Polygenic sex determination produces modular sex polymorphism in an African cichlid fish.

For many vertebrates, a single genetic locus initiates a cascade of developmental sex differences in the gonad and throughout the organism, resulting in adults with two phenotypically distinct sexes. Species with polygenic sex determination (PSD) have multiple interacting sex determination alleles segregating within a single species, allowing for more than two genotypic sexes and scenarios where sex genotype at a given locus can be decoupled from gonadal sex. Here we investigate the effects of PSD on secondary sexual characteristics in the cichlid fish Metriaclima mbenjii, where one female (W) and one male (Y) sex determination allele interact to produce siblings with four possible sex classes: ZZXX females, ZWXX females, ZWXY females, and ZZXY males. We find that PSD in M. mbenjii produces an interplay of sex linkage and sex limitation resulting in modular variation in morphological and behavioral traits. Further, the evolution or introgression of a newly acquired sex determiner creates additional axes of phenotypic variation for varied traits, including genital morphology, craniofacial morphology, gastrointestinal morphology, and home tank behaviors. In contrast to single-locus sex determination, which broadly results in sexual dimorphism, polygenic sex determination can induce higher-order sexual polymorphism. The modularity of secondary sexual characteristics produced by PSD provides context for understanding the evolutionary causes and consequences of maintenance, gain, or loss of sex determination alleles in populations.

Female Mimicry by Sneaker Males Has a Transcriptomic Signature in Both the Brain and the Gonad in a Sex-Changing Fish.

Phenotypic plasticity represents an elegant adaptive response of individuals to a change in their environment. Bluehead wrasses (Thalassoma bifasciatum) exhibit astonishing sexual plasticity, including female-to-male sex change and discrete male morphs that differ strikingly in behavior, morphology, and gonadal investment. Using RNA-seq transcriptome profiling, we examined the genes and physiological pathways underlying flexible behavioral and gonadal differences among female, dominant (bourgeois) male, and female-mimic (sneaker) male blueheads. For the first time in any organism, we find that female mimicry by sneaker males has a transcriptional signature in both the brain and the gonad. Sneaker males shared striking similarity in neural gene expression with females, supporting the idea that males with alternative reproductive phenotypes have "female-like brains." Sneaker males also overexpressed neuroplasticity genes, suggesting that their opportunistic reproductive strategy requires a heightened capacity for neuroplasticity. Bourgeois males overexpressed genes associated with socio-sexual behaviors (e.g., isotocin), but also neuroprotective genes and biomarkers of oxidative stress and aging, indicating a hitherto unexplored cost to these males of attaining the reproductively privileged position at the top of the social hierarchy. Our novel comparison of testicular transcriptomes in a fish with male sexual polymorphism associates greater gonadal investment by sneaker males with overexpression of genes involved in cell proliferation and sperm quality control. We propose that morphological female-mimicry by sneaker male teleosts entails pervasive downregulation of androgenesis genes, consistent with low androgen production in males lacking well-developed secondary sexual characters.

Sexual plasticity: A fishy tale.

Teleost fish exhibit remarkably diverse and plastic patterns of sexual development. One of the most fascinating modes of plasticity is functional sex change, which is widespread in marine fish including species of commercial importance; however, the regulatory mechanisms remain elusive. In this review, we explore such sexual plasticity in fish, using the bluehead wrasse (Thalassoma bifasciatum) as the primary model. Synthesizing current knowledge, we propose that cortisol and key neurochemicals modulate gonadotropin releasing hormone and luteinizing hormone signaling to promote socially controlled sex change in protogynous fish. Future large-scale genomic analyses and systematic comparisons among species, combined with manipulation studies, will likely uncover the common and unique pathways governing this astonishing transformation. Revealing the molecular and neuroendocrine mechanisms underlying sex change in fish will greatly enhance our understanding of vertebrate sex determination and differentiation as well as phenotypic plasticity in response to environmental influences. Mol. Reprod. Dev. 84: 171-194, 2017. © 2016 Wiley Periodicals, Inc.

Characterizing the neurotranscriptomic states in alternative stress coping styles.

BACKGROUND: Animals experience stress in many contexts and often successfully cope. Individuals exhibiting the proactive versus reactive stress coping styles display qualitatively different behavioral and neuroendocrine responses to stressors. The predisposition to exhibiting a particular coping style is due to genetic and environmental factors. In this study we explore the neurotranscriptomic and gene network biases that are associated with differences between zebrafish (Danio rerio) lines selected for proactive and reactive coping styles and reared in a common garden environment. RESULTS: Using RNA-sequencing we quantified the basal transcriptomes from the brains of wild-derived zebrafish lines selectively bred to exhibit the proactive or reactive stress coping style. We identified 1953 genes that differed in baseline gene expression levels. Weighted gene coexpression network analyses identified one gene module associated with line differences. Together with our previous pharmacological experiment, we identified a core set of 62 genes associated with line differences. Gene ontology analyses reveal that many of these core genes are implicated in neurometabolism (e.g. organic acid biosynthetic and fatty acid metabolic processes). CONCLUSIONS: Our results show that proactive and reactive stress coping individuals display distinct basal neurotranscriptomic states. Differences in baseline expression of select genes or regulation of specific gene modules are linked to the magnitude of the behavioral response and the display of a coping style, respectively. Our results expand the molecular mechanisms of stress coping from one focused on the neurotransmitter systems to a more complex system that involves an organism's capability to handle neurometabolic loads and allows for comparisons with other animal taxa to uncover potential conserved mechanisms.

Characterization and distribution of kisspeptins, kisspeptin receptors, GnIH, and GnRH1 in the brain of the protogynous bluehead wrasse (Thalassoma bifasciatum).

The kisspeptin and gonadotropin-inhibitory hormone (GnIH) systems regulate the hypothalamic-pituitary-gonadal (HPG) axis in a broad range of vertebrates through direct or indirect effects on hypothalamic/preoptic gonadotropin-releasing hormone (GnRH) neurons and pituitary gonadotropes. These systems are sensitive to environmental factors, including social conditions, and may assist in relaying environmental signals to the HPG axis in a potentially broad range of taxa. In this study, we characterized expression of kisspeptin-system genes (kiss1, kiss2, kissr1, and kissr2), gnih, and gnrh1 in the brain of the bluehead wrasse (Thalassoma bifasciatum), an important teleost model of socially-controlled sex change. We analyzed cDNA sequences and examined transcript distributions in the brain using in situ hybridization (ISH) to determine if expression occurs in reproductively-relevant and conserved regions. Expression of kiss1 was detected in the habenula, lateral hypothalamic nucleus (LHn), and preoptic area (POA), while kiss2 was expressed in the dorsal hypothalamus, with sporadic signal in the POA. Expression of kissr1 was detected in the POA, habenula, and LHn, while kissr2 expression was widespread. Gnih mRNA was detected in the posterior periventricular nucleus (NPPv), and gnrh1 neurons localized to the POA. Neurons expressing kissr2 and gnih co-regionalized in the NPPv, while kissr1, kissr2, and gnrh1 co-regionalized in the POA. Double-label ISH revealed very close proximity between kissr1 and gnrh1 neurons, suggesting potential communication between the kisspeptin and GnRH1 systems through these interneurons. These expression patterns are generally conserved and suggest that if kisspeptins do signal GnRH1 neurons, the interaction is indirect, possibly through neurons adjacent to GnRH1. With this foundation in place, future studies can help determine the interactions among these systems and whether these peptides assist in transducing social changes into a shift from female to male sexual function.

Estrogenic signaling and sociosexual behavior in wild sex-changing bluehead wrasses, Thalassoma bifasciatum.

Estrogenic signaling is an important focus in studies of gonadal and brain sexual differentiation in fishes and vertebrates generally. This study examined variation in estrogenic signaling (1) across three sexual phenotypes (female, female-mimic initial phase [IP] male, and terminal phase [TP] male), (2) during socially-controlled female-to-male sex change, and (3) during tidally-driven spawning cycles in the protogynous bluehead wrasse (Thalassoma bifasciatum). We analyzed relative abundances of messenger RNAs (mRNAs) for the brain form of aromatase (cyp19a1b) and the three nuclear estrogen receptors (ER) (ERα, ERßa, and ERßb) by qPCR. Consistent with previous reports, forebrain/midbrain cyp19a1b was highest in females, significantly lower in TP males, and lowest in IP males. By contrast, ERα and ERßb mRNA abundances were highest in TP males and increased during sex change. ERßa mRNA did not vary significantly. Across the tidally-driven spawning cycle, cyp19a1b abundances were higher in females than TP males. Interestingly, cyp19a1b levels were higher in TP males close (~1 h) to the daily spawning period when sexual and aggressive behaviors rise than males far from spawning (~10-12 h). Together with earlier findings, our results suggest alterations in neural estrogen signaling are key regulators of socially-controlled sex change and sexual phenotype differences. Additionally, these patterns suggest TP male-typical sociosexual behaviors may depend on intermediate rather than low estrogenic signaling. We discuss these results and the possibility that an inverted-U shaped relationship between neural estrogen and male-typical behaviors is more common than presently appreciated.

Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses.

Fishes exhibit remarkably diverse, and plastic, patterns of sexual development, most striking of which is sequential hermaphroditism, where individuals readily reverse sex in adulthood. How this stunning example of phenotypic plasticity is controlled at a genetic level remains poorly understood. Several genes have been implicated in regulating sex change, yet the degree to which a conserved genetic machinery orchestrates this process has not yet been addressed. Using captive and in-the-field social manipulations to initiate sex change, combined with a comparative qPCR approach, we compared expression patterns of four candidate regulatory genes among three species of wrasses (Labridae)-a large and diverse teleost family where female-to-male sex change is pervasive, socially-cued, and likely ancestral. Expression in brain and gonadal tissues were compared among the iconic tropical bluehead wrasse (Thalassoma bifasciatum) and the temperate spotty (Notolabrus celidotus) and kyusen (Parajulus poecilepterus) wrasses. In all three species, gonadal sex change was preceded by downregulation of cyp19a1a (encoding gonadal aromatase that converts androgens to oestrogens) and accompanied by upregulation of amh (encoding anti-müllerian hormone that primarily regulates male germ cell development), and these genes may act concurrently to orchestrate ovary-testis transformation. In the brain, our data argue against a role for brain aromatase (cyp19a1b) in initiating behavioural sex change, as its expression trailed behavioural changes. However, we find that isotocin (it, that regulates teleost socio-sexual behaviours) expression correlated with dominant male-specific behaviours in the bluehead wrasse, suggesting it upregulation mediates the rapid behavioural sex change characteristic of blueheads and other tropical wrasses. However, it expression was not sex-biased in temperate spotty and kyusen wrasses, where sex change is more protracted and social groups may be less tightly-structured. Together, these findings suggest that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioural sex change may be more variable.

Stress, novel sex genes, and epigenetic reprogramming orchestrate socially controlled sex change.

Bluehead wrasses undergo dramatic, socially cued female-to-male sex change. We apply transcriptomic and methylome approaches in this wild coral reef fish to identify the primary trigger and subsequent molecular cascade of gonadal metamorphosis. Our data suggest that the environmental stimulus is exerted via the stress axis and that repression of the aromatase gene (encoding the enzyme converting androgens to estrogens) triggers a cascaded collapse of feminizing gene expression and identifies notable sex-specific gene neofunctionalization. Furthermore, sex change involves distinct epigenetic reprogramming and an intermediate state with altered epigenetic machinery expression akin to the early developmental cells of mammals. These findings reveal at a molecular level how a normally committed developmental process remains plastic and is reversed to completely alter organ structures.

The Need for Speed: Neuroendocrine Regulation of Socially-controlled Sex Change.

Socially-controlled functional sex change in fishes is a dramatic example of adaptive reproductive plasticity. Functional gonadal sex change can occur within a week while behavioral sex change can begin within minutes. Significant progress has been made in understanding the neuroendocrine bases of this phenomenon at both the gonadal and the neurobiological levels, but a detailed mechanistic understanding remains elusive. We are working with sex-changing wrasses to identify evolutionarily-conserved neuroendocrine pathways underlying this reproductive adaptation. One key model is the bluehead wrasse (Thalassoma bifasciatum), in which sex change is well studied at the behavioral, ecological, and neuroendocrine levels. Bluehead wrasses show rapid increases in aggressive and courtship behaviors with sex change that do not depend on the presence of gonads. The display of male-typical behavior is correlated with the expression of arginine vasotocin, and experiments support a role for this neuropeptide. Estrogen synthesis is also critical in the process. Female bluehead wrasses have higher abundance of aromatase mRNA in the brain and gonads, and estrogen implants block behavioral sex change. While established methods have advanced our understanding of sex change, a full understanding will require new approaches and perspectives. First, contributions of other neuroendocrine systems should be better characterized, particularly glucocorticoid and thyroid signaling. Second, advances in genomics for non-traditional model species should allow conserved mechanisms to be identified with a key next-step being manipulative tests of these mechanisms. Finally, advances in genomics now also allow study of the role of epigenetic modifications and other regulatory mechanisms in the dramatic alterations across the sex-change process.

Large-scale transcriptome sequencing reveals novel expression patterns for key sex-related genes in a sex-changing fish.

BACKGROUND: Teleost fishes exhibit remarkably diverse and plastic sexual developmental patterns. One of the most astonishing is the rapid socially controlled female-to-male (protogynous) sex change observed in bluehead wrasses (Thalassoma bifasciatum). Such functional sex change is widespread in marine fishes, including species of commercial importance, yet its underlying molecular basis remains poorly explored. METHODS: RNA sequencing was performed to characterize the transcriptomic profiles and identify genes exhibiting sex-biased expression in the brain (forebrain and midbrain) and gonads of bluehead wrasses. Functional annotation and enrichment analysis were carried out for the sex-biased genes in the gonad to detect global differences in gene products and genetic pathways between males and females. RESULTS: Here we report the first transcriptomic analysis for a protogynous fish. Expression comparison between males and females reveals a large set of genes with sex-biased expression in the gonad, but relatively few such sex-biased genes in the brain. Functional annotation and enrichment analysis suggested that ovaries are mainly enriched for metabolic processes and testes for signal transduction, particularly receptors of neurotransmitters and steroid hormones. When compared to other species, many genes previously implicated in male sex determination and differentiation pathways showed conservation in their gonadal expression patterns in bluehead wrasses. However, some critical female-pathway genes (e.g., rspo1 and wnt4b) exhibited unanticipated expression patterns. In the brain, gene expression patterns suggest that local neurosteroid production and signaling likely contribute to the sex differences observed. CONCLUSIONS: Expression patterns of key sex-related genes suggest that sex-changing fish predominantly use an evolutionarily conserved genetic toolkit, but that subtle variability in the standard sex-determination regulatory network likely contributes to sexual plasticity in these fish. This study not only provides the first molecular data on a system ideally suited to explore the molecular basis of sexual plasticity and tissue re-engineering, but also sheds some light on the evolution of diverse sex determination and differentiation systems.