Seasonality and Hormonal Control of Territorial Aggression in Female Song Sparrows (Passeriformes: Emberizidae: Melospiza melodia)

Male and female song sparrows perform similar aggressive behaviors including flights, wing-wave threat displays, and growls. Females very rarely sing, however. To investigate the seasonal variation and hormonal control of territorial aggression in females, we simulated female intrusions during the pre-breeding, breeding and post-molt seasons in the field. Circulating levels of testosterone, dihydrotestosterone, estradiol, progesterone and corticosterone in females experiencing simulated intrusion were compared to those of passively netted females matched for breeding stage. Female aggressive responses to female intrusion dropped from the pre-breeding to the post-molt seasons. Levels of circulating androgens were significantly higher in control females than in females experiencing a simulated intrusion. There were no significant differences in any of the other hormones measured. Although song sparrow female-female aggression appears to be behaviorally similar to male-male aggression, the seasonal variation and hormonal support of aggression differ between the sexes.     

Two Sides of the Same Coin? Consistency in Aggression to Conspecifics and Predators in a Female Songbird

Different forms of aggression have traditionally been treated separately according to function or context (e.g. aggression towards a conspecific versus a predator). However, recent work on individual consistency in behavior predicts that different forms of aggression may be correlated across contexts, suggesting a lack of independence. For nesting birds, aggression towards both conspecifics and nest predators can affect reproductive success, yet the relationship between these behaviors, especially in females, is not known. Here we examine free-living female dark-eyed juncos (Junco hyemalis) and compare their aggressive responses towards three types of simulated intruders near the nest: a same-sex conspecific, an opposite-sex conspecific, and a nest predator. We also examine differences in the strength of response that might relate to the immediacy of the perceived threat the intruder poses for the female or her offspring. We found greater aggression directed towards a predator than a same-sex intruder, and towards a same-sex than an opposite-sex intruder, consistent with a predator being a more immediate threat than a same-sex intruder, followed by an opposite-sex intruder. We also found positive relationships across individuals between responses to a same-sex intruder and a simulated predator, and between responses to a same-sex and an opposite-sex intruder, indicating that individual females are consistent in their relative level of aggression across contexts. If correlated behaviors are mediated by related mechanisms, then different forms of aggression may be expressions of the same behavioral tendency and constrained from evolving independently.     

The role of androgen receptors in regulating territorial aggression in male song sparrows

This paper examines the role that androgen receptors (ARs) play in modulating aggressive behavior in male song sparrows, Melospiza melodia morphna. Song sparrows are seasonally breeding, territorial birds that maintain year-round territories with male-female pair bonds formed during the spring breeding season. Plasma testosterone levels peak as territories are established and mates acquired. In late summer, testosterone levels fall and remain basal during the non-breeding season. We examined the role of ARs in regulating territorial aggression in captive song sparrows under short- and long-day conditions as well as just prior to, and at the start of the breading season in freely living birds using the nonsteroidal antiandrogen flutamide to block AR function. Birds were implanted with either empty or drug filled silastic implants for 18 to 42 days and then challenged with a novel male decoy to assess the individual birds level of male-male aggression. Freely living birds remained on their home territory and underwent a simulated territorial intrusion, whereas laboratory-held birds were assessed using a laboratory simulated territorial intrusion and remained in their home cage. Experimental treatment of male song sparrows decreased aggressive behavior during the pre-breeding life history substage (March-April) in freely living birds as well as in laboratory-held birds under long-day (16L:8D) conditions. During the early breeding substage (April-May) there was no measurable effect of flutamide treatment on aggressive behavior, nor was there a difference in behavior in the (8L:16D) laboratory birds. This demonstrates that ARs are an important component of the neuroendocrine control of aggressive behavior. Given that flutamide only affected aggression during the pre-breeding substage and in LD birds, the results suggest that AR dependent control of aggressive behavior changes as song sparrow life history states change.     

Sexual differentiation and seasonal variation in response to conspecific and heterospecific acoustic signals

Interspecific territoriality is frequently reported between closely related species; however, few studies have demonstrated interspecific territoriality between distantly related species living in sympatry. We conducted playback experiments to investigate territorial behaviour in male and female White‐bellied Wrens (Uropsila leucogastra) in response to simulated conspecific and heterospecific intruders during the breeding and non‐breeding seasons. We explored whether heterospecific songs of the Happy Wren (Pheugopedius felix), a distantly related species and ecological competitor, elicited antagonistic responses from focal White‐bellied Wrens, and whether such responses differed between the sexes. We also examined whether male and female responses to conspecific and heterospecific rivals varied with season. We found that male White‐bellied Wrens always responded to conspecific song, and responded significantly more to heterospecific song compared to a control stimulus (Tropical Parula, Setophaga pitiayumi). In contrast, although female White‐bellied Wrens responded strongly to conspecific song, their response to heterospecific song did not differ significantly from the control stimulus. The proportion of males that responded to heterospecific songs and the proportion of females that responded to conspecific songs varied seasonally, showing significantly lower responses during the breeding season. The intense responses of male White‐bellied Wrens to playback of heterospecific songs suggest that they recognise ecological competitors based on their vocal signals. Furthermore, the decrease in agonistic interactions during the breeding season is in line with the hypothesis that aggressive behaviour may be detrimental to reproductive and parental activity, and the hypothesis that heterospecific animals pose less of a threat during the breeding season.     

Long-term influence of simulated territorial intrusions on dawn and dusk singing in the Winter Wren: spring versus autumn

Males of many songbird species have peaks of singing activity at dawn and dusk. Singing during those twilight periods can function in territory proclamation, and males are suggested to adjust song output to the level of intruder pressure. We used song playback during the breeding season to simulate intrusions into territories of male Winter Wrens (Troglodytes troglodytes) shortly after dawn. We then compared male singing behaviour during the dawn and dusk chorus before and 1 day after the simulated intrusion. One day after the playback, male Wrens increased their song output before sunrise, which confirms our results from a previous study on dawn singing in autumn territories. At dusk, on the evening following the playback, males slightly increased song output after sunset, but singing activity at dusk was generally very low. We found no significant changes of song output after sunrise, before sunset, and between 2 days of control without playback. These results are consistent with the hypothesis that dawn and dusk singing is important for territory defence in spring. Unlike in autumn, however, increased singing in spring at dawn and dusk could also serve to defend other resources such as fertile mates or to strengthen the pair bond after a territorial challenge. In comparison with the results on autumnal singing, male Wrens started singing earlier at dawn during the breeding season, and they generally sang more songs at dawn and immediately after playback. The increase in absolute numbers of songs sung in the morning after playback seemed greater in spring than in autumn; however, the proportional increase relative to overall song output was similar in both seasons.     

Non-invasive administration of 17β-estradiol rapidly increases aggressive behavior in non-breeding,but not breeding,male song sparrows

17β-Estradiol (E₂) acts in the brain via genomic and non-genomic mechanisms to influence physiology and behavior. There is seasonal plasticity in the mechanisms by which E₂ activates aggression, and non-genomic mechanisms appear to predominate during the non-breeding season. Male song sparrows (Melospiza melodia) display E₂-dependent territorial aggression throughout the year. Field studies show that song sparrow aggression during a territorial intrusion is similar in the non-breeding and breeding seasons, but aggression after an intrusion ends differs seasonally. Non-breeding males stop behaving aggressively within minutes whereas breeding males remain aggressive for hours. We hypothesize that this seasonal plasticity in the persistence of aggression relates to seasonal plasticity in E₂ signaling. We used a non-invasive route of E₂ administration to compare the non-genomic (within 20 min) effects of E₂ on aggressive behavior in captive non-breeding and breeding season males. E₂ rapidly increased barrier contacts (attacks) during an intrusion by 173% in non-breeding season males only. Given that these effects were observed within 20 min of E₂ administration, they likely occurred via a non-genomic mechanism of action. The present data, taken together with past work, suggest that environmental cues associated with the non-breeding season influence the molecular mechanisms through which E₂ influences behavior. In song sparrows, transient expression of aggressive behavior during the non-breeding season is highly adaptive: it minimizes energy expenditure and maximizes the amount of time available for foraging. In all, these data suggest the intriguing possibility that aggression in the non-breeding season may be activated by a non-genomic E₂ mechanism due to the fitness benefits associated with rapid and transient expression of aggression.     

Seasonality in song behaviour revisited: seasonal and annual variants and invariants in the song of the domesticated canary (Serinus canaria)

The song of the domesticated canary (Serinus canaria) is one of the most widely used models to study the neural correlates of behavioural plasticity and the mechanisms of female mate choice. However, only few studies have described the song behaviour in detail and monitored their changes throughout the year, and these data are restricted to the “Waterslager” strain. Here, we studied the song characteristics of the male common domesticated canary at different times of the year, the spring breeding and autumnal non-breeding season, and monitored the birds' songs up to the following breeding season. During breeding, males have increased plasma levels of testosterone, and songs are on average longer and consist of fewer non-repeated syllable types compared to the non-breeding season. When subsequent seasons are compared, song duration and the proportion of non-repeated syllable types change seasonally but not across years. Repertoire size remains constant throughout seasons although syllable types are exchanged. Syllable carry-over is significantly higher from one breeding season to the next than between the breeding and non-breeding season. Further, the repertoire of the breeding season contains more potentially sexually attractive syllable types than that of the non-breeding season. These data show that overall song structure is retained throughout the year while seasonality occurs in the temporal pattern and in repertoire composition.     

Seasonal Differences of Gene Expression Profiles in Song Sparrow (Melospiza melodia) Hypothalamus in Relation to Territorial Aggression

Background

Male song sparrows (Melospiza melodia) are territorial year-round; however, neuroendocrine responses to simulated territorial intrusion (STI) differ between breeding (spring) and non-breeding seasons (autumn). In spring, exposure to STI leads to increases in luteinizing hormone and testosterone, but not in autumn. These observations suggest that there are fundamental differences in the mechanisms driving neuroendocrine responses to STI between seasons. Microarrays, spotted with EST cDNA clones of zebra finch, were used to explore gene expression profiles in the hypothalamus after territorial aggression in two different seasons.

Methodology/Principal Findings

Free-living territorial male song sparrows were exposed to either conspecific or heterospecific (control) males in an STI in spring and autumn. Behavioral data were recorded, whole hypothalami were collected, and microarray hybridizations were performed. Quantitative PCR was performed for validation. Our results show 262 cDNAs were differentially expressed between spring and autumn in the control birds. There were 173 cDNAs significantly affected by STI in autumn; however, only 67 were significantly affected by STI in spring. There were 88 cDNAs that showed significant interactions in both season and STI.

Conclusions/Significance

Results suggest that STI drives differential genomic responses in the hypothalamus in the spring vs. autumn. The number of cDNAs differentially expressed in relation to season was greater than in relation to social interactions, suggesting major underlying seasonal effects in the hypothalamus which may determine the differential response upon social interaction. Functional pathway analyses implicated genes that regulate thyroid hormone action and neuroplasticity as targets of this neuroendocrine regulation.     

Overlapping and matching in the song contests of black-capped chickadees

During vocal contests, animals alter both the timing and the patterning of responses to their opponents. Time-specific responses (such as overlapping an opponent's song) and pattern-specific responses (such as matching the type of an opponent's song) may reveal information about interacting animals. Here we explore the consequences of overlapping and frequency matching during song contests of male black-capped chickadees, Poecile atricapillus. Using interactive playback, we engaged birds of high and low dominance status in vocal interactions with a simulated territorial intruder. The playback intruder either overlapped or avoided overlapping the subjects' songs and either matched or avoided matching the frequency of the subjects' songs. Individuals who were overlapped by the playback intruder showed higher variability in their song length and song timing than individuals who were not overlapped. Individuals who were frequency-matched by the playback intruder responded with more agitated responses (more flights and passes over the speaker and closer distances of closest approach) and spent more time farther away from the loudspeaker. We argue that the timing of song delivery and the choice of song type are distinct functional components of vocal interactions, where overlapping and matching songs are threatening signals that have separate consequences for opponent behaviour and song performance. High-ranking males responded at greater distances from the loudspeaker in all treatments and responded with lower agitation levels than low-ranking males. We demonstrate that males of different quality show different behavioural responses to territorial intruders, where males of high status appear reluctant to engage an intruder as intensely as males of low status.     

Aggressive interactions differentially modulate local and systemic levels of corticosterone and DHEA in a wild songbird

During the nonbreeding season, when gonadal androgen synthesis is basal, recent evidence suggests that neurosteroids regulate the aggression of male song sparrows. In particular, dehydroepiandrosterone (DHEA) is rapidly converted in the brain to androgens in response to aggressive interactions. In other species, aggressive encounters increase systemic glucocorticoid levels. However, the relationship between aggression and local steroid levels is not well understood. Here, during the breeding and nonbreeding seasons, we tested the effects of a simulated territorial intrusion (STI) on DHEA and corticosterone levels in the brachial and jugular plasma. Jugular plasma is enriched with neurosteroids and provides an indirect index of brain steroid levels. Further, during the nonbreeding season, we directly measured steroid levels in the brain and peripheral tissues. Both breeding and nonbreeding males displayed robust aggressive responses to STI. During the breeding season, STI increased brachial and jugular corticosterone levels and jugular DHEA levels. During the nonbreeding season, STI did not affect plasma corticosterone levels, but increased jugular DHEA levels. During the nonbreeding season, STI did not affect brain levels of corticosterone or DHEA. However, STI did increase corticosterone and DHEA concentrations in the liver and corticosterone concentrations in the pectoral muscle. These data suggest that 1) aggressive social interactions affect neurosteroid levels in both seasons and 2) local steroid synthesis in peripheral tissues may mobilize energy reserves to fuel aggression in the nonbreeding season. Local steroid synthesis in brain, liver or muscle may serve to avoid the costs of systemic increases in corticosterone and testosterone.     

Individual variation in response to simulated territorial challenge among territory-holding song sparrows

Lack (1946) suggested that male songbirds exhibit consistent individual differences in the vigor or manner in which they defend their territories against intrusion. The causes and consequences of such individual variation have not been incorporated into models of territoriality, however, because of a lack of experimental data confirming Lack's suggestion. In this paper, we test the possibility that male song sparrows Melospiza melodia who are successful territory holders differ consistently in the vigor with which they defend their territory, by conducting repeated song playback trials with the same set of territory-holding subjects across a breeding season. We found only relatively weak seasonal trends in responsiveness: the amount birds sang in response to playback increased significantly across the breeding season and responsiveness was generally lower when a male's social mate was egg laying. By contrast, we found extensive variation among males in how closely they approached a simulated territorial intrusion. These individual differences remained significantly consistent across four rounds of playback trials that spanned the breeding season as determined by Kendall's coefficient of concordance. Our results confirm that some individual song sparrows are consistently more vigorous than others in territory defense, at least in one conspicuous aspect of their behavior, and suggest that further work is needed to understand the nature and consequences of variation in patterns of defense among successful territory holders.     

Fighting and Mating Between Groups in a Cooperatively Breeding Mammal, the Banded Mongoose

Many cooperatively breeding animals actively defend a territory containing resources such as food and shelter, which are essential for reproduction. Some observations, however, indicate that conflicts between groups are often triggered by the attempts of males or females, or both, to gain extra‐group copulations. We studied interactions between 12 groups of banded mongooses (Mungos mungo) in Uganda to test whether the frequency of inter‐group encounters was linked to the reproductive status of females, and conducted an experiment to examine the responses of individuals to mongooses from other groups. The rate at which focal groups fought with other groups was higher when its females were in estrous, suggesting that many fights take place over access to mates. Both males and estrous females were instrumental in instigating encounters with rival groups, and extra‐group copulation in the midst of a fight was observed on three occasions. We experimentally simulated encounters with foreign individuals by presenting each of six groups with cages containing a male and a female from a rival group. Subordinate males were the first to investigate these ‘intruders’, spent more time around the intruders' cages, and were more aggressive to the intruders than either dominant males or females. Subordinate males directed more attention and aggression towards the male intruder than the female intruder. We suggest that male banded mongooses actively seek extra‐group copulations in pursuit of paternity, while females may actively seek extra‐group copulations as a way of reducing inbreeding depression.     

Seasonal specificity of hormonal, behavioral, and coloration responses to within- and between-sex encounters in male lizards (Sceloporus undulatus).

This study reports the gender and seasonal specificity of hormonal, behavioral, and coloration responses displayed by "resident" male lizards (Sceloporus undulatus) exposed to male or female "intruders" during staged encounters in outdoor enclosures. Resident males were engaged in staged encounters with males or females for 1 h per day on 9 consecutive days during the breeding and postbreeding seasons. Male-specific responses occurred during the breeding but not the postbreeding season. These included (1) a transient increase in plasma testosterone (T) that was evident on Day 4 and had subsided by Day 10, (2) behavioral displays of aggression (full shows and chases), and (3) a lightening of dorsal integumental color. Female-specific behavioral responses (nod sets) were displayed in both seasons. Season-specific responses consisted only of a transient increase in plasma corticosterone (B) during the breeding season that was evident on Day 4 and had subsided by Day 10. Pushups were displayed in response to both genders during both seasons, although the frequency of pushups was significantly higher in response to females than to males during the postbreeding season. The coloration of residents did not change in response to male intruders during the postbreeding season or to females during either season. These results define the gender and seasonal specificity of hormonal, behavioral, and coloration responses of resident male S. undulatus in social interactions with conspecifics. Thus, our results clarify the biological significance of these responses in terms of potentially aggressive versus courtship interactions and breeding versus postbreeding contexts.     

Species song discrimination in adult female song and swamp sparrows

Six female song sparrows (Melospiza melodia) and six female swamp sparrows (Melospiza georgiana) were treated with oestradiol, and their response to song measured by frequency and intensity of copulation-solicitation display. Test stimuli were synthetic songs composed of either song sparrow or swamp sparrow syllables assembled in either song-sparrow-like or swamp-sparrow-like temporal patterns. Female song sparrows responded preferentially to songs containing their own species' syllables and to songs containing their own species' temporal patterns. Swamp sparrows were also sensitive to both syllable type and temporal, pattern, in contrast to male swamp sparrows, which show no preference for swamp sparrow temporal patterns.     

Differential mechanisms for regulation of the stress response across latitudinal gradients

We examined plasticity of the stress response among three populations of the white-crowned sparrow (Zonotrichia leucophrys). These populations breed at different elevations and latitudes and thus have breeding seasons that differ markedly in length. We hypothesize that in populations where birds raise only one or rarely two broods in a season, the fitness costs of abandoning a nest are substantially larger than in closely related populations that raise up to three broods per season. Thus individuals with short breeding seasons should be less responsive to stressors and therefore less likely to abandon their young. In our study, baseline and handling-induced corticosterone levels were similar among populations, but corticosteroid-binding globulins differed, leading to a direct relationship between stress-induced free corticosteroid levels and length of breeding season. There were also population-specific differences in intracellular low-affinity (glucocorticoid-like) receptors in both liver and brain tissue. Although investigations of population-based differences in glucocorticoid secretion are common, this is the first study to demonstrate population-level differences in binding globulins. These differences could lead to dramatically different physiological and behavioral responses to stress.     

Protracted reproductive seasonality in the male giant panda (Ailuropoda melanoleuca) reflected by patterns in androgen profiles, ejaculate characteristics, and selected behaviors

The female giant panda (Ailuropoda melanoleuca) experiences a brief (24-72 h) seasonal estrus, occurring once annually in spring (February-May). Our aim was to determine the existence and temporal profile of reproductive seasonality in the male of this species. The study was facilitated by 3 yr of access to eight giant panda males living in a large breeding center in China. Seasonal periods for the male were defined on the basis of female reproductive activity as prebreeding, breeding (early, peak, late), and nonbreeding seasons. Testes size, fecal androgen excretion, ejaculated sperm density, and frequency of reproductive behaviors (i.e., locomotion, scent marking, vocalizations) increased (P < 0.05) from the prebreeding period (October 1-January 31) to the early breeding season (February 1-March 21). Testes volume and sperm concentration were maximal from March 22 through April 15, a period coinciding with maximal female breeding activity. The occurrence of male reproductive behaviors and fecal androgen concentrations began declining during peak breeding and continued from April 16 through May 31 (late breeding period), returning to nadir throughout the nonbreeding interval (June 1-September 30). Reproductive quiescence throughout the latter period was associated with basal testes size/volume and aspermic ejaculates. Our results reveal that testes morphometry, fecal androgen excretion, seminal quality, and certain behaviors integrated together clearly demonstrate reproductive seasonality in the male giant panda. The coordinated increases in testes size, androgen production, sperm density, and sexual behaviors occur over a protracted interval, likely to prepare for and then accommodate a brief, unpredictable female estrus.     

Context, Structural Variability and Distinctiveness of California Towhee (Pipilo crissalis) Vocal Duets

Avian vocal duets occur when paired birds produce temporally and structurally coordinated vocalizations. Duets are given by members of many species from taxonomically distinct lineages and show great variety in form that often reflects function. By describing the structure of vocal duets we can learn about the diversity of communication signals present in nature and also gain insight into the evolution and operation of those signals. This study quantified the usage patterns and acoustic structure of California towhee duet vocalizations, and tested the distinctiveness of duets among different pairs. California towhee duets consist of a 'squeal' vocalization that is highly unlike the species-typical call note or male advertizement song. California towhees duet an average of three times per hour during the breeding season, and all duets are accompanied by an approach response that brings duetting partners into close spatial proximity. Males and females produce duet contributions with the same syllabic structure. Individual birds produce highly variable squeal vocalizations that are distinctive enough to signal identity. California towhee duet characteristics indicate that the squeal vocalization has evolved separately from other vocal traits in this species and was promoted by natural selection as a duet-specific vocalization. Duet usage patterns and structure suggest that these communication signals function in a cooperative context.     

Perceived wintering latitude determines timing of song output in a migratory bird

Migratory bird populations frequently consist of individuals that overwinter variable distances from the breeding site. Seasonal changes in photoperiod, which varies with latitude, underlie seasonal changes in singing frequency in birds. Therefore, migratory populations that consist of individuals that overwinter at different latitudes with large overwintering ranges could experience within‐population variation in seasonal production of song. To test the influence of overwintering latitude on intrapopulation variance in song production in the spring, we subjected two groups of Eastern Song Sparrows (Melospiza melodia melodia) from the same partially migratory breeding population to different photoperiodic schedules associated with a 1,300‐km difference in overwintering location. One group remained on the natural photoperiodic schedule of the breeding site (resident group) while the other group experienced a nonbreeding photoperiod that mimicked a southern migration in the fall followed by a northern migration back to the breeding site in the spring (migratory group). We compared song output between the two groups in three different stages (nonbreeding, prebreeding, and breeding). Little singing occurred during nonbreeding stage sample dates (20 November, 6 December) for the resident group, and no singing occurred for the migrant group. During the prebreeding stage (27 January, 7 February), significantly more singing occurred in the resident group than in the migrant group. During the breeding stage (21 March, 4 April), after a simulated migration for the migrants, song output was similar in both groups. These results suggest that within‐population variation in wintering latitude may contribute to variation in seasonal changes in singing behavior, which may covary with readiness to breed. Studies utilizing confirmed migrants and residents, rather than merely simulated migrants and residents, are also needed to better understand these processes.     

Effects of corticosterone on territorial behavior of free-living male song sparrows Melospiza melodia

A group of 10 territorial male song sparrows, Melospiza melodia, were given subcutaneous implants of corticosterone in Silastic tubing. A second group of 10 territorial males were given empty implants as controls. After 18-24 hr all males were then subjected to a simulated territorial intrusion (STI) by placing a caged male song sparrow in the center of the subject's territory, and playing tape recorded songs through a speaker placed alongside. Significantly fewer males with corticosterone implants responded to STI than to controls, and the latency to respond was longer. Of the 3 experimental males that did respond to STI, all had a lower frequency of songs and did not approach the simulated intruder as closely as controls. Many males were captured 2-7 days after implantation and blood samples collected for measurement of circulating hormone levels. As expected, plasma levels of corticosterone were high in the group given corticosterone implants. However, plasma levels of luteinizing hormone (LH) were not affected by treatment with corticosterone, and although circulating levels of testosterone were depressed slightly compared with controls, they were within the normal range for territorial and breeding males. There were no differences in body mass despite greatly increased fat depots in males treated with corticosterone. These data suggest that high levels of corticosterone, similar to those measured during stressful episodes both in the laboratory and field, may suppress territorial behavior independently of the adenohypophysial-gonad axis. Since plasma levels of LH and testosterone are not depressed markedly, thus maintaining the gonads in a near functional state, renesting can begin as soon as environmental conditions ameliorate. Such mechanisms could potentially increase the probability of raising viable young after unpredictable, severe weather resulted in failure of the previous breeding attempt.     

Context-dependent effects of castration and testosterone treatment on song in male European starlings

Most seasonally breeding songbirds display dramatic seasonal fluctuations in plasma testosterone (T) levels and mate attraction behaviors, including song. However, males of some songbird species, such as the European starling (Sturnus vulgaris), continue to sing at high levels after the breeding season, when T levels are basal. In male starlings song during the breeding season functions mainly to attract mates, whereas song during the nonbreeding season appears unrelated to reproduction. This suggests that song produced in a context unrelated to female courtship, unlike song directed toward females, is not regulated by plasma T. In captive males housed in large outdoor aviaries we explored the relationship between plasma T and song produced during the breeding season within and outside a courtship context. This was achieved by determining the effects of castration and subsequent T treatment on song and mate attraction behaviors in both the presence and the absence of a female. Compared to intact males, castrated males did not show reduced song activity in the absence of a female for at least 6 months after the operation, strongly suggesting that the expression of noncourtship song is not regulated by plasma T. Likewise, we found that experimentally elevating T levels in castrated males did not affect noncourtship song rates. However, control castrated males receiving empty implants tended to show reduced noncourtship song rates after implantation. This may have been due to a suppressive effect caused by the presence of the T-implanted castrated males in the same aviary. In contrast, courtship singing was clearly controlled by plasma T: it was abolished by castration and restored by subsequent T replacement when males were housed both individually and in a group situation. High plasma levels of T also appeared necessary for the activation of three other behavioral traits critical for mate attraction, namely, nesthole occupancy, spending time (singing) in a nesthole, and carrying green nesting material into a nesthole.