Control of testes mass by androgen receptor paralogs in a cichlid.

Steroid hormones play numerous important and diverse roles in the differentiation and development of vertebrate primary and secondary reproductive characteristics. However, the exact role of androgen receptors-which bind circulating androgens-in this regulatory pathway is unclear. Teleost fishes further complicate this question by having two paralogs of the androgen receptor (ARα and ARß) resulting from a duplication of their ancestral genome. We investigated the functional role of these two ARs on adult testes mass, by eliminating receptor function of one or both paralogs using CRISPR/Cas9 genome edited Astatotilapia burtoni, an African cichlid fish. Fish with two or more functional AR alleles were more likely to be male compared to fish with one or fewer, suggesting that the two paralogs may play redundant roles in the A. burtoni sex determination system. We replicated previous work showing that fish lacking functional ARß possess testes smaller than wild-type fish, while fish lacking ARα possess testes larger than wild-type fish. However, we found novel evidence supporting a complex relationship between the two AR paralogs in the regulation of testes mass. For instance, the effects of ARα mutation on testes mass are eliminated in homozygous ARß mutants but the reverse is not true. These results suggest a dynamic relationship between these two AR paralogs where ARß functions may be permissive to ARα functions in the control of testes mass. This mechanism may contribute to the robust physiological plasticity displayed by A. burtoni and other social teleost fishes.

Modular genetic control of social status in a cichlid fish.

Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARß, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARß, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARß, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.

Behavioral evolution contributes to hindbrain diversification among Lake Malawi cichlid fish.

The evolutionary diversification of animal behavior is often associated with changes in the structure and function of nervous systems. Such evolutionary changes arise either through alterations of individual neural components ("mosaically") or through scaling of the whole brain ("concertedly"). Here we show that the evolution of a courtship behavior in Malawi cichlid fish is associated with rapid, extensive, and specific diversification of orosensory, gustatory centers in the hindbrain. We find that hindbrain volume varies significantly between species that build pit (depression) compared to castle (mound) type bowers and that this trait is evolving rapidly among castle-building species. Molecular analyses of neural activity via immediate early gene expression indicate a functional role for hindbrain structures during bower building. Finally, comparisons of bower building species in neighboring Lake Tanganyika suggest parallel patterns of neural diversification to those in Lake Malawi. Our results suggest that mosaic brain evolution via alterations to individual brain structures is more extensive and predictable than previously appreciated.

Genome-wide effects of social status on DNA methylation in the brain of a cichlid fish, Astatotilapia burtoni.

BACKGROUND: Successful social behavior requires real-time integration of information about the environment, internal physiology, and past experience. The molecular substrates of this integration are poorly understood, but likely modulate neural plasticity and gene regulation. In the cichlid fish species Astatotilapia burtoni, male social status can shift rapidly depending on the environment, causing fast behavioral modifications and a cascade of changes in gene transcription, the brain, and the reproductive system. These changes can be permanent but are also reversible, implying the involvement of a robust but flexible mechanism that regulates plasticity based on internal and external conditions. One candidate mechanism is DNA methylation, which has been linked to social behavior in many species, including A. burtoni. But, the extent of its effects after A. burtoni social change were previously unknown. RESULTS: We performed the first genome-wide search for DNA methylation patterns associated with social status in the brains of male A. burtoni, identifying hundreds of Differentially Methylated genomic Regions (DMRs) in dominant versus non-dominant fish. Most DMRs were inside genes supporting neural development, synapse function, and other processes relevant to neural plasticity, and DMRs could affect gene expression in multiple ways. DMR genes were more likely to be transcription factors, have a duplicate elsewhere in the genome, have an anti-sense lncRNA, and have more splice variants than other genes. Dozens of genes had multiple DMRs that were often seemingly positioned to regulate specific splice variants. CONCLUSIONS: Our results revealed genome-wide effects of A. burtoni social status on DNA methylation in the brain and strongly suggest a role for methylation in modulating plasticity across multiple biological levels. They also suggest many novel hypotheses to address in mechanistic follow-up studies, and will be a rich resource for identifying the relationships between behavioral, neural, and transcriptional plasticity in the context of social status.

Mechanistic target of rapamycin (mTOR) implicated in plasticity of the reproductive axis during social status transitions.

The highly conserved brain-pituitary-gonadal (BPG) axis controls reproduction in all vertebrates, so analyzing the regulation of this signaling cascade is important for understanding reproductive competence. The protein kinase mechanistic target of rapamycin (mTOR) functions as a conserved regulator of cellular growth and metabolism in all eukaryotes, and also regulates the reproductive axis in mammals. However, whether mTOR might also regulate the BPG axis in non-mammalian vertebrates remains unexplored. We used complementary experimental approaches in an African cichlid fish, Astatotilapia burtoni, to demonstrate that mTOR is involved in regulation of the brain, pituitary, and testes when males rise in rank to social dominance. mTOR or downstream components of its signaling pathway (p-p70S6K) were detected in gonadotropin-releasing hormone (GnRH1) neurons, the pituitary, and testes. Transcript levels of mtor in the pituitary and testes also varied when reproductively-suppressed subordinate males rose in social rank to become dominant reproductively-active males, a transition similar to puberty in mammals. Intracerebroventricular injection of the mTORC1 inhibitor, rapamycin, revealed a role for mTOR in the socially-induced hypertrophy of GnRH1 neurons. Rapamycin treatment also had effects at the pituitary and testes, suggesting involvement of the mTORC1 complex at multiple levels of the reproductive axis. Thus, we show that mTOR regulation of BPG function is conserved to fishes, likely playing important roles in regulating reproduction and fertility across all male vertebrates.

Hormonal regulation of social ascent and temporal patterns of behavior in an African cichlid.

For many species, social rank determines which individuals perform certain social behaviors and when. Higher ranking or dominant (DOM) individuals maintain status through aggressive interactions and perform courtship behaviors while non-dominant (ND) individuals do not. In some species ND individuals ascend (ASC) in social rank when the opportunity arises. Many important questions related to the mechanistic basis of social ascent remain to be answered. We probed whether androgen signaling regulates social ascent in male Astatotilapia burtoni, an African cichlid whose social hierarchy can be readily controlled in the laboratory. As expected, androgen receptor (AR) antagonism abolished reproductive behavior during social ascent. However, we discovered multiple AR- and status-dependent temporal behavioral patterns that typify social ascent and dominance. AR antagonism in ASC males increased the time between successive behaviors compared to DOM males. Socially ascending males, independent of AR activation, were more likely than DOM males to follow aggressive displays with another aggressive display. Further analyses revealed differences in the sequencing of aggressive and courtship behaviors, wherein DOM males were more likely than ASC males to follow male-directed aggression with courtship displays. Strikingly, this difference was driven mostly by ASC males taking longer to transition from aggression to courtship, suggesting ASC males can perform certain DOM-typical temporal behavioral patterns. Our results indicate androgen signaling is necessary for social ascent and hormonal signaling and social experience may shape the full suite of DOM-typical behavioral patterns.

Behavior-dependent cis regulation reveals genes and pathways associated with bower building in cichlid fishes.

Many behaviors are associated with heritable genetic variation [Kendler and Greenspan (2006) Am J Psychiatry 163:1683-1694]. Genetic mapping has revealed genomic regions or, in a few cases, specific genes explaining part of this variation [Bendesky and Bargmann (2011) Nat Rev Gen 12:809-820]. However, the genetic basis of behavioral evolution remains unclear. Here we investigate the evolution of an innate extended phenotype, bower building, among cichlid fishes of Lake Malawi. Males build bowers of two types, pits or castles, to attract females for mating. We performed comparative genome-wide analyses of 20 bower-building species and found that these phenotypes have evolved multiple times with thousands of genetic variants strongly associated with this behavior, suggesting a polygenic architecture. Remarkably, F1 hybrids of a pit-digging and a castle-building species perform sequential construction of first a pit and then a castle bower. Analysis of brain gene expression in these hybrids showed that genes near behavior-associated variants display behavior-dependent allele-specific expression with preferential expression of the pit-digging species allele during pit digging and of the castle-building species allele during castle building. These genes are highly enriched for functions related to neurodevelopment and neural plasticity. Our results suggest that natural behaviors are associated with complex genetic architectures that alter behavior via cis-regulatory differences whose effects on gene expression are specific to the behavior itself.

Astatotilapia burtoni: A Model System for Analyzing the Neurobiology of Behavior.

Most biomedical research is performed using a very limited number of "model" species. In part, this has resulted from a combination of full genomes, manipulation of genes, and short generation times in these species. However, the advent of low-cost sequencing and gene editing in any organism has increased the use of nontraditional organisms. Many scientists have paraphrased the adage by Krogh [ Krogh , A. ( 2018 ) Science 70 , 200 - 204 ] that for many biological problems some species will prove to be most convenient and useful to study. In particular, using organisms most suited to the specific research question can lead to novel insights about fundamental physiological, neurobiological, immunological, and neuroendocrine systems that can advance our understanding of the well-being and health of humans. In addition, such studies have led to new ideas about the evolution and mechanisms that control social behavior. Fishes constitute about 50% of all vertebrate species and are the most diverse vertebrate radiation. Here we review behavioral and neurobiological discoveries of plasticity in social behavior resulting from analysis of an African cichlid fish, showing how its unique behavioral system has facilitated a broad range of discoveries. For many future questions, Astatotilapia burtoni and other cichlid fishes may be ideally suited to study as we advance our understanding of the neural basis of social decisions.

The Value of Comparative Animal Research: Krogh's Principle Facilitates Scientific Discoveries.

Biomedical research is dominated by relatively few nonhuman animals to investigate healthy and disease conditions. Research has overrelied on these models due to their well-described genomes, the capability to control specific genes, and the high rate of reproduction. However, recent advances in large-scale molecular sequencing experiments have revealed, in some cases, the limited similarities in experimental outcomes observed in common rodents (i.e., mice) compared with humans. The value of more varied comparative animal models includes examples such as long-term body weight regulation in seasonally breeding hamsters as a means to help understand the obesity epidemic, vocal learning in songbirds to illuminate language acquisition and maintenance, and reproduction in cichlid fish to discover novel genes conserved in humans. Studying brain genes in prairie voles and cichlids advanced knowledge about social behavior. Taken together, experiments on diverse animal species highlight nontraditional systems for advancing our understanding of human health and well-being.

Rhythmic expressed clock regulates the transcription of proliferating cellular nuclear antigen in teleost retina.

Teleost fish continues to grow their eyes throughout life with the body size. In Astatotilapia burtoni, the fish retina increases by adding new retinal cells at the ciliary marginal zone (CMZ) and in the outer nuclear layer (ONL). Cell proliferation at both sites exhibits a daily rhythm in number of dividing cells. To understand how this diurnal rhythm of new cell production is controlled in retinal progenitor cells, we studied the transcription pattern of clock genes in retina, including clock1a, clock1b, bmal1a (brain and muscle ARNT-Like), and per1b (period1b). We found that these genes have a strong diurnal rhythmic transcription during light-dark cycles but not in constant darkness. An oscillation in pcna transcription was also observed during light-dark cycles, but again not in constant darkness. Our results also indicate an association between Clock proteins and the upstream region of pcna (proliferating cellular nuclear antigen) gene. A luciferase reporter assay conducted in an inducible clock knockdown cell line further demonstrated that the mutation on predicted E-Boxes in pcna promoter region significantly attenuated the transcriptional activation induced by Clock protein. These results suggested that the diurnal rhythmic expression of clock genes in A. burtoni retina could be light dependent and might contribute to the daily regulation of the proliferation of the retina progenitors through key components of cell cycle machinery, for instance, pcna.

Cognitive skills and the evolution of social systems.

How do animal social skills influence evolution? Complex animal social behaviors require many cognitive skills including individual recognition and observational learning. For social systems to evolve, these abilities need to be transmitted genetically or culturally and supported by the evolution of underlying neural systems. Because animal skill sets are so varied, it seems best to describe animal cognitive behaviors as being a social calculus that can change with experience, which has evolved to match and facilitate the complexity of the social system where it arose. That is, acquiring and using social information in response to a rapidly changing complex world leads to social competence enabling success in essential behavioral interactions. Here, we describe the remarkable suite of social skills discovered in the African cichlid fish Astatotilapia burtoni, including an attention hierarchy, male deception, transitive inference, the mechanistic bases of social dominance, female mate choice and the neural control of female reproductive behavior. The social calculus of this species is presented as an example of a potential causal factor in the evolution of sophisticated social behavior necessary for the evolutionary success of their social system.

Differential activation of vasotocin neurons in contexts that elicit aggression and courtship.

Despite continued study on the neurobiological bases of aggressive and sexual behaviors, it is still not well understood how the brain integrates social information with physiological and neural states to produce context-specific behavioral outcomes. In fishes, manipulation of endogenous levels of arginine vasotocin (AVT) through peripheral and intracerebroventricular pharmacological injections results in significant changes in social behaviors, including aggressive and reproduction-related behaviors. In addition, many features of AVT neurons have been shown to correlate with social status and associated behavioral phenotypes. In this study, we used the immediate early gene egr-1 as a marker for neuronal activity and quantified the number of AVT neurons that were positive for egr-1 mRNA by in situ hybridization in Astatotilapia burtoni males that were exposed to either a social context that would elicit aggression or to one that would elicit courtship. In these social settings, focal males readily displayed context- appropriate bouts of aggression (towards the opponent) or bouts of courting (towards females). We found that males that fought had higher levels of egr-1 expression in the preoptic area compared to courting males. A greater proportion of AVT cells was positive for egr-1 after a fight than after a bout of courting. We mapped mRNA distribution of AVT V1a receptor subtypes v1a1 and v1a2 in the brain and identified overlapping areas of expression in nuclei in the ventral telencephalon, hypothalamus and thalamus as key areas for AVT signaling in males.

Dopaminergic inhibition of gonadotropin-releasing hormone neurons in the cichlid fish Astatotilapia burtoni.

Dopamine regulates reproduction in part by modulating neuronal activity within the hypothalamic-pituitary-gonadal (HPG) axis. Previous studies suggested numerous mechanisms by which dopamine exerts inhibitory control over the HPG axis, ultimately changing the levels of sex steroids that regulate reproductive behaviors. However, it is not known whether these mechanisms are conserved across vertebrate species. In particular, it is unknown whether mechanisms underlying dopaminergic control of reproduction are shared between mammals and teleost fish. In mammals, dopamine directly inhibits gonadotropin-releasing hormone (GnRH1) hypothalamic neurons, the gatekeepers for activation of the HPG axis. Here, we demonstrate, for the first time in teleost fish, dopaminergic control of GnRH1 neurons via direct dopamine type-2-like receptor (D2R)-mediated inhibition within the hypothalamus. These results suggest that direct dopaminergic control of GnRH1 neurons via interactions in the hypothalamus is not exclusive to tetrapod reproductive control, but is likely conserved across vertebrate species.

Polygenic sex determination in the cichlid fish Astatotilapia burtoni.

BACKGROUND: The East African riverine cichlid species Astatotilapia burtoni serves as an important laboratory model for sexually dimorphic physiology and behavior, and also serves as an outgroup species for the explosive adaptive radiations of cichlid species in Lake Malawi and Lake Victoria. An astounding diversity of genetic sex determination systems have been revealed within the adaptive radiation of East African cichlids thus far, including polygenic sex determination systems involving the epistatic interaction of multiple, independently segregating sex determination alleles. However, sex determination has remained unmapped in A. burtoni. Here we present mapping results supporting the presence of multiple, novel sex determination alleles, and thus the presence of polygenic sex determination in A. burtoni. RESULTS: Using mapping in small families in conjunction with restriction-site associated DNA sequencing strategies, we identify associations with sex at loci on linkage group 13 and linkage group 5-14. Inheritance patterns support an XY sex determination system on linkage group 5-14 (a chromosome fusion relative to other cichlids studied), and an XYW system on linkage group 13, and these associations are replicated in multiple families. Additionally, combining our genetic data with comparative genomic analysis identifies another fusion that is unassociated with sex, with linkage group 8-24 and linkage group 16-21 fused in A. burtoni relative to other East African cichlid species. CONCLUSIONS: We identify genetic signals supporting the presence of three previously unidentified sex determination alleles at two loci in the species A. burtoni, strongly supporting the presence of polygenic sex determination system in the species. These results provide a foundation for future mapping of multiple sex determination genes and their interactions. A better understanding of sex determination in A. burtoni provides important context for their use in behavioral studies, as well as studies of the evolution of genetic sex determination and sexual conflicts in East African cichlids.

Identification of prohormones and pituitary neuropeptides in the African cichlid, Astatotilapia burtoni.

BACKGROUND: Cichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni, a well-studied member of the diverse African cichlid clade. RESULTS: Utilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages. CONCLUSION: We characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.

Two types of dominant male cichlid fish: behavioral and hormonal characteristics.

Male African cichlid fish, Astatotilapia burtoni, have been classified as dominant or subordinate, each with unique behavioral and endocrine profiles. Here we characterize two distinct subclasses of dominant males based on types of aggressive behavior: (1) males that display escalating levels of aggression and court females while they establish a territory, and (2) males that display a stable level of aggression and delay courting females until they have established a territory. To profile differences in their approach to a challenge, we used an intruder assay. In every case, there was a male-male confrontation between the resident dominant male and the intruder, with the intruder quickly taking a subordinate role. However, we found that dominant males with escalating aggression spent measurably more time attacking subordinates than did dominant males with stable aggression that instead increased their attention toward the females in their tank. There was no difference in the behavior of intruders exposed to either type of dominant male, suggesting that escalating aggression is an intrinsic characteristic of some dominant males and is not elicited by the behavior of their challengers. Male behavior during the first 15 min of establishing a territory predicts their aggressive class. These two types of dominant males also showed distinctive physiological characteristics. After the intruder assay, males with escalating aggression had elevated levels of 11-ketotestosterone (11-KT), testosterone, estradiol, and cortisol, while those with stable aggression did not. These observations show that the same stimulus can elicit different behavioral and endocrine responses among A. burtoni dominant males that characterize them as either escalating or stable aggressive types. Our ability to identify which individuals within a population have escalating levels of aggressive responses versus those which have stable levels of aggressive responses when exposed to the same stimulus, offers a potentially powerful model for investigating the underlying molecular mechanisms that modulate aggressive behavior.

A Neural Basis for Control of Cichlid Female Reproductive Behavior by Prostaglandin F2α.

In most species, females time reproduction to coincide with fertility. Thus, identifying factors that signal fertility to the brain can provide access to neural circuits that control sexual behaviors. In vertebrates, levels of key signaling molecules rise at the time of fertility to prime the brain for reproductive behavior [1-11], but how and where they regulate neural circuits is not known [12, 13]. Specifically, 17α,20ß-dihydroxyprogesterone (DHP) and prostaglandin F2α (PGF2α) levels rise in teleost fish around the time of ovulation [10, 14, 15]. In an African cichlid fish, Astatotilapia burtoni, fertile females select a mate and perform a stereotyped spawning routine, offering quantifiable behavioral outputs of neural circuits. We show that, within minutes, PGF2α injection activates a naturalistic pattern of sexual behavior in female A. burtoni. We also identify cells in the brain that transduce the prostaglandin signal to mate and show that the gonadal steroid DHP modulates mRNA levels of the putative receptor for PGF2α (Ptgfr). We use CRISPR/Cas9 to generate the first targeted gene mutation in A. burtoni and show that Ptgfr is necessary for the initiation of sexual behavior, uncoupling sexual behavior from reproductive status. Our findings are consistent with a model in which PGF2α communicates fertility status via Ptgfr to circuits in the brain that drive female sexual behavior. Our targeted genome modification in a cichlid fish shows that dissection of gene function can reveal basic control mechanisms for behaviors in this large family of species with diverse and fascinating social systems [16, 17].

Timing reproduction in teleost fish: cues and mechanisms.

Fish comprise half of extant vertebrate species and use a rich variety of reproductive strategies that have yielded insights into the basic mechanisms that evolved for sex. To maximize the chances of fertilization and survival of offspring, fish species time reproduction to occur at optimal times. For years, ethologists have performed painstaking experiments to identify sensory inputs and behavioral outputs of the brain during mating. Here we review known mechanisms that generate sexual behavior, focusing on the factors that govern the timing of these displays. The development of new technologies, including high-throughput sequencing and genome engineering, has the potential to provide novel insights into how the vertebrate brain consummates mating at the appropriate time.

Epigenetic DNA Methylation Linked to Social Dominance.

Social status hierarchies are ubiquitous in vertebrate social systems, including humans. It is well known that social rank can influence quality of life dramatically among members of social groups. For example, high-ranking individuals have greater access to resources, including food and mating prerogatives that, in turn, have a positive impact on their reproductive success and health. In contrast low ranking individuals typically have limited reproductive success and may experience lasting social and physiological costs. Ultimately, social rank and behavior are regulated by changes in gene expression. However, little is known about mechanisms that transduce social cues into transcriptional changes. Since social behavior is a dynamic process, we hypothesized that a molecular mechanism such as DNA methylation might play a role these changes. To test this hypothesis, we used an African cichlid fish, Astatotilapia burtoni, in which social rank dictates reproductive access. We show that manipulating global DNA methylation state strongly biases the outcomes of social encounters. Injecting DNA methylating and de-methylating agents in low status animals competing for status, we found that animals with chemically increased methylation states were statistically highly likely to ascend in rank. In contrast, those with inhibited methylation processes and thus lower methylation levels were statistically highly unlikely to ascend in rank. This suggests that among its many roles, DNA methylation may be linked to social status and more generally to social behavior.

Social Crowding during Development Causes Changes in GnRH1 DNA Methylation.

Gestational and developmental cues have important consequences for long-term health, behavior and adaptation to the environment. In addition, social stressors cause plastic molecular changes in the brain that underlie unique behavioral phenotypes that also modulate fitness. In the adult African cichlid, Astatotilapia burtoni, growth and social status of males are both directly regulated by social interactions in a dynamic social environment, which causes a suite of plastic changes in circuits, cells and gene transcription in the brain. We hypothesized that a possible mechanism underlying some molecular changes might be DNA methylation, a reversible modification made to cytosine nucleotides that is known to regulate gene function. Here we asked whether changes in DNA methylation of the GnRH1 gene, the central regulator of the reproductive axis, were altered during development of A. burtoni. We measured changes in methylation state of the GnRH1 gene during normal development and following the gestational and developmental stress of social crowding. We found differential DNA methylation within developing juveniles between 14-, 28- and 42-day-old. Following gestational crowding of mouth brooding mothers, we saw differential methylation and transcription of GnRH1 in their offspring. Taken together, our data provides evidence for social control of GnRH1 developmental responses to gestational cues through DNA methylation.