timing while also revealing ALAN's impact on seasonal reproductive timing. This study reveals that, ALAN accelerated reproductive development but maintained latitude-dependent variation in reproductive timing across both migrant and resident bird populations.

In the modern era of industrialization, illuminated nights have become a common defining feature of human-occupied environments, particularly cities. Artificial light at night (ALAN) imposes several known negative impacts on the neuroendocrine system, metabolism, and seasonal reproduction of species living in the wild. However, we know little about the impact of ALAN on populations of birds that either live year-round in the same location or move to different latitudes across seasons. To test whether ALAN has differing impact on reproductive timing of the bird populations that winter in sympatry but breed at different latitudes, we monitored sedentary and migratory male dark-eyed juncos that were or were not exposed to low intensity (~2.5 ± 0.5 lux) ALAN. All groups were held in common conditions and day length was gradually increased to mimic natural day length changes (NDL). We assessed seasonal reproductive response from initiation to termination of the breeding cycle. As expected based on earlier research, the sedentary birds exhibited earlier gonadal recrudescence and terminated breeding later than the migratory birds. In addition, resident and migrant birds exposed to ALAN initiated gonadal recrudescence earlier and terminated reproduction sooner as compared to their conspecifics experiencing NDL. Importantly, the difference in the reproductive timing of sedentary and migratory populations was maintained even when exposed to ALAN. This variation in the seasonal reproductive timing may likely have a genetic ground or early developmental effects imposed due to different latitude of origin. This study reveals first that latitude-dependent variation in reproductive timing is maintained despite exposure to ALAN, and second that ALAN accelerated reproductive development across both migrants and residents. The results corroborating relationship between latitude, population, and ALAN impact on seasonal reproductive timing, may provide a potential mechanism to test the fitness of a population and its range expansion to exploit urban environment. ### Competing Interest Statement The authors have declared no competing interest.

Individuals and populations time annual events such as migration and reproduction to match favorable times in their environment. Physiological preparations for reproduction rely on predictive cues such as day length to accurately time reproduction. In birds, preparation typically begins with light reception by the hypothalamus, which initiates multiple central and peripheral responses. We studied two closely related populations of a songbird, the dark-eyed junco, that live in a common winter environment but diverge in their timing of reproduction as spring approaches. One population is resident and initiates reproduction earlier than the other, which migrates northward prior to reproducing. We caught resident and migrant juncos from the field during early spring and collected hypothalamic and pituitary tissues. We used isobaric tandem mass tag (TMT) labeling to identify differentially expressed proteins (DEPs) as possible regulators of the seasonal divergence in reproductive timing. We found 3038 unique proteins expressed in the hypothalamus and pituitary proteome, among which we identified 75 DEPs. These were associated with hormones, neurotransmitter secretion, transport, neuropeptide synthesis, prohormone synthesis, neurogenesis, GnRH synthesis, release and stability, food intake, locomotion, and social behavior. Some of these proteins were associated with early breeding in resident juncos, and others were associated with increased food intake, fat metabolism, locomotor activity and phenology in migratory juncos. Our results provide new insight into the neuroendocrine regulation of the timing of reproduction and migration. This study provides the first evidence of a relationship between functional protein variation in the neuroendocrine tissues and seasonal divergence in reproductive timing.

The initiation of reproduction in many seasonally breeding animals is controlled by photoperiod and tends to be clinal: populations at higher latitudes breed later than those at lower latitudes, often reflecting a higher photoperiodic threshold. Migratory animals presumably time reproduction to match conditions at their breeding grounds, at least in part, by cues perceived on their wintering grounds. We asked how closely related dark-eyed junco (Junco hyemalis) populations that overwinter in sympatry but breed in allopatry respond to their shared winter environment by comparing early spring indices of readiness to migrate (fat and muscle condition) and breed (baseline and elevated testosterone). We measured stable hydrogen isotopes from feathers grown the preceding year and claws grown during winter to estimate breeding and wintering latitudes, respectively. We predicted that if reproductive initiation is adapted to the emergence of resources at their respective breeding destinations, then birds migrating to higher latitudes (slate-coloured junco; J. h. hyemalis) should delay breeding as compared with those migrating to lower latitudes (pink-sided junco; J. h. mearnsi) despite a common overwinter environment. We found higher testosterone in pink-sided juncos, consistent with earlier reproductive initiation, suggesting local adaptation in reproductive phenology is achieved through differential responses to predictive environmental cues.

Urban habitats can shape interactions between hosts and parasites by altering not only exposure rates but also within-host processes. Artificial light at night (ALAN) is common in urban environments, and chronic exposure can impair host immunity in ways that may increase infection. However, studies of causal links between this stressor, immunity, and infection dynamics are rare, particularly in migratory animals. Here, we experimentally tested how ALAN affects cellular immunity and haemosporidian parasite intensity across the annual cycle of migrant and resident subspecies of the dark-eyed junco (Junco hyemalis). We monitored an experimental group exposed to light at night and a control group under natural light/dark cycles as they passed through short days simulating early spring to longer days simulating the breeding season, followed by autumn migration. Using generalized additive mixed models, we show that ALAN increased inflammation, and leucocyte counts were greatest in early spring and autumn. At the start of the experiment, few birds had active infections based on microscopy, but PCR revealed many birds had chronic infections. ALAN increased parasitaemia across the annual cycle, with strong peaks in spring and autumn that were largely absent in control birds. As birds were kept in indoor aviaries to prevent vector exposure, this increased parasitaemia indicates relapse of chronic infection during costly life-history stages (i.e. reproduction). Although the immunological and parasitological time series were in phase for control birds, cross-correlation analyses also revealed ALAN desynchronized leucocyte profiles and parasitaemia, which could suggest a general exaggerated inflammatory response. Our study shows how a common anthropogenic influence can shape within-host processes to affect infection dynamics.

In the modern era of industrialization, illuminated nights have become a common defining feature of human-occupied environments, particularly cities. Artificial light at night (ALAN) imposes several known negative impacts on the neuroendocrine system, metabolism, and seasonal reproduction of species living in the wild. However, we know little about the impact of ALAN on populations of birds that either live year-round in the same location or move to different latitudes across seasons. To test whether ALAN has differing impact on reproductive timing of the bird populations that winter in sympatry but breed at different latitudes, we monitored sedentary and migratory male dark-eyed juncos that were or were not exposed to low intensity (~2.5 ± 0.5 lux) ALAN. All groups were held in common conditions and day length was gradually increased to mimic natural day length changes (NDL). We assessed seasonal reproductive response from initiation to termination of the breeding cycle. As expected based on earlier research, the sedentary birds exhibited earlier gonadal recrudescence and terminated breeding later than the migratory birds. In addition, resident and migrant birds exposed to ALAN initiated gonadal recrudescence earlier and terminated reproduction sooner as compared to their conspecifics experiencing NDL. Importantly, the difference in the reproductive timing of sedentary and migratory populations was maintained even when exposed to ALAN. This variation in the seasonal reproductive timing may likely have a genetic ground or early developmental effects imposed due to different latitude of origin. This study reveals first that latitude-dependent variation in reproductive timing is maintained despite exposure to ALAN, and second that ALAN accelerated reproductive development across both migrants and residents. The results corroborating relationship between latitude, population, and ALAN impact on seasonal reproductive timing, may provide a potential mechanism to test the fitness of a population and its range expansion to exploit urban environment.

NEW FINDINGS:What is the central question of this study? What are the molecular underpinnings of the seasonal adaptation in a latitudinal migratory songbird? What is the main finding and its importance? We found changes in mRNA levels after a photoperiod-induced alteration of seasonal state in a captive long-distance latitudinal avian migrant. The hypothalamus and liver transcriptomes revealed genes involved in the regulatory and functional pathways between non-migratory and migratory states. Our results provide insights into mechanisms underlying homeostasis during seasonal changes that are conserved across most species, including humans.ABSTRACT:Very little is understood about genetic mechanisms underlying the onset of spring migration in latitudinal avian migrants. To gain insight into the genetic architecture of the hypothalamus and liver tissues of a long-distance migrant, we examined and compared the transcriptome profile of captive night-migratory black-headed buntings (Emberiza melanocephala) between photoperiod-induced winter non-migratory (WnM) and spring migratory (SM) life-history states under short and long days, respectively. High-throughput 454 pyrosequenced transcripts were mapped initially with reference to the genome of two phylogenetically close species, Taeniopygia guttata and Ficedula albicollis. The F. albicollis genome gave higher annotation results and was used for further analysis. A total of 216 (78 in hypothalamus; 138 in liver) genes were found to be expressed differentially between the WnM and SM life-history states. These genes were enriched for physiological pathways that might be involved in the regulation of seasonal migrations in birds. For example, genes for the ATP binding pathway in the hypothalamus were expressed at a significantly higher level in SM than in the WnM life-history state. Likewise, upregulated genes associated with the myelin sheath and focal adhesion were enriched in the hypothalamus, and those with cell-to-cell junction, intracellular protein transport, calcium ion transport and small GTPase-mediated signal transduction were enriched in the liver. Many of these genes are a part of physiological pathways potentially involved in the regulation of seasonal migration in birds. These results show molecular changes at the regulatory and metabolic levels associated with seasonal transitions in a long-distance migrant and provide the basis for future studies aimed at unravelling the genetic control of migration in birds.

The molecular underpinnings of metabolic adaptation to seasons are poorly understood in long- distance migrants. We measured changes in physiology and performed de novo sequencing of RNA extracted from liver samples collected at 4-h intervals over a period of 24 h from a long-distance avian migrant, the blackheaded bunting (Emberiza melanocephala), during two states: photostimulated vernal migratory (M) state and photorefractory non-migratory (nM) state. The M state was differentiated from the nM state based on body fattening and weight gain, as well as on Zugunruhe, that is, nocturnal migratory restlessness in caged birds. We found that baseline blood glucose and triglyceride levels were significantly higher in the M state than the nM state; conversely, surface body temperature was higher in the nM state than the M state. In a total of 6 liver samples that were sequenced from each state, 11,246 genes were annotated, including 4448 genes that were cyclic over 24 h. We found 569 differentially expressed genes (DEGs) between the M and the nM state, and the M state showed 131 upregulated and 438 downregulated genes. These DEGs formed core gene hubs associated with specific biological processes in both the states. In addition, weighted gene coexpression network analysis revealed two discrete modules of coexpressed genes, with a significant difference in the expression pattern of metab olism-associated genes between M and nM states. These results demonstrate, for the first time, transcriptome-wide changes in the liver between two distinct physiological states and give molecular insights into seasonal metabolic adaptations in latitudinal migrants.

We hypothesized differences in molecular strategies for similar journeys that migrants undertake to reproduce in spring and to overwinter in autumn. We tested this in redheaded buntings (Emberiza bruniceps) photoinduced into spring and autumn migratory states, with winter and summer non-migratory states as controls. Compared with controls, buntings fattened, gained weight and showed Zugunruhe (nocturnal migratory restlessness) in the migratory state. Spring migration was associated with greater fat and body mass, and higher intensity of Zugunruhe, compared with autumn migration. Circulating corticosterone levels were higher in spring, while T3 levels were higher in autumn. Hypothalamic expression of thyroid hormone-responsive (dio2, dio3), light-responsive (per2, cry1, adcyap1) and th (tyrosine hydroxylase, involved in dopamine biosynthesis) genes showed significant changes with transition from non-migratory to the migratory state. There were significantly higher mRNA expressions in autumn, except for higher th levels in the spring. Furthermore, the expression patterns of dnmt3a (not dnmt3b) and tet2 genes suggested an epigenetic difference between the non-migrant and migrant periods, and the spring and autumn migrant periods. These results demonstrate for the first time seasonal transition in hypothalamic gene expressions, and suggest differences in regulatory strategies at the transcriptional level for spring and autumn migrations in songbirds.

The circadian rhythms are involved in the photostimulation of seasonal responses in migratory blackheaded buntings. Here, we investigated whether changes in daily levels and rhythm in corticosterone (cort) and insulin secretions were associated with transitions in the photoperiodic seasonal states. Buntings were exposed to short days to maintain the winter (photosensitive) non-migratory state, and to long days for varying durations to induce the premigratory, migratory (shown by migratory restlessness at night, Zugunruhe) and summer non-migratory (photorefractory) states. We monitored activity patterns, and measured plasma cort and insulin levels at six and four times, respectively, over 24h in each seasonal state. Buntings were fattened and weighed heavier, and exhibited intense nighttime activity in the migratory state. The daytime activity patterns also showed seasonal differences, with a bimodal pattern with morning and evening activity bouts only in the summer non-migratory state. Further, the average baseline hormone levels were significantly higher in premigratory and migratory than in the winter non-migratory state. Both cort and insulin levels showed a significant daily rhythm, but with seasonal differences. Whereas, cort rhythm acrophases (estimated time of peak secretion over 24h) were at night in the winter non-migratory, premigratory and migratory states, the insulin rhythm acrophases were found early in the day and night in winter and summer non-migratory states, respectively. These results suggest that changes in daily levels and rhythm in cort and insulin mediate changes in the physiology and behavior with photostimulated transition in seasonal states in migratory blackheaded buntings.

The present study investigated seasonal alterations in the daily rhythms of hypothalamic expression of genes involved in the photoperiodic regulation of annual cycles in birds. We measured the 4‐hourly mRNA expression of genes involved in the photoperiodic transduction (OPN5, EYA3, CGA, TSHβ, DIO2, DIO3) and neurosteroid‐dependent processes (AR, CYP19, ERα, ERβ) in the hypothalamus of migratory blackheaded buntings photoinduced with photosensitive, photostimulated (early and late stimulated) and photorefractory seasonal states. There were significant differences in daily mRNA profiles between the photoperiodic states. Particularly, increased CGA, TSHβ and DIO2 and decreased DIO3 mRNA levels in the early photostimulated state, compared to the photosensitive state, suggest that thyroid hormones have a role in photostimulation in buntings. Similar differences in the expression of genes coding for the aromatase enzyme (CYP19) and receptors for oestrogen (ERα, ERβ) (but not androgen; AR) indicate that there is seasonal alteration in the neuro‐oestrogen‐mediated functions. Furthermore, peak expression times of CGA, TSHβ and DIO2 genes at hours 14‐15 of the day in the early stimulated state indicated molecular regulation of the daily rhythm of photoinducibility in buntings. Most significantly, however, we found an attenuated daily rhythm in thyroid hormone modulatory genes and a switch of peak expression time from day to night in CYP19 mRNA rhythm in the subsequent late photostimulated state, although testicular maturation still persisted. These alterations in daily rhythms may have signalled the initiation of processes underlying other seasonal phenologies in parallel with the gonadal response, such as a manifestation of the night‐time flight in buntings. These results show alterations in daily rhythms underlying the transcriptional regulation of the photoperiod‐induced seasonal states in migratory blackheaded buntings.

This study investigated whether, in photoperiodic songbirds, the circadian pacemaker system (CPS) connects to the seasonal photoperiodic responses, by changes at transcriptional level in the level and 24-h rhythm of its constituent neurotransmitters. We used black-headed buntings (Emberiza melanocephala), which exhibit distinct seasonal states in captivity under appropriate photoperiods and hence served as a useful model system. Under short days, buntings remain in the photosensitive state (Pse) (winter phenotype: non-migratory, non-breeding). Under long days, however, buntings undergo through early-photostimulated (spring phenotype: pre-migratory, pre-breeding), late photostimulated (summer phenotype: migratory, breeding) and photorefractory (autumn phenotype: post-breeding) states. During all four seasonal states, we measured in the retina, pineal and hypothalamus, which together form avian CPS, 4-hourly mRNA expression of c-fos (a neuronal-activity marker) and of genes coding for neuropeptides (vasoactive intestinal peptide, vip; somatostatin, sst; neuropeptide Y, npy) and for intermediary enzymes of amino acid (glutamate: glutaminase, gls and glutamic-oxaloacetic transaminase 2, got2; GABA: glutamic acid decarboxylase, gad65) and amine (dopamine: tyrosine hydroxylase, th) neurotransmitters biosynthetic pathway. There was a significant alteration in level and 24-h pattern of mRNA expression, albeit with seasonal differences in presence, waveform parameters and phase relationship of 24-h rhythm, of different genes. Particularly, mRNA expression of all candidate genes (except hypothalamic vip, pineal gls and retinal th) was arrhythmic in late photostimulated state. These results underscore that circadian rhythm of peptide, amino acid and amine neurotransmitter biosynthesis in CPS plays a critical role in the photoperiodic regulation of seasonal states in birds.