Search

Vocal learning in songbirds is mediated by cortico-basal ganglia circuits that govern diverse functions during different stages of development. We investigated developmental changes in axonal projections to and from motor cortical regions that underlie learned vocal behavior in juvenile zebra finches (Taeniopygia guttata). Neurons in LMAN-core project to RA, a motor cortical region that drives vocal output; these RA-projecting neurons send a transient collateral projection to AId, a region adjacent to RA, during early vocal development. Both RA and AId project to a region of dorsal thalamus (DLM), which forms a feedback pathway to cortico-basal ganglia circuitry. These projections provide pathways conveying efference copy and a means by which information about vocal motor output could be reintegrated into cortico-basal ganglia circuitry, potentially aiding in the refinement of juvenile vocalizations during learning. We used tract-tracing techniques to label the projections of LMAN-core to AId and of RA to DLM in juvenile songbirds. The volume and density of terminal label in the LMAN-core→AId projection declined substantially during early stages of sensorimotor learning. In contrast, the RA→DLM projection showed no developmental change. The retraction of LMAN-core→AId axon collaterals indicates a loss of efference copy to AId and suggests that projections that are present only during early stages of sensorimotor learning mediate unique, temporally restricted processes of goal-directed learning. Conversely, the persistence of the RA→DLM projection may serve to convey motor information forward to the thalamus to facilitate song production during both learning and maintenance of vocalizations. This article is protected by copyright. All rights reserved.

Vocal learning, the ability to imitate sounds from conspecifics and the environment, is a key component of human spoken language and learned song in three independently evolved avian groups—oscine songbirds, parrots, and hummingbirds. Humans and each of these three bird clades exhibit specialized behavioral, neuroanatomical, and brain gene expression convergence related to vocal learning, speech, and song. To understand the evolutionary basis of vocal learning gene specializations and convergence, we searched for and identified accelerated genomic regions (ARs), a marker of positive selection, specific to vocal learning birds. We found avian vocal learner-specific ARs, and they were enriched in noncoding regions near genes with known speech functions or brain gene expression specializations in humans and vocal learning birds, including FOXP2, NEUROD6, ZEB2, and MEF2C, and near genes with major neurodevelopmental functions, including NR2F1, NRP2, and BCL11B. We also found enrichment near the SFARI class S genes associated with syndromic vocal communication forms of autism spectrum disorders. These findings reveal strong candidate noncoding regions near genes for the evolutionary adaptations that distinguish vocal learning species from their close vocal nonlearning relatives and provide further evidence of molecular convergence between birdsong and human spoken language.

Theoretically, seasonal changes in immune functioning in animals are shaped by the trade-off between a probability of encountering pathogens and availability of resources. We used leukocyte profile (absolute and relative leukocyte counts) as a simple measure of immune system condition to study how it changes during the transition from postjuvenile molt to autumn migration in a free-living migratory songbird, the Eurasian blackcap (Sylvia atricapilla). We observed the higher white blood cells (WBC) and lymphocyte counts in molting birds compared to migrating individuals, but we did not find differences in heterophils and ratio of heterophils to lymphocytes (HL ratio). We suppose that the high number of WBC in molting blackcaps could reflect the heightened ability of their immune system to resists infections. The lower WBC counts in migrants compared to molting birds were mostly due to reduced lymphocyte numbers, thus representing in a downregulation of specific immunity. An absence of heterophil differences between molt and migration might indicate that various components of immunity can change relatively independently (or at different pace). Fat scores had no effect on WBC counts and HL ratio. Therefore, we found no strong evidence for a resource-immune functionality trade-off during transition from postjuvenile molt to autumn migration in immature Eurasian blackcap. This study is an important step in understanding how immune system in general and leukocyte profile in particular changes in transition between life-history stages in migratory songbirds.

The ubiquitous activity of humans is a fundamental feature of urban environments affecting local wildlife in several ways. Testing the influence of human disturbance would ideally need experimental approach, however, in cities, this is challenging at relevant spatial and temporal scales. Thus, to better understand the ecological effects of human activity, we exploited the opportunity that the city-wide lockdowns due to the COVID-19 pandemic provided during the spring of 2020. We assessed changes in reproductive success of great tits (Parus major) at two urban habitats affected strikingly differently by the ‘anthropause’, and at an unaffected forest site. Our results do not support that urban great tits benefited from reduced human mobility during the lockdown. First, at one of our urban sites, the strongly (− 44%) reduced human disturbance in 2020 (compared to a long-term reference period) did not increase birds’ reproductive output relative to the forest habitat where human disturbance was low in all years. Second, in the other urban habitat, recreational human activity considerably increased (+ 40%) during the lockdown and this was associated with strongly reduced nestling body size compared to the pre-COVID reference year. Analyses of other environmental factors (meteorological conditions, lockdown-induced changes in air pollution) suggest that these are not likely to explain our results. Our study supports that intensified human disturbance can have adverse fitness consequences in urban populations. It also highlights that a few months of ‘anthropause’ is not enough to counterweight the detrimental impacts of urbanization on local wildlife populations.

Motor learning is a core aspect of human life and appears to be ubiquitous throughout the animal kingdom. Dopamine, a neuromodulator with a multifaceted role in synaptic plasticity, may be a key signaling molecule for motor skill learning. Though typically studied in the context of reward-based associative learning, dopamine appears to be necessary for some types of motor learning. Mesencephalic dopamine structures are highly conserved among vertebrates, as are some of their primary targets within the basal ganglia, a subcortical circuit important for motor learning and motor control. With a focus on the benefits of cross-species comparisons, this review examines how “model-free” and “model-based” computational frameworks for understanding dopamine’s role in associative learning may be applied to motor learning. The hypotheses that dopamine could drive motor learning either by functioning as a reward prediction error, through passive facilitating of normal basal ganglia activity, or through other mechanisms are examined in light of new studies using humans, rodents, and songbirds. Additionally, new paradigms that could enhance our understanding of dopamine’s role in motor learning by bridging the gap between the theoretical literature on motor learning in humans and other species are discussed.

The genetic profile of vertebrate pallia has long driven debate on homology across distantly related clades. Based on an expression profile of the orphan nuclear receptor NR4A2 in mouse and chicken brains, Puelles et al. (The Journal of Comparative Neurology, 2016, 524, 665–703) concluded that the avian lateral mesopallium is homologous to the mammalian claustrum, and the medial mesopallium homologous to the insula cortex. They argued that their findings contradict conclusions by Jarvis et al. (The Journal of Comparative Neurology, 2013, 521, 3614–3665) and Chen et al. (The Journal of Comparative Neurology, 2013, 521, 3666–3701) that the hyperpallium densocellare is instead a mesopallium cell population, and by Suzuki and Hirata (Frontiers in Neuroanatomy, 2014, 8, 783) that the avian mesopallium is homologous to mammalian cortical layers 2/3. Here, we find that NR4A2 is an activity‐dependent gene and cannot be used to determine brain organization or species relationships without considering behavioral state. Activity‐dependent NR4A2 expression has been previously demonstrated in the rodent brain, with the highest induction occurring within the claustrum, amygdala, deep and superficial cortical layers, and hippocampus. In the zebra finch, we find that NR4A2 is constitutively expressed in the arcopallium, but induced in parts of the mesopallium, and in sparse cells within the hyperpallium, depending on animal stimulus or behavioral state. Basal and induced NR4A2 expression patterns do not discount the previously named avian hyperpallium densocellare as dorsal mesopallium and conflict with proposed homology between the avian mesopallium and mammalian claustrum/insula at the exclusion of other brain regions. Broadly, these findings highlight the importance of controlling for behavioral state and neural activity to genetically define brain cell population relationships within and across species., In 2016, Puelles et al. sought to determine the relationship of avian and mammalian pallial regions using the expression profile of the NR4A2 nuclear orphan receptor, concluding that the lateral avian mesopallium was most like the mammalian claustrum, not layer 2 of the cortex as found by Suzuki and Hirata (Frontiers in Neuroanatomy, 2014, 8, 783). However, we found that the NR4A2 expression patterns were characteristic of an activity‐dependent gene. Looking at different behavioral contexts in the zebra finch, we found induced expression of NR4A2 in regions across the pallium associated with visual, somatosensory, and motor activity, confounding and contradicting previous findings. Accordingly, we do not believe NR4A2 is a suitable gene for assessing homologous brain anatomy without understanding the animal's behavioral state.

Eastern equine encephalitis virus (EEEV) is the most pathogenic arbovirus endemic to the United States. Studies have demonstrated Florida’s role as a regional reservoir for the virus and its ability to support year-round transmission. Previous research has developed risk index models for mapping locations most at risk for EEEV transmission. We compared vector abundance, vector feeding behavior, potential host species, and fauna presence at high versus low–moderate risk sites during the winter and spring. Predicted high-risk sites had a significantly greater abundance of mosquitoes overall, including Culiseta melanura (Coquillett) (Diptera: Culicidae), the primary enzootic vector of EEEV. Twenty host species were identified from Cs. melanura bloodmeals, with the majority taken from avian species. Culiseta melanura largely fed upon the Northern Cardinal (Cardinalis cardinalis (Passeriformes: Cardinalidae)), which accounted for 20–24.4% of the bloodmeals obtained from this species in years 1 and 2, respectively. One EEEV-positive mosquito pool (Cs. melanura) and nine EEEV seropositive sentinel chickens were confirmed during winter-spring collections from high-risk sites; no seropositive chickens nor mosquito pools were found at the low–moderate risk sites. These results suggest that high-risk sites for EEEV activity are characterized by habitats that support populations of Cs. melanura and which may also provide ample opportunities to feed upon Northern Cardinals. The overall low level of mosquito populations during the winter also suggests that control of Cs. melanura populations in winter at high-risk sites may prove effective in reducing EEEV transmission during the peak summer season.

As a major physiological mechanism involved in cellular renewal and repair, immune function is vital to the body's capacity to support tissue maintenance and organismal survival. Because immune defenses can be energetically expensive, the activities of metabolically active organs, such as the liver, are predicted to increase during infection by most pathogens. However, some pathogens are immunosuppressive, which might reduce the metabolic capacities of select organs to suppress immune response.

Microfilariae (MF) are the immature stages of filarial nematode parasites and inhabit the blood and dermis of all classes of vertebrates, except fish. Concurrent ingestion of MF and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Shortly after being ingested, MF penetrate the mosquito’s midgut and may introduce virus into the mosquito’s hemocoel, creating a disseminated viral infection much sooner than normal. This phenomenon is known as microfilarial enhancement. Both American Robins and Common Grackles harbor MF—that is, Eufilaria sp. and Chandlerella quiscali von Linstow (Spirurida: Onchocercidae), respectively. We compared infection and dissemination rates in Culex pipiens L. mosquitoes that fed on birds with and without MF infections that had been infected with West Nile virus (WNV). At moderate viremias, about 107 plaque-forming units (pfu)/ml of blood, there were no differences in infection or dissemination rates among mosquitoes that ingested viremic blood from a bird with or without microfilaremia. At high viremias, >108.5 pfu/ml, mosquitoes feeding on a microfilaremic Grackle with concurrent viremia had significantly higher infection and dissemination rates than mosquitoes fed on viremic Grackles without microfilaremia. Microfilarial enhancement depends on the specific virus, MF, and mosquito species examined. How virus is introduced into the hemocoel by MF differs between the avian/WNV systems described here (i.e., leakage) and various arboviruses with MF of the human filarid, Brugia malayi (Brug) (Spirurida: Onchocercidae) (i.e., cotransport). Additional studies are needed to determine if other avian species and their MF are involved in the microfilarial enhancement of WNV in nature.

In order to assess the effects of chitosan supplementation on immune function, antioxidant enzyme activities and histological changes in Leiothrix lutea exposed to acute heat stress, 80 healthy adult birds were randomly divided into five experimental groups. The normal‐temperature group (NTG) was maintained at 21°C and fed the basic diet. The treatment groups were fed the basic diet supplemented with 0%, 0.1%, 0.5% and 1.0% chitosan, respectively, in normal‐temperature environment for 30 days and then exposed to heat (35°C and 40% relative humidity) for 3 hr. The results showed that the immune function and anti‐oxidative enzyme activities in L. lutea in heat‐stressed environment were enhanced by chitosan supplementation, whereas oxidative damage of tissues and cells were alleviated. The results revealed that addition of 0.5% chitosan to the diet may be optimal, playing a key role in meeting the demands of captive‐bred L. lutea in high‐temperature environments. This may constitute a useful feeding strategy in accordance with the behavioural selection of wild L. lutea, and could effectively promote ex situ conservation.

Songbirds learn vocalizations by hearing and practicing songs. As song develops, the tempo becomes faster and more precise. In the songbird brain, discrete nuclei form interconnected myelinated circuits that control song acquisition and production. The myelin sheath increases the speed of action potential propagation by insulating the axons of neurons and by reducing membrane capacitance. As the brain develops, myelin increases in density, but the time course of myelin development across discrete song nuclei has not been systematically studied in a quantitative fashion. We tested the hypothesis that myelination develops differentially across time and song nuclei. We examined myelin development in the brains of the zebra finch (Taeniopygia guttata) from chick at posthatch day (d) 8 to adult (up to 147 d) in five major song nuclei: HVC (proper name), robust nucleus of the arcopallium (RA), Area X, lateral magnocellular nucleus of the anterior nidopallium, and medial portion of the dorsolateral thalamic nucleus (DLM). All of these nuclei showed an increase in the density of myelination during development but at different rates and to different final degrees. Exponential curve fits revealed that DLM showed earlier myelination than other nuclei, and HVC showed the slowest myelination of song nuclei. Together, these data show differential maturation of myelination in different portions of the song system. Such differential maturation would be well placed to play a role in regulating the development of learned song.

PurposeThe main aim of our study was to examine morphological differentiation between and within sex of hen fleas—Ceratophyllus gallinae(Schrank, 1803) population collected from Eurasian blue tit (Cyanistes caeruleusLinnaeus, 1758), inhabiting nest boxes and to determine the morphological parameters differentiating this population.MethodsA total of 296 fleas were collected (148 females and 148 males), determined to species and sex, then the following characters were measured in each of the examined fleas: body length, body width, length of head, width of head, length of comb, height of comb, length of tarsus, length of thorax and length of abdomen.ResultsThe comparison of body size showed the presence of two groups among female and male life forms of the hen flea, which mostly differed in length of abdomen, whereas the length of head and tarsus III were less variable.ConclusionTill now, the only certain information is the presence of two adult life forms ofC. gallinae. The genesis of their creation is still unknown and we are not able to identify the mechanism responsible for the morphological differentiation of fleas collected from the same host. In order to find answer to this question, future research in the field of molecular taxonomy is required.

Usutu virus (USUV) is a zoonotic arbovirus causing avian mass mortalities. The first outbreak in North-Western Germany occurred in 2018. This retrospective analysis focused on combining virological and pathological findings in birds and immunohistochemistry. 25 common blackbirds, one great grey owl, and one kingfisher collected from 2011 to 2018 and positive for USUV by qRT-PCR were investigated. Macroscopically, most USUV infected birds showed splenomegaly and hepatomegaly. Histopathological lesions included necrosis and lymphohistiocytic inflammation within spleen, Bursa fabricii, liver, heart, brain, lung and intestine. Immunohistochemistry revealed USUV antigen positive cells in heart, spleen, pancreas, lung, brain, proventriculus/gizzard, Bursa fabricii, kidney, intestine, skeletal muscle, and liver. Analysis of viral genome allocated the virus to Europe 3 or Africa 2 lineage. This study investigated whether immunohistochemical detection of double-stranded ribonucleic acid (dsRNA) serves as an alternative tool to detect viral intermediates. Tissue samples of six animals with confirmed USUV infection by qRT-PCR but lacking viral antigen in liver and spleen, were further examined immunohistochemically. Two animals exhibited a positive signal for dsRNA. This could indicate either an early state of infection without sufficient formation of virus translation products, occurrence of another concurrent virus infection or endogenous dsRNA not related to infectious pathogens and should be investigated in more detail in future studies.

Neonicotinoids are insecticides widely used as seed treatments that appear to have multiple negative effects on birds at a diversity of biological scales. Adult birds exposed to a low dose of imidacloprid, one of the most commonly used neonicotinoids, presented reduced fat stores, delayed migration and potentially altered orientation. However, little is known on the effect of imidacloprid on birds growth rate despite studies that have documented disruptive effects of low imidacloprid doses on thyroid gland communication. We performed a $$2 \times 2$$ 2 × 2 factorial design experiment in Zebra finches, in which nestling birds were exposed to a very low dose (0.205 mg kg body $$\hbox {mass}^{-1}$$ mass - 1 ) of imidacloprid combined with food restriction during posthatch development. During the early developmental period, imidacloprid exposure resulted in an improvement of body condition index in treated nestlings relative to controls. Imidacloprid also led to compensatory growth in food restricted nestlings. This early life neonicotinoid exposure also carried over to adult age, with exposed birds showing higher lean mass and basal metabolic rate than controls at ages of 90–800 days. This study presents the first evidence that very low-dose neonicotinoid exposure during early life can permanently alter adult phenotype in birds.

SUMMARY Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory “sensing-and-alarm” function and ~42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights germane to control of liver infections., In brief Lefebvre et al. describe the dynamics and mechanisms by which circulating memory CD8 T cells infiltrate the liver to control local malaria and bacterial infection. This work suggests that circulating memory CD8 T cells could be useful targets for developing vaccines and therapeutics for malaria and other liver-specific pathogens., Graphical abstract

Eurasian jays have been reported to protect their caches by responding to cues about either the visual perspective or current desire of an observing conspecific, similarly to other corvids. Here, we used established paradigms to test whether these birds can – like humans – integrate multiple cues about different mental states and perform an optimal response accordingly. Across five experiments, which also include replications of previous work, we found little evidence that our jays adjusted their caching behaviour in line with the visual perspective and current desire of another agent, neither by integrating these social cues nor by responding to only one type of cue independently. These results raise questions about the reliability of the previously reported effects and highlight several key issues affecting reliability in comparative cognition research., eLife digest Eurasian jays, Garrulus glandarius, are members of the crow family. These large-brained birds hide food when it is abundant, and eat it later, when it is scarce. Previous studies have found that jays avoid theft by other jays by carefully deciding what food to hide, and where. In one study, they preferred to hide their food behind an opaque barrier, rather than a transparent one, when another jay was watching. In a second study, they preferred to hide food that the watching jay had already eaten enough of, and thus did not want. These studies suggest that jays have flexible cognitive skills when it comes to protecting their food. They respond to whether a potential thief can see their hiding place and to how much a thief might want the food they are stashing. The next question is, can Eurasian jays combine these two pieces of information? For example, if a jay has two types of food they could hide when another jay is present, but only has one place to hide them (either in view or out-of-view of the other jay), does the first jay prefer to stash the food that the second jay has already eaten, and therefore does not want anymore, only when the hiding place is visible to second jay? To find out, Amodio et al. watched Eurasian jays hiding macadamia nuts or peanuts in the presence of another jay. In the first setup, jays were given one food to hide and two possible hiding places, one opaque and one transparent, while being watched by a jay that had either had its fill of the food, or not tried it. In the second setup, jays were given both foods to hide, but only had one place to hide them (either transparent or opaque); while being watched by a jay that had eaten enough of one of the foods. Contrary to expectations, the jays did not seem to be able to combine the information about what the other jay could see and what it had been eating. In fact, they seemed unable to respond to either piece of information. When Amodio et al. repeated the original experiments, the jays did not seem to prefer to hide food out of sight, or to hide food that the watcher had already eaten. These results raise questions about the repeatability of experiments on food hiding strategies in birds of the crow family. It suggests that previous findings should be further investigated, potentially to identify important factors that might affect the repeatability of food-hiding tactics. Repeating the experiments may show how best to investigate behavioural patterns in jays in the future.

Adaptations resulting from co-evolutionary interactions between avian brood parasites and their hosts have been extensively studied, yet the physiological mechanisms underlying antiparasitic host defences remain little known. Prolactin, one of the main hormones involved in the regulation of avian parental behaviour, might play a key role in the orchestration of the host responses to avian brood parasitism. Given the positive association between prolactin and parental behaviour during incubation, decreasing prolactin levels are expected to facilitate egg-rejection decisions. We tested this prediction by implanting Eurasian blackbird (Turdus merula) females with an inhibitor of prolactin secretion, bromocriptine mesylate, to experimentally decrease their plasma prolactin levels. Bromocriptine mesylate-implanted individuals ejected mimetic model eggs at higher rates, and showed shorter latency to egg ejection, than placebo-treated birds. To our knowledge, this is the first experimental evidence that behavioural host defences against avian brood parasitism are mediated by prolactin.

Two Eurasian Blackbirds (Turdus merula) from central Italy were found with severe cyclocoelid trematodosis associated with airsacculitis. The birds were submitted with severe respiratory distress; one died shortly after hospitalization, while the second bird was euthanized. At necropsy, a massive presence of cyclocoelid flukes was observed in the coelomic cavity and air sacs of both birds. The air sacs were diffusely opaque, thickened, and covered by scant fibrinous exudate mixed with numerous parasites. Histologically, the air sacs showed diffuse and severe oedema with fibrinous exudate. Diffuse mononucleated and heterophilic infiltration mixed with multiple granulomas contained degenerated trematodes. Morishitium polonicum was identified using morphologic keys and molecular analysis of extracted DNA. Infections caused by M. polonicum are poorly documented in blackbirds and the findings in these birds support the pathogenic role of this trematode as a potential cause of death in blackbirds in Italy. Extended epidemiologic surveys are required to properly assess the potential importance of M. polonicum as a life-threatening pathogen in Blackbird populations.

In the vocal learning model, the juvenile first memorizes a model sound, and the imprinted memory gradually converts into vocal-motor output during the sensorimotor integration. However, early acquired memory may not precisely represent the fine structures of a model sound. How do juveniles ensure precise model imitation? Here we show that juvenile songbirds develop an auditory learning program by actively and attentively engaging with tutor’s singing during the sensorimotor phase. The listening/approaching behavior requires previously acquired model memory and the individual variability of approaching behavior correlates with the precision of tutor song imitation. Moreover, it is modulated by dopamine and associated with forebrain regions for sensory processing. Overall, precise vocal learning may involve two steps of auditory processing: a passive imprinting of model memory occurs during the early sensory period; the previously acquired memory then guides an active and selective engagement of the re-exposed model to fine tune model imitation. Wan-Chun Liu et al. demonstrate that the sensory phase of vocal learning in zebra finches is split across two stages: (1) passive listening and formation of a memory, and (2) active listening and behavioral engagement of juveniles with adult tutors. Furthermore, they show that approach behavior is correlated with song imitation quality, and immediate early gene expression in the caudal medial nidopallium linked to auditory behavior.

When anthropogenic noise occurs simultaneously with an acoustic signal or cue, it can be difficult for an animal to interpret the information encoded within vocalizations. However, limited research has focused on how anthropogenic noise affects the identification of acoustic communication signals. In songbirds, research has also shown that black-capped chickadees (Poecile atricapillus) will shift the pitch and change the frequency at which they sing in the presence of anthropogenic, and experimental noise. Black-capped chickadees produce several vocalizations; their fee-bee song is used for mate attraction and territorial defence, and contains information about dominance hierarchy and native geographic location. Previously, we demonstrated that black-capped chickadees can discriminate between individual female chickadees via their fee-bee songs. Here we used an operant discrimination go/no-go paradigm to discern whether the ability to discriminate between individual female chickadees by their song would be impacted by differing levels of anthropogenic noise. Following discrimination training, two levels of anthropogenic noise (low: 40 dB SPL; high: 75 dB SPL) were played with stimuli to determine how anthropogenic noise would impact discrimination. Results showed that even with low-level noise (40 dB SPL) performance decreased and high-level (75 dB SPL) noise was increasingly detrimental to discrimination. We learned that perception of fee-bee songs does change in the presence of anthropogenic noise such that birds take significantly longer to learn to discriminate between females, but birds were able to generalize responding after learning the discrimination. These results add to the growing literature underscoring the impact of human-made noise on avian wildlife, specifically the impact on perception of auditory signals.

How sound is generated in the hummingbird syrinx is largely unknown despite their complex vocal behavior. To fill this gap, syrinx anatomy of four North American hummingbird species were investigated by histological dissection and contrast-enhanced microCT imaging, as well as measurement of vocalizations in a heliox atmosphere. The placement of the hummingbird syrinx is uniquely located in the neck rather than inside the thorax as in other birds, while the internal structure is bipartite with songbird-like anatomical features, including multiple pairs of intrinsic muscles, a robust tympanum and several accessory cartilages. Lateral labia and medial tympaniform membranes consist of an extracellular matrix containing hyaluronic acid, collagen fibers, but few elastic fibers. Their upper vocal tract, including the trachea, is shorter than predicted for their body size. There are between-species differences in syrinx measurements, despite similar overall morphology. In heliox, fundamental frequency is unchanged while upper-harmonic spectral content decrease in amplitude, indicating that syringeal sounds are produced by airflow-induced labia and membrane vibration. Our findings predict that hummingbirds have fine control of labia and membrane position in the syrinx; adaptations that set them apart from closely related swifts, yet shows convergence in their vocal organs with those of oscines.

Birdsong, like human speech, consists of a sequence of temporally precise movements acquired through vocal learning. The learning of such sequential vocalizations depends on the neural function of the motor cortex and basal ganglia. However, it is unknown how the connections between cortical and basal ganglia components contribute to vocal motor skill learning, as mammalian motor cortices serve multiple types of motor action and most experimentally tractable animals do not exhibit vocal learning. Here, we leveraged the zebra finch, a songbird, as an animal model to explore the function of the connectivity between cortex-like (HVC) and basal ganglia (area X), connected by HVC((X)) projection neurons with temporally precise firing during singing. By specifically ablating HVC((X)) neurons, juvenile zebra finches failed to copy tutored syllable acoustics and developed temporally unstable songs with less sequence consistency. In contrast, HVC((X))-ablated adults did not alter their learned song structure, but generated acoustic fluctuations and responded to auditory feedback disruption by the introduction of song deterioration, as did normal adults. These results indicate that the corticobasal ganglia input is important for learning the acoustic and temporal aspects of song structure, but not for generating vocal fluctuations that contribute to the maintenance of an already learned vocal pattern.

Wild birds are frequently exposed to the zoonotic tick-borne bacteria Borrelia burgdorferi sensu lato (s.l.), and some bird species act as reservoirs for some Borrelia genospecies. Studying the tropism of Borrelia in the host, how it is sequestered in different organs, and whether it is maintained in circulation and/or in the host's skin is important to understand pathogenicity, infectivity to vector ticks and reservoir competency.We evaluated tissue dissemination of Borrelia in blackbirds (Turdus merula) and great tits (Parus major), naturally and experimentally infected with Borrelia genospecies from enzootic foci. We collected both minimally invasive biological samples (feathers, skin biopsies and blood) and skin, joint, brain and visceral tissues from necropsied birds. Infectiousness of the host was evaluated through xenodiagnoses and infection rates in fed and moulted ticks. Skin biopsies were the most reliable method for assessing avian hosts' Borrelia infectiousness, which was supported by the agreement of infection status results obtained from the analysis of chin and lore skin samples from necropsied birds and of their xenodiagnostic ticks, including a significant correlation between the estimated concentration of Borrelia genome copies in the skin and the Borrelia infection rate in the xenodiagnostic ticks. This confirms a dermatropism of Borrelia garinii, B. valaisiana and B. turdi in its avian hosts. However, time elapsed from exposure to Borrelia and interaction between host species and Borrelia genospecies may affect the reliability of skin biopsies. The blood was not useful to assess infectiousness of birds, even during the period of expected maximum spirochetaemia. From the tissues sampled (foot joint, liver, spleen, heart, kidney, gut and brain), Borrelia was detected only in the gut, which could be related with infection mode, genospecies competition, genospecies-specific seasonality and/or excretion processes. This study received financial support from Fundação para a Ciência e a Tecnologia by the strategic program of MARE (MARE - UID/MAR/04292/2019), the fellowship to Ana Cláudia Norte (SFRH/BPD/108197/2015) and from the Portuguese National Institute of Health Doutor Ricardo Jorge. Dieter Heylen is funded by the Marie Sklodowska-Curie Actions (EU-Horizon 2020, Individual Global Fellowship, project no. 799609), the Fund for Scientific Research – Flanders (FWO) and the Bill & Melinda Gates Foundation. info:eu-repo/semantics/publishedVersion

The keen visual systems of birds have been relatively well-studied. The foundations of avian vision rest on their cone and rod photoreceptors. Most birds use four cone photoreceptor types for color vision, a fifth cone for achromatic tasks, and a rod for dim-light vision. The cones, along with their oil droplets, and rods are conserved across birds – with the exception of a few shifts in spectral sensitivity – despite taxonomic, behavioral and ecological differences. Here, however, we describe a novel photoreceptor organelle in a group of New World flycatchers (Empidonax spp.) in which the traditional oil droplet is replaced with a complex of electron-dense megamitochondria surrounded by hundreds of small, orange oil droplets. The photoreceptors with this organelle were unevenly distributed across the retina, being present in the central region (including in the fovea), but absent from the retinal periphery and the area temporalis of these insectivorous birds. Of the many bird species with their photoreceptors characterized, only the two flycatchers described here (E. virescens and E. minimus) possess this unusual retinal structure. We discuss the potential functional significance of this unique sub-cellular structure, which might provide an additional visual channel for these small predatory songbirds.

We investigated at the transcriptional level the role of daily rhythm in melatonin secretion in seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala), which when exposed to short (SP) and long (LP) photoperiods exhibits distinct seasonal life-history states (LHSs). We reproduced the seasonal LHS by subjecting buntings to SP (8 h light: 16 h darkness, 8 L:16D), which maintained the nonmigratory/ nonbreeding phenotype, and to LP (16 L:8D), which induced the premigratory/ prebreeding, migratory/ breeding and nonmigratory/ postbreeding phenotypes. Plasma melatonin measured at 4 h intervals showed loss of the daily rhythm in the LP-induced premigratory/ prebreeding and migratory/ breeding LHSs. Subsequently, mRNA expression of genes coding for the aryl-alkamine-N-acetyltransferase (AANAT; the rate-liming enzyme of melatonin biosynthesis) and for the receptors for melatonin (Mel1A, Mel1B and Mel1C) was examined in the retina, pineal and hypothalamus; the interacting independent circadian clocks comprising the songbird circadian timing system. Except AANAT that was not amplified in the hypothalamus, we found significant alterations in both, the level and persistence of 24 h rhythm in mRNA expression of all genes, albeit with photoperiod and seasonal differences between three circadian clock tissues. Particularly, 24 h mRNA expression pattern of all genes, except retinal Mel1A, lacked a significant daily rhythm in the LP-induced migratory/ breeding LHS. These results underscore the overall importance of the circadian rhythm in the role of melatonin in photoperiodically-controlled seasonal responses in migratory songbirds.

The enhanced electrochemical activity of nanostructured materials is readily exploited in energy devices, but their utility in scalable and human-compatible implantable neural interfaces can significantly advance the performance of clinical and research electrodes. We utilize low-temperature selective dealloying to develop scalable and biocompatible one-dimensional platinum nanorod (PtNR) arrays that exhibit superb electrochemical properties at various length scales, stability, and biocompatibility for high performance neurotechnologies. PtNR arrays record brain activity with cellular resolution from the cortical surfaces in birds and nonhuman primates. Significantly, strong modulation of surface recorded single unit activity by auditory stimuli is demonstrated in European Starling birds as well as the modulation of local field potentials in the visual cortex by light stimuli in a nonhuman primate and responses to electrical stimulation in mice. PtNRs record behaviorally and physiologically relevant neuronal dynamics from the surface of the brain with high spatiotemporal resolution, which paves the way for less invasive brain−machine interfaces.

Many urban areas have elevated soil lead concentrations due to prior large-scale use of lead in products such as paint and automobile gasoline. This presents a potential problem for the growing numbers of wildlife living in urbanized areas as lead exposure is known to affect multiple physiological systems, including the nervous system, in vertebrate species. In humans and laboratory animals, low-level lead exposure is associated with neurological impairment, but less is known about how lead may affect the behavior of urban wildlife. We focused on the Northern Mockingbird Mimus polyglottos, a common, omnivorous North American songbird, to gain insights into how lead may affect the physiology and behavior of urban wildlife. We predicted that birds living in neighborhoods with high soil lead concentrations would (a) exhibit elevated lead concentrations in their blood and feathers, (b) exhibit lower body condition, (c) exhibit less diverse and consistent vocal repertoires, and (d) behave more aggressively during simulated conspecific territorial intrusions compared to birds living in neighborhoods with lower soil lead concentrations. Controlling for other habitat differences, we found that birds from areas of high soil lead had elevated lead concentrations in blood and feathers, but found no differences in body condition or vocal repertoires. However, birds from high lead areas responded more aggressively during simulated intrusions. These findings indicate that sub-lethal lead exposure may be common among wildlife living in urban areas, and that this exposure is associated with increased aggression. Better understanding of the extent of the relationship between lead exposure and aggression and the consequences this could have for survival and reproduction of wild animals are clear priorities for future work in this and other urban ecosystems.

Food limitation is a universal stressor for wildlife populations and is increasingly exacerbated by human activities. Anthropogenic environmental change can significantly alter the availability and quality of food resources for reservoir hosts and impact host–pathogen interactions in the wild. The state of the host's nutritional reserves at the time of infection is a key factor influencing infection outcomes by altering host resistance. Combining experimental and model-based approaches, we investigate how an environmental stressor affects host resistance to West Nile virus (WNV). Using American robins (Turdus migratorius), a species considered a superspreader of WNV, we tested the effect of acute food deprivation immediately prior to infection on host viraemia. Here, we show that robins food deprived for 48 h prior to infection, developed higher virus titres and were infectious longer than robins fed normally. To gain an understanding about the epidemiological significance of food-stressed hosts, we developed an agent-based model that simulates transmission dynamics of WNV between an avian host and the mosquito vector. When simulating a nutritionally stressed host population, the mosquito infection rate rose significantly, reaching levels that represent an epidemiological risk. An understanding of the infection disease dynamics in wild populations is critical to predict and mitigate zoonotic disease outbreaks.

Summary The motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle-specific force. Birdsong is an excellent model system for understanding acquisition and maintenance of complex fine motor skills, but we know surprisingly little about how the motor pool controlling the syrinx is organized and how MU recruitment drives changes in vocal output. Here we developed an experimental paradigm to measure MU size distribution using spatiotemporal imaging of intracellular calcium concentration in cross-sections of living intact syrinx muscles. We combined these measurements with muscle stress and an in vitro syrinx preparation to determine the control resolution of fundamental frequency (fo), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 40%–50% innervating ≤3 and 13%–17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm2) known to skeletal vertebrate muscle, small force steps by the major fo controlling muscle provide control of 50-mHz to 7.3-Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.

Sex differences in ornamentation are common and, in species with conventional sex roles, are generally thought of as stable, due to stronger sexual selection on males. Yet, especially in gregarious species, ornaments can also have non-sexual social functions, raising the possibility that observed sex differences in ornamentation are plastic. For example, females may invest in costly ornamentation more plastically, to protect body and reproductive ability in more adverse ecological conditions. We tested this hypothesis with experimental work on the mutually-ornamented common waxbill (Estrilda astrild), supplementing their diets either with pigmentary (lutein, a carotenoid) or non-pigmentary (vitamin E) antioxidants, or alleviating winter cold temperature. We found that both lutein and vitamin E supplementation increased red bill colour saturation in females, reaching the same mean saturation as males, which supports the hypothesis that female bill colour is more sensitive to environmental or physiological conditions. The effect of vitamin E, a non-pigment antioxidant, suggests that carotenoids were released from their antioxidant functions. Alleviating winter cold did not increase bill colour saturation in either sex, but increased the stability of female bill colour over time, suggesting that female investment in bill colour is sensitive to cold-mediated stress. Together, results show that waxbill bill sexual dichromatism is not stable. Instead, sexual dichromatism can be modulated, and even disappear completely, due to ecology-mediated plastic adjustments in female bill colour.

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 tshβ and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1β 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.

Sexual signals are archetypes of contingent evolution: hyper-diverse across species, often evolving fast and in unpredictable directions. It is unclear to which extent their evolutionary unpredictability weakens deterministic evolution, or takes place bounded by deterministic patterns of trait evolution. We compared the evolution of sound frequency in sexual signals (advertisement songs) and non-sexual social signals (calls) across > 500 genera of the crown songbird families. Contrary to the acoustic adaptation hypothesis, we found no evidence that forest species used lower sound frequencies in songs or calls. Consistent with contingent evolution in song, we found lower phylogenetic signal for the sound frequency of songs than calls, which suggests faster and less predictable evolution, and found unpredictable direction of evolution in lineages with longer songs, which presumably experience stronger sexual selection on song. Nonetheless, the most important deterministic pattern of sound frequency evolution—its negative association with body size—was stronger in songs than calls. This can be explained by songs being longer-range signals than most calls, and thus using sound frequencies that animals of a given size produce best at high amplitude. Results indicate that sexual selection can increase aspects of evolutionary contingency while strengthening, rather than weakening, deterministic patterns of evolution.

Singing occurs in songbirds of both sexes, but some species show typical degrees of sex-specific performance. We studied the transcriptional sex differences in the HVC, a brain nucleus critical for song pattern generation, of the forest weaver (Ploceus bicolor), the blue-capped cordon-bleu (Uraeginthus cyanocephalus), and the canary (Serinus canaria), which are species that show low, medium, and high levels of sex-specific singing, respectively. We observed persistent sex differences in gene expression levels regardless of the species-specific sexual singing phenotypes. We further studied the HVC transcriptomes of defined phenotypes of canary, known for its testosterone-sensitive seasonal singing. By studying both sexes of canaries during both breeding and nonbreeding seasons, nonbreeding canaries treated with testosterone, and spontaneously singing females, we found that the circulating androgen levels and sex were the predominant variables associated with the variations in the HVC transcriptomes. The comparison of natural singing with testosterone-induced singing in canaries of the same sex revealed considerable differences in the HVC transcriptomes. Strong transcriptional changes in the HVC were detected during the transition from nonsinging to singing in canaries of both sexes. Although the sex-specific genes of singing females shared little resemblance with those of males, our analysis showed potential functional convergences. Thus, male and female songbirds achieve comparable singing behaviours with sex-specific transcriptomes.

Synopsis Most animals experience reproductive transitions in their lives; for example, reaching reproductive maturity or cycling in and out of breeding condition. Some reproductive transitions are abrupt, while others are more gradual. In most cases, changes in communication between the sexes follow the time course of these reproductive transitions and are typically thought to be coordinated by steroid hormones. We know a great deal about hormonal control of communication behaviors in birds and frogs, as well as the central neural control of these behaviors. There has also been significant interest in the effects of steroid hormones on central nervous system structures that control both the production and reception of communication signals associated with reproductive behaviors. However, peripheral sensory structures have typically received less attention, although there has been growing interest in recent years. It is becoming clear that peripheral sensory systems play an important role in reproductive communication, are plastic across reproductive conditions, and, in some cases, this plasticity may be mediated by steroid hormones. In this article, we discuss recent evidence for the role of peripheral auditory structures in reproductive communication in birds and frogs, the plasticity of the peripheral auditory system, and the role of steroid hormones in mediating the effects of the peripheral auditory system on reproductive communication. We focus on both seasonal and acute reproductive transitions, introduce new data on the role of hormones in modulating seasonal patterns, and make recommendations for future work.

Movement-related neuronal discharge in ventral tegmental area (VTA) and ventral pallidum (VP) is inconsistently observed across studies. One possibility is that some neurons are movement-related and others are not. Another possibility is that the precise behavioral conditions matter - that a single neuron can be movement related under certain behavioral states but not others. We recorded single VTA and VP neurons in birds transitioning between singing and non-singing states, while monitoring body movement with microdrive-mounted accelerometers. Many VP and VTA neurons exhibited body movement-locked activity exclusively when the bird was not singing. During singing, VP and VTA neurons could switch off their tuning to body movement and become instead precisely time-locked to specific song syllables. These changes in neuronal tuning occurred rapidly at state boundaries. Our findings show that movement-related activity in limbic circuits can be gated by behavioral context.Significance statementNeural signals in the limbic system have long been known to represent body movements as well as reward. Here we show that single neurons dramatically change their tuning to movements when a bird starts to sing.

SUMMARYIncreases in the scale and complexity of behavioral data pose an increasing challenge for data analysis. A common strategy involves replacing entire behaviors with small numbers of handpicked, domain-specific features, but this approach suffers from several crucial limitations. For example, handpicked features may miss important dimensions of variability, and correlations among them complicate statistical testing. Here, by contrast, we apply the variational autoencoder (VAE), an unsupervised learning method, to learn features directly from data and quantify the vocal behavior of two model species: the laboratory mouse and the zebra finch. The VAE converges on a parsimonious representation that outperforms handpicked features on a variety of common analysis tasks, enables the measurement of moment-by-moment vocal variability on the timescale of tens of milliseconds in the zebra finch, provides strong evidence that mouse ultrasonic vocalizations do not cluster as is commonly believed, and captures the similarity of tutor and pupil birdsong with qualitatively higher fidelity than previous approaches. In all, we demonstrate the utility of modern unsupervised learning approaches to the quantification of complex and high-dimensional vocal behavior.

Signals containing parameter trade-offs are likely to be honest indicators of signaler quality because they are difficult to produce. Signals with a trill-rate/bandwidth trade-off have been described for many songbird species, one mouse, and one non-human primate species. However, there were no reports about whether there is a vocal performance trade-off in social calls of bats. This study investigated (1) a possible vocal performance trade-off in territorial calls of male Great Himalayan leaf-nosed bats, Hipposideros armiger, recorded from 9 locations in south China, and (2) the relationships between vocal performance (vocal deviation and consistency) and caller's quality (body mass) to determine whether vocal performance honestly indicates a caller's quality. Vocal deviation measures the deviation of a call relative to an extreme call and vocal consistency measures the spectral consistency across a string of syllables. Our results showed a significant negative correlation between syllable repetition rate and frequency bandwidth, suggesting a vocal performance trade-off similar to the one in songbirds. Further, there was a significant negative relationship between body mass and vocal deviation, but no significant correlation between body mass and vocal consistency. This study provides the first empirical evidence for a vocal performance trade-off of social calls in bats, and the potential for the level of performance to indicate caller quality.

Mating system theory predicts that social polygyny—when one male forms pair bonds with two females—may evolve by female choice in species with biparental care. Females will accept a polygynous male if the benefit of mating with a male providing high-quality genes or rearing resources outweighs the cost of sharing mate assistance in parental care. Based on this rationale, we hypothesise that the population frequency of social polygyny (FSP) varies due to changes in mate sharing costs caused by changing environmental conditions. We predicted that: (1) polygamous females (i.e. mated with a polygynous male) pay a survival cost compared to monogamous females; (2) FSP would be higher in years with better rearing conditions and (3) the difference in survival rates between monogamous and polygamous females would be small following years with higher FSP. We tested these predictions using regression and multistate analyses of capture-recapture data of pied flycatchers, Ficedula hypoleuca, in central Spain collected over 26 years (1990–2016). Monogamous females had a higher mean survival rate than polygamous females (prediction 1), but there was no difference in survival between polygynous and monogamous males. In addition, FSP was positively associated with annual reproductive success (a proxy of the quality of rearing conditions—prediction 2). Finally, following years with high FSP, the survival of polygamous females was similar to that of monogamous females (prediction 3), while the chance of breeding in a polygamous state for 2 years in a row increased for both males and females. Our findings suggest that fluctuating environmental conditions may be a necessary but neglected aspect of understanding social polygyny mechanisms. © 2022, The Author(s)., Spanish Ministry of Economy and Competitiveness CGL2014-55969-P and CGL2015-70639-P, Hungarian National Research, Development and Innovation Office. K-129215, Spanish MECD (FJCI-2015-24579), ARAID foundation, National Research, Development and Innovation Office (NKFIH) grant nr. K-139992.

Acoustic signals serve communication within and across species throughout the animal kingdom. Studying the genetics, evolution, and neurobiology of acoustic communication requires annotating acoustic signals: segmenting and identifying individual acoustic elements like syllables or sound pulses. To be useful, annotations need to be accurate, robust to noise, and fast.We here introduceDAS, a method that annotates acoustic signals across species based on a deep-learning derived hierarchical presentation of sound. We demonstrate the accuracy, robustness, and speed ofDASusing acoustic signals with diverse characteristics from insects, birds, and mammals.DAScomes with a graphical user interface for annotating song, training the network, and for generating and proofreading annotations. The method can be trained to annotate signals from new species with little manual annotation and can be combined with unsupervised methods to discover novel signal types.DASannotates song with high throughput and low latency, allowing realtime annotations for closed-loop experimental interventions. Overall,DASis a universal, versatile, and accessible tool for annotating acoustic communication signals.

Maternal effects via hormonal transfer from the mother to the offspring provide a tool to translate environmental cues to the offspring. Experimental manipulations of maternally transferred hormones have yielded increasingly contradictory results, which may be explained by differential effects of hormones under different environmental contexts. Yet context-dependent effects have rarely been experimentally tested. We therefore studied whether maternally transferred thyroid hormones (THs) exert context-dependent effects on offspring survival and physiology by manipulating both egg TH levels and post-hatching nest temperature in wild pied flycatchers (Ficedula hypoleuca) using a full factorial design. We found no clear evidence for context-dependent effects of prenatal THs related to postnatal temperature on growth, survival and potential underlying physiological responses (plasma TH levels, oxidative stress and mitochondrial density). We conclude that future studies should test for other key environmental conditions, such as food availability, to understand potential context-dependent effects of maternally transmitted hormones on offspring, and their role in adapting to changing environments.

Experimental manipulation has established testosterone as a potent, pleiotropic regulator coordinating morphology, physiology and behavior. However, the relationship of field-sampled, unmanipulated testosterone concentrations with traits of interest is often equivocal. Circulating testosterone varies over the course of the day, and recent reports indicate that testosterone is higher during the night in diurnal songbirds. Yet, most field studies sample testosterone during the morning. Sampling at times when levels and individual variation are low may be one reason relationships between testosterone and other traits are not always observed. Testosterone is regulated by the hypothalamic-pituitary-gonadal axis, with gonadotropin-releasing hormone (GnRH) initiating the endocrine cascade. Research has examined GnRH-induced testosterone levels with traits of interest, yet the relevance of these induced levels and their relationship with endogenously produced levels are not fully clear. Using photostimulated male great tits (Parus major) we tested the hypotheses that circulating testosterone levels peak during the night and that GnRH-induced testosterone concentrations are positively related to nightly testosterone peaks. Blood was sampled during first, middle or last third of night. One week later, baseline and GnRH-induced testosterone levels were sampled during mid-morning. Morning baseline testosterone levels were low compared with night-sampled levels that peaked during the first third of the night. Further, GnRH-induced testosterone was strongly positively correlated with levels observed during the first third of the night. These data suggest that morning testosterone samples likely do not reflect an individual's endogenous peak. Instead, GnRH-induced testosterone levels do approximate an individual's nightly peak and may be an alternative for birds that cannot easily be sampled at night in the field. These findings are likely to have implications for research aimed at relating traits of interest with natural variation in sex steroid hormone levels.