Neuroendocrine pathways that regulate social behavior are remarkably conserved across divergent taxa. The neuropeptides arginine vasotocin/vasopressin (AVT/AVP) and their receptor V1a mediate aggression, space use, and mating behavior in male vertebrates. The hormone prolactin (PRL) also regulates social behavior across species, most notably paternal behavior. Both hormone systems may be involved in the evolution of monogamous mating systems. We compared AVT, AVT receptor V1a2, PRL, and PRL receptor PRLR1 gene expression in the brains as well as circulating androgen concentrations of free-living reproductively active males of two closely related North American cichlid species, the monogamous Herichthys cyanoguttatus and the polygynous Herichthys minckleyi. We found that H. cyanoguttatus males bond with a single female and together they cooperatively defend a small territory in which they reproduce. In H. minckleyi, a small number of large males defend large territories in which they mate with several females. Levels of V1a2 mRNA were higher in the hypothalamus of H. minckleyi, and PRLR1 expression was higher in the hypothalamus and telencephalon of H. minckleyi. 11-ketotestosterone levels were higher in H. minckleyi, while testosterone levels were higher in H. cyanoguttatus. Our results indicate that a highly active AVT/V1a2 circuit(s) in the brain is associated with space use and social dominance and that pair bonding is mediated either by a different, less active AVT/V1a2 circuit or by another neuroendocrine system. ?? 2013 Elsevier Inc.

The roles of estrogen and androgens in male social behavior are well studied, but little is known about how these hormones contribute to behavior in a social hierarchy. Here we test the role of aromatase, the enzyme that converts testosterone into estradiol, in mediating aggression and reproductive behavior in male Astatotilapia burtoni, an African cichlid fish that displays remarkable plasticity in social behavior. We first measured aromatase expression in subordinate and dominant males in brain regions that regulate social behavior and found that subordinate males have higher aromatase expression than dominant males in the magnocellular and gigantocellular regions of the preoptic area. Next, we functionally tested the role of aromatase in regulating behavior by intraperitoneally injecting dominant males with either saline or fadrozole (FAD), an aromatase inhibitor, and found that FAD treatment decreases aggressive, but not reproductive, behaviors compared to saline controls. To determine the underlying physiological and molecular consequences of FAD treatment, we measured estradiol and testosterone levels from plasma and brain aromatase expression in FAD and saline treated dominant males. We found that estradiol levels decreased and testosterone levels increased in response to FAD treatment. Moreover, FAD treated males had increased aromatase expression in the gigantocellular portion of the POA, possibly a compensatory response. Overall, our results suggest aromatase is a key enzyme that promotes aggression in A. burtoni males through actions in the preoptic area. ?? 2013 Elsevier Inc.

Mate choice is fundamental to sexual selection, yet little is known about underlying physiological mechanisms that influence female mating decisions. We investigated the endocrine underpinnings of female mate choice in the African cichlid Astatotilapia burtoni, a non-seasonal breeder. In addition to profiling behavioral and hormonal changes across the female reproductive cycle, we tested two hypotheses regarding possible factors influencing female mate choice. We first asked whether female mate choice is influenced by male visual and/or chemical cues. A. burtoni females were housed for one full reproductive cycle in the center of a dichotomous choice apparatus with a large (attractive) or small (unattractive) conspecific male on either side. Females associated mostly with small, less attractive males, but on the day of spawning reversed their preference to large, attractive males, with whom they mated almost exclusively, although this choice depended on the relative amount of androgens released into the water by small males. We next asked whether male behavior or androgen levels change in relation to the stimulus females' reproductive state. We found that stimulus male aggression decreased and reproductive displays increased as the day of spawning approached. Moreover male testosterone levels changed throughout the females' reproductive cycle, with larger males releasing more testosterone into the water than small males. Our data suggest that female association in a dichotomous choice assay is only indicative of the actual mate choice on the day of spawning. Furthermore, we show that male behavior and hormone levels are dependent on the reproductive state of conspecific females. ?? 2012 Elsevier Inc.

Across vertebrates, the mesolimbic reward system is a highly conserved neural network that serves to evaluate the salience of environmental stimuli, with dopamine as the neurotransmitter most relevant to its function. Although brain regions in the dopaminergic reward system have been well characterized in mammals, homologizing these brain areas with structures in teleosts has been controversial, especially for the mesencephalo-diencephalic dopaminergic cell populations. Here we examine the neurochemical profile of five dopaminergic cell groups (Vc, POA, PPr, TPp, pTn) in the model cichlid Astatotilapia burtoni to better understand putative homology relationships between teleosts and mammals. We characterized in the adult brain the expression patterns of three genes (etv5, nr4a2, and pitx3) that either specify dopaminergic cell fate or maintain dopaminergic cell populations. We then determined whether these genes are expressed in dopaminergic cells. We find many striking similarities in these gene expression profiles between dopaminergic cell populations in teleosts and their putative mammalian homologs. Our results suggest that many of these dopaminergic cell groups are indeed evolutionarily ancient and conserved across vertebrates. ?? 2013 Elsevier B.V.

Individuals integrate information about their environment into adaptive behavioural responses, yet how different sensory modalities contribute to these decisions and where in the brain this integration occurs is not well understood. We presented male cichlid fish (Astatotilapia burtoni) with sensory information in three social contexts: intruder challenge, reproductive opportunity and a socially neutral situation. We then measured behavioural and hormonal responses along with induction of the immediate early gene c-Fos in candidate forebrain regions. In the intruder challenge context, males were exposed to either a visual stimulus of a dominant male, the putative male pheromone androstenedione, or both. We found that, compared to the neutral context, a visual stimulus was necessary and sufficient for an aggressive response, whereas both chemical and visual stimuli were needed for an androgen response. In the reproductive opportunity context, males were exposed to either a visual stimulus of a receptive female, a progesterone metabolite (female pheromone) only, or both. We further found that the visual stimulus is necessary and sufficient for an androgen response in the reproductive opportunity context. In the brain, we observed c-Fos induction in response to a visual challenge stimulus specifically in dopaminergic neurones of area Vc (the central region of the ventral telencephalon), a putative striatal homologue, whereas presentation of a chemical stimulus did not induce c-Fos induction in the intruder challenge context. Our results suggest that different sensory cues are processed in a social context-specific manner as part of adaptive decision-making processes.

Understanding how complex organisms function as integrated units that constantly interact with their environment is a long-standing chal- lenge in biology. To address this challenge, organismal biology reveals general organizing principles of physiological systems and behavior—in particular, in complex multicellular animals. Organismal biology also focuses on the role of individual variability in the evolutionary main- tenance of diversity. To broadly advance these frontiers, cross-compatibility of experimental designs, methodological approaches, and data interpretation pipelines represents a key prerequisite. It is now possible to rapidly and systematically analyze complete genomes to elucidate genetic variation associated with traits and conditions that define individuals, populations, and species. However, genetic variation alone does not explain the varied individual physiology and behavior of complex organisms. We propose that such emergent properties of complex organisms can best be explained through a renewed emphasis on the context and life-history dependence of individual phenotypes to comple- ment genetic data.

Hormones play an important role in the regulation of reproductive behavior. Here, we examined the effects of the fatty acid derivative prostaglandin F2α (PGF2) on female sexual behavior as well as the interaction between PGF2-induced mating behavior with male courtship display in the lek-breeding African cichlid fish, Astatotilapia burtoni. In a two-way choice paradigm, we found that nonreproductive females preferred to associate with smaller, less aggressive males over larger, more aggressive males. However, PGF2-treated females dramatically reversed their preference to larger males. In a second experiment, PGF2 treatment dramatically increased sexual behavior in nonreproductive females as measured by time spent in the bower of the stimulus male, even when the female and the stimulus male were separated by a transparent divider. This effect was even more pronounced when the stimulus males were exposed to the putative female pheromone 17α,20β-progesterone (17α,20β-P). Under full-contact conditions, only PGF2-treated females visited a stimulus male's bower, where they even displayed circling behavior usually only seen during spawning. Interestingly, male performance prior to PGF2 treatment predicted female sexual response. Our study demonstrates the importance of PGF2 in the control of female reproductive behavior in interaction with male performance.

An individual's position in a social hierarchy profoundly affects behavior and physiology through interactions with community members, yet little is known about how the brain contributes to status differences between and within the social states or sexes. We aimed to determine sex-specific attributes of social status by comparing circulating sex steroid hormones and neural gene expression of sex steroid receptors in dominant and subordinate male and female Astatotilapia burtoni, a highly social African cichlid fish. We found that testosterone and 17??-estradiol levels are higher in males regardless of status and dominant individuals regardless of sex. Progesterone was found to be higher in dominant individuals regardless of sex. Based on pharmacological manipulations in males and females, progesterone appears to be a common mechanism for promoting courtship in dominant individuals. We also examined expression of androgen receptors, estrogen receptor ??, and the progesterone receptor in five brain regions that are important for social behavior. Most of the differences in brain sex steroid receptor expression were due to sex rather than status. Our results suggest that the parvocellular preoptic area is a core region for mediating sex differences through androgen and estrogen receptor expression, whereas the progesterone receptor may mediate sex and status behaviors in the putative homologs of the nucleus accumbens and ventromedial hypothalamus. Overall our results suggest sex differences and similarities in the regulation of social dominance by gonadal hormones and their receptors in the brain. ?? 2013 Elsevier Inc.

Steroid hormones play an important role in modulating behavioral responses to various social stimuli. It has been suggested that variation in the hormonal regulation of behavior across species is associated with social organization and/or mating system. In order to further elucidate the interplay of hormones and behavior in social situations, we exposed males of the monogamous convict cichlid Amatitliana nigrofasciata to three social stimuli: gravid female, intruder male, and a nonsocial stimulus. We used a repeated measure design to create behavioral profiles and explore how sex steroid hormones respond to and regulate social behavior. Results show distinct behavioral responses to different social situations, with circulating 11-ketotestosterone increasing in response to social stimuli. Pharmacological manipulations using specific androgen and estrogen receptor agonists and antagonists exposed complex control over digging behavior in the social opportunity context. In the social challenge context, aggressive behaviors decreased in response to blocking the androgen receptor pathway. Our results extend our understanding of sex steroid regulation of behavioral responses to social stimulation. ?? 2013 Elsevier Inc.

Sex steroid hormones coordinate neurotransmitter systems in the male brain to facilitate sexual behavior. Although neurotransmitter release in the male brain has been well documented, little is known about how androgens orchestrate changes in gene expression of neurotransmitter receptors. We used male whiptail lizards (Cnemidophorus inornatus) to investigate how androgens alter neurotransmitter-related gene expression in brain regions involved in social decision making. We focused on three neurotransmitter systems involved in male-typical sexual behavior, including the N-methyl-d-aspartate (NMDA) glutamate receptor, nitric oxide and dopamine receptors. Here, we show that in androgen-treated males, there are coordinated changes in neurotransmitter-related gene expression. In androgen-implanted castrates compared with blank-implanted castrates (control group), we found associated increases in neuronal nitric oxide synthase gene expression in the nucleus accumbens (NAcc), preoptic area and ventromedial hypothalamus, a decrease of NR1 gene expression (obligate subunit of NMDA receptors) in the medial amygdaloid area and NAcc and a decrease in D1 and D2 dopamine receptor gene expression in the NAcc. Our results support and expand the current model of androgen-mediated gene expression changes of neurotransmitter-related systems that facilitate sexual behavior in males. This also suggests that the proposed evolutionarily ancient reward system that reinforces sexual behavior in amniote vertebrates extends to reptiles.

Nonapeptides and their receptors have important functions in mediating social behavior across vertebrates. Where these nonapeptides are synthesized in the brain has been studied extensively in most vertebrate lineages, yet we know relatively little about the neural distribution of nonapeptide receptors outside of mammals. As nonapeptides play influential roles in behavioral regulation in all vertebrates, including teleost fish, we mapped the distributions of the receptors for arginine vasotocin (AVT; homolog of arginine vasopressin) and isotocin (IST; homolog of oxytocin/mesotocin) throughout the forebrain of Astatotilapia burtoni, an African cichlid fish with behavioral phenotypes that are plastic and reversible based on the immediate social environment. We characterized the distribution of the AVT V1a2 receptor (V1aR) and the IST receptor (ITR) using both immunohistochemistry for protein detection and in situ hybridization for mRNA detection, as well as AVT and IST using immunohistochemistry. Expression of the neuropeptide receptors was widely distributed throughout the fore- and midbrain, including the proposed teleost homologs of the mammalian amygdala complex, striatum, hypothalamus, and ventral tegmental area. We conclude that although the location of nonapeptide synthesis is restricted compared to tetrapod vertebrates, the distribution of nonapeptide receptors is highly conserved across taxa. Our results significantly extend our knowledge of where nonapeptides act in the brains of teleosts to mediate social transitions and behavior. ?? 2012 Elsevier B.V.

The diverse cichlid species flocks of the East African lakes provide a classical example of adaptive radiation. Territorial aggression is thought to influence the evolution of phenotypic diversity in this system. Most vertebrates mount hormonal (androgen, glucocorticoid) responses to a territorial challenge. These hormones, in turn, influence behavior and multiple aspects of physiology and morphology. Examining variation in competition-induced hormone secretion patterns is thus fundamental to an understanding of the mechanisms of phenotypic diversification. We test here the hypothesis that diversification in male aggression has been accompanied by differentiation in steroid hormone levels. We studied two pairs of sibling species from Lake Victoria belonging to the genera Pundamilia and Mbipia. The two genera are ecologically differentiated, while sibling species pairs differ mainly in male color patterns. We found that aggression directed toward conspecific males varied between species and across genera: Pundamilia nyererei males were more aggressive than Pundamilia pundamilia males, and Mbipia mbipi males were more aggressive than Mbipia lutea males. Males of both genera exhibited comparable attack rates during acute exposure to a novel conspecific intruder, while Mbipia males were more aggressive than Pundamilia males during continuous exposure to a conspecific rival, consistent with the genus difference in feeding ecology. Variation in aggressiveness between genera, but not between sibling species, was reflected in androgen levels. We further found that M. mbipi displayed lower levels of cortisol than M. lutea. Our results suggest that concerted divergence in hormones and behavior might play an important role in the rapid speciation of cichlid fishes. ?? 2012 Elsevier Inc.

Animals evaluate and respond to their social environment with adaptive decisions. Revealing the neural mechanisms of such decisions is a major goal in biology. We analyzed expression profiles for 10 neurochemical genes across 12 brain regions important for decision-making in 88 species representing five vertebrate lineages. We found that behaviorally relevant brain regions are remarkably conserved over 450 million years of evolution. We also find evidence that different brain regions have experienced different selection pressures, because spatial distribution of neuroendocrine ligands are more flexible than their receptors across vertebrates. Our analysis suggests that the diversity of social behavior in vertebrates can be explained, in part, by variations on a theme of conserved neural and gene expression networks.

Social environment can affect the expression of sex-typical behavior in both males and females. Males of the African cichlid species Astatotilapia burtoni have long served as a model system to study the neural, endocrine, and molecular basis of socially plastic dominance behavior. Here we show that in all-female communities of A. burtoni, some individuals acquire a male-typical dominance phenotype, including aggressive territorial defense, distinctive color patterns, and courtship behavior. Furthermore, dominant females have higher levels of circulating androgens than either subordinate females or females in mixed-sex communities. These male-typical traits do not involve sex change, nor do the social phenotypes in all-female communities differ in relative ovarian size, suggesting that factors other than gonadal physiology underlie much of the observed variation. In contrast to the well-studied situation in males, dominant and subordinate females do not differ in the rate of somatic growth. Dominant females are not any more likely than subordinates to spawn with an introduced male, although they do so sooner. These results extend the well known extraordinary behavioral plasticity of A. burtoni to the females of this species and provide a foundation for uncovering the neural and molecular basis of social dominance behavior while controlling for factors such as sex, gonadal state and growth. ?? 2012 Elsevier Inc.

While the survival value of paternal care is well understood, little is known about its physiological basis. Here we investigate the neuroendocrine contributions to paternal care in the monogamous cichlid, Amatitlania nigrofasciata. We first explored the dynamic range of paternal care in three experimental groups: biparental males (control fathers housed with their mate), single fathers (mate removed), or lone males (mate and offspring removed). We found that control males gradually increase paternal care over time, whereas single fathers increased care immediately after mate removal. Males with offspring present had lower levels of circulating 11-ketotestosterone (11-KT) yet still maintained aggressive displays toward brood predators. To determine what brain regions may contribute to paternal care, we quantified induction of the immediate early gene c-Fos, and found that single fathers have more c-Fos induction in the forebrain area Vv (putative lateral septum homologue), but not in the central pallium (area Dc). While overall preoptic area c-Fos induction was similar between groups, we found that parvocellular preoptic isotocin (IST) neurons in single fathers showed increased c-Fos induction, suggesting IST may facilitate the increase of paternal care after mate removal. To functionally test the role of IST in regulating paternal care, we treated biparental males with an IST receptor antagonist, which blocked paternal care. Our results indicate that isotocin plays a significant role in promoting paternal care, and more broadly suggest that the convergent evolution of paternal care across vertebrates may have recruited similar neuroendocrine mechanisms. ?? 2012 Elsevier Inc..

The factors promoting the evolution of parental care strategies have been extensively studied in experiment and theory. However, most attempts to examine parental care in an evolutionary context have evaluated broad taxonomic categories. The explosive and recent diversifications of East African cichlid fishes offer exceptional opportunities to study the evolution of various life history traits based on species-level phylogenies. The Xenotilapia lineage within the endemic Lake Tanganyika cichlid tribe Ectodini comprises species that display either biparental or maternal only brood care and hence offers a unique opportunity to study the evolution of distinct parental care strategies in a phylogenetic framework. In order to reconstruct the evolutionary relationships among 16 species of this lineage we scored 2,478 Amplified Fragment Length Polymorphisms (AFLPs) across the genome. We find that the Ectodini genus Enantiopus is embedded within the genus Xenotilapia and that during 2.5 to 3 million years of evolution within the Xenotilapia clade there have been 3-5 transitions from maternal only to biparental care. While most previous models suggest that uniparental care (maternal or paternal) arose from biparental care, we conclude from our species-level analysis that the evolution of parental care strategies is not only remarkably fast, but much more labile than previously expected.

Across taxa, individuals must respond to a dynamic social environment of challenges and opportunities on multiple biological levels, including behavior, hormone profiles, and gene expression. We investigated the response to a complex social environment including both territorial challenges and reproductive opportunities in the African cichlid fish Astatotilapia burtoni (Burton's mouthbrooder), a species well-known for its phenotypic plasticity. Male A. burtoni are either socially dominant or subordinate and can transition between the two phenotypes. We used this transition to simultaneously study changes in aggression, reproductive behavior, testosterone and estradiol levels, gonadal histology, and testes expression of three genes involved in testosterone synthesis. We have found that males immediately become aggressive and increase testosterone levels when they become dominant in this paradigm of challenge and opportunity. Reproductive behavior and estradiol increase slightly later but are also up-regulated within 24. h. Increases in steroid hormone levels are accompanied by an increase in expression of steroidogenic acute regulatory protein (StAR), the rate-limiting enzyme during testosterone synthesis, as well as an increase in testis maturation as measured by histological organization. Reproductive behavior was found to correlate with female gravidity, suggesting that males were able to perceive reproductive opportunity. Our study demonstrates the rapid plasticity at multiple levels of biological organization that animals can display in response to changes in their complex social environment. ?? 2012 Elsevier Inc.

Social interactions require knowledge of the environment and status of others, which can be acquired indirectly by observing the behavior of others. When being observed, animals can also alter their signals based on who is watching. Here we observed how male cichlid fish (Astatotilapia burtoni) behave when being watched in two different contexts. In the first, we show that aggressive and courtship behaviors displayed by subordinate males depends critically on whether dominant males can see them, and in the second, we manipulated who was watching aggressive interactions and showed that dominant males will change their behavior depending on audience composition. In both cases, when a more dominant individual is out of view and the audience consists of more subordinate individuals, those males signal key social information to females by displaying courtship and dominant behaviors. In contrast, when a dominant male is present, males cease both aggression and courtship. These data suggest that males are keenly aware of their social environment and modulate their aggressive and courtship behaviors strategically for reproductive and social advantage.

Social status strongly affects behavior and physiology, in part mediated by gonadal hormones, although how each sex steroid acts across levels of biological organization is not well understood. We examine the role of sex steroids in modulating social behavior in dominant (DOM) and subordinate (SUB) males of a highly social fish, Astatotilapia burtoni. We first used agonists and antagonists to each sex steroid receptor and found that androgens and progestins modulate courtship behavior only in DOM, whereas estrogens modulate aggressive behavior independent of social status. We then examined the hormonal and physiological responses to sex steroid receptor antagonist treatment and uncovered substantial changes in circulating steroid hormone levels and gonad size only in SUB, not in DOM. Consistent with status-based physiological sensitivities to drug manipulation, we found that neuropeptide and steroid receptor gene expression in the preoptic area was sensitive only in SUB. However, when we compared the transcriptomes of males that received either vehicle or an estrogen receptor antagonist, 8.25% of all genes examined changed expression in DOM in comparison with only 0.56% in SUB. Finally, we integrate behavior, physiology, and brain gene expression to infer functional modules that underlie steroid receptor regulation of behavior. Our work suggests that environmentally induced changes at one level of biological organization do not simply affect changes of similar magnitude at other levels, but that instead very few key pathways likely serve as conduits for executing plastic responses across multiple levels.

Previous winning experience increases the probability of winning a subsequent contest. However, it is not clear whether winning probability is affected only by the outcome of the contest (winning or losing) or whether fighting experience itself is also sufficient to induce this effect. We investigated this question in the East African cichlid fish Pundamilia spec. To create an unresolved conflict we allowed males to fight their own mirror image prior to a real fight against a size-matched non-mirror-stimulated control male. When males fight their own mirror image, the image's response corresponds to the action of the focal animal, creating symmetrical fighting conditions without the experience of losing or winning. We found that mirror-stimulated males were more likely to win an ensuing contest than control males. Interestingly, in this species mirror stimulation also induced an increase in circulating androgens, which is consistent with the hypothesis that stimulation of these sex steroids during aggressive encounters may prepare the animal for subsequent encounters. Our results suggest that fighting experience alone coupled with an androgen response, increases the likelihood of winning, even in the absence of a winning experience. ?? 2011 Elsevier Inc.