Charles Ziegenfus

TESTOSTERONE AND AVIAN LIFE HISTORIES: EFFECTS OF EXPERIMENTALLY ELEVATED TESTOSTERONE ON BEHAVIOR AND CORRELATES OF FITNESS IN THE DARK-EYED JUNCO (JUNCO HYEMALIS)

Hormones influence many aspects of organismal behavior, physiology, and morphology, and thus hormones may lie at the root of many life-history trade-offs. By manipulating hormones we can create novel phenotypes (i.e., perform phenotypic engineering) and attempt to relate phenotypic variation to fitness. We report the effect of testosterone treatment on parental behavior and vocal behavior of adult male dark-eyed juncos. Testosterone partially suppressed paternal behavior and increased the frequency of song. When we compared treated males and controls for nine potential correlates of fitness (offspring growth and survival to the age of 10 d, condition of females, length of the interval between consecutive nestings, size of subsequent clutches and broods, mate retention within and between breeding seasons, and survival rate), we found no statistical differences. In some measures treated males outperformed controls, but in most the reverse was true. The power of some of our tests was not great enough to detect small differences. At this stage of our investigation, three interpretations of our results seem almost equally probable: (1) a broad range of behavioral phenotypes is selectively neutral in the junco, (2) male parental behavior is beneficial to males only in some years or habitats, or (3) we have yet to measure the correlates of fitness that are most strongly affected by the behavioral changes induced by elevated testosterone.

Testosterone and avian life histories: the effect of experimentally elevated testosterone on corticosterone and body mass in dark-eyed juncos.

To assess whether alterations in the normal pattern of testosterone (T) secretion might be beneficial or detrimental, we studied a breeding population of dark-eyed juncos in which we elevated T experimentally and measured its effect on potential correlates of fitness. We treated both free-living and captive males with implants that were either empty (C-males, controls) or packed with T (T-males, experimentals). Timing of implant varied and was designed to mimic natural peak breeding levels except that peaks were either prolonged or premature. We bled the birds at recapture and analyzed their plasma, and that of their female mates, for T and corticosterone (B). We also measured body mass and fat score in free-living T- and C-males. In the field, T-implants elevated T and kept it elevated for at least a month. Experimental males also had higher B than controls. In captives, the effect of the implants on plasma T was detectable within 24 hr. B in captive T-males was again higher than in captive C-males. In females, neither T nor B differed between mates of T- and C-males. T-males implanted in early spring lost more mass between implant and recapture in late spring than did controls and also had lower fat scores when recaptured. When implants were inserted in summer, treatment did not influence mass. Elevated T in early spring apparently hastened the transition from the winter to the breeding mode of fat storage. We suggest that prolonged elevation of testosterone might be selected against because of the association between T and B. Premature elevation of T might be costly because of the resultant loss of mass and fat reserves, which could lead to mortality when spring snowstorms prevent access to food.

Testosterone affects reproductive success by influencing extra–pair fertilizations in male dark–eyed juncos (Aves: Junco hyemalis)

Monogamous male birds typically allocate less effort to courtship and more to parental behaviour than males of polygynous species. The seasonal pattern of testosterone (T) secretion varies accordingly. Monogamous males exhibit a spring peak in plasma T followed by lower levels during the parental phase, while males of polygynous species continue to court females and maintain T at higher levels. To determine whether testosterone underlies the trade–off between mating and parental effort, we treated male dark–eyed juncos (Junco hyemalis) with exogenous T and compared the reproductive success (RS) of T–treated males (T–males) to that of controls. T–males had lower apparent annual RS than controls, probably because elevated T reduced parental care. Nevertheless, annual genetic RS of the treatment groups was similar because (i) T–males suffered fewer losses in genetic RS due to extra–pair fertilizations (EPFs), and (ii) T–males gained more genetic RS through their own EPFs. This is the first hormonal manipulation of an avian phenotype shown to have influenced male RS through EPFs. Together with other studies, it suggests that testosterone may have mediated the evolution of inter– and intraspecific differences in allocation of reproductive effort to mate attraction and parental care.

The Relative Impact of Extra-Pair Fertilizations on Variation in Male and Female Reproductive Success in Dark-Eyed Juncos (Junco hyemalis)

Differences among species in breeding system and degree of sex- ual dimorphism are thought to be mirrored by species-level differences in past or present sexual selection. The greater the deviation from monogamy, the greater the potential influence of sexual selection on the sex that is more variable in reproductive success (RS). The recent discovery of extra-pair fertilizations (EPFs) in apparently (i.e., socially) monogamous bird species, and the fact that EPFs have the potential to increase variance in RS of such species, led us to quantify EPFs in Dark-eyed Juncos and to explore the relative impact of EPFs on male and female variation in RS. We obtained season-long measures of frequency of EPFs, numbers of mates acquired, and apparent RS (young produced in nests of social mates) and genetic RS for 50 male and 45 female juncos studied during two breeding seasons in Virginia, USA. Forty-two of the 50 males produced offspring and 19 of these 42 (45.2%) lost paternity to EPFs. Thirty-eight of the 45 females produced offspring, and 13 of the 38 (34.2%) had at least one young sired by EPFs. There were no cases of conspecific brood parasitism. Of 187 young, 53 (28%) were sired by EPFs. Males that gained EPFs rarely suffered losses, and males that were victims of EPFs rarely gained by them. The result was that some males were more successful than others. We calculated variance in male and female apparent and genetic RS and mating success (MS, number of mates per season). We also calculated statistical dependence of RS on mating success. Among males, apparent reproductive success was less variable (standardized vari- ance (SV) = 0.55) than genetic success (SV = 0.72), indicating that EPFs in- creased variance in male RS. The sex difference in variance in genetic success (male SV = 0.72, female SV = 0.51) was considerably less than the sex difference in variance in mating success (male SV = 0.65, female SV = 0.37). Interestingly, in both males and females, genetic RS increased with number of mates, suggesting that sexual selection, when defined as the statistical dependence of RS on MS, was acting on both sexes. To our knowledge this is the first study to report EPF- based sexual selection in a socially monogamous female songbird. Although an increase in RS with MS will clearly cause selection to favor attributes of individ-

Testosterone Affects Neural Gene Expression Differently in Male and Female Juncos: A Role for Hormones in Mediating Sexual Dimorphism and Conflict

Despite sharing much of their genomes, males and females are often highly dimorphic, reflecting at least in part the resolution of sexual conflict in response to sexually antagonistic selection. Sexual dimorphism arises owing to sex differences in gene expression, and steroid hormones are often invoked as a proximate cause of sexual dimorphism. Experimental elevation of androgens can modify behavior, physiology, and gene expression, but knowledge of the role of hormones remains incomplete, including how the sexes differ in gene expression in response to hormones. We addressed these questions in a bird species with a long history of behavioral endocrinological and ecological study, the dark-eyed junco (Junco hyemalis), using a custom microarray. Focusing on two brain regions involved in sexually dimorphic behavior and regulation of hormone secretion, we identified 651 genes that differed in expression by sex in medial amygdala and 611 in hypothalamus. Additionally, we treated individuals of each sex with testosterone implants and identified many genes that may be related to previously identified phenotypic effects of testosterone treatment. Some of these genes relate to previously identified effects of testosterone-treatment and suggest that the multiple effects of testosterone may be mediated by modifying the expression of a small number of genes. Notably, testosterone-treatment tended to alter expression of different genes in each sex: only 4 of the 527 genes identified as significant in one sex or the other were significantly differentially expressed in both sexes. Hormonally regulated gene expression is a key mechanism underlying sexual dimorphism, and our study identifies specific genes that may mediate some of these processes.

Potential for sexual conflict assessed via testosterone-mediated transcriptional changes in liver and muscle of a songbird

Males and females can be highly dimorphic in metabolism and physiology despite sharing nearly identical genomes, and both sexes respond phenotypically to elevated testosterone, a steroid hormone that alters gene expression. Only recently has it become possible to learn how a hormone such as testosterone affects global gene expression in non-model systems, and whether it affects the same genes in males and females. To investigate the transcriptional mechanisms by which testosterone exerts its metabolic and physiological effects on the periphery, we compared gene expression by sex and in response to experimentally elevated testosterone in a well-studied bird species, the dark-eyed junco (Junco hyemalis). We identified 291 genes in the liver and 658 in the pectoralis muscle that were differentially expressed between males and females. In addition, we identified 1727 genes that were differentially expressed between testosterone-treated and control individuals in at least one tissue and sex. Testosterone treatment altered the expression of only 128 genes in both males and females in the same tissue, and 847 genes were affected significantly differently by testosterone treatment in the two sexes. These substantial differences in transcriptional response to testosterone suggest that males and females may employ different pathways when responding to elevated testosterone, despite the fact that many phenotypic effects of experimentally elevated testosterone are similar in both sexes. In contrast, of the 121 genes that were affected by testosterone treatment in both sexes, 78% were regulated in the same direction (e.g. either higher or lower in testosterone-treated than control individuals) in both males and females. Thus, it appears that testosterone acts through both unique and shared transcriptional pathways in males and females, suggesting multiple mechanisms by which sexual conflict can be mediated.

HOMING SUCCESS OF MIGRANT VERSUS NONMIGRANT DARK-EYED JUNCOS (JUNCO HYEMALIS)

Abstract Within a population, the evolution of migratory behavior is accompanied by a suite of physiological, behavioral, and cognitive adaptations. Spatial memory is associated with the hippocampus in mammals and birds; in some cases, hippocampal neuroanatomy correlates with differences in behavior. In a recent study, a migratory subspecies of sparrow, the Dark-eyed Junco (Junco hyemalis hyemalis), performed better on room-scale spatial memory tests than did a nonmigratory conspecific. Migrants collected after migration also possessed greater hippocampal neuron density, which suggests a neurological basis for differences in spatial memory and a link between migratory behavior and enhanced spatial memory. It is likely that homing behavior, like migration, relies to some extent on spatial memory. In some instances, spatial memory performance has generalized across spatial scales, with pronounced differences at larger scales. We tested whether differences in spatial memory between migrants and nonmigrants, previously observed at a room scale, were detectable at a landscape scale; specifically, we investigated whether differences in homing ability could be detected after displacements of 1–40 km. We found no difference in number of returning individuals or in duration of return. Our results suggest that homing in this species may not rely on aspects of spatial memory that differed in aviary tests.

Avian olfactory displays: a hypothesis for the function of bill-wiping in a social context

Bill-wiping, or the scraping by a bird of its bill along a substrate, has been observed in social contexts and cited as an irrelevant displacement activity. However, several behaviors once categorized as displacement behaviors have since been shown to serve adaptive functions. Here, we hypothesize that bill-wiping may function in social interactions by releasing odors from the waxy residue of preen oil on the bill. We assessed behavioral context associated with bill-wiping by comparing the frequency of bill-wiping by free-living male songbirds when presented with a caged male or female conspecific paired with playback; males bill-wiped significantly more often in response in a courtship context and in a mate-guarding context than in a territorial context. Bill-wiping frequency correlated with courtship behaviors, such as tail spreading and singing short-range songs, but not with aggressive behaviors. We also noted attributes of individuals that engaged in bill-wiping during courtship and found that younger, smaller males performed this behavior more frequently than older, larger males. Finally, we conducted a captive Y-maze experiment to test whether dried preen oil residue would be more detectable if it were manipulated (scratched to potentially release odor) or unmanipulated. Preliminary evidence suggests that males could be more responsive to manipulated preen oil, though stronger tests are needed. Taken together, our results suggest a functional hypothesis: bill-wiping during courtship may be an olfactory display that releases odor that may be detected by potential mates and rivals. We conclude by suggesting ways in which future work can test the olfactory display hypothesis.