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International audience; Species that have sex determined by environmental conditions during development (i.e., environmental sex determination [ESD]) are especially vulnerable to environmental change, including altered stress levels, habitat loss, and species translocations. These factors can produce multigenerational trends in population size and eco-evolutionary dynamics not captured by existing theory based on lifetime reproductive success (R0). Here, we extend ESD theory to use per capita growth rate r as a more appropriate measure of evolutionary success (fitness), and we demonstrate the importance of this change when males and females can differ in maturation times and when maturation times vary with local conditions (plasticity). In these cases, we show that primary and secondary sex ratios may be strongly biased; that optimal maturation times, when locally plastic, depend on the balance between mortality and growth effects; and that plasticity of maturation times can ameliorate fitness costs of increasing environmental stress

The view that has genotypic sex determination and environmental sex determination as mutually exclusive states in fishes and reptiles has been contradicted by the discovery that chromosomal sex and environmental influences can co-exist within the same species, hinting at a continuum of intermediate states. Systems where genes and the environment interact to determine sex present the opportunity for sex reversal to occur, where the phenotypic sex is the opposite of that predicted by their sex chromosome complement. The skink Bassiana duperreyi has XX/XY sex chromosomes with sex reversal of the XX genotype to a male phenotype, in laboratory experiments, and in field nests, in response to exposure to cold incubation temperatures. Here we studied the frequency of sex reversal in adult populations of B. duperreyi in response to climatic variation, using elevation as a surrogate for environmental temperatures. We demonstrate sex reversal in the wild for the first time in adults of a reptile species with XX/XY sex determination. The highest frequency of sex reversal occurred at the highest coolest elevation location, Mt Ginini (18.46%) and decreased in frequency to zero with decreasing elevation. We model the impact of this under Fisher’s frequency-dependent selection to show that, at the highest elevations, populations risk the loss of the Y chromosome and a transition to temperature-dependent sex determination. This study contributes to our understanding of the risks of extinction from climate change in species subject to sex reversal by temperature, and will provide focus for future research to test on-the-ground management strategies to mitigate the effects of climate in local populations.

Transitions from environmental sex determination (ESD) to genotypic sex determination (GSD) require an intermediate step of sex reversal, i.e., the production of individuals with a mismatch between the ancestral genotypic and the phenotypic sex. Among amniotes, the sole well-documented transition in this direction was shown in the laboratory in the central bearded dragon, Pogona vitticeps, where very high incubation temperatures led to the production of females with the male-typical (ZZ) genotype. These sex-reversed females then produced offspring whose sex depended on the incubation temperature. Sex-reversed animals identified by molecular and cytogenetic markers were also reported in the field, and their increasing incidence was speculated as a climate warming-driven transition in sex determination. We show that the molecular and cytogenetic markers normally sex-linked in P. vitticeps are also sex-linked in P. henrylawsoni and P. minor, which points to quite ancient sex chromosomes in this lineage. Nevertheless, we demonstrate, based on a crossing experiment with a male bearded dragon who possesses a mismatch between phenotypic sex and genotype, that the used cytogenetic and molecular markers might not be reliable for the identification of sex reversal. Sex reversal should not be considered as the only mechanism causing a mismatch between genetic sex-linked markers and phenotypic sex, which can emerge also by other processes, here most likely by a rare recombination between regions of sex chromosomes which are normally sex-linked. We warn that sex-linked, even apparently for a long evolutionary time, and sex-specific molecular and cytogenetic markers are not a reliable tool for the identification of sex-reversed individuals in a population and that sex reversal has to be verified by other approaches, particularly by observation of the sex ratio of the progeny.

For species with environmental sex determination, understanding the relationship between reproduction and environmental factors is important for predicting their reproductive output. Here, we study intra-annual variation in green turtle nesting during the 2010–2016 seasons at Penang Island (5°16′28″–5°28′15″N 100°10′52″–100°11′55″E), Malaysia. The additive modelling on a monthly-basis number of nests shows that fluctuation in the number of nests relates to temperature in addition to month of year, rather than precipitation. The number of nests tended to be higher in response to higher temperature during March–July, whereas the lower temperature during August–February also tended to result in a relatively higher number of nests. Concentration of nests during March–July resulted from a shorter inter-nesting interval during warm temperatures, whereas relatively low temperatures may homogenize the temporal distribution of the number of nests. This study provides fundamental information for green turtle nesting seasonality in response to environmental change.

Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

Sex reversal at high temperatures during embryonic development (e.g., ZZ females) provides the opportunity for new genotypic crosses (e.g., ZZ male × ZZ female). This raises the alarming possibility that climatic warming could lead to the loss of an entire chromosome-one member of the sex chromosome pair (the Y or W)-and the transition of populations to environmental sex determination (ESD). Here we examine the evolutionary dynamics of sex-determining systems exposed to climatic warming using theoretical models. We found that the loss of sex chromosomes is not an inevitable consequence of sex reversal. A large frequency of ZZ sex reversal (50% reversal from male to female) typically divides the outcome between loss of the ZW genotype and the stable persistence of ZZ males, ZW females and ZZ females. The amount of warming associated with sex chromosome loss depended on several features of wild populations-environmental fluctuation, immigration, heritable variation in temperature sensitivity and differential fecundity of sex-reversed individuals. Chromosome loss was partially or completely buffered when sex-reversed individuals suffered a reproductive fitness cost, when immigration occurred or when heritable variation for temperature sensitivity existed. Thus, under certain circumstances, sex chromosomes may persist cryptically in systems where the environment is the predominant influence on sex.

With or without sex chromosomes, sex determination is a synthesis of many molecular events that drives a community of cells towards a coordinated tissue fate. In this review, we will consider how a sex determination pathway can be engaged and stabilized without an inherited genetic determinant. In many reptilian species, no sex chromosomes have been identified, yet a conserved network of gene expression is initiated. Recent studies propose that epigenetic regulation mediates the effects of temperature on these genes through dynamic post-transcriptional, post-translational and metabolic pathways. It is likely that there is no singular regulator of sex determination, but rather an accumulation of molecular events that shift the scales towards one fate over another until a threshold is reached sufficient to maintain and stabilize one pathway and repress the alternative pathway. Investigations into the mechanism underlying sex determination without sex chromosomes should focus on cellular processes that are frequently activated by multiple stimuli or can synthesize multiple inputs and drive a coordinated response. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

Amniotes possess variability in sex determination, ranging from environmental sex determination to genotypic sex determination with differentiated sex chromosomes. Differentiated sex chromosomes have emerged independently several times. Their noteworthy convergent characteristic is the evolutionary stability, documented among amniotes in mammals, birds, and some lineages of lizards, snakes and turtles. Combining the analysis of multiple partial transcriptomes with the comparison of copy gene numbers between male and female genomes, we uncovered partial gene content of the highly differentiated ZZ/ZW sex chromosomes in the gecko genus Paroedura. The differentiated ZZ/ZW sex chromosomes of these geckos share genes with the part of the chicken chromosome 4 homologous with the XX/XY sex chromosomes of viviparous mammals and the ZZ/ZW sex chromosomes of lacertid lizards, as well as with the chicken chromosome 15, homologous with the XX/XY sex chromosomes of iguanas and ZZ/ZW sex chromosomes of softshell turtles. Along with other analogous cases, this finding reinforces the observation that particular chromosomes are repeatedly coopted for the function of sex chromosomes in amniotes. Notably, according to the phylogenetic distribution, the subclade of the genus Paroedura represents a rare case of the reversal of the for a considerable evolutionary time highly differentiated ZZ/ZW sex chromosomes back to poorly differentiated state.

Background and Aims The ability of individuals to change sex during their lifetime is known as environmental sex determination (ESD). This represents a unique life history trait, allowing plants to allocate resources differentially to male and female functions across lifetimes, potentially maximizing fitness in response to changing environmental or internal cues. In this study, Acer pensylvanicum, a species with an unconfirmed sex determination system, was investigated to see what patterns in sex expression existed across multiple years, if there were sex-based differences in growth and mortality, and whether this species conformed to theoretical predictions that females are larger and in better condition. Methods Patterns of sex expression were documented over 4 years in a phenotypically subdioecious A. pensylvanicum population located in New Jersey, USA, and data on size, mortality, health and growth were collected. A machine-learning algorithm known as a boosted classification tree was used to develop a model to predict the sex of a tree based on its condition, size and previous sex. Results In this study, 54 % of the trees switched sex expression during a 4-year period, with 26 % of those trees switching sex at least twice. Consistently monoecious trees could change relative sex expression by as much as 95 %. Both size and condition were influential in predicting sex, with condition exerting three times more relative influence than size on expressed sex. Contrary to theoretical predictions, the model showed that full female sex expression did not increase with size. Healthy trees were more likely to be male; predicted female sex expression increased with deteriorating health. Growth rate negatively correlated with multiple years of female sex expression. Populations maintained similar male-skewed sex ratios across years and locations and may result from differential mortality: 75 % of dead trees flowered female immediately before death. Conclusions This study shows conclusively that A. pensylvanicum exhibits ESD and that femaleness correlates with decreased health, in contrast to prevailing theory. The mortality findings advance our understanding of puzzling non-equilibrium sex ratios and life history trade-offs resulting from male and female sex expression.

Fisherian sex-ratio theory predicts sexual species should have a balanced primary sex ratio. However, organisms with environmental sex determination (ESD) are particularly vulnerable to experiencing skewed sex ratios when environmental conditions vary. Theoretical work has modeled sex-ratio dynamics for animals with ESD with regard to 2 traits predicted to be responsive to sex-ratio selection: 1) maternal oviposition behavior and 2) sensitivity of embryonic sex determination to environmental conditions, and much research has since focused on how these traits influence offspring sex ratios. However, relatively few studies have provided estimates of univariate quantitative genetic parameters for these 2 traits, and the existence of phenotypic or genetic covariances among these traits has not been assessed. Here, we leverage studies on 3 species of reptiles (2 turtle species and a lizard) with temperature-dependent sex determination (TSD) to assess phenotypic covariances between measures of maternal oviposition behavior and thermal sensitivity of the sex-determining pathway. These studies quantified maternal behaviors that relate to nest temperature and sex ratio of offspring incubated under controlled conditions. A positive covariance between these traits would enhance the efficiency of sex-ratio selection when primary sex ratio is unbalanced. However, we detected no such covariance between measures of these categories of traits in the 3 study species. These results suggest that maternal oviposition behavior and thermal sensitivity of sex determination in embryos might evolve independently. Such information is critical to understand how animals with TSD will respond to rapidly changing environments that induce sex-ratio selection.

The evolution of sex determination mechanisms and sex chromosomes has always been a central research topic in evolutionary biology. Because the sex determination process occurs during the early stage of development, and the regulation of sex-linked genes often involves non-coding RNAs and epigenetic modifications, this topic is an interdisciplinary hotspot with developmental biology and molecular biology. This review elaborates on the importance and origin of sex, numerous ways of sex determination, and the mechanisms of sex chromosome evolution. We will summarize the reported sex-determining genes and introduce the population genetic models under which the sex chromosomes evolve without homologous recombination. To date, only a few sex-determining genes have been discovered, but they have already exhibited a great diversity beyond biologists’ expectation. Future research directions will focus on identifying more upstream sex-determining genes for plants and animals and their downstream sex-determining pathways. New genome research and gene knockout techniques will develop other appropriate model organisms in this area, and to ultimately address the basic biological questions that why different organisms need to evolve such a great variety of sex-determining ways and how they transit to each other.

The Indian garden lizard, Calotes versicolor, lacks cytologically recognizable sex chromosomes, and its mechanism of sex determination is unclear. We evaluated genotype-to-sex-phenotype association using RAD-seq in wild-caught males and females, 30 of each sex. Of 210,736 unique, 96-nt long RAD-tags, 48% contained polymorphisms, 23% of which were present in at least 40 of 60 individuals. Twenty one RAD-tags neared, but none achieved, the inclusion criteria for sex enrichment, as expected if C. versicolor lacks highly differentiated sex chromosomes. Three RAD-tags with alleles most strongly associated with sex tended to be heterozygous in females and to lack male-specific alleles, suggesting a ZW female/ZZ male system. Putative female alleles, however, were present in some males and lacking from some females, suggesting either recombination between these markers and the sex locus or sex reversal due to environmental or genetic factors. Paired-end, 250-nt reads from 1 male provided a fragmented draft genome assembly. Four sex-associated RAD-tags were identical to portions of 4 unique C. versicolor genomic contigs rather than linked to a single putative sex-linked region. The lack of strongly sex-linked loci coupled with weak evidence for temperature-associated sex determination intensifies the need for further investigation of the puzzling sex determination mechanism in C. versicolor.

Parker, Baker, and Smith provided the first robust theory explaining why anisogamy evolves in parallel in multicellular organisms. Anisogamy sets the stage for the emergence of separate sexes, and for another phenomenon with which Parker is associated: sperm competition. In outcrossing taxa with separate sexes, Fisher proposed that the sex ratio will tend towards unity in large, randomly mating populations due to a fitness advantage that accrues in individuals of the rarer sex. This creates a vast excess of sperm over that required to fertilize all available eggs, and intense competition as a result. However, small, inbred populations can experience selection for skewed sex ratios. This is widely appreciated in haplodiploid organisms, in which females can control the sex ratio behaviorally. In this review, we discuss recent research in nematodes that has characterized the mechanisms underlying highly skewed sex ratios in fully diploid systems. These include self-fertile hermaphroditism and the adaptive elimination of sperm competition factors, facultative parthenogenesis, non-Mendelian meiotic oddities involving the sex chromosomes, and environmental sex determination. By connecting sex ratio evolution and sperm biology in surprising ways, these phenomena link two “seminal” contributions of G. A. Parker.

Sex ratio theory predicts both mean sex ratio and variance under a range of population structures. Here, we compare two genera of phoretic nematodes (Parasitodiplogaster and Ficophagus spp.) associated with twelve fig-pollinating wasp species in Panama. The host wasps exhibit classic Local Mate Competition: only inseminated females disperse from natal figs, and their offspring form mating pools that consist of scores of the adult offspring contributed by one or a few foundress mothers. In contrast, in both nematode genera, only sexually undifferentiated juveniles disperse, and their mating pools routinely consist of eight or fewer adults. Across all mating pool sizes, the sex ratios observed in both nematode genera are consistently female-biased (~0.34 males), which is markedly less female-biased than is often observed in the host wasps (~0.10 males). In further contrast with their hosts, variances in nematode sex ratios are also consistently precise (significantly less than binomial). The constraints associated with predictably small mating pools within highly subdivided populations appear to select for precise sex ratios that contribute both to the reproductive success of individual nematodes, and to the evolutionary persistence of nematode species. We suggest that some form of environmental sex determination underlies these precise sex ratios.

WOS:000618603800001; Sex allocation research has primarily focused on offspring sex-ratio adjustment by mothers. Yet, fathers also benefit from producing more of the sex with greater fitness returns. Here, we review the state-of-the art in the study of male-driven sex allocation and, counter to the current paradigm, we propose that males can adaptively influence offspring sex ratio through a wide variety of mechanisms. This includes differential production and motility of X- versus Y-bearing sperms in mammals, variation in seminal fluid composition in haplo-diploid invertebrates, and epigenetic mechanisms in some fish and lizards exhibiting environmental sex determination. Conflicts of interest between mothers and fathers over offspring sex ratios can emerge, although many more studies are needed in this area. While many studies of sex allocation have focused on adaptive explanations with little attention to mechanisms, and vice versa, the integration of these two topics is essential for understanding male-driven sex allocation.

Studying environmental sex determination (ESD) in cichlids provides a phylogenetic and comparative approach to understand the evolution of the underlying mechanisms, their impact on the evolution of the overlying systems, and the neuroethology of life history strategies. Natural selection normally favors parents who invest equally in the development of male and female offspring, but evolution may favor deviations from this 50:50 ratio when environmental conditions produce an advantage for doing so. Many species of cichlids demonstrate environmental sex determination in response to water chemistry (temperature, pH, and oxygen concentration). The relative strengths of, and the exact interactions between, these factors varies between congeners, demonstrating genetic variation in sensitivity. The presence of sizable proportions of the less common sex towards the environmental extremes in most species strongly suggests the presence of some genetic sex determining loci acting in parallel with the ESD factors. Sex determination and differentiation in these species does not seem to result in the organization of a final and irreversible sexual fate, so much as a life-long on-going battle between competing male determining and female determining genetic and hormonal networks governed by epigenetic factors. We discuss what is, and is not, known about the epigenetic mechanism behind the differentiation of both gonads and sex differences in the brain. Beyond the well studied tilapia species, the two best-studied dwarf cichlid systems showing environmental sex determination are the South American genus Apistogramma, and the West African genus Pelvicachromis. Both these species demonstrate male morphs with alternative reproductive tactics. We discuss the further neuroethology opportunities such systems provide to the study of epigenetics of alternative life history strategies and other behavioral variation.

Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.

In this review, we consider the insight that has been gained through theoretical examination of environmental sex determination (ESD) and thermolability – how theory has progressed our understanding of the ecological and evolutionary dynamics associated with ESD, the transitional pathways between different modes of sex determination, and the underlying mechanisms. Following decades of theory on the adaptive benefits of ESD, several hypotheses seem promising. These hypotheses focus on the importance of differential fitness (sex-specific effects of temperature on fitness) in generating selection for ESD, but highlight alternative ways differential fitness arises: seasonal impacts on growth, sex-specific ages of maturation, and sex-biased dispersal. ESD has the potential to generate biased sex ratios quite easily, leading to complex feedbacks between the ecology and evolution of ESD. Frequency-dependent selection on sex acts on ESD-related traits, driving local adaptation or plasticity to restore equilibrium sex ratio. However, migration and overlapping generations (“mixing”) diminish local adaptation and leave each cohort/population with the potential for biased sex ratios. Incorporating mechanism into ecology and evolution models reveals similarities between different sex-determining systems. Dosage and gene regulatory network models of sexual development are beginning to shed light on how temperature sensitivity and thresholds may arise. The unavoidable temperature sensitivity in sex-determining systems inherent to these models suggests that evolutionary transitions between genotypic sex determination (GSD) and temperature-dependent sex determination, and between different forms of GSD, are simple and elegant. Theoretical models are often best-served by considering a single piece of a puzzle; however, there is much to gain from reflecting on all of the pieces together in one integrative picture.

Squamate reptiles show high diversity in sex determination ranging from environmental sex determination to genotypic sex determination with varying degrees of differentiation of sex chromosomes. Unfortunately, we lack even basic information on sex determination mode in several lineages of squamates, which prevents full understanding of their diversity and evolution of sex determination. One of the reptilian lineages with missing information on sex determination is the family Gerrhosauridae, commonly known as the plated lizards. Several species of gerrhosaurids have been studied in the past by conventional cytogenetic methods, but sex-specific differences were not identified. In this study, we applied both conventional and molecular cytogenetic methods to metaphases from both sexes of the Peters’ keeled plated lizard (Tracheloptychus petersi). We identified accumulations of rDNA loci in a pair of microchromosomes in metaphases from males, but only in a single microchromosome in females. The restriction of the observed heterozygosity to females suggests a putative ZZ/ZW system of sex chromosomes, which represents the first report of sex chromosomes in a gerrhosaurid lizard. The lack of sex-specific signals in all other cytogenetic methods implies that the sex chromosomes of T. petersi are poorly differentiated in sequence content.

Environmental sex determination (ESD) is common among ectothermic vertebrates. The stress axis and production of stress hormones (corticosteroids) regulates ESD in fish, but evidence of a similar influence in reptiles is sparse and conflicting. The central bearded dragon (Pogona vitticeps) has a system of sex determination involving the interplay between sex chromosomes (ZZ/ZW female heterogamety) and the thermal environment. High egg incubation temperatures induce sex reversal of the ZZ genotype, feminizing chromosomally male individuals. Here we show that corticosterone elevation is not associated with sex reversal in the central bearded dragon, either during embryonic development or adulthood. We also demonstrate experimentally that sex determination is not affected by corticosterone injection into the yolk. This strongly suggests that stress axis upregulation by high temperature during incubation does not cause sex reversal in P. vitticeps. Our work is in general agreement with other research in reptiles, which suggests that the stress axis does not mediate sex in reptiles with ESD. Alternative biological systems may be responsible for capturing environmental conditions during reptile development, such as cellular calcium and redox regulation or the action of temperature-sensitive splicing factors.

The adaptive significance of temperature-dependent sex determination (TSD) in reptiles remains unknown decades after TSD was first identified in this group. Concurrently, there is growing concern about the effect that rising temperatures may have on species with TSD, potentially producing extremely biased sex ratios or offspring of only one sex. The current state-of the-art in TSD research on sea turtles is reviewed here and, against current paradigm, it is proposed that TSD provides an advantage under warming climates. By means of coadaptation between early survival and sex ratios, sea turtles are able to maintain populations. When offspring survival declines at high temperatures, the sex that increases future fecundity (females) is produced, increasing resilience to climate warming. TSD could have helped reptiles to survive mass extinctions in the past via this model. Flaws in research on sex determination in sea turtles are also identified and it is suggested that the development of new techniques will revolutionize the field.

Noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) have been extensively studied in biological processes such as disease development, cell proliferation but remained unclear in sex differentiation in organisms. In this study, the transcriptome profiles were comparatively analyzed between male and female gonads in Mauremys mutica. A total of 8237 differentially expressed genes (DEGs), 9573 DE lncRNAs, 84 DE circRNAs and 665 DE miRNAs were identified between male and female gonads. Through gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of the DE RNAs, it was revealed that the DE mRNAs were majorly involved in GO terms, such as 'reproduction', 'reproductive process' and the pathways of 'focal adhesion' and 'oocyte meiosis'. In addition, a co-expression network showed that the expression of gametogenesis and sex differentiation related genes, including dmrt3a, tdrd7, sox14, etc were closely associated with the levels of their corresponding ncRNAs. Intriguingly, the dmrt1 circRNA and its target mRNA were detected upregulated both in adult testis and male producing temperature (MPT) embryos. Our findings demonstrated the sexually dimorphic expression profiles of mRNAs and ncRNAs in turtle gonads, which will provide the index to find out the molecular mechanisms behind the sex differentiation in turtles, even in other environmental sex determination (ESD) species.

A fit animal must develop testes or ovaries, with brain and physiology to match. In species with alternative male morphs this coordination of development across tissues operates within sexes as well as between. For Pelvicachromis pulcher, an African cichlid in which early pH exposure influences both sex and alternative male morph, we sequence both copies of aromatase (cyp19a1), a key gene for sex determination. We analyze gene expression and epigenetic state, comparing gonad and brain tissue from females, alternative male morphs, and fry. Relative to brain, we find elevated expression of the A-copy in the ovaries but not testes. Methylation analysis suggests strong epigenetic regulation, with one region specifying sex and another specifying tissue. We find elevated brain expression of the B-copy with no sex or male morph differences. B-copy methylation follows that of the A-copy rather than corresponding to B-copy expression. In 30-day old fry, we see elevated B-copy expression in the head, but we do not see the expected elevated A-copy expression in the trunk that would reflect ovarian development. Interestingly, the A-copy epialleles that distinguish ovaries from testes are among the most explanatory patterns for variation among fry, suggesting epigenetic marking of sex prior to differentiation and thus laying the groundwork for mechanistic studies of epigenetic regulation of sex and morph differentiation.

Of the 356 species of turtles worldwide, approximately 61% are threatened or already extinct. Turtles are among the most threatened of the major groups of vertebrates, in general, more so than birds, mammals, fishes or even the much besieged amphibians. Reasons for the dire situation of turtles worldwide include the familiar list of impacts to other species including habitat destruction, unsustainable overexploitation for pets and food, and climate change (many turtles have environmental sex determination). Two notable characteristics of pre-Anthropocene turtles were their massive population sizes and correspondingly high biomasses, the latter among the highest values (over 855 kilograms per hectare) ever reported for animals. As a result of their numerical dominance, turtles have played important roles as significant bioturbators of soils, infaunal miners of sea floors, dispersers and germination enhancers of seeds, nutrient cyclers, and consumers. The collapse of turtle populations on a global scale has greatly diminished their ecological roles.

The mangrove rivulus fish (Kryptolebias marmoratus) is an emerging model for research in the biological sciences. The species is androdioecious, with populations consisting predominantly of self-fertilizing hermaphrodites and a low abundance of males, but no females. Males arise either through environmental sex determination at the embryonic stage or environmentally driven sex change from hermaphrodite at the adult stage. Marked sexual dimorphisms in external morphological characters accompany gonadal transitions, including the loss of a caudal ocellus (‘eyespot’) and the development of orange color in males. How well external morphological features predict internal gonadal morphology, however, has not been systematically examined across the geographical range of this species. Our goal was to identify reliable external morphological predictors of animals possessing an ovotestis (hermaphrodite) or a testis (male) and to see if the strength of prediction is consistent throughout rivulus’ geographical range. We demonstrate that the state of the ocellus and, to a greater extent, orange coloration are key predictors of internal gonadal state. However, the degree to which these characteristics are predictive of internal gonadal state was region-specific, with the ocellus and orange status of animals from Bahamas and Belize, respectively, having a lower probability of forecasting internal gonadal state than those from other regions. These results will help to streamline laboratory studies by allowing unambiguous assignment of sex, and will assist field studies by providing greater opportunity to determine sex ratios in wild populations.

In sexually plastic Acer pensylvanicum, determination of sex can occur extremely late, within three weeks of spring flowering. Physical damage causing complete vascular tissue severance results in increased female expression. Species with environmental sex determination are rare amongst angiosperms but widely distributed across taxa. The timing of floral development in species that change sex based on environmental cues is unexplored. We investigated the timing of differentiation of sexual organs in buds of Acer pensylvanicum, an understory tree in eastern North America with environmental sex determination. We collected branches from individuals at three collection times in the early spring of 2016 and kept them in a warm greenhouse until anthesis. All individuals exhibited complete or partial female inflorescences in the greenhouse in one or more collection. However, none of these same individuals produced only female flowers in the field. Unlike many other woody species that differentiate bud sexual primordia 9–12 months prior to flowering, A. pensylvanicum may differentiate the sexual organs in its flower buds as late as three weeks prior to anthesis. In a separate series of branch collections in 2017, we found that the stress response to cutting leads to increased female sex expression in branches, while earlier warm temperatures (e.g., those caused by growing in a protected greenhouse environment) or increased carbohydrate availability does not. Given the labile sex determination system of A. pensylvanicum, the ability to delay differentiation of buds into male or female until shortly before spring flowering would allow individual trees to respond to sex-determining damage cues as late as mid-spring. This supports the hypothesis that A. pensylvanicum may not exhibit the lag-time characteristic of temperate spring and early-summer flowering woody species and may change sex expression in response to stress.

The sex of an organism can be determined by its genetics or its early environment. Across the animal kingdom, genetic sex determination (GSD) is far more common than environmental sex determination (ESD). Here, we propose an explanation for this pattern: the coupling of genes that bias offspring sex ratios towards one sex with genes that are beneficial in that sex but costly in the other. Gradual strengthening of the sex-specific tendency of this association eventuates in a neo-sex chromosome; that is, GSD. Our model predicts to which system of heterogamety ESD will evolve when nesting behaviour is an important determinant of brood sex ratios. It explains the puzzling observation in some GSD species of sex reversal induced by extreme environments. The model also suggests an approach to discovering sex-determining genes in ESD species.

In recent years, scientists have begun to pay serious attention to the hypothesis that human parental coital rates around the time of conception causally influences the sexes of subsequent births. In this paper, the grounds of the argument are outlined. The point is important because, if the hypothesis were credible, it can potentially explain one of the best established (and otherwise unexplained) epidemiological features of sex ratio at birth - its rises during and just after World Wars 1 and 2 insofar as increased coital rates increase the ratio. Moreover, the greater the understanding of the variations of sex ratio at birth, the greater will be the understanding of the causes of those selected diseases associated with unusual sex ratios at birth (testicular cancer, hepatitis B, Toxoplasma gondii, and, perhaps, prostatic cancer).

The phenology of egg production and oviposition in organisms affects survival and development of neonates and thus, both offspring and maternal fitness. In addition, in organisms with environmental sex determination, clutch phenology can affect hatchling sex ratios with attendant effects on population demography. The rapid rate of contemporary climate change might disrupt reproductive phenologies that evolved to match environmental conditions. To better understand the response of clutch phenology to annual and long-term changes in climate, we studied a population of Sonoran Desert Tortoises (Gopherus morafkai) in Arizona in 1993 and 1997–2005, specifically quantifying three phenophases, including (1) the estimated time of appearance of shelled eggs in females, (2) the estimated time that eggs were last visible in X-radiographs, and (3) the duration of the interval between the first two events. The mean date for appearance of shelled eggs was 6 June, and the mean date they were last visible was 26...

In amniotes (mammals, reptiles and birds) we can find two major sex-determining modes: environmental sex determination (ESD), largely in the form of temperature-dependent sex determination, and genotypic sex determination (GSD). It is evident that in reptiles there were multiple transitions between these two modes, but their directions are often difficult to reconstruct. However, it seems that GSD to ESD transitions were much less frequent. Such a transition demands production of sex-reversed individuals, i.e. individuals with a mismatch between gonadal and genotypic sex, which have to be viable and fertile in spite of lacking sex-specific parts of genomes linked to Y or W chromosomes. Up to now, there is only a single well-documented transition from the ancestral GSD to ESD among amniotes: a laboratory experiment with the bearded dragon, Pogona vitticeps. The gonadal females with male-typical ZZ genotype induced by high incubation temperatures in this species possessed masculinized morphology and behaviour. However, it is not clear whether these masculinized phenotypic traits can be attributed to the lack of female-specific W chromosome, or to extreme incubation temperatures. In this contribution, we describe the results of the experiments aiming to develop a system allowing disentangling the effects of temperature and genotype by hormonal manipulations, specifically by the application of fadrozole and exogenous estradiol (E2) to developing eggs in a captive bred population of the bearded dragon. We documented that both high incubation temperature and E2 application at normal incubation temperature can induce development of ZZ sex-reversed females in this laboratory population and that these females are able to lay eggs. On the other hand, the application of fadrozole led either to high hatchling mortality or to development of individuals with enlarged hemipenes, but did not induce sex reversal. The developed system will be useful in future studies to fully utilize the very valuable bearded dragon model for general understanding of potential limits and constraints of GSD to ESD transitions.

Zebrafish (Danio rerio) is extensively used in research; however the mechanisms that control this species sex determination are still poorly understood. In the latest decades, it has been established that zebrafish sex is determined by genetic factors on a polygenic basis, as various candidate genes with sex dimorphic expression, as well sex-linked loci have been identified in different zebrafish strains. However, it has been evidenced that sex determination in this species is also influenced by environmental factors. For instance, temperature can have a crucial role in zebrafish sex determination. Likewise, the exposure to endocrine disrupting chemicals (EDCs), the most studied zebrafish sex changing factor, can strongly influence the course of sex differentiation and unbalance the sex ratio of zebrafish populations. Despite this, so far the influence of environmental factors is still less understood and only few studies have addressed this topic. Therefore, this review intends to gather current knowledge on the environmental factors involved in sex determination of zebrafish and identify important gaps in this research area. Briefly, the current understanding on zebrafish sex related genetics is also addressed.

A master sex-determining gene, the Y chromosome-linked anti-Müllerian hormone (amhy) gene, has been described in two New World atheriniform species but little is known on the distribution, evolution, and function(s) of this gene in other Atheriniformes. Interestingly, amhy has been found to coexist with temperature-dependent sex determination (TSD), providing a unique opportunity to explore the interplay between genotypic and environmental sex determination. In this study, the search for an amhy homolog was extended to an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae (Atherinidae). The full sequences, including the coding and noncoding regions, of the autosomal amh (amha) and a putative amhy were obtained. The deduced Amha and Amhy proteins comprised 511 and 340 amino acids (aa), respectively. PCR analysis with genomic DNA from wild adults and from laboratory-reared juveniles revealed a high, but not complete association of ∼95% between amhy and maleness. The spatiotemporal expression of amhy and amha during gonadal sex differentiation was analyzed by qRT-PCR and in situ hybridization (ISH). amhy transcription (in amhy-positive larvae) started before and peaked during histological differentiation of the gonads whereas amha was negligible during the same period in both genotypes. These results demonstrate that the amhy, although with some structural differences in relation to the amhy of some New World atheriniforms, is strongly associated with maleness and probably important for testicular development in this Old World atheriniform. Thus, amhy is a candidate sex determination gene in cobaltcap silverside and it will be key to scrutinize the mechanism of sex determination in this species.

This review is the first assembling information on intersexuality in aquatic invertebrates, from freshwater to estuarine and marine environments. Intersex is a condition whereby an individual of a gonochorist (separate sexes) species has oocytes or distinct stages of spermatogonia, at varying degrees of development, within the normal gonad of the opposite gender (i.e. spermatocytes in the ovary or oocytes in the testis), often involving alterations in the gonadal structure, reproductive tract or external genitalia. By the end of 2016 we found approximately 340 records of aquatic invertebrate species evidencing signs of intersexuality (or imposex), all comprised within the Phyla Mollusca and Arthropoda. Gastropod molluscs are by far the group with more examples documented (256 species), followed by crustaceans, i.e., decapods, copepods and amphipods. To our knowledge no further cases of intersexuality were known concerning other invertebrate taxa. Despite some reports suggesting that a baseline level of intersexuality may occur naturally in some populations, the causes are multifaceted and mostly linked with environmental contamination by estrogenic and organotin endocrine disrupting chemicals (EDCs), parasitism, and genetic/environmental sex determination abnormalities. A more comprehensive discussion about the origin of intersexuality, prevalence and causes, knowledge gaps and future research directions in the light of new omics scientific advances (genomics, proteomics and transcriptomics) is also provided. The lack of studies linking molecular responses of invertebrate intersex individuals to multiple stressors represents a true challenge to be further investigated in the future.

The study of temperature-dependent sex determination (TSD) in vertebrates has attracted major scientific interest. Recently, concerns for species with TSD in a warming world have increased because imbalanced sex ratios could potentially threaten population viability. In contrast, relatively little attention has been given to the direct effects of increased temperatures on successful embryonic development. Using 6603 days of sand temperature data recorded across 6 years at a globally important loggerhead sea turtle rookery-the Cape Verde Islands-we show the effects of warming incubation temperatures on the survival of hatchlings in nests. Incorporating published data (n = 110 data points for three species across 12 sites globally), we show the generality of relationships between hatchling mortality and incubation temperature and hence the broad applicability of our findings to sea turtles in general. We use a mechanistic approach supplemented by empirical data to consider the linked effects of warming temperatures on hatchling output and on sex ratios for these species that exhibit TSD. Our results show that higher temperatures increase the natural growth rate of the population as more females are produced. As a result, we project that numbers of nests at this globally important site will increase by approximately 30% by the year 2100. However, as incubation temperatures near lethal levels, the natural growth rate of the population decreases and the long-term survival of this turtle population is threatened. Our results highlight concerns for species with TSD in a warming world and underline the need for research to extend from a focus on temperature-dependent sex determination to a focus on temperature-linked hatchling mortalities.

An eulimid gastropod, Megadenus atrae n. sp., endoparasitic in the cloacal chamber of the black sea cucumber Holothuria atra Jaeger is described from Okinawa, Japan, as the fifth species of the genus. Conspecific specimens have also been found from southeast India, northeast Australia and New Caledonia. The generic assignment is justified by the presence of (i) a thick, long proboscis that bears a large fold (pseudopallium) near the base and a collar-like structure at the middle, (ii) a thin, globose shell that is covered by the pseudopallium, and (iii) sexual dimorphism with the female generally larger than the male. The new species is distinguishable from the four previously described congeners by its cauldron-shaped pseudopallium, moderately-developed collar of the proboscis and rounded basal lip of the shell. The comparisons of the size and sex of solitary and paired individuals support a previous hypothesis that the species of Megadenus Rosén, 1910 are protandrous with environmental sex determination. The present species occurs mostly as monogamous pairs despite its very low population density, implying that the presence of a conspecific individual acts as a cue for larval settlement. Both mechanisms would increase individual reproductive success in such permanent parasites with low prevalence and abundance as the species of Megadenus.

Environmental sex determination occurs in many organisms, however the means by which environmental stimuli are translated into endocrine messages remains poorly understood. The N-methyl-ᴅ-aspartate receptor (NMDAR) was evaluated as a candidate neural sensor of environmental signals linking environmental cues to endocrine responses using the crustacean Daphnia pulex. NMDAR agonists, modulators, and antagonists were evaluated for their ability to impact D. pulex male sex determination during early stages of reproductive maturity under conditions that simulated seasonal change. The antagonists MK-801 and desipramine significantly increased male sex determination. Both chemicals are also modulators of serotonergic and noradrenergic systems, thus, we evaluated several modulators of monoamine neurotransmission in an effort to discern which signaling pathways might contribute to male sex determination. Compounds that altered serotonergic signaling also stimulated male sex determination. The involvement of the glutamate and monoamine signaling in male sex determination was supported by the increase in mRNA levels of related receptors and transporters under conditions that stimulate male sex determination. Further, mRNA levels of components of the terminal endocrine pathway responsible for male sex determination were also elevated under stimulatory conditions. Overall, we provide evidence that glutamatergic and serotonergic systems function upstream of the endocrine regulation of male sex determination in early life stage daphnids.

13 pages, 4 figures, 2 tables, Epigenetics integrates genomic and environmental information to produce a given phenotype. Here, the model of Conserved Epigenetic Regulation of Sex (CERS) is discussed. This model is based on our knowledge on genes involved in sexual development and on epigenetic regulation of gene expression activation and silencing. This model was recently postulated to be applied to the sexual development of fish, and it states that epigenetic and gene expression patterns are more associated with the development of a particular gonadal phenotype, e.g., testis differentiation, rather than with the intrinsic or extrinsic causes that lead to the development of this phenotype. This requires the existence of genes with different epigenetic modifications, for example, changes in DNA methylation levels associated with the development of a particular sex. Focusing on DNA methylation, the identification of CpGs, the methylation of which is linked to sex, constitutes the basis for the identification of Essential Epigenetic Marks (EEM). EEMs are defined as the number and identity of informative epigenetic marks that are strictly necessary, albeit perhaps not sufficient, to bring about a specific, measurable, phenotype of interest. Here, we provide a summary of the genes where DNA methylation has been investigated so far, focusing on fish. We found that cyp19a1a and dmrt1, two key genes for ovary and testis development, respectively, consistently show an inverse relationship between their DNA methylation and expression levels, thus following CERS predictions. However, in foxl2a, a pro-female gene, and amh, a pro-male gene, such relationship is not clear. The available data of other genes related to sexual development such as sox9, gsdf, and amhr2 are also discussed. Next, we discuss the use of CERS to make testable predictions of how sex is epigenetically regulated and to better understand sexual development, as well as the use of EEMs as tools for the diagnosis and prognosis of sex. We argue that CERS can aid in focusing research on the epigenetic regulation of sexual development not only in fish but also in vertebrates in general, particularly in reptiles with temperature sex-determination, and can be the basis for possible practical applications including sex control in aquaculture and also in conservation biology, This study was supported by the Spanish Ministry of Science grants AGL2016–787107-R “Epimark” to FP and AGL2015-73864-JIN “Ambisex” to LR. DA was supported by an Epimark contract, AV and NS-B were supported by Spanish government scholarships (BES-2014-069051 and BES-2017-079744, respectively); LR and JM-P were supported by Ambisex contracts

Many reptiles and some fish determine offspring sex by environmental cues such as incubation temperature. The mechanism by which environmental signals are captured and transduced into specific sexual phenotypes has remained unexplained for over 50 years. Indeed, environmental sex determination (ESD) has been viewed as an intractable problem because sex determination is influenced by a myriad of genes that may be subject to environmental influence. Recent demonstrations of ancient, conserved epigenetic processes in the regulatory response to environmental cues suggest that the mechanisms of ESD have a previously unsuspected level of commonality, but the proximal sensor of temperature that ultimately gives rise to one sexual phenotype or the other remains unidentified. Here, we propose that in ESD species, environmental cues are sensed by the cell through highly conserved ancestral elements of calcium and redox (CaRe) status, then transduced to activate ubiquitous signal transduction pathways, or influence epigenetic processes, ultimately to drive the differential expression of sex genes. The early evolutionary origins of CaRe regulation, and its essential role in eukaryotic cell function, gives CaRe a propensity to be independently recruited for diverse roles as a 'cellular sensor' of environmental conditions. Our synthesis provides the first cohesive mechanistic model connecting environmental signals and sex determination pathways in vertebrates, providing direction and a framework for developing targeted experimentation.

All species of sea turtles exhibit a cooler male/warmer female temperature-sex ratio response. Field and experimental studies on loggerhead sea turtle sex ratios suggest that increased sand moisture impacts sea turtle sex ratios with, and perhaps beyond, a cooling effect. This study examines how varying sand moisture impacts the embryo’s response to temperature. Across three years, loggerhead (Caretta caretta) sea turtle eggs were incubated at temperatures ranging from 28.0 °C to 33.0 °C. Groups of eggs were incubated in one of three volumetric moisture contents: low, medium, or high. Temperatures inside the group of eggs were recorded throughout incubation. Hatchlings were raised for 2–3 months and sex was identified laparoscopically. We calculated temperature response curves for groups of eggs incubated at each moisture level. Pivotal temperatures did not different among eggs incubated in different sand moistures. The transitional ranges of temperatures (TRT) for eggs incubated in high moisture and low moisture were narrower than the TRT for eggs incubated in medium moisture. The results of this study are crucial for understanding how sea turtle embryos respond to temperature directly or indirectly under different moisture conditions. Current sex ratio predictions rely on the embryos response to temperature only and may inaccurately estimate sex ratios especially during periods of heavy rainfall or drought.

Premise Environmental sex determination (ESD) is a rare sex determination system in which individuals may switch sex expression throughout their lifetimes in response to environmental factors. In sexually stable species, individuals usually bear more female flowers if the plants are larger, have greater access to limiting resources, or are in better condition. Research regarding sexually plastic species with ESD and how resources correlate with sex expression is limited. Furthermore, most research investigates resources at the population level, failing to account for resources available to individuals for growth, maintenance, or reproduction. Methods Acer pensylvanicum is a species that is known to switch sex. Using twig samples collected during 2014-2016 in December and May, we analyzed resource status in the form of stored nonstructural carbohydrates (NSCs) and compared this with expressed sex. Results We found that females had higher sugar concentrations than males. Furthermore, males changing expression to female had higher sugar concentrations during the prior winter than did males remaining male. We found that size was not a key predictor: neither male nor female-flowering individuals increased NSC concentrations with size. Dying female trees had high concentrations of NSCs throughout the dying process and only manifested reduced NSCs once dead. Conclusions This is the first study showing significant correlations between NSCs and sex expression in a plant species with ESD. These findings support the hypothesis that sex switching could be a consequence of increased resource availability and that the high female mortality of A. pensylvanicum populations is likely not a direct result of carbon starvation.

Premise Plant sex is usually fixed, but in rare cases, sex expression is flexible and may be influenced by environmental factors. Theory links female sex expression to better health, but manipulative work involving the experimental change of health via injury is limited, particularly in sexually plastic species. A better understanding of mechanisms influencing shifts in sex is essential to our understanding of life history theory regarding trade-offs in sex allocation and differential mortality. Methods We investigated the relationship between physiological stress and sex expression in sexually plastic striped maple trees (Acer pensylvanicum) by inflicting damage of various intensities (crown pruning, defoliation, and hydraulic restriction). We then monitored the sex expression of injured and control individuals for 2 years to assess the extent to which injury may cue changes in sex expression. Results We found that severe damage such as full defoliation or severe pruning increased odds of changing sex to female and decreased odds of changing to male. In fact, no pruned male trees flowered male 2 years later, while all males in the control group flowered partially or fully male. After full defoliation, trees had 4.5 times higher odds of flowering female. Not all injury is equal; less-severe physical trauma did not affect the frequency of sex change to femaleness. Conclusions This work demonstrates that physical trauma in striped maple appears to exhibit a threshold effect in which only the most stressful of physiological cues instigate changes in sex expression, a phenomenon previously unknown, and that damage stress is strongly correlated with switching to femaleness. These findings have implications for population sex ratios and sustainability within an increasing stressful climate regime.

Over the last several decades zebrafish (Danio rerio) has become a major model organism for the study of vertebrate development and physiology. Given this, it may be surprising how little is known about the mechanism that zebrafish use to determine sex. While zebrafish are a gonochoristic species (having two sexes) that do not switch sex as adults, it was appreciated early on that sex ratios obtained from breeding lab domesticated lines were not typically a 1:1 ratio of male and female, suggesting that sex was not determined by a strict chromosomal mechanism. Here we will review the recent progress toward defining the genetic mechanism for sex determination in both wild and domesticated zebrafish.

The pejerrey is an atherinopsid species from South America that presents a combination of genotypic and environmental (temperature-dependent) sex determination whereby low and high temperatures induce feminization and masculinization, respectively. Masculinization involves a heat-induced stress response leading to increased circulating cortisol and androgens. We tested whether crowding would elicit a similar response as high temperature and affect the sex ratios of pejerrey. Larvae with XX and XY genotypes were reared at 15, 62 and 250 larvae/L in 0.4, 1.6, and 6.4 L containers during a period considered critical for sex determination at 25 °C, a mixed-sex promoting temperature. Fish were analysed at 3–7 weeks for whole-body cortisol and 11-ketotestosterone (11-KT) titer and hydroxy-steroid dehydrogenase (hsd11b2) mRNA transcript abundance, and after completion of gonadal sex differentiation (10–14 weeks) for determination of phenotypic and genotypic sex mismatches. Crowding was associated with depressed growth, higher cortisol and 11-KT titers, increased hsd11b2 transcription, and increased frequency of masculinization compared to intermediate and/or low rearing densities. Perceived crowding (by rearing in containers with mirror-finish, reflecting walls) also caused masculinization. These results suggest the possibility that other environmental factors besides temperature can also affect sex determination in pejerrey and that a stress response leading to increased cortisol and androgen levels, which is potentially perceived by the brain, may be a common feature among different forms of environmental sex determination in this species.

Sex determination is the first step toward the establishment of phenotypic sex in most vertebrates. Aquatic poikilotherms such as teleost fishes exhibit a high diversity of sex-determination mechanisms and gonadal phenotypes that are remarkably plastic and responsive to a variety of environmental factors (e.g., water temperature, pH, salinity, photoperiod, population density). This chapter reviews current knowledge of genotypic and environmental sex determination systems in fishes with special reference to Atheriniformes-one of the best-characterized taxa in this field-and offers perspectives to guide and stimulate further research.

Understanding gonadal development in lampreys is complicated by their complex life cycle, the long period during which their gonads remain histologically undifferentiated, and their lack of any close living relatives. This chapter synthesizes the available information related to lamprey sex determination, sex differentiation, sexual maturation, and sex steroids, and it identifies key research needs. A detailed review of lamprey sex ratios shows that: (1) adult lampreys (i.e., during the upstream migration or at spawning) exhibit a small but consistent excess of males in virtually all species studied (with significantly female-biased sex ratios noted only in sea lamprey in the three upper Great Lakes following initiation of sea lamprey control); (2) larval sex ratios are generally at parity or with an excess of females; (3) transformers collected above barriers or following lampricide treatment tend to be male biased in the earliest age classes to metamorphose; and (4) there is spatial and temporal variation in sex ratio during the parasitic feeding phase, but overall sex ratio is less male biased than during the adult phase, suggesting that females suffer higher mortality just prior to or during sexual maturation. The shift in sex ratio observed in the upper Great Lakes following initiation of control led to suggestions of environmental sex determination (ESD), specifically density-dependent sex determination, but evidence for ESD in lampreys is equivocal. Sex ratios did not become female biased in the lower Great Lakes, and all five lakes now show a slight excess of males even though abundance has been low and relatively stable for the past several decades. Furthermore, although a significant relationship between larval density and sex ratio has been observed in two non-parasitic species in the southeastern United States, significant relationships between larval density and sex ratio are not evident among contemporaneous sea lamprey populations (i.e., before or after the initiation of control). ESD, usually temperature-dependent sex determination, has been reported in a number of fish species, but no fish species with exclusively ESD have been identified to date. Skewed sex ratios may result from environmental influences on genetic sex determination rather than strict ESD, and the nature of the genotype × environment interactions can differ among populations and over time. However, apart from ruling out “the usual suspects” (i.e., genes implicated in sex determination in other vertebrates), nothing is known regarding the possible genetic basis of sex determination in lampreys. In contrast, many of the genes involved in the sex differentiation process (i.e., development of the undifferentiated gonad into an ovary or testis) tend to be conserved among vertebrates, and initial studies suggest that at least some of the same genes are involved in gonadal development in lampreys. Understanding the factors influencing lamprey sex determination and differentiation has been complicated by lack of knowledge regarding the critical sex differentiation period. Lampreys are sometimes said to pass through an initial female stage or female intersexual stage, because mitosis and meiosis appear to occur in most larvae regardless of future sex. However, meiosis and oocyte growth are more synchronized and extensive in female larvae, and the extent to which oocytes develop and regress in presumptive males either varies among individuals and species or reflects differences in the degree to which these transient processes are detected. Ovarian differentiation is generally thought to be complete at ~1 and 2–3 years of age in non-parasitic and most parasitic lamprey species, respectively, and at 4–5 years in the anadromous sea lamprey. Later and more prolonged mitosis in parasitic species permits elaboration of a larger stock of oocytes, and persistence of a limited number of undifferentiated germ cells in some parasitic species may allow further oocyte recruitment in large larvae. In all species, testicular differentiation occurs at or around the onset of metamorphosis, at which time resumption of mitosis in the remaining undifferentiated germ cells produces spermatogonia. In vivo biopsy studies showed that sea lamprey gonads can remain labile as long as undifferentiated germ cells remain in the gonads (i.e., after the apparent completion of ovarian differentiation, but up until differentiation of the remaining germ cells at the end of the larval stage). The presence of “atypical” gonads (which often developed into typical males in biopsied larvae) in sea lamprey from both the Great Lakes and Atlantic drainages is consistent with delayed gonadal differentiation but requires further study. Despite the apparent lability of the lamprey gonad, hormonal sex control has not been successful. Non-parasitic lampreys begin maturing during the latter stages of metamorphosis; in contrast, parasitic species remain sexually immature until they approach the end of the juvenile feeding phase, and sexual maturation proceeds during the non-trophic spawning migration. Although the rate of maturation varies among species, depending on the duration of migration, all species and life history types appear to converge again during final maturation ~1–2 months before spawning. Oocytes begin to approach their size at maturity (~1 mm in virtually all species), and, at ovulation, the oocytes (now typically called eggs) are synchronously released into the body cavity. The mature ovary constitutes ~25–35% of a female’s total body weight, regardless of species, but the total number of eggs (fecundity) increases approximately with the cubic power of body length so that fecundity in the largest anadromous parasitic species (e.g., mean 172,000 in sea lamprey) is almost two orders of magnitude higher than that of the much smaller non-parasitic species. The mature testis constitutes ~2–10% of a male’s body weight, with gonadosomatic index (GSI) appearing to be higher in males of non-parasitic species compared to parasitic species, although absolute testis size is still much higher in parasitic species. The study of lamprey steroidogenesis and steroid receptors is contributing to our understanding of the evolution of steroid hormones as transcriptions factors in vertebrates, but much still needs to be learned regarding the role of sex steroids in lamprey sex differentiation and sexual maturation.

11 pages, 5 figures, supplementary information https://doi.org/10.3389/fgene.2019.01128, The sex of Chinese tongue sole (Cynoglossus semilaevis) is determined by both genetic sex determination (GSD) and environmental sex determination (ESD), making it an ideal model to study the relationship between sex-determination and temperature. In the present study, transcriptomes of undifferentiated gonads from genetic females and males, as well as differentiated gonads from males, females, and pseudomales under high and normal temperature treatments were generated for comparative transcriptomic analysis. A mean of 68.24 M high-quality clean reads was obtained for each library. Differentially expressed genes (DEGs) between different sexes and environmental treatments were identified, revealing that the heat shock protein gene family was involved in the high temperature induced sex reversal. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in pseudomale and genetic female comparison included neuroactive ligand-receptor interaction, cortisol synthesis and secretion, and steroid hormone biosynthesis. Furthermore, weighted gene co-expression network analyses were conducted on all samples, and two modules were positive correlated with pseudomale under high temperature. An illustrated protein-protein interaction map of the module identified a hub gene, hsc70. These findings provide insights into the genetic network that is involved in sex determination and sexual differentiation, and improve our understanding of genes involved in sex reversal under high temperature, This work was supported by National Nature Science Foundation of China (31722058, 31802275, and 31472269), National Key R&D Program of China (2018YFD0900301), the AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology (2017ASTCP-ES06), the Taishan Scholar Project Fund of Shandong of China to CS, the National Ten-Thousands Talents Special Support Program to CS, and the International Scientific Partnership Program ISPP at King Saud University (No. 0050)