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Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka ( Oryzias latipes ) mutants deficient in teleost-specific androgen receptor ohnologs ( ara and arb ). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage. How has the genome duplication impacted the diversification of sexual characteristics in the teleost lineage? This study shows that androgen receptor ohnologs in medaka appear to have diverged in their roles for regulating morphological and behavioural sexual characteristics after loss from an ancestral role in spermatogenesis.

Background The American alligator ( Alligator mississippiensis ) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD. Results Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B , and transcription factor C/EBPA . Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX . Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9 , and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted. Conclusions Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

Juvenile hormone is an essential regulator of major developmental and life history events in arthropods. Most of the insects use juvenile hormone III as the innate juvenile hormone ligand. By contrast, crustaceans use methyl farnesoate. Despite this difference that is tied to their deep evolutionary divergence, the process of this ligand transition is unknown. Here we show that a single amino-acid substitution in the receptor Methoprene-tolerant has an important role during evolution of the arthropod juvenile hormone pathway. Microcrustacea Daphnia pulex and D. magna share a juvenile hormone signal transduction pathway with insects, involving Methoprene-tolerant and steroid receptor coactivator proteins that form a heterodimer in response to various juvenoids. Juvenile hormone-binding pockets of the orthologous genes differ by only two amino acids, yet a single substitution within Daphnia Met enhances the receptor’s responsiveness to juvenile hormone III. These results indicate that this mutation within an ancestral insect lineage contributed to the evolution of a juvenile hormone III receptor system. Juvenile hormone (JH) is a key regulator of development both in insects and the crustacea Daphnia pulex and D. magna. Here, Miyakawa et al. investigate the evolutionary significance of a single amino-acid variation between crustacea and insects in the JH receptor gene, Methoprene-tolerant.

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na + reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se . We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

Temperature-dependent sex determination (TSD), commonly found among reptiles, is a sex determination mode in which the incubation temperature during a critical temperature sensitive period (TSP) determines sexual fate of the individual rather than the individual’s genotypic background. In the American alligator ( Alligator mississippiensis ), eggs incubated during the TSP at 33 °C (male producing temperature: MPT) yields male offspring, whereas incubation temperatures below 30 °C (female producing temperature: FPT) lead to female offspring. However, many of the details of the underlying molecular mechanism remains elusive and the molecular link between environmental temperature and sex determination pathway is yet to be elucidated. Here we show the alligator TRPV4 ortholog (AmTRPV4) to be activated at temperatures proximate to the TSD-related temperature in alligators and using pharmacological exposure, we show that AmTRPV4 channel activity affects gene expression patterns associated with male differentiation. This is the first experimental demonstration of a link between a well-described thermo-sensory mechanism, TRPV4 channel and its potential role in regulation of TSD in vertebrates, shedding unique new light on the elusive TSD molecular mechanism.

Background The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex . Results To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N -methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids. Conclusions We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex . This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.

Steroid hormone receptor family provides an example of evolution of diverse transcription factors through whole-genome duplication (WGD). However, little is known about how their functions have been evolved after the duplication. Teleosts present a good model to investigate an accurate evolutionary history of protein function after WGD, because a teleost-specific WGD (TSGD) resulted in a variety of duplicated genes in modern fishes. This study focused on the evolution of androgen receptor (AR) gene, as two distinct paralogs, ARα and ARβ, have evolved in teleost lineage after TSGD. ARα showed a unique intracellular localization with a higher transactivation response than that of ARβ. Using site-directed mutagenesis and computational prediction of protein–ligand interactions, we identified two key substitutions generating a new functionality of euteleost ARα. The substitution in the hinge region contributes to the unique intracellular localization of ARα. The substitution on helices 10/11 in the ligand-binding domain possibly modulates hydrogen bonds that stabilize the receptor–ligand complex leading to the higher transactivation response of ARα. These substitutions were conserved in Acanthomorpha (spiny-rayed fish) ARαs, but not in an earlier branching lineage among teleosts, Japanese eel. Insertion of these substitutions into ARs from Japanese eel recapitulates the evolutionary novelty of euteleost ARα. These findings together indicate that the substitutions generating a new functionality of teleost ARα were fixed in teleost genome after the divergence of the Elopomorpha lineage. Our findings provide a molecular explanation for an adaptation process leading to generation of the hyperactive AR subtype after TSGD.

Background The gene doublesex ( dsx ) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes ( DapmaDsx1 and DapmaDsx2 ) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females . D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. Results We identified homologs of both dsx genes from, D. pulex , D. galeata , and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa . The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5’ upstream regulatory elements are preserved in D . magna and D . pulex . Conclusions The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.

Sexual development and reproduction are largely linked to epigenetic changes in many fish species. However, understanding of epigenetic regulation in scombrid species, such as tunas and mackerels, is limited. This study investigates DNA methylation’s impact on cyp19a expression, crucial for estrogen synthesis, focusing on chub mackerel. Given the commercial significance of scombrids and susceptibility of marine fish to environmental changes, elucidating epigenetic mechanisms, particularly in the context of global warming, becomes imperative. We aimed to generalize observations from chub mackerel to other scombrids. Additionally, we studied DNA methylation patterns across fish with different sexual systems to understand aromatase regulation’s phenotypic plasticity. Our in silico analysis revealed highly conserved promoter sequences within scombrids, sharing TFBS like Foxl2, FOS::JUN, ESRR, and Sox3, while CpG content varies. This indicates a conserved regulatory network controlling gene expression. We found sexual dimorphism in DNA methylation, with males hypermethylated and aromatase expression downregulated. Despite similar dnmt1 expression, tet1 , tet2 , and tet3 were higher expressed in females, suggesting that the observed DNA methylation patterns are maintained through active demethylation rather than differential methylation. Gonochoristic Japanese anchovy and protogynous bamboo leaf wrasse displayed similar trends, but species-specific methylation patterns highlight DNA methylation’s complex role in gonadal changes. In vitro assays confirmed methylation’s regulatory role and identified an SF-1 binding site relevant for promoter activation in chub mackerel. Another studied SF-1 site, present in both chub mackerel and bamboo leaf wrasse, showed regulatory effects, indicating potential similar regulatory mechanisms for cyp19a expression. Overall, our findings suggest that while global methylation affects cyp19a transcription, the variation in CpG density and location could be introducing nuances in its epigenetic regulation. This study contributes to our understanding of the cyp19a regulation in fish gonad maturation.

Background The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna , these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments. Results We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene ( Dll ), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene. Conclusions We developed a microinjection system for RNAi studies in D. pulex . These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.