Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism.

The evolution of social behavior depends on genetic changes, yet, how genomic variation manifests itself in behavioral diversity is still largely unresolved. Chromosomal inversions can play a pivotal role in producing distinct behavioral phenotypes, in particular, when inversion genes are functionally associated with hormone synthesis and signaling. Male ruffs exhibit alternative reproductive tactics (ARTs) with an autosomal inversion determining two alternative morphs with clear behavioral and hormonal differences from the ancestral morph. We investigated hormonal and transcriptomic differences in the pituitary and gonads. Using a GnRH challenge, we found that the ability to synthesize testosterone in inversion carriers is severely constrained, whereas the synthesis of androstenedione, a testosterone precursor, is not. Inversion morphs were able to produce a transient increase in androstenedione following the GnRH injection, supporting the view that pituitary sensitivity to GnRH is comparable to that of the ancestral morph. We then performed gene expression analyses in a second set of untreated birds and found no evidence of alterations to pituitary sensitivity, gonadotropin production or gonad sensitivity to luteinizing hormone or follicle-stimulating hormone across morphs. Inversion morphs also showed reduced progesterone receptor expression in the pituitary. Strikingly, in the gonads, inversion morphs over-expressed STAR, a gene that is located outside of the inversion and responsible for providing the cholesterol substrate required for the synthesis of sex hormones. In conclusion, our results suggest that the gonads determine morph-specific differences in hormonal regulation.

Social monogamy vs. polyandry: ecological factors associated with sex roles in two closely related birds within the same habitat.

Why mainly males compete and females take a larger share in parental care remains an exciting question in evolutionary biology. Role-reversed species are of particular interest, because such 'exceptions' help to test the rule. Using mating systems theory as a framework, we compared the reproductive ecology of the two most contrasting coucals with regard to sexual dimorphism and parental care: the black coucal with male-only care and the biparental white-browed coucal. Both species occur in the same lush habitat and face similar ecological conditions, but drastically differ in mating system and sexual dimorphism. Black coucals were migratory and occurred at high breeding densities. With females being obligatory polyandrous and almost twice as heavy as males, black coucals belong to the most extreme vertebrates with reversed sexual dimorphism. Higher variance in reproductive success in fiercely competing females suggests that sexual selection is stronger in females than in males. In contrast, resident white-browed coucals bred at low densities and invariably in pairs. They were almost monomorphic and the variance in reproductive success was similar between the sexes. Black coucals were more likely to lose nests than white-browed coucals, probably facilitating female emancipation of parental care in black coucals. We propose that a combination of high food abundance, high population density, high degree of nest loss and male bias in the adult sex ratio represent ecological conditions that facilitate role reversal and polyandry in coucals and terrestrial vertebrates in general.

The number of life-history stages does not influence the androgen responsiveness to male-male interactions: sedentary and migratory black redstarts (Phoenicurus ochruros) do not elevate testosterone in response to simulated territorial intrusions.

High plasma levels of testosterone at the beginning of the breeding season adjust male physiology for mating and promote territorial behavior in birds. Conversely intra-sexual competition may elicit a temporary increase in circulating testosterone. Male black redstarts (Phoenicurus ochruros) from migratory populations show the expected increase in baseline testosterone during early breeding, but circulating testosterone levels do not change in response to male-male interactions. Because sedentary populations express fewer life-history stages they may be more flexible in timing of life-history stages and more responsive to environmental modulation of hormone concentrations. Therefore, we tested whether the androgen responsiveness to male-male interactions differs between migratory (6 life-history stages) and sedentary black redstarts (3 life-history stages) during early breeding, predicting that in contrast to migratory birds, sedentary birds would modulate testosterone in response to simulated territorial intrusions (STI). In contrast to our prediction, sedentary males did not modulate post-capture testosterone levels in response to simulated territorial intrusions. Males of both populations increased testosterone after an injection of gonadotropin releasing hormone (GnRH), demonstrating that they were capable of increasing testosterone. Interestingly, in sedentary males the GnRH injection elicited a higher testosterone response in STI males than in control males. The two populations did not differ in their behavioral response to the STIs, except that sedentary males spent less time close to the decoy. In combination with previous data from black redstarts and other socially monogamous and biparental birds our current study adds to the growing evidence that current theory regarding hormone-behavior relationship needs to be refined.

Independence among physiological traits suggests flexibility in the face of ecological demands on phenotypes.

Phenotypic flexibility allows animals to adjust their physiology to diverse environmental conditions encountered over the year. Examining how these varying traits covary gives insights into potential constraints or freedoms that may shape evolutionary trajectories. In this study, we examined relationships among haematocrit, baseline corticosterone concentration, constitutive immune function and basal metabolic rate in red knot Calidris canutus islandica individuals subjected to experimentally manipulated temperature treatments over an entire annual cycle. If covariation among traits is constrained, we predict consistent covariation within and among individuals. We further predict consistent correlations between physiological and metabolic traits if constraints underlie species-level patterns found along the slow-fast pace-of-life continuum. We found no consistent correlations among haematocrit, baseline corticosterone concentration, immune function and basal metabolic rate either within or among individuals. This provides no evidence for constraints limiting relationships among these measures of the cardiovascular, endocrine, immune and metabolic systems in individual red knots. Rather, our data suggest that knots are free to adjust individual parts of their physiology independently. This makes good sense if one places the animal within its ecological context where different aspects of the environment might put different pressures on different aspects of physiology.

Difficulties and special issues associated with field research in behavioral neuroendocrinology.

Classical behavioral neuroendocrinology has focused on a limited number of domestic mammals and birds. The model systems used in these studies represent a very small proportion of the diversity of hormone-behavior interactions found in nature. In the last three decades, an increasing number of researchers have concentrated their efforts on studying behavioral neuroendocrinology of wild animals. Field behavioral neuroendocrinology presents a series of challenges ranging from the design of the experiments to sample preservation and transportation. The constraints of field conditions limit the number of factors that can be controlled for and the questions that can be addressed. On the other side, many behaviors can be studied only in the field, and only a few species can be kept in captivity. Thus, field studies are necessary to understand the complexity and variety of interactions between hormones, brain, and behavior. In this article, we will review some of the peculiarities and challenges of field behavioral neuroendocrinology, including solutions for some of the most commonly encountered technical issues.

Social, state-dependent and environmental modulation of faecal corticosteroid levels in free-ranging female spotted hyenas.

Little is known about to what extent the sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis may be state dependent and vary in the same species between environments. Here we tested whether the faecal corticosteroid concentrations of matrilineal adult female spotted hyenas are influenced by social and reproductive status in adjacent ecosystems and whether they vary between periods with and without social stress. Females in the Serengeti National Park frequently become socially subordinate intruders in other hyena territories by undertaking long-distance foraging trips to migratory herds, whereas in the Ngorongoro Crater they usually forage inside their own small territories on resident prey. The faecal corticosteroid concentrations in Serengeti females were significantly higher than in Ngorongoro females. Energy expenditure by lactation is exceptionally high in spotted hyenas and this may be reflected in their corticosteroid levels. The faecal corticosteroid levels in both populations were higher in lactating than in non-lactating females. During periods of social stability, faecal corticosteroid concentrations increased in non-lactating females but not in lactating females as social status declined. Lactating Serengeti females had significantly higher faecal corticosteroid concentrations during periods with acute severe social stress than during periods without, indicating that the HPA axis is sensitive to social stimuli even in lactating females. So far few studies have used non-invasive monitoring methods for assessing social stress in freeranging animals. This study demonstrates for the first time, to the authors' knowledge, that corticosteroid concentrations may differ between periods with and without social stress for a free-ranging female mammal and that the modulating effect of social status may depend on reproductive status.

Androgens and the role of female "hyperaggressiveness" in spotted hyenas (Crocuta crocuta).

It has been suggested that female dominance in spotted hyenas evolved due to selection for "hyperaggressive" androgenized females. According to this view, virilized external genitalia of female hyenas developed as a byproduct of selection for "androgen-facilitated social dominance." The evidence that female hyenas have higher androgen levels than males or other female mammals is inconclusive. We compared concentrations of testosterone (T), 5alpha-dihydrotestosterone (DHT), and androstenedione (AE) from a population in the Serengeti. Females had significantly lower T and DHT levels than predispersal and postdispersal males. AE levels did not significantly differ between females and postdispersal males, but were significantly lower in predispersal males. Our results, and those from studies that have demonstrated that male and female hyenas experience similar levels of maternal androgens during fetal development, provide little support for a theory of selection for female dominance and hyperaggressiveness through enhanced secretion of androgens. Our data are consistent with an alternative view that female virilization is a byproduct of selection for precocial aggressive cubs of both sexes. According to this view, high investment in lactation favored selection for accelerated fetal development, high neonatal aggression, and facultative siblicide.

Noninvasive fecal monitoring of glucocorticoids in spotted hyenas, Crocuta crocuta.

The aim of this study was to validate a method for measuring glucocorticoids noninvasively in feces of spotted hyenas (Crocuta crocuta). Three established enzyme immunoassays (EIA) for cortisol, corticosterone, and 11-oxoetiocholanolone were tested, but proved unsatisfactory. A new EIA using another corticosterone antibody was established and was used for all subsequent analyses; this EIA was validated by demonstrating parallelism between serial dilutions of spotted hyena fecal extracts and dilutions of standard corticosterone and by the recovery of corticosterone added to fecal extracts. High-performance liquid chromatography (HPLC) fractions analyzed by EIA showed various immunoreactive substances with polarities of unconjugated steroids. The physiological relevance of fecal glucocorticoid metabolites was further validated by demonstrating that (1) injection of exogenous ACTH to four males and two females led to a significant increase in fecal glucocorticoid metabolites within 24-50 h, (2) the translocation of a male spotted hyena to a new enclosure resulted in a fivefold increase compared to baseline concentrations, and (3) agonistic social interactions and physical conflict resulted in large increases of fecal glucocorticoid metabolites in both protagonists. Fecal steroid assessment is therefore of use in monitoring adrenal activity in spotted hyenas.