Multiple behavioral mechanisms shape development in a highly social cichlid fish.

Early-life social experiences shape adult phenotype, yet the underlying behavioral mechanisms remain poorly understood. We manipulated early-life social experience in the highly social African cichlid fish Astatotilapia burtoni to investigate the effects on behavior and stress axis function in juveniles. Juveniles experienced different numbers of social partners in stable pairs (1 partner), stable groups (6 fish; 5 partners), and socialized pairs (a novel fish was exchanged every 5 days; 5 partners). Treatments also differed in group size (groups vs. pairs) and stability (stable vs. socialized). We then measured individual behavior and water-borne cortisol to identify effects of early-life experience. We found treatment differences in behavior across all assays: open field exploration, social cue investigation, dominant behavior, and subordinate behavior. Treatment did not affect cortisol. Principal components (PC) analysis revealed robust co-variation of behavior across contexts, including with cortisol, to form behavioral syndromes sensitive to early-life social experience. PC1 (25.1 %) differed by social partner number: juveniles with more partners (groups and socialized pairs) were more exploratory during the social cue investigation, spent less time in the territory, and were more interactive as dominants. PC5 (8.5 %) differed by stability: socialized pairs were more dominant, spent less time in and around the territory, were more socially investigative, and had lower cortisol than stable groups or pairs. Observations of the home tanks provided insights into the social experiences that may underlie these effects. These results contribute to our understanding of how early-life social experiences are accrued and exert strong, lasting effects on phenotype.

Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.

Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.

Vasopressin mediates nonapeptide and glucocorticoid signaling and social dynamics in juvenile dominance hierarchies of a highly social cichlid fish.

Early-life social experience can strongly affect adult behavior, yet the behavioral mechanisms underlying developmental trajectories are poorly understood. Here, we use the highly social cichlid, Burton's Mouthbrooder (Astatotilapia burtoni) to investigate juvenile social status and behavior, as well as the underlying neuroendocrine mechanisms. We placed juveniles in pairs or triads and found that they readily establish social status hierarchies, with some group structural variation depending on group size, as well as the relative body size of the group members. Next, we used intracerebroventricular injections to test the hypothesis that arginine vasopressin (AVP) regulates juvenile social behavior and status, similar to adult A. burtoni. While we found no direct behavioral effects of experimentally increasing (via vasotocin) or decreasing (via antagonist Manning Compound) AVP signaling, social interactions directed at the treated individual were significantly altered. This group-level effect of central AVP manipulation was also reflected in a significant shift in whole brain expression of genes involved in nonapeptide signaling (AVP, oxytocin, and oxytocin receptor) and the neuroendocrine stress axis (corticotropin-releasing factor (CRF), glucocorticoid receptors (GR) 1a and 1b). Further, social status was associated with the expression of genes involved in glucocorticoid signaling (GR1a, GR1b, GR2, mineralocorticoid receptor), social interactions with the dominant fish, and nonapeptide signaling activity (AVP, AVP receptor V1aR2, OTR). Together, our results considerably expand our understanding of the context-specific emergence of social dominance hierarchies in juveniles and demonstrate a role for nonapeptide and stress axis signaling in the regulation of social status and social group dynamics.

Early-life social environment alters juvenile behavior and neuroendocrine function in a highly social cichlid fish.

Early-life experiences can shape adult behavior, with consequences for fitness and health, yet fundamental questions remain unanswered about how early-life social experiences are translated into variation in brain and behavior. The African cichlid fish Astatotilapia burtoni, a model system in social neuroscience, is well known for its highly plastic social phenotypes in adulthood. Here, we rear juveniles in either social groups or pairs to investigate the effects of early-life social environments on behavior and neuroendocrine gene expression. We find that both juvenile behavior and neuroendocrine function are sensitive to early-life effects. Behavior robustly co-varies across multiple contexts (open field, social cue investigation, and dominance behavior assays) to form a behavioral syndrome, with pair-reared juveniles towards the end of syndrome that is less active and socially interactive. Pair-reared juveniles also submit more readily as subordinates. In a separate cohort, we measured whole brain expression of stress and sex hormone genes. Expression of glucocorticoid receptor 1a was elevated in group-reared juveniles, supporting a highly-conserved role for the stress axis mediating early-life effects. The effect of rearing environment on androgen receptor α and estrogen receptor α expression was mediated by treatment duration (1 vs. 5 weeks). Finally, expression of corticotropin-releasing factor and glucocorticoid receptor 2 decreased significantly over time. Rearing environment also caused striking differences in gene co-expression, such that expression was tightly integrated in pair-reared juveniles but not group-reared or isolates. Together, this research demonstrates the important developmental origins of behavioral phenotypes and identifies potential behavioral and neuroendocrine mechanisms.

A Role for Oxytocin-Like Receptor in Social Habituation in a Teleost.

Oxytocin (OT) mediates social habituation in rodent model systems, but its role in mediating this effect in other vertebrates is unknown. We used males of the African cichlid fish, Astatotilapia burtoni, to investigate two aspects of isotocin (IT; an OT homolog) signaling in social habituation. First, we examined the expression of IT receptor 2 (ITR2) as well as two immediate early genes in brain regions implicated in social recognition. Next, we examined IT neuron activity using immunohistochemistry. Patterns of gene expression in homologs of the amygdala and hippocampus implicate IT signaling in these regions in social habituation to a territorial neighbor. In the preoptic area, the expression of the ITR2 subtype and IT neuron activity respond to the presence of a male, independent of familiarity. Our results implicate IT in mediating social habituation in a teleost.

Ancestral androgenic differentiation pathways are repurposed during the evolution of adult sexual plasticity.

Although early exposure to androgens is necessary to permanently organize male phenotype in many vertebrates, animals that exhibit adult sexual plasticity require mechanisms that prevent early fixation of genital morphology and allow for genital morphogenesis during adult transformation. In Lythrypnus dalli, a teleost fish that exhibits bi-directional sex change, adults display dimorphic genitalia morphology despite the absence of sex differences in the potent fish androgen 11-ketotestosterone. Based on conserved patterns of vertebrate development, two steroid-based mechanisms may regulate the early development and adult maintenance of dimorphic genitalia; local androgen receptor (AR) and steroidogenic enzyme expression. Consistent with the ancestral pattern of AR expression during the multipotential phase of differentiation, juvenile differentiation into either sex involved high mesenchymal AR expression. In adults, AR expression was high throughout the male genitalia, but low or absent in females. Consistent with the hypothesis that adult sexual plasticity repurposes pathways from primary differentiation, we show that adults with transitioning genitalia also exhibited higher AR expression relative to females. Local androgen biosynthesis may also participate in genitalia transformation, as transitioning adults had greater 11ß-HSD-like immunoreactivity in the epithelial layer of the dorsal lumen compared to both sexes. By administering an AR antagonist to adult males, we show AR is necessary to maintain male-typical morphology. In a species that is resistant to early sexual canalization, early androgenic differentiation mechanisms are consistent with other vertebrates and the tissue-specific regulation of AR expression appears to be repurposed in adulthood to allow for transitions between sexual phenotypes.

Juvenile social status predicts primary sex allocation in a sex changing fish.

Both individual sex and population sex ratio can affect lifetime reproductive success. As a result, multiple mechanisms have evolved to regulate sexual phenotype, including adult sex change in fishes. While adult sex change is typically socially regulated, few studies focus on the non-chromosomal mechanisms regulating primary sex allocation. We investigated primary sex determination in the bluebanded goby (Lythrypnus dalli), a bidirectionally sex-changing fish. Of the studies investigating primary sex determination in species with adult sex change, this is the first to incorporate the roles of social status and size, key factors for determining adult sex allocation. For L. dalli, adult sex is regulated by social status: dominants are male; subordinates are female. In social groups of laboratory-reared juveniles, we demonstrate that status also predicts primary sex. Dominant juveniles developed male-typical genitalia, and their gonads contained significantly less ovarian tissue than subordinates, which developed female-typical genitalia. To better understand natural development, we quantified the distribution of juveniles and adults on the reef and analyzed genital papilla and gonad morphology in a sample of wild-caught juveniles. Juveniles were observed in various social environments, and most grouped with other juveniles and/or adults. The majority of field-caught juveniles had female-typical genitalia and bisexual, female-biased gonads. These data are consistent with a single mechanism that regulates sexual phenotype throughout life. Social status could first cause and then maintain through adulthood a female-biased population, allowing individuals to regulate sex based on local conditions, which is important for optimizing lifetime reproductive success.

Agonistic reciprocity is associated with reduced male reproductive success within haremic social networks.

While individual variation in social behaviour is ubiquitous and causes social groups to differ in structure, how these structural differences affect fitness remains largely unknown. We used social network analysis of replicate bluebanded goby (Lythrypnus dalli) harems to identify the reproductive correlates of social network structure. In stable groups, we quantified agonistic behaviour, reproduction and steroid hormones, which can both affect and respond to social/reproductive cues. We identified distinct, optimal social structures associated with different reproductive measures. Male hatching success (HS) was negatively associated with agonistic reciprocity, a network structure that describes whether subordinates 'reciprocated' agonism received from dominants. Egg laying was associated with the individual network positions of the male and dominant female. Thus, males face a trade-off between promoting structures that facilitate egg laying versus HS. Whether this reproductive conflict is avoidable remains to be determined. We also identified different social and/or reproductive roles for 11-ketotestosterone, 17ß-oestradiol and cortisol, suggesting that specific neuroendocrine mechanisms may underlie connections between network structure and fitness. This is one of the first investigations of the reproductive and neuroendocrine correlates of social behaviour and network structure in replicate, naturalistic social groups and supports network structure as an important target for natural selection.

Contextual modulation of social and endocrine correlates of fitness: insights from the life history of a sex changing fish.

Steroid hormones are critical regulators of reproductive life history, and the steroid sensitive traits (morphology, behavior, physiology) associated with particular life history stages can have substantial fitness consequences for an organism. Hormones, behavior and fitness are reciprocally associated and can be used in an integrative fashion to understand how the environment impacts organismal function. To address the fitness component, we highlight the importance of using reliable proxies of reproductive success when studying proximate regulation of reproductive phenotypes. To understand the mechanisms by which the endocrine system regulates phenotype, we discuss the use of particular endocrine proxies and the need for appropriate functional interpretation of each. Lastly, in any experimental paradigm, the responses of animals vary based on the subtle differences in environmental and social context and this must also be considered. We explore these different levels of analyses by focusing on the fascinating life history transitions exhibited by the bi-directionally hermaphroditic fish, Lythrypnus dalli. Sex changing fish are excellent models for providing a deeper understanding of the fitness consequences associated with behavioral and endocrine variation. We close by proposing that local regulation of steroids is one potential mechanism that allows for the expression of novel phenotypes that can be characteristic of specific life history stages. A comparative species approach will facilitate progress in understanding the diversity of mechanisms underlying the contextual regulation of phenotypes and their associated fitness correlates.

A mechanism for rapid neurosteroidal regulation of parenting behaviour.

While systemic steroid hormones are known to regulate reproductive behaviour, the actual mechanisms of steroidal regulation remain largely unknown. Steroidogenic enzyme activity can rapidly modulate social behaviour by influencing neurosteroid production. In fish, the enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) synthesizes 11-ketotestosterone (KT, a potent teleost androgen) and deactivates cortisol (the primary teleost glucocorticoid), and both of these steroid hormones can regulate behaviour. Here, we investigated the role of neurosteroidogenesis in regulating parenting in a haremic bidirectionally hermaphroditic fish, Lythrypnus dalli, where males provide all requisite parental care. Using an in vitro assay, we found that an 11ß-HSD inhibitor, carbenoxolone (CBX), reduced brain and testicular KT synthesis by 90% or more. We modulated neurosteroid levels in parenting males via intracerebroventricular injection of CBX. Within only 20 min, CBX transiently eliminated parenting behaviour, but not other social behaviour, suggesting an enzymatic mechanism for rapid neurosteroidal regulation of parenting. Consistent with our proposed mechanism, elevating KT levels rescued parenting when paired with CBX, while cortisol alone did not affect parenting. Females paired with the experimental males opportunistically consumed unattended eggs, which reduced male reproductive success by 15%, but some females also exhibited parenting behaviour and these females had elevated brain KT. Brain KT levels appear to regulate the expression of parenting behaviour as a result of changes in neural 11ß-HSD activity.

Stress and serial adult metamorphosis: multiple roles for the stress axis in socially regulated sex change.

Socially regulated sex change in teleost fishes is a striking example of social status information regulating biological function in the service of reproductive success. The establishment of social dominance in sex changing species is translated into a cascade of changes in behavior, physiology, neuroendocrine function, and morphology that transforms a female into a male, or vice versa. The hypothalamic-pituitary-interrenal axis (HPI, homologous to HP-adrenal axis in mammals and birds) has been hypothesized to play a mechanistic role linking status to sex change. The HPA/I axis responds to environmental stressors by integrating relevant external and internal cues and coordinating biological responses including changes in behavior, energetics, physiology, and morphology (i.e., metamorphosis). Through actions of both corticotropin-releasing factor and glucocorticoids, the HPA/I axis has been implicated in processes central to sex change, including the regulation of agonistic behavior, social status, energetic investment, and life history transitions. In this paper, we review the hypothesized roles of the HPA/I axis in the regulation of sex change and how those hypotheses have been tested to date. We include original data on sex change in the bluebanded goby (Lythyrpnus dalli), a highly social fish capable of bidirectional sex change. We then propose a model for HPA/I involvement in sex change and discuss how these ideas might be tested in the future. Understanding the regulation of sex change has the potential to elucidate evolutionarily conserved mechanisms responsible for translating pertinent information about the environment into coordinated biological changes along multiple body axes.

Behavioral and physiological responses to central administration of corticotropin-releasing factor in the bluebanded goby (Lythrypnus dalli).

Central manipulation of neuromodulators is critical to establishing causal links between brain function and behavioral output. The absence of a rigorous method of evaluating intracerebroventricular (i.c.v.) injection efficacy in small model organisms is one reason why peripheral administration of neuroactive substances is more common. We use the bluebanded goby (Lythrypnus dalli), a small, highly social fish, to 1) validate our method of i.c.v. injection by testing the hypothesis that corticotropin-releasing factor (CRF) elevates ventilation rate (VR) and 2) propose a novel bioassay using basal physiology and behavior during recovery from anesthesia/i.c.v. administration to assess injection efficacy, neuromodulator activity, and procedural confounds. Central CRF administration significantly increased ventilation rate, demonstrating successful delivery of CRF to the brain. There were no significant differences in cortisol among treatments. The injection procedure did, however, decouple the temporal relationship between the initiation of ventilation and time to regain equilibrium present in control fish. Importantly, neither i.c.v. vehicle nor CRF injection affected the initiation of ventilation, disrupted the stereotyped recovery pattern following anesthesia, or initiated an endocrine stress response. Taken together, we suggest that 1) i.c.v. injection can be effectively used to manipulate central levels of CRF in L. dalli and 2) physiological and behavioral recovery from anesthesia may be used to evaluate injection/technique efficacy. We will use these data in future studies as a measure of effective CRF delivery, to allow for appropriate recovery from i.c.v. injection, and to better evaluate independent effects of CRF on social and/or sexual behavior.