Molecular mechanisms of phenotypic plasticity in Astatotilapia burtoni

textThe ability of an animal to respond and adapt to stimuli is necessary for its survival and involves plasticity and coordination of multiple levels of biological organization, including behavior, tissue organization, hormones, and gene expression. Each of these levels of response is complex, and none of them responds to stimuli in isolation. Thus, to understand how each system responds, it is necessary to consider its role in the context of the entire organism. Here, I have used the African cichlid fish Astatotilapia burtoni and its extraordinary phenotypic plasticity to investigate how animals respond to a change in social status from subordinate to dominant and attempted to integrate these multiple levels of biological response, as well as the roles of several candidate neuromodulators,. First, I have described how male A. burtoni become more aggressive and reproductive during their transition to dominance as well as increasing circulating levels of testosterone and estradiol and the histological organization of their testes. I then mapped the distribution of expression of two behaviorally relevant neuropeptides, arginine vasotocin and isotocin, and their respective receptors, throughout the A. burtoni brain, and found that they were highly expressed in several brain areas important for social behavior and decision-making. I then investigated the role of arginine vasotocin in social status and behavior via pharmacological manipulation and qPCR, showing the importance of arginine vasotocin in controlling the transition to dominance. Lastly, I investigated the role of aromatase, testosterone, and estradiol in male A. burtoni, both in stable dominant males and in males as they transition to dominance, using pharmacological manipulation and quantitative radioactive in situ hybridization, illustrating that estradiol synthesis during dominance is dependent on aromatase activity and necessary for aggressive behavior.Cellular and Molecular Biolog

Inhibition of Aromatase Induces Partial Sex Change in a Cichlid Fish: Distinct Functions for Sex Steroids in Brains and Gonads

Sex steroids are major drivers of sexual development and also responsible for the maintenance of the established gender. Especially fishes exhibit great plasticity and less conservation in sex determination and sexual development compared to other vertebrate groups. In addition, fishes have a constant sex steroid production throughout their entire lifespan, which makes them particularly susceptible to interferences with the endogenous sex steroid system. This susceptibility has recently been used to show that inhibition of the key enzyme of estrogen synthesis, aromatase Cyp19a1, can induce functional sex reversal even in adult fish. Here, we investigated the impact of the aromatase inhibitor (AI) fadrozole in adult females of the East African cichlid fish Astatotilapia burtoni. Using gene expression, phenotypic measurements, behavioral experiments, and hormone measurements, we assessed if females treated with fadrozole develop a male-like phenotype. We found that AI treatment has a different effect on gene expression in the gonad compared to the brain, the 2 tissues mostly implicated in sexual development. In contrast to observations in other gonochoristic species, A. burtoni ovaries cannot be transformed into functional testis by AI. However, rapid changes towards a male-like phenotype can be induced with AI in coloration, hormone levels, and behavior

Social regulation of reproduction in male cichlid fishes

© 2014 Elsevier Inc. Social interactions and relative positions within a dominance hierarchy have helped shape the evolution of reproduction in many animals. Since reproduction is crucial in all animals, and rank typically regulates access to reproductive opportunities, understanding the mechanisms that regulate socially-induced reproductive processes is extremely important. How does position in a dominance hierarchy impact an individual\u27s reproductive behavior, morphology, and physiology? Teleost fishes, and cichlids in particular, are ideally-suited models for studying how social status influences reproduction on multiple levels of biological organization. Here I review the current knowledge on the reproductive behavioral and physiological consequences of relative position in a dominance hierarchy, with a particular focus on male cichlids. Dominant and subordinate social status is typically associated with distinct differences in activity along the entire hypothalamic-pituitary-gonadal axis. Further, when transitions in social status occur between subordinate and dominant individuals, there are plastic changes from whole-organism behavior to molecular-level gene expression modifications that occur quickly. These rapid changes in behavior and physiology have allowed cichlids the flexibility to adapt to and thrive in their often dynamic physical and social environments. Studies in cichlid fishes have, and will continue, to advance our understanding of how the social environment can modulate molecular, cellular, and behavioral outcomes relevant to reproductive success. Future studies that take advantage of the extreme diversity in mating systems, reproductive tactics, and parental care strategies within the cichlid group will help generate hypotheses and careful experimental tests on the mechanisms governing the social control of reproduction in many vertebrates

Social Opportunity Causes Rapid Transcriptional Changes in the Social Behaviour Network of the Brain in an African Cichlid Fish

Animals constantly integrate external stimuli with their own internal physiological state to make appropriate behavioural decisions. Little is known, however, about where in the brain the salience of these signals is evaluated, or which neural and transcriptional mechanisms link this integration to adaptive behaviours. We used an African cichlid fish Astatotilapia burtoni to test the hypothesis that a new social opportunity activates the conserved \u27social behaviour network\u27 (SBN), a collection of brain nuclei known to regulate social behaviours across vertebrates. We measured mRNA levels of immediate early genes (IEGs) in microdissected brain regions as a proxy for neuronal activation, and discovered that IEGs were higher in all SBN nuclei in males that were given an opportunity to rise in social rank compared to control stable subordinate and dominant individuals. Furthermore, because the presence of sex-steroid receptors is one defining criteria of SBN nuclei, we also tested whether social opportunity or status influenced androgen and oestrogen receptor mRNA levels within these same regions. There were several rapid region-specific changes in receptor mRNA levels induced by social opportunity, most notably in oestrogen receptor subtypes in areas that regulate social aggression and reproduction, suggesting that oestrogenic signalling pathways play an important role in regulating male status. Several receptor mRNA changes occurred in regions with putative homologies to the mammalian septum and extended amygdala, two regions shared by SBN and reward circuits, suggesting an important role in the integration of social salience, stressors, hormonal state and adaptive behaviours. We also demonstrated increases in plasma sex- and stress-steroids at 30 min after a rise in social rank. This rapid endocrine and transcriptional response suggests that the SBN is involved in the integration of social inputs with internal hormonal state to facilitate the transition to dominant status, which ultimately leads to improved fitness for the previously reproductively-suppressed individual. © 2012 British Society for Neuroendocrinology

Social context and status affect behavior, physiology and brain activity of the highly social cichlid fish, Astatotilapia burtoni

Group living confers many benefits while at the same time exposing group members to intense competition for resources and status within the group. Monitoring the social environment involves not only participating in many fast paced social interactions but also monitoring the relationships and status of other group members. In chapter 1, we use a network analysis approach to study replicate communities of the cichlid fish, Astatotilapia burtoni, which live in fast-paced social groups with a complex dominance hierarchy among males. We found several correlations between an individual’s behavior, their position in the social hierarchy and the social environment. In addition, we found that community properties are a key parameter that predicts both cortisol and testosterone levels. In chapter 2 we extend this paradigm and look at the effects of social status within a community on brain activity as measured by cytochrome oxidase histochemistry. In addition, we examined brain activity in transitioning animals as measured by cytochrome oxidase and egr-1 induction. We find that social status has a variety of subtle effects on brain activity. Finally, we tested the role of the sex steroid hormones estradiol and testosterone on social transition using an aromatase inhibitor as well as an androgen receptor antagonist. We found very few effects of blocking the nuclear androgen pathway. Blocking aromatase resulted in a hyperaggressive phenotype suggesting that estradiol plays an important role in the transition process in this species.Ecology, Evolution and Behavio

Steroid receptor expression in the fish inner ear varies with sex, social status, and reproductive state

<p>Abstract</p> <p>Background</p> <p>Gonadal and stress-related steroid hormones are known to influence auditory function across vertebrates but the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown. The presence of steroid receptors in the ear suggests a direct pathway for hormones to act on the peripheral auditory system, but little is known about which receptors are expressed in the ear or whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA expression levels of multiple steroid receptor subtypes (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα, ARβ; corticosteroid receptors: GR2, GR1a/b, MR) and aromatase in the main hearing organ of the inner ear (saccule) in the highly social African cichlid fish <it>Astatotilapia burtoni</it>, and tested whether these receptor levels were correlated with circulating steroid concentrations.</p> <p>Results</p> <p>We show that multiple steroid receptor subtypes are expressed within the main hearing organ of a single vertebrate species, and that expression levels differ between the sexes. We also show that steroid receptor subtype-specific changes in mRNA expression are associated with reproductive phase in females and social status in males. Sex-steroid receptor mRNA levels were negatively correlated with circulating estradiol and androgens in both males and females, suggesting possible ligand down-regulation of receptors in the inner ear. In contrast, saccular changes in corticosteroid receptor mRNA levels were not related to serum cortisol levels. Circulating steroid levels and receptor subtype mRNA levels were not as tightly correlated in males as compared to females, suggesting different regulatory mechanisms between sexes.</p> <p>Conclusions</p> <p>This is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mRNA expression in the peripheral auditory system of any single vertebrate. Our data suggest that changes in steroid receptor mRNA expression in the inner ear could be a regulatory mechanism for physiological state-dependent auditory plasticity across vertebrates.</p

Reproductive status regulates expression of sex steroid and GnRH receptors in the olfactory bulb

Neuromodulators including gonadotropin-releasing hormone (GnRH) and sex steroids help integrate an animal\u27s internal physiological state with incoming external cues, and can have profound effects on the processing of behaviorally relevant information, particularly from the olfactory system. While GnRH and steroid receptors are present in olfactory processing regions across vertebrates, little is known about whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA levels of two GnRH receptors (GnRH-R1, GnRH-R2), five sex steroid receptors (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα, ARβ), and aromatase in the olfactory bulb of the highly social African cichlid fish Astatotilapia burtoni. We asked whether these receptor levels changed with reproductive condition in females, or with social status, which regulates reproductive capacity in males. Our results reveal that mRNA levels of multiple sex steroid, GnRH receptor subtypes, and aromatase in the olfactory bulb vary with sex, social status in males, and reproductive condition in females, which highlights the potential importance of changing receptor levels in fine-tuning the olfactory system during the reproductive cycle. Further, steroid receptor mRNA levels were positively correlated with circulating steroid levels in males, but negatively correlated in females, suggesting different regulatory control between sexes. These results provide support for the hypothesis that the first-order olfactory relay station is a substrate for both GnRH and sex steroid modulation, and suggest that changes in receptor levels could be an important mechanism for regulating reproductive, social, and seasonal plasticity in olfactory perception observed across vertebrates. © 2010 Elsevier B.V

Neuroendocrine regulation of social Interactions in a cichlid fish

Tese apresentada para cumprimentos dos requisitos necessários à obtenção do grau de Doutor em Biologia do Comportamento apresentada no ISPA - Instituto Universitário no ano de 2019.O estudo do comportamento animal e em particular do comportamento social tem atraído investigadores desde há muito tempo. Todos os animais interagem com os outros, característica fundamental para a sua sobrevivência e reprodução. No entanto, para obter uma total compreensão do comportamento social, é necessária a integração de seus vários componentes. Com esta tese, pretendemos clarificar este tópico, estudando como o cérebro controla o comportamento através da ação conjunta de seus circuitos neurais, genes e moléculas, e também como o ambiente social de forma recíproca influencia o cérebro. Baseado neste objetivo e usando a tilápia de Moçambique (Oreochromis mossambicus) como espécie modelo, num primeiro estudo investigámos como o comportamento social é controlado por uma rede dinâmica de regiões cerebrais, a Social Decision Making Network (SDMN). Aqui, tentámos entender quais são as pistas específicas que desencadeiam mudanças no padrão de ativação dessa rede neural, usando lutas entre machos. Os nossos resultados sugerem que é a avaliação mútua do comportamento de combate que impulsiona mudanças temporárias no estado do SDMN, e não a avaliação do resultado da luta ou apenas a expressão de comportamento agressivo. Em seguida, explorámos a modulação hormonal do comportamento social, em particular pelo neuropeptídeo vasotocina. Para isso, manipulámos o sistema da vasotocina injetando vasotocina e um antagonista específico dos receptores de vasotocina V1A em machos. Para distinguir se a vasotocina afeta o comportamento isoladamente ou em combinação com andrógenios, conduzimos esta experiência em peixes castrados e peixes controlo. Curiosamente, descobrimos que a vasotocina afetou o comportamento dos machos em relação às fêmeas, mas não em relação aos machos, e que os andrógenios e a vasotocina modularam a agressividade dos machos em relação às fêmeas. Em seguida, procurámos compreender como as interações sociais afetam os sistemas neuroendócrinos. Nesse sentido, utilizámos um paradigma de intrusões territoriais para avaliar os padrões temporais dos níveis de andrógenios e tentámos relacioná-los ao fenótipo comportamental de cada indivíduo. Obtivemos padrões distintos de resposta androgénica às interações sociais devido a diferenças individuais subjacentes em sua extensão de resposta. Este estudo oferece uma importante contribuição para a área de investigação, fornecendo possíveis razões para as discrepâncias associadas à hipótese de desafio, o principal modelo em endocrinologia comportamental que descreve a relação entre andrógenios e interações sociais. Finalmente, pensa-se que os andrógenios respondem às interações sociais como forma de preparar os indivíduos para outras interações. Assim, tentámos descobrir como um aumento de andrógenios no sangue afeta o cérebro. Para esse efeito, injetámos peixes com andrógenios e estudámos as mudanças transcriptómicas que ocorrem no cérebro usando a técnica de RNAseq, permitindo uma compreensão mais detalhada do efeito dos andrógenios no cérebro. Em suma, o comportamento social é complexo e depende de vários fatores internos e externos. Os resultados desta tese fornecem um contributo significativo para pesquisas futuras.The study of animal behavior and in specific of social behavior has attracted researchers for a long time. All animals interact with others, a feature which is fundamental to their survival and reproduction. However, to get a complete understanding of social behavior, the integration of its various components is required. In this thesis, we aimed to shed light on this topic, studying how the brain controls behavior through the concerted action of its neural circuits, genes and molecules, and also how the social environment feedbacks and impacts the brain. Grounded upon this objective and using the Mozambique tilapia (Oreochromis mossambicus) as a model species, in a first study we investigated how social behavior is controlled by a dynamic network of brain regions, the Social Decision Making Network (SDMN). Here, we tried to understand what are the specific cues that trigger changes in the pattern of activation of this neural network, by using staged fights between males. Our results suggest that it is the mutual assessment of relative fighting behavior that drives acute changes in the state of the SDMN, and not the assessment of fight outcome or just the expression of aggressive behavior. Then, we explored the hormonal modulation of social behavior, in particular of the neuropeptide vasopressin. For this purpose, we manipulated the vasotocin system by injecting vasotocin and a specific antagonist of vasotocin receptors V1A in males. To distinguish if vasotocin affected behavior alone or in combination with androgens, we conducted this experiment in both castrated and control fish. Interestingly, we found that vasotocin affected the behavior of males towards females but not towards males and that both androgens and vasotocin modulated aggressiveness towards females. Next, we sought to comprehend how social interactions affect neuroendocrine systems. In that sense, we used a paradigm of territorial intrusions to assess temporal patterns of androgen levels and tried to relate them to the behavioral phenotype of each individual. We obtained distinct patterns of androgen response to social interactions due to underlying individual differences in their scope for response. This study makes an important contribution to the field by providing possible reasons for discrepancies associated with the Challenge Hypothesis, the major framework in behavioral endocrinology which describes the relationship between androgens and social interactions. Finally, it is believed that androgens respond to social interactions as a way to prepare individuals for further interactions. Thus, we tried to uncover how an androgen surge in the blood affects the brain. To accomplish this, we injected fish with androgens and studied brain transcriptomic changes with the RNAseq technique, allowing the achievement of a thorough understanding of the effect of androgens on the brain. In sum, social behavior is complex and dependent on several internal and external factors. The findings from this thesis provide significant insights for future research.Fundação para a Ciência e Tecnologi

Social regulation of male reproductive plasticity in an African cichlid fish

Social interactions with the outcome of a position in a dominance hierarchy can have profound effects on reproductive behavior and physiology, requiring animals to integrate environmental information with their internal physiological state; but how is salient information from the animal\u27s dynamic social environment transformed into adaptive behavioral, physiological, and molecular-level changes? The African cichlid fish, Astatotilapia burtoni, is ideally suited to understand socially controlled reproductive plasticity because activity of the male reproductive (brainpituitary gonad) axis is tightly linked to social status. Males form hierarchies in which a small percentage of brightly colored dominant individuals have an active reproductive axis, defend territories, and spawn with females, while the remaining males are subordinate, drably colored, do not hold a territory, and have a suppressed reproductive system with minimal opportunities for spawning. These social phenotypes are plastic and quickly reversible, meaning that individual males may switch between dominant and subordinate status multiple times within a lifetime. Here, we review the rapid and remarkable plasticity that occurs along the entire reproductive axis when males rise in social rank, a transition that has important implications for the operational sex ratio of the population. When males rise in rank, transformations occur in the brain, pituitary, circulation, and testes over short time-scales (minutes to days). Changes are evident in overt behavior, as well as modifications at the physiological, cellular, and molecular levels that regulate reproductive capacity. Widespread changes triggered by a switch in rank highlight the significance of external social information in shaping internal physiology and reproductive competence. © The Author 2013. All rights reserved

The genetics of sexual development in East African cichlid fishes

Sexual development leads to the formation of males and females. This process can be controlled genetically, environmentally or by a combination thereof. Genetic sex determination is based on single- or polygenic actions, either linked to sex chromosomes or independent from them, while environmental sex determination includes factors such as temperature, density or social status. Teleost fishes, as the most species-rich clade of vertebrates, feature an overwhelming diversity of sex-determining mechanisms. In contrast to most mammals, where the presence or absence of one single gene defines the sexual fate, several master sex regulators have been identified in fish. Together with the description of complex polygenic regulations and the discovery of new functions of presumably conserved down-stream factors, fish exhibit a greater plasticity in generating males and females than previously thought. East African cichlid fishes are extremely diverse in terms of ecology, morphology and breeding behavior and therefore represent a prime model system in evolutionary biology in general, and in the study of sexual development in particular. Several sex determination systems have been described, which differ sometimes even between closely related species in this fish family. In my thesis, I investigated a set of candidate genes for sexual development in cichlids. More specifically, we studied these genes in adult cichlid species and in one of them also during early male development. Differential gene expression patterns, measured in brain and gonad tissue, were observed between cichlid lineages and within the same species over time. One striking case is the shift in gene expression of the aromatase cyp19a1. In vertebrates, and in all teleosts investigated so far, cyp19a1A and cyp19a1B are expressed in the ovaries and the brain, respectively. In the species rich and extremely diverse haplochromine lineage, we found an overexpression of cyp19a1B in the testis. In another study, we show that Astatotilapia burtoni, a member haplochromine lineage, most likely has an XX-XY sex determination system. Making use of hormonally sex-reversed fish and subsequent crossing experiments, we establish all-male broods, which offered an ideal platform to investigate gene expression of early testis and brain genes. In addition, we identified several promising candidates for genetic sex determiners in this species