Photoperiodically driven transcriptome-wide changes in the hypothalamus reveal transcriptional differences between physiologically contrasting seasonal life-history states in migratory songbirds.

We investigated time course of photoperiodically driven transcriptional responses in physiologically contrasting seasonal life-history states in migratory blackheaded buntings. Birds exhibiting unstimulated winter phenotype (photosensitive state; responsive to photostimulation) under 6-h short days, and regressed summer phenotype (photorefractory state; unresponsiveness to photostimulation) under 16-h long days, were released into an extended light period up to 22 h of the day. Increased tshß and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1ß mRNA levels in blood confirmed photoperiodic induction by hour 18 in photosensitive birds. Further, at hours 10, 14, 18 and 22 of light exposure, the comparison of hypothalamus RNA-Seq results revealed transcriptional differences within and between states. Particularly, we found reduced expression at hour 14 of transthyretin and proopiomelanocortin receptor, and increased expression at hour 18 of apolipoprotein A1 and carbon metabolism related genes in the photosensitive state. Similarly, valine, leucine and isoleucine degradation pathway genes and superoxide dismutase 1 were upregulated, and cocaine- and amphetamine-regulated transcript and gastrin-releasing peptide were downregulated in the photosensitive state. These results show life-history-dependent activation of hypothalamic molecular pathways involved in initiation and maintenance of key biological processes as early as on the first long day.

Seasonal reproductive state determines gene expression in the hypothalamus of a latitudinal migratory songbird during the spring and autumn migration.

We investigated gonadal effects on hypothalamic transcription of genes in sham-operated and castrated redheaded buntings photostimulated into spring and autumn migratory states. RNA-Seq results showed testes-dependent differences between spring and autumn migratory states. In particular, differentially expressed genes enriched G-protein-coupled receptor and calcium-ion signaling pathways during spring and autumn states, respectively. qPCR assay showed attenuated gabra5, ttr, thra and thrb expressions, suggesting reduced GABA and thyroid hormone effects on photo-sexual response in spring. In spring castrates, reduced npy, tac1 and nrcam and increased ank3 expression suggested testicular effects on the appetite, prolactin release and neuronal functions, whereas in autumn castrates, reduced rasgrp1, grm5 and grin1, and increased mras expression suggested testicular effects on the ras, G-protein and glutamate signaling pathways. Castration-induced reciprocal switching of pomc and pdyn expressions suggested effects on the overall homeostasis in both seasons. These results demonstrate transcriptome-wide changes, with season-dependent roles of testes in songbird migration.

Exploration of tissue-specific gene expression patterns underlying timing of breeding in contrasting temperature environments in a song bird.

BACKGROUND: Seasonal timing of breeding is a life history trait with major fitness consequences but the genetic basis of the physiological mechanism underlying it, and how gene expression is affected by date and temperature, is not well known. In order to study this, we measured patterns of gene expression over different time points in three different tissues of the hypothalamic-pituitary-gonadal-liver axis, and investigated specifically how temperature affects this axis during breeding. We studied female great tits (Parus major) from lines artificially selected for early and late timing of breeding that were housed in two contrasting temperature environments in climate-controlled aviaries. We collected hypothalamus, liver and ovary samples at three different time points (before and after onset of egg-laying). For each tissue, we sequenced whole transcriptomes of 12 pools (n = 3 females) to analyse gene expression. RESULTS: Birds from the selection lines differed in expression especially for one gene with clear reproductive functions, zona pellucida glycoprotein 4 (ZP4), which has also been shown to be under selection in these lines. Genes were differentially expressed at different time points in all tissues and most of the differentially expressed genes between the two temperature treatments were found in the liver. We identified a set of hub genes from all the tissues which showed high association to hormonal functions, suggesting that they have a core function in timing of breeding. We also found ample differentially expressed genes with largely unknown functions in birds. CONCLUSIONS: We found differentially expressed genes associated with selection line and temperature treatment. Interestingly, the latter mainly in the liver suggesting that temperature effects on egg-laying date may happen down-stream in the physiological pathway. These findings, as well as our datasets, will further the knowledge of the mechanisms of tissue-specific avian seasonality in the future.

Fine-tuning of seasonal timing of breeding is regulated downstream in the underlying neuro-endocrine system in a small songbird.

The timing of breeding is under selection in wild populations as a result of climate change, and understanding the underlying physiological processes mediating this timing provides insight into the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in the timing of breeding in females are reflected in differences in candidate gene expression and, if so, whether these differences occur in the upstream (hypothalamus) or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg laying, which were housed in climate-controlled aviaries and went through two breeding cycles within 1 year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on the timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with the timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues, with the most pronounced differences in the hypothalamus. Differences between time points, and early- and late-laying females, were found exclusively in the ovary and liver. Altogether, we show that fine-tuning of the seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system.

Seasonally sympatric but allochronic: differential expression of hypothalamic genes in a songbird during gonadal development.

Allochrony, the mismatch of reproductive schedules, is one mechanism that can mediate sympatric speciation and diversification. In songbirds, the transition into breeding condition and gonadal growth is regulated by the hypothalamic-pituitary-gonadal (HPG) axis at multiple levels. We investigated whether the difference in reproductive timing between two seasonally sympatric subspecies of dark-eyed juncos (Junco hyemalis) was related to gene expression along the HPG axis. During the sympatric pre-breeding stage, we measured hypothalamic and testicular mRNA expression of candidate genes via qPCR in captive male juncos. For hypothalamic mRNA, we found our earlier breeding subspecies had increased expression of gonadotropin-releasing hormone (GnRH) and decreased expression of androgen receptor, oestrogen receptor alpha and mineralocorticoid receptor (MR). Subspecies did not differ in expression of hypothalamic gonadotropin-inhibitory hormone (GnIH) and glucocorticoid receptor (GR). While our earlier breeding subspecies had higher mRNA expression of testicular GR, subspecies did not differ in testicular luteinizing hormone receptor, follicle-stimulating hormone receptor or MR mRNA expression levels. Our findings indicate increased GnRH production and decreased hypothalamic sensitivity to sex steroid negative feedback as factors promoting differences in the timing of gonadal recrudescence between recently diverged populations. Differential gene expression along the HPG axis may facilitate species diversification under seasonal sympatry.

Difference in control between spring and autumn migration in birds: insight from seasonal changes in hypothalamic gene expression in captive buntings.

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.

Temporal expression of clock genes in central and peripheral tissues of spotted munia under varying light conditions: Evidence for circadian regulation of daily physiology in a non-photoperiodic circannual songbird species.

We investigated if the duration and/or frequency of the light period affect 24-h rhythm of circadian clock genes in central and peripheral tissues of a non-photoperiodic songbird, the spotted munia (Lonchura punctulata), in which a circannual rhythm regulates the reproductive cycle. We monitored activity-rest pattern and measured 24-h mRNA oscillation of core clock (Bmal1, Clock, Per2, Cry1 and Cry2) and clock-controlled (E4bp4, Rorα and Rev-erbα) genes in the hypothalamus, retina, liver and gut of spotted munia subjected to an aberrant light-dark (LD) cycle (3.5L:3.5D; T7, T = period length of LD cycle) and continuous light (LL, 24L:0D), with controls on 24-h LD cycle (T24, 12L:12D). Munia exhibited rhythmic activity-rest pattern with period matched to T7 or T24 under an LD cycle and were arrhythmic with a scattered activity pattern and higher activity duration under LL. At the transcriptional level, both clock and clock-controlled genes showed a significant 24-h rhythm in all four tissues (except Clock in the liver) under 12L:12D, suggesting a conserved tissue-level circadian time generation in spotted munia. An exposure to 3.5L:3.5D or LL induced arrhythmicity in transcriptional oscillation of all eight genes in the hypothalamus (except Rev-erbα) and liver (except Bmal1 and Rev-erbα under T7 and Cry1 under LL). In the retina, however, all genes showed arrhythmic 24-h mRNA expression under LL, but not under T7 (except in E4bp4 and Rorα). Interestingly, unlike in the liver, Bmal1, Per2, Cry1, Rorα and Rev-erbα mRNA expressions were rhythmic in the gut under both T7 (except Rorα) and LL conditions. These results showed variable relationship of internal circadian clocks with the external light environment and suggested a weak coupling of circadian clocks between the central (hypothalamus and retina) and peripheral (liver and gut) tissues. We suggest tissue-level circadian clock regulation of daily physiology and behavior in the spotted munia.

Concurrent hypothalamic gene expression under acute and chronic long days: Implications for initiation and maintenance of photoperiodic response in migratory songbirds.

Hypothalamic expression of the thyroid hormone (TH) responsive gonadostimulatory (eya3, cga, tshß, dio2, dio3, gnrh, gnih) and neurosteroid pathway genes (androgen receptor [ar], aromatase [cyp19], estrogen receptor [er] α and ß) was examined in photosensitive redheaded buntings exposed to 2 (acute, experiment 1) or 12 (chronic, experiment 2) long days (16L:8D). Experiment 2 also included a photorefractory group. Acute long days caused a significant increase in eya3, cga, tshß, dio2 and gnrh and decrease in dio3 mRNA levels. eya3, cga and tshß expressions were unchanged after the chronic long days. We also found increased cyp19, erα and erß mRNA levels after acute, and increased cyp19 and decreased erß levels after the chronic long-day exposure. Photorefractory buntings showed expression patterns similar to that in the photosensitive state, except for high gnrh and gnih and low dio3 mRNA levels. Consistent with gene expression patterns, there were changes in fat deposition, body mass, testis size, and plasma levels of testosterone, tri-iodothyronine and thyroxine. These results show concurrent photostimulation of the TH-signalling and neurosteroid pathways, and extend the idea, based on differences in gene expression, that transitions in seasonal photoperiodic states are accomplished at the transcriptional levels in absolute photorefractory species.

Pinealectomy abolishes circadian behavior and interferes with circadian clock gene oscillations in brain and liver but not retina in a migratory songbird.

In songbirds, the pineal gland is part of the multi-oscillatory circadian timing system, with participating component oscillators in the eyes and hypothalamus. This study investigated the role of the pineal gland in development of the nighttime migratory restlessness (Zugunruhe) and generation of circadian gene oscillations in the retina, brain and liver tissues in migratory redheaded buntings (Emberiza bruniceps). Pinealectomized (pinx) and sham-operated buntings entrained to short days (8h light: 16h darkness, 8L:16D) were sequentially exposed for 10days each to stimulatory long days (13L: 11D) and constant dim light (LLdim; a condition that tested circadian rhythm persistence). Whereas activity-rest pattern was monitored continuously, the mRNA expressions of clock genes (bmal1, clock, npas2, per2, cry1, rorα, reverα) were measured in the retina, hypothalamus, telencephalon, optic tectum and liver tissues at circadian times, CT, 1, 6, 13, 17 and 21 (CT 0, activity onset) on day 11 of the LLdim. The absence of the pineal gland did not affect the development of long-day induced Zugunruhe but caused decay of the circadian rhythm in Zugunruhe as well as the clock gene oscillations in the hypothalamus, but not in the retina. Further, there were variable effects of pinealectomy in the peripheral brain and liver tissue circadian gene oscillations, notably the persistence of per 2 and cry1 (optic tectum), rorα (telencephalon) and npas2 (liver) mRNA oscillations in pinx birds. We suggest the pineal gland dependence of the generation of circadian gene oscillations in the hypothalamus, not retina, and peripheral brain and liver tissues in migratory redheaded buntings.

Circadian timing in central and peripheral tissues in a migratory songbird: dependence on annual life-history states.

Predictable seasonal change in photoperiod triggers a sequential change in the daily activity-rest pattern, adaptive for migration in several bird species. The night-migratory black-headed bunting (Emberiza melanocephala) is day active under short photoperiods (8 h light:16 h dark, short day sensitive). Under long photoperiods (16 h light:8 h dark), the buntings are initially day active (long day premigratory) but subsequently become intensely night active (long day migratory) and after few weeks again return to a day active pattern (long day refractory). However, it is unclear how the daily expression of circadian genes changes during photoperiod-induced seasonal life-history states (LHSs). We measured period 2 (Per2), cryptochrome 1 (Cry1), brain and muscle arnt-like protein 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) mRNA expressions in various neural and peripheral tissues of buntings in different LHSs and discovered differences of ∼2 to 6 h in the phase and 2- to 4-fold in amplitude of circadian oscillations of Per2, Cry1, and Bmal1 between photoperiod-induced LHSs. Phase relationship in mRNA oscillations was altered between oscillator components in the circadian pacemaker system (retina, pineal, hypothalamus) as well as in the peripheral (liver, muscle) tissues. These results show for the first time altered waveforms of clock gene expressions in all tissues in parallel with behavioral shifts and suggest the involvement of circadian system in photoperiod induction of seasonal LHSs in a migratory species.

Hypothalamic gene switches control transitions between seasonal life history states in a night-migratory photoperiodic songbird.

This study investigated photoperiodic plasticity in hypothalamic expression of genes implicated in the photoperiodic light perception (rhodopsin, melanopsin, neuropsin and peropsin), transduction (pax6, bmal1, clock, per2 and casr), induction (eya3, tshß, dio2 and dio3, gnrh and gnih) and metabolism (NPY, sirtuin1, foxO1, hmgcr, citrate synthase and dehydrogenases) in photosensitive and photorefractory redheaded buntings. There was a significant increase in eya3, tsh ß, dio2, pax6 and rhodopsin and decrease in dio3 mRNA expression at hour 15 and/or 19 on the day photosensitive buntings were subjected to a 13- or 16 h, but not to 8- and 11 h light exposure. Downstream reproductive and metabolic gene expression was not altered, except for an increase in those genes coding for succinate and malate dehydrogenase enzymes involved in lipogenesis. Photorefractory buntings had high dio3 mRNA expression which significantly declined after 1 short day exposure, suggesting possible involvement of dio3 in the maintenance of photorefractoriness. Positive correlation of rhodopsin on eya 3 and tshß indicates its role in photoperiodic timing, perhaps involving the peropsin and pax6 genes. These results suggest that rapid switching of hypothalamic gene expression underlies photoperiod-induced seasonal plasticity and regulates transitions from photosensitive to photostimulated and from photorefractory to photosensitive states in migratory songbirds.

Annual life history-dependent gene expression in the hypothalamus and liver of a migratory songbird: insights into the molecular regulation of seasonal metabolism.

Birds seasonally switch from one life history state (LHS) to another to maximize their fitness. Accordingly, they exhibit distinct differences in their physiological and behavioral phenotypes between seasons. Possible molecular mechanisms underlying changes through the seasons have scarcely been examined in migratory birds. The present study measured key genes suggested to be involved in the metabolic regulation of 4 photoperiodically induced seasonal LHSs in a long-distance migratory songbird, the blackheaded bunting (Emberiza melanocephala). Buntings were held under short days (8 h light:16 h darkness, 8L:16D), during which they maintained the winter nonmigratory phenotype. Then they were exposed for several weeks to long days (13L:11D). Differences in the activity-rest pattern, body fattening and weight gain, testis size, organ (heart, intestine) weights, and blood glucose and triglyceride levels confirmed that buntings sequentially exhibited spring migration-linked premigratory, migratory, and postmigratory LHSs under long days. The mRNA levels of circadian genes involved in metabolism (Bmal1, Clock, Npas2, Rorα, and Rev-erbα) and of genes that encode for proteins/enzymes involved in the regulation of glucose (Sirt1, FoxO1, Glut1, and Pygl) and lipids (Hmg-CoA; Pparα, Pparγ; Fasn and Acaca) showed LHS-dependent changes in their light-dark expression patterns in the hypothalamus and liver. These initial results on genetic regulation of metabolism in a migratory species extend the idea that the transitions between LHSs in a seasonal species are accomplished by changes at multiple regulatory levels. Thus, these findings promise new insights into the mechanism(s) of adaptation to seasons in higher vertebrates.

Daily expression of six clock genes in central and peripheral tissues of a night-migratory songbird: evidence for tissue-specific circadian timing.

In birds, independent circadian clocks reside in the retina, pineal, and hypothalamus, which interact with each other and produce circadian time at the functional level. However, less is known of the molecular clockwork, and of the integration between central and peripheral clocks in birds. The present study investigated this, by monitoring the timed expression of five core clock genes (Per2. Cry1. Cry2. Bmal1, and Clock) and one clock-controlled gene (E4bp4) in a night-migratory songbird, the redheaded bunting (rb; Emberiza bruniceps). The authors first partially cloned these six genes, and then measured their 24-h profiles in central (retina, hypothalamus) and peripheral (liver, heart, stomach, gut, testes) tissues, collected at six times (zeitgeber time 2 [ZT2], ZT6, ZT11, ZT13, ZT18, and ZT23; ZT0 = lights on) from birds (n = 5 per ZT) on 12 h:12 h light-dark cycle. rbPer2. rbCry1. rbBmal1, and rbClock were expressed with a significant rhythm in all the tissues, except in the retina (only rbClock) and testes. rbCry2, however, had tissue-specific expression pattern: a significant rhythm in the hypothalamus, heart, and gut, but not in the retina, liver, stomach, and testes. rbE4bp4 had a significant mRNA rhythm in all the tissues, except retina. Further, rbPer2 mRNA peak was phase aligned with lights on, whereas rbCry1. rbBmal1, and rbE4bp4 mRNA peaks were phase aligned with lights off. rbCry2 and rbClock had tissue-specific scattered peaks. For example, both rbCry2 and rbClock peaks were close to rbCry1 and rbBmal1 peaks, respectively, in the hypothalamus, but not in other tissues. The results are consistent with the autoregulatory circadian feedback loop, and indicate a conserved tissue-level circadian time generation in buntings. Variable phase relationships between gene pairs forming positive and negative limbs of the feedback loop may suggest the tissue-specific contribution of individual core circadian genes in the circadian time generation.

Neural steroid sensitivity and aggression: comparing individuals of two songbird subspecies.

Hormones coordinate the expression of complex phenotypes and thus may play important roles in evolutionary processes. When populations diverge in hormone-mediated phenotypes, differences may arise via changes in circulating hormones, sensitivity to hormones or both. Determining the relative importance of signal and sensitivity requires consideration of both inter- and intrapopulation variation in hormone levels, hormone sensitivity and phenotype, but such studies are rare, particularly among closely related taxa. We compared males of two subspecies of the dark-eyed junco (Junco hyemalis) for territorial aggression and associations among behaviour, circulating testosterone (T), and gene expression of androgen receptor (AR), aromatase (AROM) and oestrogen receptor α in three behaviourally relevant brain regions. Thus, we examined the degree to which evolution may shape behaviour via changes in plasma T as compared with key sex steroid binding/converting molecules. We found that the white-winged junco (J. h. aikeni) was more aggressive than the smaller, less ornamented Carolina junco (J. h. carolinensis). The subspecies did not differ in circulating testosterone, but did differ significantly in the abundance of AR and AROM mRNA in key areas of the brain. Within populations, both gene expression and circulating T co-varied significantly with individual differences in aggression. Notably, the differences identified between populations were opposite to those predicted by the patterns among individuals within populations. These findings suggest that hormone-phenotype relationships may evolve via multiple pathways, and that changes that have occurred over evolutionary time do not necessarily reflect standing physiological variation on which current evolutionary processes may act.

Neural sensitivity to sex steroids predicts individual differences in aggression: implications for behavioural evolution.

Testosterone (T) regulates many traits related to fitness, including aggression. However, individual variation in aggressiveness does not always relate to circulating T, suggesting that behavioural variation may be more closely related to neural sensitivity to steroids, though this issue remains unresolved. To assess the relative importance of circulating T and neural steroid sensitivity in predicting behaviour, we measured aggressiveness during staged intrusions in free-living male and female dark-eyed juncos (Junco hyemalis). We compared aggressiveness to plasma T levels and to the abundance of androgen receptor (AR), aromatase (AROM) and oestrogen receptor alpha (ORα) mRNA in behaviourally relevant brain areas (avian medial amygdala, hypothalamus and song control regions). We also asked whether patterns of covariation among behaviour and endocrine parameters differed in males and females, anticipating that circulating T may be a better predictor of behaviour in males than in females. We found that circulating T related to aggressiveness only in males, but that gene expression for ORα, AR and AROM covaried with individual differences in aggressiveness in both sexes. These findings are among the first to show that individual variation in neural gene expression for three major sex steroid-processing molecules predicts individual variation in aggressiveness in both sexes in nature. The results have broad implications for our understanding of the mechanisms by which aggressive behaviour may evolve.

Testing alternative hypotheses for evolutionary diversification in an African songbird: rainforest refugia versus ecological gradients.

Geographic isolation in rainforest refugia and local adaptation to ecological gradients may both be important drivers of evolutionary diversification. However, their relative importance and the underlying mechanisms of these processes remain poorly understood because few empirical studies address both putative processes in a single system. A key question is to what extent is divergence in signals that are important in mate and species recognition driven by isolation in rainforest refugia or by divergent selection across ecological gradients? We studied the little greenbul, Andropadus virens, an African songbird, in Cameroon and Uganda, to determine whether refugial isolation or ecological gradients better explain existing song variation. We then tested whether song variation attributable to refugial or ecological divergence was biologically meaningful using reciprocal playback experiments to territorial males. We found that much of the existing song variation can be explained by both geographic isolation and ecological gradients, but that divergence across the gradient, and not geographic isolation, affects male response levels. These data suggest that ecologically divergent traits, independent of historical isolation during glacial cycles, can promote reproductive isolation. Our study provides further support for the importance of ecology in explaining patterns of evolutionary diversification in ecologically diverse regions of the planet.

Memory in the making: localized brain activation related to song learning in young songbirds.

Songbird males learn to sing their songs from an adult 'tutor' early in life, much like human infants learn to speak. Similar to humans, in the songbird brain there are separate neural substrates for vocal production and for auditory memory. In adult songbirds, the caudal pallium, the avian equivalent of the auditory association cortex, has been proposed to contain the neural substrate of tutor song memory, while the song system is involved in song production as well as sensorimotor learning. If this hypothesis is correct, there should be neuronal activation in the caudal pallium, and not in the song system, while the young bird is hearing the tutor song. We found increased song-induced molecular neuronal activation, measured as the expression of an immediate early gene, in the caudal pallium of juvenile zebra finch males that were in the process of learning to sing their songs. No such activation was found in the song system. Molecular neuronal activation was significantly greater in response to tutor song than to novel song or silence in the medial part of the caudomedial nidopallium (NCM). In the caudomedial mesopallium, there was significantly greater molecular neuronal activation in response to tutor song than to silence. In addition, in the NCM there was a significant positive correlation between spontaneous molecular neuronal activation and the strength of song learning during sleep. These results suggest that the caudal pallium contains the neural substrate for tutor song memory, which is activated during sleep when the young bird is in the process of learning its song. The findings provide insight into the formation of auditory memories that guide vocal production learning, a process fundamental for human speech acquisition.

Carotenoid availability in diet and phenotype of blue and great tit nestlings.

Carotenoids are biologically active pigments of crucial importance for the development of avian embryos and nestlings. Thus parental ability to provide nestlings with a carotenoid-rich diet may enhance offspring fitness. However, very little is known about the possible effects of carotenoid availability in the diet on growing nestlings in natural populations. We experimentally manipulated dietary intake of carotenoids by nestlings of two closely related passerine species, the great tit Parus major and the blue tit Parus caeruleus, and measured nestling antioxidants, body condition, immunity and plumage colour. There was no detectable increase in plasma carotenoids after treatment in carotenoid-fed nestlings of either species despite regular supply of dietary carotenoids. However, in carotenoid-fed blue tit nestlings, plasma vitamin E concentration increased with plasma carotenoid concentration, while that was not the case for control nestlings. In both species, there was no significant effect of carotenoid supply on immune function. Carotenoid supplementation enhanced yellow feather colour in great tit nestlings only. In both species a strong effect of carotenoid supply was found on body condition with an increase in body mass for small carotenoid-fed nestlings compared to similarly sized control nestlings. Dietary availability of carotenoids may thus have important fitness consequences for tits. We hypothesise that the difference in effect of dietary carotenoids on the two species is due to relatively larger clutch size and higher growth rates of blue tits compared to great tits, leading to blue tit nestlings being more in need of carotenoids for antioxidant function than great tit nestlings.

Proximate mechanisms of variation in the carotenoid-based plumage coloration of nestling great tits (Parus major L.).

Many vertebrates use carotenoid-based signals in social or sexual interactions. Honest signalling via carotenoids implies some limitation of carotenoid-based colour expression among phenotypes in the wild, and at least five limiting proximate mechanisms have been hypothesized. Limitation may arise by carotenoid-availability, genetic constraints, body condition, parasites, or detrimental effects of carotenoids. An understanding of the relative importance of the five mechanisms is relevant in the context of natural and sexual selection acting on signal evolution. In an experimental field study with carotenoid supplementation, simultaneous cross-fostering, manipulation of brood size and ectoparasite load, we investigated the relative importance of these mechanisms for the variation in carotenoid-based coloration of nestling great tits (Parus major). Carotenoid-based plumage coloration was significantly related to genetic origin of nestlings, and was enhanced both in carotenoid-supplemented nestlings, and nestlings raised in reduced broods. We found a tendency for ectoparasite-induced limitation of colour expression and no evidence for detrimental effects of carotenoids on growth pattern, mortality and recruitment of nestlings to the local breeding population. Thus, three of the five proposed mechanisms can generate individual variation in the expression of carotenoid-based plumage coloration in the wild and thus could maintain honesty in a trait potentially used for signalling of individual quality.

Hypothalamic circadian organization in birds. II. Clock gene expression.

While the site of the major circadian pacemaker in mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus, is very well characterized, little is known about hypothalamic circadian organization in birds. This paper reviews recent findings on clock gene expression in the hypothalamus of several bird species focusing on circadian pPer2 expression in the house sparrow. In contrast to mammals, rhythmic Per2 gene expression in the house sparrow hypothalamus is not restricted to a single cell group but occurs in two distinct hypothalamic nuclei, the SCN and the lateral hypothalamic nucleus (LHN). The complex temporal and spatial distribution of pPer2 expression suggests a longitudinal compartmentalization of the SCN with period gene expression being initiated in the most rostral portion before lights on. In the lateral hypothalamus, phasing of pPer2-rhythmicity appeared delayed. In pinealectomized house sparrows, the overall circadian pPer2 expression pattern is maintained indicating that rhythmic pPer2 transcription in the SCN and LHN of the house sparrow are not driven by the pineal gland. Rather, they reflect the activity of autonomous hypothalamic circadian oscillators. Certain changes in peak expression levels and the expression phase, however, suggest that the pineal melatonin rhythm affects both the phase and the amplitude of rhythmic hypothalamic pPer2 expression.