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.     

Effects of testosterone on cell-mediated and humoral immunity in non-breeding adult European starlings

One of the primary assumptions of the immunocompetence hypothesisis that testosterone is immunosuppressive. Although many studiesin birds and mammals have supported this assumption, conflictingresults have been reported in a variety of species. We investigatedthe effects of testosterone manipulation on both cell-mediatedand humoral immunity in adult songbirds, European starlings(Sturnus vulgaris). Male and female starlings were wild-caught,housed in the laboratory, and implanted with either empty silasticcapsules or capsules containing testosterone. Six weeks after implantation, humoral immunity was assessed by injecting thebirds with a novel antigen, keyhole limpet hemocyanin, andmeasuring specific antibody responses 10 and 15 days latervia an enzyme-linked immunosorbant assay. Cell-mediated immunitywas assessed 7 weeks after implantation via intradermal injectionof the T-cell mitogen phytohemagglutinin into the wing web and measuring the degree of swelling 24 h later. Antibody responsesto antigenic challenge were significantly suppressed in testosterone-treatedfemales 10 days post-injection and in both sexes 15 days post-injection.Furthermore, there was a significant inverse relationship betweenindividual variability in antibody responsiveness and plasmatestosterone concentrations. Cell-mediated responses to phytohemagglutininstimulation were also significantly suppressed in testosterone-treatedmales compared to same-sex controls. Testosterone treatmentsignificantly increased plasma corticosterone concentrations compared to controls, and the possibility of this effect mediatingthe immunosuppressive effects of testosterone is discussed.The present study is among the first to demonstrate testosterone-inducedsuppression of both cell-mediated and humoral immunity in aspecies of songbird.     

Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings

Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory.     

Song acquisition in photosensitive and photorefractory male European starlings

We tested the ability of 1-year-old European starlings (Sturnus vulgaris) to acquire songs while in different physiological states. Photorefractory males, with low testosterone levels, learned songs as completely and as accurately as photosensitive males in full reproductive condition. This indicates that song acquisition in 1-year-old males does not depend on high levels of androgens. The ability to learn songs during the nonbreeding season may reflect the high song output of potential tutors through most of the year, including the photorefractory period, and may facilitate increases in song repertoire size in adulthood.     

Status-appropriate singing behavior,testosterone and androgen receptor immunolabeling in male European starlings (Sturnus vulgaris)

Vocalizations convey information about an individual's motivational, internal, and social status. As circumstances change, individuals respond by adjusting vocal behavior accordingly. In European starlings, a male that acquires a nest site socially dominates other males and dramatically increases courtship song. Although circulating testosterone is associated with social status and vocal production it is possible that steroid receptors fine-tune status-appropriate changes in behavior. Here we explored a possible role for androgen receptors. Male starlings that acquired nest sites produced high rates of courtship song. For a subset of males this occurred even in the absence of elevated circulating testosterone. Immunolabeling for androgen receptors (ARir) was highest in the medial preoptic nucleus (POM) in males with both a nest site and elevated testosterone. For HVC, ARir was higher in dominant males with high testosterone (males that sang longer songs) than dominant males with low testosterone (males that sang shorter songs). ARir in the dorsal medial portion of the nucleus intercollicularis (DM) was elevated in males with high testosterone irrespective of dominance status. Song bout length related positively to ARir in POM, HVC and DM, and testosterone concentrations related positively to ARir in POM and DM. Results suggest that the role of testosterone in vocal behavior differs across brain regions and support the hypothesis that testosterone in POM underlies motivation, testosterone in HVC relates to song quality, and testosterone in DM stimulates vocalizations. Our data also suggest that singing may influence AR independent of testosterone and that alternative androgen-independent pathways regulate status-appropriate singing behavior.     

Song predicts immunocompetence in male European starlings (Sturnus vulgaris)

According to the immunocompetence handicap hypothesis, sexually selected characteristics predict immune function and this relationship is mediated by testosterone. In the present study, we investigated whether bird song could predict immunity in European starlings (Sturnus vulgaris). We recorded the singing and reproductive behaviours of 16 adult male starlings in an outdoor aviary and then assessed their cell-mediated and humoral immunity in vivo. The males were observed in groups of four for 2 h each day over a 4-day period. For each male, the number of songs produced was recorded and the average song-bout length was computed. Next, cell-mediated and humoral immunity were assessed via cutaneous swelling responses to the T-cell mitogen phytohaemagglutinin and antibody responses to a novel antigen, keyhole limpet haemocyanin. Song rate and song-bout length were positively correlated with cell-mediated and humoral immunity, respectively. Additionally, a negative relationship between plasma testosterone concentration and antibody response was observed. These data demonstrate that male starling song can be used as a predictor of immunocompetence, with more robust singers exhibiting enhanced immunity. Whether this relationship is mediated directly by testosterone requires further investigation.     

Melatonin,immunity and cost of reproductive state in male European starlings.

The effects of reproductive condition and exogenous melatonin on immune function were investigated in castrated European starlings, Sturnus vulgaris. Photorefractory and photostimulated starlings exposed to long days were implanted with melatonin or with blank capsules. Photostimulated starlings with blank capsules exhibited reduced splenocyte proliferation in response to the T-cell mitogen, concanavalin A, compared with the other long-day birds. Exogenous melatonin prevented the suppression of immune function by photostimulation. Photorefractory starlings, with or without melatonin implants, exhibited enhanced immune function compared with photostimulated starlings implanted with blanks. This enhancement was not mediated by endogenous melatonin, but appeared to be related to changes in reproductive state. In addition to the traditional costs of reproduction in birds (e.g. raising of young), there may be a cost of the reproductive state of starlings (i.e. whether they are photorefractory or photostimulated). These data are, we believe, the first to indicate a direct effect of reproductive state on immune function that is independent of both photoperiod (i.e., changes in the duration of melatonin secretion) and gonadal steroids.     

Effects of marijuana on testosterone in male subjects

Clinical studies have given contradictory reports on the effect of smoking marijuana on the plasma levels of testosterone in males. A reanalysis of existing data established that testosterone levels are depressed both after smoking one marijuana cigarette and after intravenous infusion of delta-9-tetrahydrocannabinol, a pharmacologically active component of marijuana. Simulation of the marijuana interaction, under the assumption that delta-9-tetrahydrocannabinol inhibits testosterone production or secretion, suggests a minimum of 24 hours are required for testosterone to return to pre-smoking levels. A series of clinical studies are specified to clarify the nature of the interaction.     

Differences in the social context of song production in captive male and female European starlings.

Studies on singing behavior in Oscine focus essentially on males and are carried out during the breeding season. Singing in females appears rare and is not well documented. However, females of several species can produce a complex song. Does this lack of data correspond to a real difference in males and females or to a non appropriate context of observation? We studied the vocal and social behavior of captive male and female European starlings during two periods: breeding and non-breeding periods. Our results indicated that females sang mostly in a non-breeding context: their singing behavior was strongly diminished when nestboxes were present in the aviary. Moreover, females sang more frequently when their closest neighbor was a female whereas males sang mostly when they had no immediate neighbor. These results indicate a difference between males and females for the context of song production.     

Age- and behavior-related variation in volumes of song control nuclei in male European starlings

There is considerable interindividual variation in the volumes of song control nuclei. Sex and physiological condition appear to contribute to these differences; however, these factors alone do not account for all of the variation. Studies have attempted to relate differences in song behavior (i.e., song repertoire size) to variation in song nucleus volume, but have met with mixed success. In this article, two studies are presented that used male European starlings (Sturnus vulgaris) to explore the relationship between song nuclei volumes and age-related differences in song behavior and interindividual variation in song behavior in adults. The results of the first study showed that song repertoire size and song bout length were significantly greater in older adult than in yearling males. In addition, the volumes of the high vocal center (HVC) and nucleus robustus archistriatalis (RA) were significantly larger in older adults than yearlings. Area X of the parolfactory lobe did not differ significantly in volume between the two age classes. In the second study, both HVC and RA volume correlated positively with song bout length but not repertoire size among adult birds. Based on these results a new hypothesis is presented that states that variation in song nuclei volumes in starlings relates more to the amount of song produced than to the number of song types stored in memory. © 1996 John Wiley & Sons, Inc.     

Breeding-context-dependent relationships between song and cFOS labeling within social behavior brain regions in male European starlings (Sturnus vulgaris)

Male European starlings (Sturnus vulgaris) sing throughout the year, but the social factors that motivate singing behavior differ depending upon the context in which song is produced. In a non-breeding context (when testosterone concentrations are low), starlings form large, mixed-sex flocks and song is involved in flock cohesion and perhaps maintenance of social hierarchies. In contrast, in a breeding context (when testosterone concentrations are high), male song plays a direct role in mate attraction. How the nervous system ensures that song production occurs in an appropriate context in response to appropriate stimuli is not well understood. The song control system regulates song production, learning, and, to some extent, perception; however, these nuclei do not appear to regulate the social context in which song is produced. A network of steroid hormone sensitive nuclei of the basal forebrain and midbrain regulates social behavior. The present study used the immediate early gene cFOS to explore possible involvement of these regions in context-dependent song production. Numbers of cFOS-labeled cells in the medial bed nucleus of the stria terminalis, anterior hypothalamus, and ventromedial nucleus of the hypothalamus related positively only to song produced in a breeding context. In contrast, numbers of cFOS-labeled cells in three zones of the lateral septum related positively only to song produced in a non-breeding context. Taken together, these data suggest differential regulation of male starling song by social behavior nuclei depending upon the breeding context in which it is produced.     

Influence of testosterone metabolites on song-control system neuroplasticity during photostimulation in adult European starlings (Sturnus vulgaris)

The song-control system is a network of discrete nuclei in the songbird brain that controls the production and learning of birdsong and exhibits some of the best-studied neuroplasticity found in the adult brain. Photoperiodic growth of the song-control system during the breeding season is driven, at least in part, by the gonadal steroid testosterone. When acting on neural tissue, however, testosterone can be metabolized into 5α-dihydrotestosterone (DHT) or 17β-estradiol (E2), which activate different hormonal signaling pathways. By treating adult starlings with both testosterone metabolites and metabolite antagonists, we attempted to isolate the effects of androgen and estrogen treatment on neuroplasticity during photostimulation in male and female European starlings (Sturnus vulgaris). Photostimulation resulted in a large HVC volume typical of the breeding season in all treatments independent of hormone treatment. E2 had additional effects on HVC growth by reducing neuron density and enhancing early survival of new neurons recruited to HVC in females but did not significantly affect HVC volume. Conversely, DHT reduced the migration of new neurons, assessed by the expression of doublecortin, to HVC. DHT also increased syrinx mass and maintained RA (robust nucleus of the arcopallium) cytoarchitecture in the presence of aromatase inhibitors. In addition, we document the first evidence of sex-specific neuroplastic responses of the song-control system to androgens and estrogens. These findings suggest that the contributions of DHT and E2 signaling in songbird neuroplasticity may be regulated by photoperiod and that future studies should account for species and sex differences in the brain.     

DHEA and estradiol levels in brain,gonads, adrenal glands,and plasma of developing male and female European starlings

Traditionally, sexual differentiation of the brain was thought to be driven by gonadal hormones, particularly testosterone (T). However, recent studies in songbirds suggest that other steroids may also be important. For example, dehydroepiandrosterone (DHEA) can be synthesized by the gonads, adrenal glands, and/or brain and locally metabolized into T and 17β-estradiol (E₂). Here, we examined DHEA and E₂ levels in the brain, peripheral tissues, and plasma of wild European starlings (Sturnus vulgaris). In Study 1, samples were collected from males and females at P0 (day of hatch), P6, and P8. In Study 2, samples were collected at P4. At P0, DHEA levels in the diencephalon were higher in males than females. DHEA levels were generally high in the gonads and adrenals, and they were higher in testes than ovaries at P8. Further, E₂ levels were non-detectable in most brain samples, suggesting that DHEA was not metabolized to E₂ or that locally produced E₂ was rapidly inactivated. At P4, DHEA levels in telencephalic regions were lower in males than females. Taken together, these data suggest that sex differences in peripheral DHEA secretion and neural DHEA metabolism at specific ages during development might play a role in sexual differentiation of the songbird brain.     

Age-dependent changes in plasma and brain cholinesterase activities of house wrens and European starlings

We determined age-dependent changes in plasma and brain cholinesterase (ChE) activity for two species of passerines: house wren (Troglodytes aedon) and European starling (Sturnus vulgaris, starling). In plasma from nestlings of both species, total ChE activity increased with age, acetycholinesterase (AChE, EC 3.1.1.7) activity declined rapidly immediately after hatching, and butyrylcholinesterase (BChE, EC 3.1.1.8) activity increased steadily. For both species, total ChE and BChE activities and the BChE:AChE ratio in plasma were significantly greater in adults than nestlings suggesting trends observed in nestlings continue post fledging. In older nestlings and adults, AChE activity in plasma was significantly greater and BChE:AChE ratio less in house wrens than starlings. For house wrens as compared with starlings, ChE activity in brain increased at a significantly greater rate with age in nestlings and was significantly greater in adults. However, ChE activity in brain was similar at fledging for both species suggesting that the increase in ChE in brain is more directly related to ontogeny than chronologic age in nestlings of passerines. For both species, ChE activity increased significantly with brain weight of nestlings but not adults. House wrens hold similar patterns of age-dependent change in ChE activity in common with starlings but also exhibit differences in AChE activity in plasma that should be considered as a factor potentially affecting their relative toxicologic response to ChE inhibitors.     

Female aggression and the maintenance of monogamy: female behaviour predicts male mating status in European starlings

Any reduction in the fitness of a breeding female induced by the settlement of additional females with her mate creates a conflict between the sexes over mating system. In birds, females are often aggressive towards other females but few studies have been able to quantify the importance of female-female aggression for the maintenance of monogamy. This study of the European starling, Sturnus vulgaris, quantifies male and female behaviour towards a potential prospecting female, presented in a cage during the pre-laying period, and relates it to the subsequent mating status of the male. A solitary breeding male was given the opportunity to attract an additional mate, which almost half of the males did. No biometric characters of the male or female were related to the subsequent mating status. Males demonstrated mate-attraction behaviour towards the caged female but the behaviour of the male did not predict the likelihood to attract an additional female. However, the proportion of time that the female spent near the potential settler was related to mating status, indicating that females that reacted more strongly towards a potential female competitor maintained their monogamous status. These results suggest that female behaviour may play an important role in shaping the mating system of facultatively polygynous species.     

Castration does not decrease nonreproductive aggression in yearling male European starlings (Sturnus vulgaris)

In the nonbreeding season, some bird species express high levels of aggression despite basal plasma testosterone (T) concentrations. Consequently, nonreproductive aggression is believed to be independent of plasma T. In the present study, we investigated the effect of castration on nonreproductive aggressive behavior in yearling male European starlings (Sturnus vulgaris). We paired castrated (Cx) with control (C) males in dyadic trials during three test periods (December, January-February, and March-April), and by using an infrared camera, we defined which male was the most aggressive one when both males were competing over access to a nest box to roost in. During each of the three periods, Cx males behaved more aggressively than C males but differences between groups did not reach significance. When analyzing the results of the three periods together, Cx males were found to be significantly more aggressive than C males. Likewise, when considering only the second and third period (when plasma T levels of C males were significantly higher than those of Cx males) the same result was obtained. Furthermore, in the third period, aggression tended to be lower than in the first and second period, although T concentrations of C males were highest in this period. Our data thus clearly show that nonreproductive aggression in yearling male starlings is independent of gonadal sex steroids and suggest it even increases by castration.     

Circannual variations in plasma luteinizing hormone levels in castrated male European starlings (Sturnus vulgaris)

Intact and castrated male European starlings were held for about 2 years in a constant 12-hr photoperiod and constant temperature conditions. At 1- to 2-month intervals, testicular width was measured by laparotomy, and blood samples were taken for analysis of plasma luteinizing hormone (LH). Most of the control birds went through at least one circannual cycle of testicular width and plasma LH concentration. In the castrates, a similar proportion of birds went through circannual LH cycles with periods indistinguishable from those of the controls. It is concluded that the testes and their hormones are not essential components of the mechanism that generates circannual gonadal cycles in male European starlings.     

Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris)

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.