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Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Sexual dimorphisms in trait expression are widespread among animals and are especially pronounced in ornaments and weapons of sexual selection, which can attain exaggerated sizes. Expression of exaggerated traits is usually male-specific and nutrition sensitive. Consequently, the developmental mechanisms generating sexually dimorphic growth and nutrition-dependent phenotypic plasticity are each likely to regulate the expression of extreme structures. Yet we know little about how either of these mechanisms work, much less how they might interact with each other. We investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle Cyclommatus metallifer, focusing on doublesex gene function and its interaction with juvenile hormone (JH) signaling. doublesex genes encode transcription factors that orchestrate male and female specific trait development, and JH acts as a mediator between nutrition and mandible growth. We found that the Cmdsx gene regulates sex differentiation in the stag beetle. Knockdown of Cmdsx by RNA-interference in both males and females produced intersex phenotypes, indicating a role for Cmdsx in sex-specific trait growth. By combining knockdown of Cmdsx with JH treatment, we showed that female-specific splice variants of Cmdsx contribute to the insensitivity of female mandibles to JH: knockdown of Cmdsx reversed this pattern, so that mandibles in knockdown females were stimulated to grow by JH treatment. In contrast, mandibles in knockdown males retained some sensitivity to JH, though mandibles in these individuals did not attain the full sizes of wild type males. We suggest that moderate JH sensitivity of mandibular cells may be the default developmental state for both sexes, with sex-specific Dsx protein decreasing sensitivity in females, and increasing it in males. This study is the first to demonstrate a causal link between the sex determination and JH signaling pathways, which clearly interact to determine the developmental fates and final sizes of nutrition-dependent secondary-sexual characters.

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.

Chloroplasts are organelles composed of sub‐organellar compartments—stroma, thylakoids, and starch granules—and are surrounded by outer and inner envelope membranes (OEM and IEM, respectively). The chloroplast OEM and IEM play key roles not only as a barrier separating the chloroplast components from the cytosol but also in the interchange of numerous metabolites and proteins between the chloroplast interior and the cytosol. Fluorescent protein markers for the chloroplast OEM have been widely used to visualize the outermost border of chloroplasts. However, the use of marker proteins requires an established cellular genetic transformation method, which limits the plant species in which marker proteins can be used. Moreover, the high accumulation of OEM marker proteins often elicits abnormal morphological phenotypes of the OEM. Because the OEM can currently only be visualized using exogenous marker proteins, the behaviors of the chloroplast and/or its OEM remain unknown in wild‐type cells of various plant species. Here, we visualized the OEM using live‐cell staining with the fluorescent dyes rhodamine B and Nile red in several plant species, including crops. We propose rhodamine B and Nile red as new tools for visualizing the chloroplast OEM in living plant cells that do not require genetic transformation. Significance Statement We established a live‐cell imaging method to visualize the chloroplast outer envelope membrane by staining living cells with fluorescent dyes. This method does not require genetic transformation and allows the observation of the chloroplast outer envelope membrane in various plant species.

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.

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.

Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, \"neckteeth,\" in response to chemical cues or signals, referred to as \"kairomones,\" in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms underlying the recognition of environmental changes, which form the basis of phenotypic plasticity.

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.

Most hymenopteran species exhibit conspicuous sexual dimorphism due to ecological differences between the sexes. As hymenopteran genomes, under the haplodiploid genetic system, exhibit quantitative differences between sexes while remaining qualitatively identical, sexual phenotypes are assumed to be expressed through sex-specific gene usage. In the present study, the molecular basis for expression of sexual dimorphism in a queenless ant, Diacamma sp., which exhibits a distinct color dimorphism, was examined. Worker females of the species appear bluish-black, while winged males exhibit a yellowish-brown body color. Initially, observations of the pigmentation processes during pupal development revealed that black pigmentation was present in female pupae but not in males, suggesting that sex-specific melanin synthesis was responsible for the observed color dimorphism. Therefore, five orthologs of the genes involved in the insect melanin synthesis (yellow, ebony, tan, pale and dopa decarboxylase) were subcloned and their spatiotemporal expression patterns were examined using real-time quantitative RT-PCR. Of the genes examined, yellow, which plays a role in black melanin synthesis in insects, was expressed at higher levels in females than in males throughout the entire body during the pupal stage. RNA interference of yellow was then carried out in order to determine the gene function, and produced females with a more yellowish, brighter body color similar to that of males. It was concluded that transcriptional regulation of yellow was responsible for the sexual color dimorphism observed in this species.

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.