The effect of environmental variation on the relationship between survival and risk-taking behaviour in a migratory songbird.

Temporal changes in environmental conditions may play a major role in the year-to-year variation in fitness consequences of behaviours. Identifying environmental drivers of such variation is crucial to understand the evolutionary trajectories of behaviours in natural contexts. However, our understanding of how environmental variation influences behaviours in the wild remains limited. Using data collected over 14 breeding seasons from a collared flycatcher (Ficedula albicollis) population, we examined the effect of environmental variation on the relationship between survival and risk-taking behaviour, a highly variable behavioural trait with great evolutionary and ecological significance. Specifically, using annual recapture probability as a proxy of survival, we evaluated the specific effect of predation pressure, food availability, and mean temperature on the relationship between annual recapture probability and risk-taking behaviour (measured as flight initiation distance [FID]). We found a negative trend, as the relationship between annual recapture probability and FID decreased over the study years and changed from positive to negative. Specifically, in the early years of the study, risk-avoiding individuals exhibited a higher annual recapture probability, whereas in the later years, risk-avoiders had a lower annual recapture probability. However, we did not find evidence that any of the considered environmental factors mediated the variation in the relationship between survival and risk-taking behaviour.

Responses in the breeding parameters of the collared flycatcher to the changing climate.

Global climate change involves various aspects of climate, including precipitation changes and declining surface wind speeds, but studies investigating biological responses have often focused on the impacts of rising temperatures. Additionally, related long-term studies on bird reproduction tend to concentrate on breeding onset, even though other aspects of breeding could also be sensitive to the diverse weather aspects. This study aimed to explore how multiple aspects of breeding (breeding onset, hatching delay, breeding season length, clutch size, fledgling number) were associated with different weather components. We used an almost four-decade-long dataset to investigate the various aspects of breeding parameters of a collared flycatcher (Ficedula albicollis) population in the Carpathian Basin. Analyses revealed some considerable associations, for example, breeding seasons lengthened with the amount of daily precipitation, and clutch size increased with the number of cool days. Parallel and opposing changes in the correlated pairs of breeding and weather parameters were also observed. The phenological mismatch between prey availability and breeding time slightly increased, and fledgling number strongly decreased with increasing mistiming. Our results highlighted the intricate interplay between climate change and the reproductive patterns of migratory birds, emphasizing the need for a holistic approach. The results also underscored the potential threats posed by climate change to bird populations and the importance of adaptive responses to changing environmental conditions.

Individual differences in song plasticity in response to social stimuli and singing position.

Individual animals can react to the changes in their environment by exhibiting behaviors in an individual-specific way leading to individual differences in phenotypic plasticity. However, the effect of multiple environmental factors on multiple traits is rarely tested. Such a complex approach is necessary to assess the generality of plasticity and to understand how among-individual differences in the ability to adapt to changing environments evolve. This study examined whether individuals adjust different song traits to varying environmental conditions in the collared flycatcher (Ficedula albicollis), a passerine with complex song. We also aimed to reveal among-individual differences in behavioral responses by testing whether individual differences in plasticity were repeatable. The presence of general plasticity across traits and/or contexts was also tested. To assess plasticity, we documented (1) short-scale temporal changes in song traits in different social contexts (after exposition to male stimulus, female stimulus or without stimuli), and (2) changes concerning the height from where the bird sang (singing position), used as a proxy of predation risk and acoustic transmission conditions. We found population-level relationships between singing position and both song length (SL) and complexity, as well as social context-dependent temporal changes in SL and maximum frequency (MF). We found among-individual differences in plasticity of SL and MF along both the temporal and positional gradients. These among-individual differences in plasticity were repeatable. Some of the plastic responses correlated across different song traits and environmental gradients. Overall, our results show that the plasticity of bird song (1) depends on the social context, (2) exists along different environmental gradients, and (3) there is evidence for trade-offs between the responses of different traits to different environmental variables. Our results highlight the need to consider individual differences and to investigate multiple traits along multiple environmental axes when studying behavioral plasticity.

ChiroVox: a public library of bat calls.

Recordings of bat echolocation and social calls are used for many research purposes from ecological studies to taxonomy. Effective use of these relies on identification of species from the recordings, but comparative recordings or detailed call descriptions to support identification are often lacking for areas with high biodiversity. The ChiroVox website (https://www.chirovox.org) was created to facilitate the sharing of bat sound recordings together with their metadata, including biodiversity data and recording circumstances. To date, more than 30 researchers have contributed over 3,900 recordings of nearly 200 species, making ChiroVox the largest open-access bat call library currently available. Each recording has a unique identifier that can be cited in publications; hence the acoustic analyses are repeatable. Most of the recordings available through the website are from bats whose species identities are confirmed, so they can be used to determine species in recordings where the bats were not captured or could not be identified. We hope that with the help of the bat researcher community, the website will grow rapidly and will serve as a solid source for bat acoustic research and monitoring.

The effect of social environment on bird song: listener-specific expression of a sexual signal.

Animal signals should consistently differ among individuals to convey distinguishable information about the signalers. However, behavioral display signals, such as bird song are also loaded with considerable within-individual variance with mostly unknown function. We hypothesized that the immediate social environment may play a role in mediating such variance component, and investigated in the collared flycatcher (Ficedula albicollis) if the identity and quality of listeners could affect song production in signalers. After presenting territorial males with either a female or male social stimulus, we found in the subsequent song recordings that the among-stimulus effects corresponded to non-zero variance components in several acoustic traits indicating that singing males are able to plastically adjust their songs according to stimulus identity. Male and female stimuli elicited different responses as the identity of the female stimuli affected song complexity only, while the identity of male stimuli altered also song length, maximum frequency, and song rate. The stimulus-specific effect on song in some cases decreased with time, being particularly detectable right after the removal of the stimulus and ceasing later, but this pattern varied across the sex of the stimulus and the song traits. We were able to identify factors that can explain the among-stimulus effects (e.g., size and quality of the stimuli) with roles that also varied among song traits. Our results confirm that the variable social environment can raise considerable variation in song performance, highlighting that within-individual plasticity of bird song can play important roles in sexual signaling.

The Effect of Vasopressin Antagonists on Maternal-Separation-Induced Ultrasonic Vocalization and Stress-Hormone Level Increase during the Early Postnatal Period.

In adults, vasopressin exerts an anxiogenic effect, but less is known about the perinatal period. As a sign of distress, rat pups emit ultrasonic vocalizations when they are separated from their mothers, known as maternal separation-induced ultrasonic vocalization (MS-USV). Previously, reduced MS-USV was reported in 7-8-day-old genetically vasopressin-deficient Brattleboro rats. Here, we aimed to examine the contributing vasopressin receptor (VR) subtypes using Wistar pups. MS-USV was recorded for 10 min, 30 min after vasopressin (V) 1aR, V1bR or V2R antagonist treatment (SR49059, SSR149415, SR121463B; 3, 10 and 30 mg/kg, intraperitoneal). Sedation was studied by the righting reflex and negative geotaxis, and finally, the stress hormone levels were measured by radioimmunoassay. The vasopressin-deficient pups showed decreased MS-USV and adrenocorticotropin levels even after a saline injection, with unchanged corticosterone levels. Thirty mg/kg of V1aR-antagonist increased the corticosterone levels. All V1bR antagonist doses decreased the MS-USV and adrenocorticotropin, while 10 + 10 mg/kg of V1aR and V1bR antagonists decreased MS-USV without influencing the stress hormones. Three mg/kg of V2R antagonist enhanced MS-USV, while 30 mg/kg increased the stress hormone levels. We confirmed that vasopressin deficiency already caused anxiolytic effects in pups. V1bRs are the most important player in connection with their adrenocorticotropin (ACTH)-regulatory role, but a combination of V1aR and V1bR antagonists might be also beneficial through other mechanisms, reducing the possibility of side effects. In contrast, antagonizing the V2Rs may be stressful due to an induction of imbalance in saltwater homeostasis.

Sequential organization of birdsong: relationships with individual quality and fitness.

Many vocalizing animals produce the discrete elements of their acoustic signals in a specific sequential order, but we know little about the biological relevance of this ordering. For that, we must characterize the degree by which individuals differ in how they organize their signals sequentially and relate these differences to variation in quality and fitness. In this study, we fulfilled these tasks in male collared flycatchers (Ficedula albicollis). We characterized the sequential order of syllables with a network analysis approach and studied the consistency of network variables on distinct time scales (within day, between days, and between years), and assessed their relationship with such quality indicators like age, body condition, arrival date, and fitness related proxies like survival to the next year and pairing success. We found that the syllables were associated nonrandomly with one another and both the frequency differences of consecutive syllables and the number of motif types were higher in the original than in randomized syllable sequences. Average degree and small-worldness showed considerable among-individual differences and decreasing repeatability with increasing time scale. Furthermore, we found relationships between male age and average degree among and within individuals. Accordingly, older males produce syllable sequences by using common syllables less often than younger individuals. However, the network variables showed no relationship with fitness-related variables. In conclusion, the sequential organization of birdsong has the potential to encode individual-specific characteristics, which thus could be used as signal in social interactions and thus potentially could be subject to sexual selection.

Auditory perception of self and others in zebra finches: evidence from an operant discrimination task.

Vocal communication is essential for social interactions in many animal species. For this purpose, an animal has to perceive vocal signals of conspecifics and is often also required to discriminate conspecifics. The capacity to discriminate conspecifics is particularly important in social species in which individuals interact repeatedly. In addition, auditory perception of self plays an important role for vocal learners. A vocal learner has to memorise vocalisations of conspecifics and to subsequently modify its own vocalisations in order to match the memorised vocalisations. Here, we investigated auditory perception of self and others in zebra finches (Taeniopygia guttata), a highly gregarious songbird species and vocal learner. We used laboratory colonies in which founder males had been previously trained to produce the same song type. This resulted in artificial dialects in the song of founders and their offspring. We investigated whether those birds would be able to discriminate between familiar and unfamiliar conspecifics based on song. Furthermore, we examined whether they would classify their own song as familiar or unfamiliar. We found that birds were able to discriminate between songs of familiar versus unfamiliar conspecifics, despite the fact that all songs were imitations of the same song type. This suggests that such discrimination is possible even based on songs with a high acoustic similarity. None of the subjects classified their own song as unfamiliar. Three out of eight males classified their own song as familiar. Thus zebra finches might recognise their own song. Further experiments are needed to confirm such self-recognition.

Valproate Exposure in ovo Attenuates the Acquisition of Social Preferences of Young Post-hatch Domestic Chicks.

Embryonic exposure to valproic acid (VPA) is known to produce sociability deficits, resembling human autistic phenotypes, in several vertebrate species. Animals living in groups prefer the proximity of peers and have the ability to perceive and to respond to social signals for modifying behavior. Chicks of Galliform birds, known to display early preference behaviors, have been used extensively for adaptive learning studies. Young precocial birds seem to be useful models also for studying the effect of embryonic VPA treatment. Here, domestic chicken eggs were injected with sodium valproate (200 µl of 35 µmol/L solution) or with vehicle (distilled water) on the 14th day of incubation. After hatching, the chicks were tested for one-trial passive avoidance learning at day 1, vocalization due to isolation as a measure of stress level (day 2), approach preference to large versus small groups of age-matched conspecifics (day 5), and to those with normal versus blurred head features (day 7). In addition, we tested the preference of birds to conspecifics reared in group versus those reared in isolation (day 9), as well as the preference of chicks to familiar versus non-familiar conspecifics (day 21). Our findings confirm previous reports concerning an adverse effect of VPA on embryonic development, including a tendency for aborted or delayed hatching and, occasionally, for locomotor disorders in a small percentage of birds (eliminated from later studies). Otherwise, VPA treatment did not impair motor activity or distress level. Memory formation for the aversive stimulus and discrimination of colors were not impaired by VPA treatment either. Innate social predispositions manifested in approach preferences for the larger target group or for the birds with natural facial features remained unaffected by VPA exposure. The most prominent finding was attenuation of social exploration in VPA-exposed birds (expressed as the frequency of positional switches between two stimulus chicks after the first choice), followed by a deficit in the recognition of familiar conspecifics, unfolding at the end of the third week. Social exploration and recognition of familiar individuals are the key elements impaired at this stage. The results underline the importance of early social exploration in ASD.

Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry.

Complex motor skills take considerable time and practice to learn. Without continued practice the level of skill performance quickly degrades, posing a problem for the timely utilization of skilled motor behaviors. Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird, while manipulating skill performance by consecutively administrating and withdrawing testosterone. We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance. We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC, a reduction that is not reversed when birds stop singing. These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills, enabling a rapid recovery of motor performance under environmental time constraints.

MHC-mediated sexual selection on birdsong: Generic polymorphism, particular alleles and acoustic signals.

Several hypotheses predict that the major histocompatibility complex (MHC) drives mating preference in females. Olfactory, colour or morphological traits are often found as reliable signals of the MHC profile, but the role of avian song mediating MHC-based female choice remains largely unexplored. We investigated the relationship between several MHC and acoustic features in the collared flycatcher (Ficedula albicollis), a European passerine with complex songs. We screened a fragment of the class IIB second exon of the MHC molecule, of which individuals harbour 4-15 alleles, while considerable sequence diversity is maintained at the population level. To make statistical inferences from a large number of comparisons, we adopted both null-hypothesis testing and effect size framework in combination with randomization procedures. After controlling for potential confounding factors, neither MHC allelic diversity nor the presence of particular alleles was associated remarkably with the investigated qualitative and quantitative song traits. Furthermore, genetic similarity among males based on MHC sequences was not reflected by the similarity in their song based on syllable content. Overall, these results suggest that the relationship between features of song and the allelic composition and diversity of MHC is not strong in the studied species. However, a biologically motivated analysis revealed that individuals that harbour an MHC allele that impairs survival perform songs with broader frequency range. This finding suggests that certain aspects of the song may bear reliable information concerning the MHC profile of the individuals, which can be used by females to optimize mate choice.

Acoustic mirrors as sensory traps for bats.

Sensory traps pose a considerable and often fatal risk for animals, leading them to misinterpret their environment. Bats predominantly rely on their echolocation system to forage, orientate, and navigate. We found that bats can mistake smooth, vertical surfaces as clear flight paths, repeatedly colliding with them, likely as a result of their acoustic mirror properties. The probability of collision is influenced by the number of echolocation calls and by the amount of time spent in front of the surface. The echolocation call analysis corroborates that bats perceive smooth, vertical surfaces as open flyways. Reporting on occurrences with different species in the wild, we argue that it is necessary to more closely monitor potentially dangerous locations with acoustic mirror properties (such as glass fronts) to assess the true frequency of fatalities around these sensory traps.

Sex-dependent role of vesicular glutamate transporter 3 in stress-regulation and related anxiety phenotype during the early postnatal period.

Stress and related disorders are in the focus of interest and glutamate is one of the most important neurotransmitters that can affect these processes. Glutamatergic neurons are characterized by vesicular glutamate transporters (VGluT1-3) among which vGluT3 is unique contributing to the non-canonical, neuromodulatory effect of glutamate. We aimed to study the role of vGluT3 in stress axis regulation and related anxiety during the early postnatal period using knockout (KO) mice with special focus on sex differences. Anxiety was explored on postnatal day (PND) 7-8 by maternal separation-induced ultrasonic vocalization (USV). Stress-hormone levels were detected 60 min after intraperitoneal lipopolysaccharide (LPS) injection 7 days later. Both genotypes gained weight, but on PND 14-15 KO mice pups had smaller body weight compared to wild type (WT). vGluT3 KO mice reacted to an immune stressor with enhanced adrenocorticotropin (ACTH) and corticosterone secretion compared to WT. Although there was a tendency for enhanced anxiety measured by more emitted USV, this did not reach the level of significance. The only sex-related effect was the enhanced corticosterone reactivity in male pups. For the HPA axis regulation in neonates vGluT3 expression seems to be dispensable under basal conditions, but is required for optimal response to immune stressors, most probably through an interaction with other neurotransmitters. Disturbance of the fine balance between these systems may result in a borderline enhanced anxiety-like behavior in vGluT3 KO pups.

Among-year variation in the repeatability, within- and between-individual, and phenotypic correlations of behaviors in a natural population.

When mean behaviors correlate among individuals, they form behavioral syndromes. One way to understand the evolution of such a group-level phenomenon is to compare horizontally patterns of correlations among populations (or species) or follow longitudinally the same population over years in the light of parallel differences in the environment. We applied the longitudinal approach to 8-year field data and analyzed phenotypic correlations, and their within- and between-individual components, among three behaviors (novelty avoidance, aggression, and risk-taking) in male collared flycatchers, Ficedula albicollis, in a meta-analytic framework. The phenotypic correlation between novelty avoidance and aggression varied heterogeneously (it was positive in some years, while it was negative in other years), while the other pair-wise correlations were consistently positive over the study period. We investigated four potential socio-ecological factors, and found evidence that the among-year alterations in the demographic structure of the population (density, age composition) can be responsible for the heterogeneous effect sizes. Comparing within- and between-individual correlations across pairs of traits, we found that the correlation between aggression and risk-taking at the among-individual level was the strongest suggesting that this relationship has the highest potential to form a behavioral syndrome. Within-year repeatabilities varied among traits, but were systematically higher than between-year repeatabilities. Our study highlights on an empirical basis that there can be several biological and statistical reasons behind detecting a phenotypic correlation in a study, but only few of these imply that fixed behavioral syndromes are maintained in a natural population. In fact, some correlations seem to be shaped by environmental fluctuations.

Female mate choice can drive the evolution of high frequency echolocation in bats: a case study with Rhinolophus mehelyi.

Animals employ an array of signals (i.e. visual, acoustic, olfactory) for communication. Natural selection favours signals, receptors, and signalling behaviour that optimise the received signal relative to background noise. When the signal is used for more than one function, antagonisms amongst the different signalling functions may constrain the optimisation of the signal for any one function. Sexual selection through mate choice can strongly modify the effects of natural selection on signalling systems ultimately causing maladaptive signals to evolve. Echolocating bats represent a fascinating group in which to study the evolution of signalling systems as unlike bird songs or frog calls, echolocation has a dual role in foraging and communication. The function of bat echolocation is to generate echoes that the calling bat uses for orientation and food detection with call characteristics being directly related to the exploitation of particular ecological niches. Therefore, it is commonly assumed that echolocation has been shaped by ecology via natural selection. Here we demonstrate for the first time using a novel combined behavioural, ecological and genetic approach that in a bat species, Rhinolophus mehelyi: (1) echolocation peak frequency is an honest signal of body size; (2) females preferentially select males with high frequency calls during the mating season; (3) high frequency males sire more off-spring, providing evidence that echolocation calls may play a role in female mate choice. Our data refute the sole role of ecology in the evolution of echolocation and highlight the antagonistic interplay between natural and sexual selection in shaping acoustic signals.

The relationship between DRD4 polymorphisms and phenotypic correlations of behaviors in the collared flycatcher.

There is increasing evidence that the genetic architecture of exploration behavior includes the dopamine receptor D4 gene (DRD4). Such a link implies that the within-individual consistency in the same behavior has a genetic basis. Behavioral consistency is also prevalent in the form of between-individual correlation of functionally different behaviors; thus, the relationship between DRD4 polymorphism and exploration may also be manifested for other behaviors. Here, in a Hungarian population of the collared flycatcher, Ficedula albicollis, we investigate how males with distinct DRD4 genotypes differ in the consistent elements of their behavioral displays during the courtship period. In completely natural conditions, we assayed novelty avoidance, aggression and risk-taking, traits that were previously shown repeatable over time and correlate with each other, suggesting that they could have a common mechanistic basis. We identified two single-nucleotide polymorphisms (SNP554 and SNP764) in the exon 3 of the DRD4 gene by sequencing a subsample, then we screened 202 individuals of both sexes for these SNPs. Focusing on the genotypic variation in courting males, we found that "AC" heterozygote individuals at the SNP764 take lower risk than the most common "AA" homozygotes (the "CC" homozygotes were not represented in our subsample of males). We also found a considerable effect size for the relationship between SNP554 polymorphism and novelty avoidance. Therefore, in addition to exploration, DRD4 polymorphisms may also be associated with the regulation of behaviors that may incur fear or stress. Moreover, polymorphisms at the two SNPs were not independent indicating a potential role for genetic constraints or another functional link, which may partially explain behavioral correlations.

Global warming alters sound transmission: differential impact on the prey detection ability of echolocating bats.

Climate change impacts the biogeography and phenology of plants and animals, yet the underlying mechanisms are little known. Here, we present a functional link between rising temperature and the prey detection ability of echolocating bats. The maximum distance for echo-based prey detection is physically determined by sound attenuation. Attenuation is more pronounced for high-frequency sound, such as echolocation, and is a nonlinear function of both call frequency and ambient temperature. Hence, the prey detection ability, and thus possibly the foraging efficiency, of echolocating bats and susceptible to rising temperatures through climate change. Using present-day climate data and projected temperature rises, we modelled this effect for the entire range of bat call frequencies and climate zones around the globe. We show that depending on call frequency, the prey detection volume of bats will either decrease or increase: species calling above a crossover frequency will lose and species emitting lower frequencies will gain prey detection volume, with crossover frequency and magnitude depending on the local climatic conditions. Within local species assemblages, this may cause a change in community composition. Global warming can thus directly affect the prey detection ability of individual bats and indirectly their interspecific interactions with competitors and prey.

Trawling bats exploit an echo-acoustic ground effect.

A water surface acts not only as an optic mirror but also as an acoustic mirror. Echolocation calls emitted by bats at low heights above water are reflected away from the bat, and hence the background clutter is reduced. Moreover, targets on the surface create an enhanced echo. Here, we formally quantified the effect of the surface and target height on both target detection and -discrimination in a combined laboratory and field approach with Myotis daubentonii. In a two-alternative, forced-choice paradigm, the bats had to detect a mealworm and discriminate it from an inedible dummy (20 mm PVC disc). Psychophysical performance was measured as a function of height above either smooth surfaces (water or PVC) or above a clutter surface (artificial grass). At low heights above the clutter surface (10, 20, or 35 cm), the bats' detection performance was worse than above a smooth surface. At a height of 50 cm, the surface structure had no influence on target detection. Above the clutter surface, also target discrimination was significantly impaired with decreasing target height. A detailed analysis of the bats' echolocation calls during target approach shows that above the clutter surface, the bats produce calls with significantly higher peak frequency. Flight-path reconstruction revealed that the bats attacked an target from below over water but from above over a clutter surface. These results are consistent with the hypothesis that trawling bats exploit an echo-acoustic ground effect, in terms of a spatio-temporal integration of direct reflections with indirect reflections from the water surface, to optimize prey detection and -discrimination not only for prey on the water but also for some range above.

The relationship between syllable repertoire similarity and pairing success in a passerine bird species with complex song.

Repertoire size, i.e. the number of unique song elements that an individual possesses, is thought to be an important target of female preference. However, the use of repertoire size reflects how researchers work with complex songs; while it does not necessary describe biological functions, as listeners of song may also rely on song composition. Specific syllables may have coherent consequences for mate attraction because they are costly to produce, mediate syllable sharing or indicate the dialect of origin. We tested for the relationship between song composition and pairing success in the collared flycatcher (Ficedula albicollis). We applied a tree-clustering method to hierarchically classify males based on the degree of repertoire overlap, and then used a phylogenetic approach to assess the degree by which pairing speed matches the hierarchically structured song data. We found that males using similar syllables also find a breeding partner at a similar speed. Partitioning the variance components of pairing speed, we detected that the consequences of particular syllables for mating are repeatable across males. When assessing the role of repertoire similarity in mediating direct syllable sharing, we derived a positive relationship between the physical distance between pairs of males and their repertoire overlap implying that neighboring males avoid copying each other's song. Finally, we were unable to demonstrate that syllables related to higher mating success are more common in the population, which would support mechanisms based on female preference for local songs. Our results imply that individual-specific song organization may be relevant for sexual selection.

Seasonal changes in courtship behavior, plasma androgen levels and in hypothalamic aromatase immunoreactivity in male free-living European starlings (Sturnus vulgaris).

In songbirds from temperate latitudes, singing during spring has an essential role in mate attraction, while during the non-breeding season it is connected to territorial aggression and/or maintaining dominance hierarchies or flock cohesion. Courtship behavior is regulated by plasma testosterone (T) levels. Other androgens, like dehydroepiandrosterone (DHEA) could be responsible for aggression. The aromatization of androgens in the brain is an essential step in mediating their effects on behavior. Our goal was to determine whether the seasonal changes in male courtship behavior (measured by average song bout length and wing-waving/flicking) are related to seasonal changes in androgen activity (measured by plasma T, DHEA levels) and aromatase (ARO) immunoreactivity in the preoptic area/medial preoptic nucleus (POA/POM) of free-living male starlings. DHEA increased during pair formation, decreased at nesting and remained at low levels. The number of ARO cells - in line with the T levels - increased during the courtship and nesting periods, but outside the breeding season it was low. Song bout length showed a similar pattern, namely the peak was reached during the courtship period, and after that males stopped singing when chicks started to hatch. Short and fast wing-flicking and wing-waving behavior was observed only during the breading season. Summarizing, we have found that song bout length of male starlings changes parallel with plasma T levels and ARO immunoreactivity in the POA/POM. Furthermore, DHEA levels were low during the sexually inactive period which suggests that other mechanisms could be involved in the aggressive non-courtship behavior/vocalization in these birds.