Daphnia uses its circadian clock for short-day recognition in environmental sex determination.

Some organisms have developed a mechanism called environmental sex determination (ESD), which allows environmental cues, rather than sex chromosomes or genes, to determine offspring sex.1,2,3,4 ESD is advantageous to optimize sex ratios according to environmental conditions, enhancing reproductive success.5,6 However, the process by which organisms perceive and translate diverse environmental signals into offspring sex remains unclear. Here, we analyzed the environmental perception mechanism in the crustacean, Daphnia pulex, a seasonal (photoperiodic) ESD arthropod, capable of producing females under long days and males under short days.7,8,9,10 Through breeding experiments, we found that their circadian clock likely contributes to perception of day length. To explore this further, we created a genetically modified daphnid by knocking out the clock gene, period, using genome editing. Knockout disrupted the daphnid's ability to sustain diel vertical migration (DVM) under constant darkness, driven by the circadian clock, and leading them to produce females regardless of day length. Additionally, when exposed to an analog of juvenile hormone (JH), an endocrine factor synthesized in mothers during male production, or subjected to unfavorable conditions of high density and low food availability, these knockout daphnids produced males regardless of day length, like wild-type daphnids. Based on these findings, we propose that recognizing short days via the circadian clock is the initial step in sex determination. This recognition subsequently triggers male production by signaling the endocrine system, specifically via the JH signal. Establishment of a connection between these two processes may be the crucial element in evolution of ESD in Daphnia.

Effects of polycaprolactone degradation products on the water flea, Daphnia magna: Carbodiimide additives have acute and chronic toxicity.

Plastics have benefited our lives in many ways, but their long persistence in the environment causes serious problems. Rapid decomposition and detoxification of plastics after use are significant challenges. As a possible solution, biodegradable plastics have attracted attention, and for environmental risk assessment research on polymer toxicity, use of indicator organisms, like water fleas and fish, has increased globally. However, such research often focuses on standardized substances without considering changes in toxicity due to plastic degradation products. Additionally, tests generally focus on acute toxicity, while long-term effects on organismal reproduction and lifespan are largely unknown. Understanding the impact of degraded polymers on biological activities is crucial for accurate risk assessment. In this study, we investigated the biological toxicity of substances generated during degradation of polycaprolactone (PCL), a common biodegradable plastic, using the indicator organism, Daphnia magna. We examined PCL, oligocaprolactones (OCLs), and monomers resulting from polymer cleavage, as well as carbodiimides, added during polyester synthesis. As a result, PCL, which is insoluble in water, reduced individual survival and total number of offspring at an exposure concentration of 100 mg/L, while no toxicity was observed for water-soluble degradation products, OCLs, and monomers. Furthermore, carbodiimides, which are expected to be released during PCL degradation, showed strong toxicity, significantly reducing individual survival and total number of offspring at 0.1-10 mg/L. These findings suggest that changes in physical properties due to polymer degradation and release of additives can significantly alter their toxicity.

Transformer gene regulates feminization under two complementary sex determination loci in the ant, Vollenhovia emeryi.

Organisms that reproduce sexually have evolved well-organized mechanisms to determine two sexes. Some hymenopterans (such as ants, bees, and wasps) have a complementary sex-determination system in which heterozygosity at one CSD locus induces female development, whereas hemi- or homozygosity at the locus induces male development. This system can generate a high cost of inbreeding, as individuals that are homozygous at the locus become sterile, diploid males. On the other hand, some hymenopterans have evolved a multi-locus, complementary, sex-determination system in which heterozygosity in at least one CSD locus induces female development. This system effectively reduces the proportion of sterile diploid males; however, how these multipleprimary signals based on CSD pass through a molecular cascade to regulate downstream genes has remained unclear. To clarify this matter, we used a backcross to investigate the molecular cascade in the ant, Vollenhovia emeryi, with two CSD loci. Here we show by gene disruption that transformer (tra) is necessary for proper feminization. Expression analysis of tra and doublesex (dsx) showed that heterozygosity in at least one of the two CSD loci is sufficient to promote female sex determination. Analysis of overexpression suggested that female-type Tra protein promotes splicing of tra pre-mRNA to female isoform by a positive-regulatory-feedback loop. Our data also showed that tra affects splicing of dsx. We conclude that two-loci sex determination system in V. emeryi evolved based on tra-dsx splicing cascade that is well conserved in other insect species. Finally, we suggest a cascade model to arrive at a binary determination of sex under multiple primary signals.

Effect of Oligomers Derived from Biodegradable Polyesters on Eco- and Neurotoxicity.

Biodegradable polymers are eco-friendly materials and have attracted attention for use in a sustainable society because they are not accumulated in the environment. Although the characteristics of biodegradable polymers have been assessed well, the effects of their degradation products have not. Herein, we comprehensively evaluated the chemical toxicities of biodegradable polyester, polycaprolactone (PCL), and synthetic oligocaprolactones (OCLs) with different degrees of polymerization. While the PCL did not show any adverse effects on various organisms, high levels of shorter OCLs and the monomer (1 µg/mL for freshwater microorganisms and 1 mg/mL for marine algae and mammalian cells) damaged the tested organisms, including freshwater microorganisms, marine algae, and mammalian cells, which indicated the toxicities of the degradation products under unnaturally high concentrations. These results highlight the need for a further understanding of the effects of the degradation products resulting from biodegradable polyesters to ensure a genuinely sustainable society.

Live-cell imaging of the chloroplast outer envelope membrane using fluorescent dyes.

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.

Developmental Staging of Sexual Egg Formation in Daphnia pulex: Unmated Females Resorb Meiotic Oocytes to Resist Starvation.

Under favorable conditions, daphnids produce only female neonates by parthenogenesis, while they produce male neonates and start sexual reproduction when they detect cues signaling a deteriorating environment. Identifying the regulatory mechanisms of such cyclical parthenogenesis is important for understanding how organisms adapt to environments and expand their habitats. However, most previous studies using the model species Daphnia magna and Daphnia pulex have focused on production of male offspring (sex determination), whereas the process of meiosis induction in females has not been investigated. Here, we report a simple experimental method to induce meiosis effectively in D. pulex females. Through observations using the new method, we describe the process of sexual reproduction along an individual developmental time course. Meiotic oocytes are oviposited only when females mate within a certain time window, and failure to mate within that window results in subsequent resorption of oocytes, a measure that may increase resistance to starvation. These results further our understanding of regulatory mechanisms and evolutionary processes in the complicated life-history of Daphnia.

CmLec4, a lectin from the fungus Cordyceps militaris, controls host infection and fruiting body formation.

Fungi belonging to the Ascomycete genus Cordyceps are endoparasitoids and parasites, mainly of insects and other arthropods. Cordyceps militaris has been used as a therapeutic drug for cancer patients. However, the infection, parasitism, and fruiting body formation mechanisms of this fungus are still unknown. Based on our hypothesis that lectin(s) is involved in the interaction between the C. militaris fungi and insects, we partially purified and characterized a new lectin from C. militaris, designated CmLec4. In addition, we searched for substance(s) in the infected silkworm extracts that could bind to CmLec4, and succeeded in purifying the sex-specific storage protein 2 as a specific binding target. To examine function of the binding protein during the process of parasitism, we investigated the effect of recombinant CmLec4 on silkworms by inoculating the protein into silkworm pupae, and found that it significantly delayed emergence compared to the control. Furthermore, cmlec4 gene knockout strains constructed in this study produced markedly lower amounts of fruiting body than the wild-type strain. All the results revealed that the lectin CmLec4 produced by C. militaris would be involved in the infection into silkworm and fruiting body formation from the host.

Juvenile hormone synthesis and signaling disruption triggering male offspring induction and population decline in cladocerans (water flea): Review and adverse outcome pathway development.

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

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.

Two insulin-like peptides may regulate egg production in opposite directions via juvenile hormone signaling in the queenless ant Pristomyrmex punctatus.

In the major eusocial species of Hymenoptera, the regulatory mechanisms controlling queen/worker differentiation and exclusive reproduction by queens have been studied extensively. These studies have shown that insulin/insulin-like growth factors and juvenile hormones (JHs) act as key endocrine factors. However, although considerable knowledge has accumulated in this area, large disparities in the regulatory mechanisms governing caste differentiation have been observed in different hymenopteran taxa to date. We focused on the queenless ant Pristomyrmex punctatus (Hymenoptera: Formicidae), which exhibits the simplest type of sociality and in which reproductive tasks (egg production) are distributed among morphologically and genetically identical workers. To elucidate the molecular mechanisms underlying reproduction in P. punctatus, we analyzed the correlations between the gene expression profiles of a reproductive marker gene, vitellogenin (PripuVTG1), and candidate regulatory genes comprising the major components of the JH and insulin/insulin-like growth factor signaling pathways that are involved in the regulation of reproduction upstream of JH signaling. Expression of insulin-like peptide 1 (PripuILP1) and JH signaling-related genes was negatively correlated with PripuVTG1 expression. On the contrary, insulin-like peptide 2 (PripuILP2a) was positively correlated with PripuVTG1. These findings suggest that an equilibrium perhaps controlled by switches in JH signaling exists between these two ILP paralogs, and that these interactions are important for regulating reproduction. Our findings are expected to be useful for understanding how various modes of sociality have evolved in insects.

Establishment of a high-sensitivity reporter system in mammalian cells for detecting juvenoids using juvenile hormone receptors of Daphnia pulex.

Environmental waters are polluted by various chemicals originating from human activities. Recently, the environmental risk of juvenile hormones (JHs) to aquatic microcrustaceans has been recognized by risk assessors and researchers. JH is a major arthropod hormone that regulates molting and reproduction and has analogs that have been used as insect growth regulators. JHs are known to disturb the sex determination system of Daphnia, which is a keystone animal in limnetic ecosystems and is not the target of extermination. To assess the risk of contaminant chemicals and to protect biodiversity, reliable methods for detecting such chemicals are essential. In this study, we attempted to establish a practical in vitro reporter assay system for detecting chemicals with JH activity. Using a newly constructed reporter vector (modified from the JH response element of Tribolium castaneum Krüppel homolog 1, which is a major JH responsive gene in insects), strong JH-dependent transcriptional activity (>40-fold activation) was found in Chinese hamster ovary cells that express JH receptors of Daphnia pulex. Dose-response analysis conducted on several JH and non-JH chemicals revealed that the established reporter assay system has strict specificity to JH chemicals, and the half maximum effective concentration (EC50 ) was between 10-7 and 10-9  m. These results suggest that the new system is a rapid and economical method for assessing the environmental risk of JH-active chemicals.

A self-marker-like protein governs hemocyte allorecognition in Halocynthia roretzi.

BACKGROUND: Self-incompatibility, fusion/non-fusion reactions, and contact reactions (CRs) have all been identified as allorecognition phenomena in ascidians. CR is a reaction characteristic of the hemocytes of Halocynthia roretzi, whereby they release phenol oxidase (PO) upon contact with non-self hemocytes. Thus, these cells may represent a primitive form of the vertebrate immune system. In the present study, we focused on the CR of H. roretzi hemocytes and sought to identify self-marker proteins that distinguish between self and non-self cells. RESULTS: We initially generated a CR-inducing monoclonal antibody against the complete hemocyte membrane-protein complement (mAb11B16B10). This antibody was identified based on the differential induction of PO activity in individual organisms. The level of PO activity induced by this antibody in individual ascidians was consistent with the observed CR-induced PO activity. mAb11B16B10 recognized a series of 12 spots corresponding to a 100-kDa protein, with differing isoelectric points (pIs). A comparison of the 2D electrophoresis gels of samples from CR-reactive/non-reactive individuals revealed that some spots in this series in hemocytes were common to the CR-non-inducible individuals, but not to CR-inducible individuals. We cloned the corresponding gene and named it Halocynthia roretzi self-marker-like protein-1 (HrSMLP1). This gene is similar to the glycoprotein DD3-3 found in Dictyostelium, and is conserved in invertebrates. CONCLUSION: We generated a CR-inducing monoclonal antibody (mAb11B16B10) that recognized a series of novel membrane proteins (HrSMLP1) in the hemocytes of H. roretzi. The combination of expressed spots of HrSMLP1 distinguishes non-self cells from self cells with respect to CR inducibility. Given that the HrSMLP1 gene is a single gene, it may represent a novel type of self-marker protein with a role in CR.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi.

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

The doublesex gene integrates multi-locus complementary sex determination signals in the Japanese ant, Vollenhovia emeryi.

A female diploid, male haploid sex determination system (haplodiploidy) is found in hymenopteran taxa, such as ants, wasps, bees and sawflies. In this system, a single, complementary sex-determination (sl-CSD) locus functions as the primary sex-determination signal. In the taxa that has evolved this system, females and males are heterozygous and hemi/homozygous at the CSD locus, respectively. While the sl-CSD system enables females to alter sex ratios in the nest, it carries a high cost in terms of inbreeding, as individuals that are homozygous at the CSD locus become sterile diploid males. To counter this risk, some of hymenopteran species have evolved a multi-locus CSD (ml-CSD) system, which effectively reduces the proportion of sterile males. However, the mechanism by which these multiple primary signals are integrated and how they affect the terminal sex-differentiation signal of the molecular cascade have not yet been clarified. To resolve these questions, we examined the molecular cascade in the Japanese ant Vollenhovia emeryi, which we previously confirmed has two CSD loci. Here, we showed that the sex-determination gene, doublesex (dsx), which is highly conserved among phylogenetically distant taxa, is responsible for integrating two CSD signals in V. emeryi. After identifying and characterizing dsx, genotypes containing two CSD loci and splicing patterns of dsx were found to correspond to the sexual phenotype, suggesting that two primary signals are integrated into dsx. These findings will facilitate future molecular and functional studies of the sex determination cascade in V. emeryi, and shed light on the evolution and diversification of sex determination systems in insects.

Juvenile hormone-independent function of Krüppel homolog 1 in early development of water flea Daphnia pulex.

Elaborate regulation of insect metamorphosis is the consequence of physiological cooperation among multiple endocrine factors such as juvenile hormones (JHs) and ecdysteroids. Hormone-induced transcription factors play important roles in substantive interactions between hormonal signaling pathways. In insects, zinc finger transcription factor Krüppel homolog 1 (Kr-h1) is a key gene of the endocrine signaling pathway in which it is directly upregulated by JH receptor Methoprene-tolerant (Met) in the presence of JH and then regulates multiple downstream factors, including components of the ecdysteroid signaling pathway. Although JH also plays a role in various biological phenomena in other arthropod species, little is known about the molecular basis of the JH signaling pathway. Here we cloned Kr-h1 from a branchiopod crustacean, Daphnia pulex, (DappuKr-h1) and analyzed its expression profile and developmental function together with consideration of its relationship to the JH signaling pathway. We suggest that DappuKr-h1 lacks JH responsiveness and regulatory relationship with the JH receptor. Moreover our loss-of-function analysis revealed that maternal mRNA of DappuKr-h1 plays a critical role in early development independent from the JH signaling pathway. These findings provide insights about whether and how the JH signaling pathway influenced evolution, leading to greater diversity in phylum Arthropoda.

Ecdysteroid and juvenile hormone biosynthesis, receptors and their signaling in the freshwater microcrustacean Daphnia.

The two essential insect hormones, ecdysteroids and juvenile hormones, are possessed not only by insects, but also widely by arthropods, and regulate various developmental and physiological processes. In contrast to the abundant information about molecular endocrine mechanisms in insects, the knowledge of non-insect arthropod endocrinology is still limited. In this review, we summarize recent reports about the molecular basis of these two major insect hormones in the freshwater microcrustacean Daphnia, a keystone taxon in limnetic ecology and a bioindicator in environmental studies. Comprehensive comparisons of endocrine signaling pathways between insects and daphnids may shed light on the regulatory mechanisms of various biological phenomena and, moreover, evolutionary processes of arthropod species.

Comparative luciferase assay for establishing reliable in vitro screening system of juvenile hormone agonists.

The cultured cell-based in vitro assay using the stringency of ligand-receptor interactions is typically useful for screening certain hormone agonists from among a very large number of molecules. However, ligands are frequently altered or modified through evolution; indeed, even in the same receptor orthologs, different ligand sensitivity profiles are considered to arise among species and/or taxa. Such ligand transition has been observed in juvenile hormone (JH), one of the most important endocrine factors in arthropods. To understand the molecular basis of ligand selectivity alteration in hormone receptors, we compared the amino acid sequences and ligand selectivity of the JH receptor, Methoprene-tolerant (Met), among three insects (Drosophila melanogaster, Aedes aegypti and Tribolium castaneum) and one crustacean (Daphnia pulex). Compared with D. pulex, we found that the receptors of the three insects showed a higher sensitivity to JH III, which is the major innate JH ligand in insects. Furthermore, point mutation analysis in Met sequences revealed a candidate amino acid residue that is important for increasing JH sensitivity in insects. Amino acid mutations in Met may have affected changes in ligand selectivity intermittently over the course of the evolution of the JH-signaling pathway. These findings are useful to improve the existing (developing) cultured cell-based assay system and may shed light on the relationship between functional diversification in hormonal signaling and the molecular evolution of hormone receptors. Copyright © 2017 John Wiley & Sons, Ltd.

The function of appendage patterning genes in mandible development of the sexually dimorphic stag beetle.

One of the defining features of the evolutionary success of insects is the morphological diversification of their appendages, especially mouthparts. Although most insects share a common mouthpart ground plan, there is remarkable diversity in the relative size and shapes of these appendages among different insect lineages. One of the most prominent examples of mouthpart modification can be found in the enlargement of mandibles in stag beetles (Coleoptera, Insecta). In order to understand the proximate mechanisms of mouthpart modification, we investigated the function of appendage-patterning genes in mandibular enlargement during extreme growth of the sexually dimorphic mandibles of the stag beetle Cyclommatus metallifer. Based on knowledge from Drosophila and Tribolium studies, we focused on seven appendage patterning genes (Distal-less (Dll), aristaless (al), dachshund (dac), homothorax (hth), Epidermal growth factor receptor (Egfr), escargot (esg), and Keren (Krn). In order to characterize the developmental function of these genes, we performed functional analyses by using RNA interference (RNAi). Importantly, we found that RNAi knockdown of dac resulted in a significant mandible size reduction in males but not in female mandibles. In addition to reducing the size of mandibles, dac knockdown also resulted in a loss of the serrate teeth structures on the mandibles of males and females. We found that al and hth play a significant role during morphogenesis of the large male-specific inner mandibular tooth. On the other hand, knockdown of the distal selector gene Dll did not affect mandible development, supporting the hypothesis that mandibles likely do not contain the distal-most region of the ancestral appendage and therefore co-option of Dll expression is unlikely to be involved in mandible enlargement in stag beetles. In addition to mandible development, we explored possible roles of these genes in controlling the divergent antennal morphology of Coleoptera.

Neverland regulates embryonic moltings through the regulation of ecdysteroid synthesis in the water flea Daphnia magna, and may thus act as a target for chemical disruption of molting.

Embryo development in arthropods is accompanied by a series of moltings. A cladoceran crustacean Daphnia magna molts three times before reaching first instar neonate during embryogenesis. Previous studies argued ecdysteroids might regulate D. magna embryogenesis. However, no direct evidence between innate ecdysteroids fluctuation and functions has been forthcoming. Recently, we identified genes involved in ecdysteroid synthesis called, neverland (neverland1 and neverland 2) and shade and in the ecdysteroid degradation (Cyp18a1). To understand the physiological roles of ecdysteroids in D. magna embryos, we performed expression and functional analyzes of those genes. Examining innate ecdysteroids titer during embryogenesis showed two surges of ecdysteroids titer at 41 and 61 h after oviposition. The first and second embryonic moltings occurred at each ecdysteroid surge. Expression of neverland1 and shade began to increase before the first peak in ecdysteroid. Knockdown of neverland1 or shade by RNAi technique caused defects in embryonic moltings and subsequent development. The ecdysteroids titer seemingly decreased in nvd1-knowckdown embryos. Knockdown of Cyp18a1 resulted in early embryonic lethality before the first molting. Our in situ hybridization analysis revealed that nvd1 was prominently expressed in embryonic gut epithelium suggesting the site for an initial step of ecdysteroidgenesis, a conversion of cholesterol to 7-dehydrocholesterol and possibly for ecdysone production. Taken together, de novo ecdysteroid synthesis by nvd1 in the gut epithelial cells stimulates molting, which is indispensable for D. magna embryo development. These findings identify neverland as a possible target for chemicals, including various pesticides that are known to disrupt molting, development and reproduction. Copyright © 2016 John Wiley & Sons, Ltd.

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation.

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.