Experimental manipulation of a signal trait reveals complex phenotype-behaviour coordination.

Animals use morphological signals such as ornamental traits or weaponry to mediate social interactions, and the extent of signal trait elaboration is often positively associated with reproductive success. By demonstrating relationships between signal traits and fitness, researchers often make inferences about how behaviour operates to shape those outcomes. However, detailed information about fine-scale individual behaviour, and its physiological basis, can be difficult to obtain. Here we show that experimental manipulations to exaggerate a signal trait (plumage colour) and concomitant changes in testosterone and stress-induced corticosterone levels altered social interactivity between manipulated males and their social mates. On average, darkened males did not have higher levels of interactivity than unmanipulated males; however, males who experienced a greater shift in colour (pale to dark), a larger, positive change in testosterone levels, and a dampened stress-induced corticosterone response had a larger increase in the number of interactions with their social mate post-manipulation compared to pre-manipulation. This work provides new insights into the integration and real-time flexibility of multivariate phenotypes and direct evidence for the role of social interactions in pair bond maintenance.

Polycyclic aromatic hydrocarbon exposure in seaside sparrows (Ammodramus maritimus) following the 2010 Deepwater Horizon oil spill.

The seaside sparrow (Ammodramus maritimus) is an abundant and permanent resident of coastal salt marshes impacted by the 2010 BP Deepwater Horizon oil spill. Such terrestrial species are often overlooked in the aftermath of marine spills, despite the potential for long-term oil exposure. We sampled the livers of seaside sparrows residing in oiled and unoiled sites from 2011 to 2014 and quantified expression of cytochrome p450 1A (CYP1A), a gene involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). In August 2011, CYP1A expression was markedly higher in birds from an oiled site compared to an unoiled site, but differences had disappeared by June 2012. In June 2013, CYP1A expression was elevated compared to 2012 levels on all sites, including those collected from sites that had not been directly oiled during the spill. This rise in CYP1A expression was possibly due to Hurricane Isaac, which made landfall near our sites between the 2012 and 2013 sampling periods. CYP1A expression was significantly attenuated again in June 2014. We also collected sediment samples from the same marshes for a total concentration analysis of PAHs. The PAH concentrations in sediment samples exhibited a similar pattern to the CYP1A data, supporting the link between marsh PAHs and bird CYP1A expression. These results indicate that contamination from marine oil spills can immediately extend to terrestrial ecosystems, and that storms, weather, or other factors may influence subsequent spatial and temporal oil exposure for several additional years.

Lipid signaling and fat storage in the dark-eyed junco.

Seasonal hyperphagia and fattening promote survivorship in migratory and wintering birds, but reduced adiposity may be more advantageous during the breeding season. Factors such as photoperiod, temperature, and food predictability are known environmental determinants of fat storage, but the underlying neuroendocrine mechanisms are less clear. Endocannabinoids and other lipid signaling molecules regulate multiple aspects of energy balance including appetite and lipid metabolism. However, these functions have been established primarily in mammals; thus the role of lipid signals in avian fat storage remains largely undefined. Here we examined relationships between endocannabinoid signaling and individual variation in fat storage in captive white-winged juncos (Junco hyemalis aikeni) following a transition to long-day photoperiods. We report that levels of the endocannabinoid 2-arachidonoylglycerol (2-AG), but not anandamide (AEA), in furcular and abdominal fat depots correlate negatively with fat mass. Hindbrain mRNA expression of CB1 endocannabinoid receptors also correlates negatively with levels of fat, demonstrating that fatter animals experience less central and peripheral endocannabinoid signaling when in breeding condition. Concentrations of the anorexigenic lipid, oleoylethanolamide (OEA), also inversely relate to adiposity. These findings demonstrate unique and significant relationships between adiposity and lipid signaling molecules in the brain and periphery, thereby suggesting a potential role for lipid signals in mediating adaptive levels of fat storage.

Gonads and the evolution of hormonal phenotypes.

Hormones are dynamic signaling molecules that influence gene activity and phenotype, and they are thus thought to play a central role in phenotypic evolution. In vertebrates, many fitness-related traits are mediated by the hormone testosterone (T), but the mechanisms by which T levels evolve are unclear. Here, we summarize a series of studies that advance our understanding of these mechanisms by comparing males from two subspecies of dark-eyed junco (Junco hyemalis) that differ in aggression, body size, and ornamentation. We first review our research demonstrating population differences in the time-course of T production, as well as findings that point to the gonad as a major source of this variation. In a common garden, the subspecies do not differ in pituitary output of luteinizing hormone, but males from the more androgenized subspecies have greater gonadal gene expression for specific steroidogenic enzymes, and they may be less sensitive to feedback along the hypothalamo-pituitary-gonadal (HPG) axis. Furthermore, we present new data from a common garden study demonstrating that the populations do not differ in gonadal sensitivity to gonadotropin-inhibitory hormone (i.e., GnIH receptor mRNA abundance), but the more androgenized subspecies expresses less gonadal mRNA for glucocorticoid receptor and mineralocorticoid receptor, suggesting altered cross-talk between the hypothalamo-pituitary-gonadal and -adrenal axes as another mechanism by which these subspecies have diverged in T production. These findings highlight the diversity of mechanisms that may generate functional variation in T and influence hormone-mediated phenotypic evolution.

Divergence along the gonadal steroidogenic pathway: Implications for hormone-mediated phenotypic evolution.

Across a range of taxa, hormones regulate suites of traits that influence survival and reproductive success; however, the mechanisms by which hormone-mediated traits evolve are still unclear. We hypothesized that phenotypic divergence might follow from differential regulation of genes encoding key steps in hormone biosynthesis and thus the rate of hormone production. We tested this hypothesis in relation to the steroid hormone testosterone by comparing two subspecies of junco (Junco hyemalis) in the wild and in captivity. These subspecies have diverged over the last 10-15kyears in multiple testosterone-mediated traits, including aggression, ornamentation, and body size. We show that variation in gonadal gene expression along the steroid biosynthetic pathway predicts phenotypic divergence within and among subspecies, and that the more androgenized subspecies exhibits a more prolonged time-course of elevated testosterone following exogenous stimulation. Our results point to specific genes that fulfill key conditions for phenotypic evolution because they vary functionally in their expression among individuals and between populations, and they map onto population variation in phenotype in a common garden. Our findings therefore build an important bridge between hormones, genes, and phenotypic evolution.

Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex.

Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1-CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females.

Population genetics of seaside Sparrow (Ammodramus maritimus) subspecies along the gulf of Mexico.

Seaside Sparrows (Ammodramus maritimus) along the Gulf of Mexico are currently recognized as four subspecies, including taxa in Florida (A. m. juncicola and A. m. peninsulae) and southern Texas (Ammodramus m. sennetti), plus a widespread taxon between them (A. m. fisheri). We examined population genetic structure of this "Gulf Coast" clade using microsatellite and mtDNA data. Results of Bayesian analyses (Structure, GeneLand) of microsatellite data from nine locations do not entirely align with current subspecific taxonomy. Ammodramus m. sennetti from southern Texas is significantly differentiated from all other populations, but we found evidence of an admixture zone with A. m. fisheri near Corpus Christi. The two subspecies along the northern Gulf Coast of Florida are significantly differentiated from both A. m. sennetti and A. m. fisheri, but are not distinct from each other. We found a weak signal of isolation by distance within A. m. fisheri, indicating this population is not entirely panmictic throughout its range. Although continued conservation concern is warranted for all populations along the Gulf Coast, A. m. fisheri appears to be more secure than the far smaller populations in south Texas and the northern Florida Gulf Coast. In particular, the most genetically distinct populations, those in Texas south of Corpus Christi, occupy unique habitats within a very small geographic range.

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.

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.