Kimberly A. Rosvall

Neural sensitivity to sex steroids predicts individual differences in aggression: implications for behavioural evolution

Testosterone (T) regulates many traits related to fitness, including aggression. However, individual variation in aggressiveness does not always relate to circulating T, suggesting that behavioural variation may be more closely related to neural sensitivity to steroids, though this issue remains unresolved. To assess the relative importance of circulating T and neural steroid sensitivity in predicting behaviour, we measured aggressiveness during staged intrusions in free-living male and female dark-eyed juncos (Junco hyemalis). We compared aggressiveness to plasma T levels and to the abundance of androgen receptor (AR), aromatase (AROM) and oestrogen receptor alpha (ORα) mRNA in behaviourally relevant brain areas (avian medial amygdala, hypothalamus and song control regions). We also asked whether patterns of covariation among behaviour and endocrine parameters differed in males and females, anticipating that circulating T may be a better predictor of behaviour in males than in females. We found that circulating T related to aggressiveness only in males, but that gene expression for ORα, AR and AROM covaried with individual differences in aggressiveness in both sexes. These findings are among the first to show that individual variation in neural gene expression for three major sex steroid-processing molecules predicts individual variation in aggressiveness in both sexes in nature. The results have broad implications for our understanding of the mechanisms by which aggressive behaviour may evolve.

Life history trade-offs and behavioral sensitivity to testosterone: an experimental test when female aggression and maternal care co-occur.

Research on male animals suggests that the hormone testosterone plays a central role in mediating the trade-off between mating effort and parental effort. However, the direct links between testosterone, intrasexual aggression and parental care are remarkably mixed across species. Previous attempts to reconcile these patterns suggest that selection favors behavioral insensitivity to testosterone when paternal care is essential to reproductive success and when breeding seasons are especially short. Females also secrete testosterone, though the degree to which similar testosterone-mediated trade-offs occur in females is much less clear. Here, I ask whether testosterone mediates trade-offs between aggression and incubation in females, and whether patterns of female sensitivity to testosterone relate to female life history, as is often the case in males. I experimentally elevated testosterone in free-living, incubating female tree swallows (Tachycineta bicolor), a songbird with a short breeding season during which female incubation and intrasexual aggression are both essential to female reproductive success. Testosterone-treated females showed significantly elevated aggression, reduced incubation temperatures, and reduced hatching success, relative to controls. Thus, prolonged testosterone elevation during incubation was detrimental to reproductive success, but females nonetheless showed behavioral sensitivity to testosterone. These findings suggest that the relative importance of both mating effort and parental effort may be central to understanding patterns of behavioral sensitivity in both sexes.

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.

Neural steroid sensitivity and aggression: comparing individuals of two songbird subspecies

Hormones coordinate the expression of complex phenotypes and thus may play important roles in evolutionary processes. When populations diverge in hormone‐mediated phenotypes, differences may arise via changes in circulating hormones, sensitivity to hormones or both. Determining the relative importance of signal and sensitivity requires consideration of both inter‐ and intrapopulation variation in hormone levels, hormone sensitivity and phenotype, but such studies are rare, particularly among closely related taxa. We compared males of two subspecies of the dark‐eyed junco (Junco hyemalis) for territorial aggression and associations among behaviour, circulating testosterone (T), and gene expression of androgen receptor (AR), aromatase (AROM) and oestrogen receptor α in three behaviourally relevant brain regions. Thus, we examined the degree to which evolution may shape behaviour via changes in plasma T as compared with key sex steroid binding/converting molecules. We found that the white‐winged junco (J. h. aikeni) was more aggressive than the smaller, less ornamented Carolina junco (J. h. carolinensis). The subspecies did not differ in circulating testosterone, but did differ significantly in the abundance of AR and AROM mRNA in key areas of the brain. Within populations, both gene expression and circulating T co‐varied significantly with individual differences in aggression. Notably, the differences identified between populations were opposite to those predicted by the patterns among individuals within populations. These findings suggest that hormone–phenotype relationships may evolve via multiple pathways, and that changes that have occurred over evolutionary time do not necessarily reflect standing physiological variation on which current evolutionary processes may act.

Proximate perspectives on the evolution of female aggression: good for the gander, good for the goose?

Female–female aggression often functions in competition over reproductive or social benefits, but the proximate mechanisms of this apparently adaptive behaviour are not well understood. The sex steroid hormone testosterone (T) and its metabolites are well-established mediators of male–male aggression, and several lines of evidence suggest that T-mediated mechanisms may apply to females as well. However, a key question is whether mechanisms of female aggression primarily reflect correlated evolutionary responses to selection acting on males, or whether direct selection acting on females has made modifications to these mechanisms that are adaptive in light of female life history. Here, I examine the degree to which female aggression is mediated at the level of T production, target tissue sensitivity to T, or downstream genomic responses in order to test the hypothesis that selection favours mechanisms that facilitate female aggression while minimizing the costs of systemically elevated T. I draw heavily from avian systems, including the dark-eyed junco (Junco hyemalis), as well as other organisms in which these mechanisms have been well studied from an evolutionary/ecological perspective in both sexes. Findings reveal that the sexes share many behavioural and hormonal mechanisms, though several patterns also suggest sex-specific adaptation. I argue that greater attention to multiple levels of analysis—from hormone to receptor to gene network, including analyses of individual variation that represents the raw material of evolutionary change—will be a fruitful path for understanding mechanisms of behavioural regulation and intersexual coevolution.

Examining sources of variation in HPG axis function among individuals and populations of the dark-eyed junco

Gonadal steroids are important mediators of traits relevant to fitness, and thus may be targets of selection. However, more knowledge is needed about sources of variation along the endocrine axes that may contribute to functional variation in steroid levels. In a controlled captive environment, we studied males of two closely related subspecies of the dark-eyed junco (Junco hyemalis) that differ in testosterone-related phenotype, asking whether they also differ in testosterone (T), and assessing the contribution of the sequential links of the hypothalamic-pituitary-gonadal axis. When males of both subspecies were challenged with gonadotropin-releasing hormone (GnRH), they were similar in circulating luteinizing hormone (LH) and T responses. When challenged with exogenous LH, they again produced levels of T similar to one another, and to the levels produced in response to GnRH. However, the smaller, less ornamented, and less aggressive subspecies had greater abundance of mRNA for LH receptor in the testes and for androgen receptor in the rostral hypothalamus, suggesting potential differences in regulatory feedback. We suggest that circulating hormone levels may be less prone to evolutionary change than the responsiveness of individual hormone targets. Among individuals, T titers were highly repeatable whether males were challenged with GnRH or with LH, but LH produced in response to GnRH did not covary with T produced in response to LH. Testis mass, but not LH receptor transcript abundance, predicted individual variation in T responses. These data implicate the gonad, but not the pituitary, as an important source of individual variation in T production.

Robust behavioral effects of song playback in the absence of testosterone or corticosterone release.

Some species of songbirds elevate testosterone in response to territorial intrusions while others do not. The search for a general explanation for this interspecific variation in hormonal response to social challenges has been impeded by methodological differences among studies. We asked whether song playback alone is sufficient to bring about elevation in testosterone or corticosterone in the dark-eyed junco (Junco hyemalis), a species that has previously demonstrated significant testosterone elevation in response to a simulated territorial intrusion when song was accompanied by a live decoy. We studied two populations of juncos that differ in length of breeding season (6-8 vs. 14-16 weeks), and conducted playbacks of high amplitude, long-range song. In one population, we also played low amplitude, short-range song, a highly potent elicitor of aggression in juncos and many songbirds. We observed strong aggressive responses to both types of song, but no detectable elevation of plasma testosterone or corticosterone in either population. We also measured rise in corticosterone in response to handling post-playback, and found full capacity to elevate corticosterone but no effect of song class (long-range or short-range) on elevation. Collectively, our data suggest that males can mount an aggressive response to playback without a change in testosterone or corticosterone, despite the ability to alter these hormones during other types of social interactions. We discuss the observed decoupling of circulating hormones and aggression in relation to mechanisms of behavior and the cues that may activate the HPA and HPG axes.

Sources of variation in HPG axis reactivity and individually consistent elevation of sex steroids in a female songbird

Understanding sources of individual differences in steroid hormone production has important implications for the evolution of reproductive and social behaviors. In females in particular, little is known about the mechanistic sources of these individual differences, despite established linkages between sex steroids and a variety of fitness-related traits. Using captive female dark-eyed juncos (Junco hyemalis) from two subspecies, we asked how variation in different components of the hypothalamo-pituitary-gonadal (HPG) axis related to variation in testosterone production among females, and we compared females to males in multiple components of the HPG axis. We demonstrated consistent individual differences in testosterone elevation in response to challenges with luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH). These hormone challenges led to more LH production but less testosterone production in females than males, and the sexes differed in some but not all measures of sensitivity to hormones along the HPG axis. Similar to findings in males, variation in testosterone production among females was not related to variation in LH production, gonadal LH-receptor mRNA abundance, or hypothalamic abundance of androgen receptor mRNA or aromatase mRNA. Rather, the primary source of individual variation in circulating steroids appears to the gonad, a conclusion further supported by positive correlations between testosterone and estradiol production. Unlike males, females did not differ by subspecies in any of the endocrine parameters that we assessed, suggesting some degree of independent evolution between the two sexes. Our results highlight the sources of physiological variation that may underlie the evolution of hormone-mediated phenotypes in females.

Highly context-specific activation of the HPG axis in the dark-eyed junco and implications for the challenge hypothesis.

One of the best studied hormone-behavior interactions is the transient rise in testosterone (T) associated with male-male aggression. However, recent research on songbirds has demonstrated numerous exceptions to this pattern.One species previously thought to elevate T in response to a simulated territorial intrusion is the dark-eyed junco (Junco hyemalis). Here, we show that under most circumstances male juncos do not elevate circulating T or CORT levels in response to social stimuli, despite being physiologically capable of elevating T as indicated by their response to GnRH. The lack of hormonal response was found regardless of the sex of the social stimulus (singing male vs. soliciting female), its sensory modality (song only, song + live lure, song + taxidermic mount), or the timecourse of sampling. Notably, males did elevate T levels when exposed to a simulated territorial intrusion in the days following simulated predation of their chicks. Whether the high T seen in these narrow circumstances represents stage-dependent social modulation of T or re-activation of male reproductive physiology in preparation for re-nesting (i.e. socially independent T modulation) remains to be determined. It is clear, however, that activation of the HPG axis is highly context-specific for male juncos. These results highlight important and unresolved issues regarding the socially mediated component of the challenge hypothesis and how it relates to the evolution of hormone-mediated traits.

Next steps for understanding the selective relevance of female-female competition

INTRODUCTION After decades of neglect, recent empirical research on exaggerated female traits (e.g., ornaments, armaments, aggression, acoustic signals, etc.) has revived interest in this widespread but poorly understood phenomenon, and shown that these traits often function in the context of femalefemale competition (West-Eberhard, 1983; Amundsen, 2000; Clutton-Brock, 2009; Rosvall, 2011a; Stockley and BroJorgensen, 2011; Rubenstein, 2012 [Theme issue]; Stockley and Campbell, 2013 [Theme issue]). However, recent reviews have emphasized the applicability of sexual vs. social selection, rather than rigorously examining the role of different ecological contexts in shaping the evolution of traits used in competitive contexts (hereafter, “competitive traits”) in females. Thus, we still lack a solid understanding of the ecological and evolutionary mechanisms driving the evolution of female trait expression, in particular whether, how, and why these mechanisms vary among species, and between the sexes. It is our opinion that two critical issues impede our understanding the evolution of competitive traits in females. (1) The field has yet to investigate the ecological and evolutionary mechanisms that underlie interspecific and intersexual variation in the expression of these traits. This is perhaps due to a perceived “apples and oranges” problem stemming from the observation that animals compete over a wide variety of resources that vary by species or sex. However, by focusing on the relationships between fitness currencies and the resources over which animals compete, we can empirically compare the strength and direction of selection across species and sexes. (2) To date, research has primarily focused on the fitness costs or benefits of female competitive traits. As with many questions in behavioral and evolutionary ecology, quantifying how costs and benefits interact is essential to furthering our understanding of the evolution of competitive traits. Here, our goal is to draw attention to these solutions in order to spur more efficient and transformative progress.