Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.     

Water pH during early development influences sex ratio and male morph in a West African cichlid fish,Pelvicachromis pulcher

Environmental sex determination (ESD) is one of the most striking examples of phenotypic plasticity. Individuals from species that exhibit ESD can develop as either males or females depending on the particular environmental conditions they experience during early development. In fish, ESD species often show a relatively subtle effect of environment, resulting in a substantial number of both sexes being produced in both male- and female-biasing conditions, rather than the unisex clutches that are typical of many reptiles. This less dramatic form of ESD allows the opportunity to study the effects of sexual differentiation on within-sex variation in behavior and morphology by comparing same-sex individuals produced in male- and female-biasing conditions. Here, we confirm that sex determination in the West African cichlid, Pelvicachromis pulcher, is influenced by pH during early development. We show that pH also affects the ratio of two alternative male reproductive types with the polygynous morph being overproduced in male-biasing conditions and the monogamous male morph being overproduced in female-biasing conditions. Our results suggest that the sexual differentiation process may be an important force in maintaining individual variation in behavior and reproductive tactics.     

VARIATION IN ENVIRONMENTAL AND GENOTYPIC SEX-DETERMINING MECHANISMS ACROSS A LATITUDINAL GRADIENT IN THE FISH,MENIDIA MENIDIA

Models of environmental sex determination (ESD) usually assume that genetic influences on sex are polygenic, but the validity of this (or any other) form of genotype-environment interaction is virtually unknown. In the Atlantic silverside, Menidia menidia, sex is determined by an interaction between temperature and genotype and the response of sex ratio to temperature differs among populations from different latitudes. We examined the genetic basis of this pattern by measuring among family variation in the proportion of females, F/(F + M), within and among high (21°C) and low (15°C) temperatures for two populations: one from Nova Scotia (NS) where the level of ESD is low, and another from South Carolina (SC) where the level of ESD is high. In NS fish, temperature had a significant influence on sex ratio in only 1 of 23 families. The distribution of the fraction of females within temperatures for families from NS was highly heterogeneous and tended to fall into distinct classes (0.0, 0.25, 0.5, 1.0) like that expected from Mendelian segregation of a major sex factor(s). In contrast, temperature had a highly significant influence on sex ratio in all SC families examined (N = 24). Family sex ratios within temperatures were highly heterogeneous and, at least at 15°C, did not conform to simple Mendelian ratios. At 21°C, the proportion of females in most SC families was near zero and so the underlying sex tendencies of different families could not be discerned. Based on a previous study, mid-latitude fish appear to have an intermediate form of sex determination: simple Mendelian sex-ratio patterns exist and there is a moderate thermal influence on sex ratio in most but not all families. We suggest that sex determination in M. menidia is controlled by an interaction between major genetic factors, polygenic factors, and temperature and that the relative importance of each component differs with latitude. High latitude populations appear to have evolved a major sex-determining factor(s) that overrides the effect of temperature, and this factor(s) is lacking in low latitude populations.     

Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization

In fish species with temperature-dependent sex determination (TSD) or genotypic sex determination plus temperature effects (GSD + TE), temperature can either affect sex differentiation or determine the sex. However, it is unknown if epigenetic control of cyp19a1a expression is critical for high temperature induced masculinization in the freshwater fish Nile tilapia. We analyzed the cyp19a1a DNA methylation levels in three age groups and found that they were lower in females than in males. At 8 months of age, males had DNA methylation levels of the cyp19a1a promoter that were almost twice as high as those of females. Exposure to high temperatures increased the cyp19a1a promoter DNA methylation levels from 30.87 ± 4.56% to 48.34 ± 0.92% (P = 0.035) in females and from 50.33 ± 7.38% to 51.66 ± 4.75% in males (P = 0.867). The increases in the cyp19a1a promoter DNA methylation levels were associated with the mRNA expression levels and might play a role in promoting gonadal differentiation in high temperature induced group females toward the male pathway. Western blot analysis revealed that the cyp19a1a protein expression levels in females significantly declined after high temperature treatment; only a slight decline was recorded in male fish. These results reveal that epigenetic control of cyp19a1a mRNA and protein expression is related to the environmental temperature and sex ratios in fish with TSD or GSD + TE.     

Population structure leads to male‐biased population sex ratios under environmental sex determination

Spatial structure has been shown to favor female‐biased sex allocation, but current theory fails to explain male biases seen in many taxa, particularly those with environmental sex determination (ESD). We present a theory and accompanying individual‐based simulation model that demonstrates how population structure leads to male‐biased population sex ratios under ESD. Our simulations agree with earlier work showing that the high productivity of female‐producing habitats creates a net influx of sex‐determining alleles into male‐producing habitats, causing larger sex ratio biases, and lower productivity in male‐producing environments (Harts et al. 2014). In contrast to previous findings, we show that male‐biasing habitats disproportionately impact the global sex ratio, resulting in stable male‐biased population sex ratios under ESD. The failure to detect a male bias in earlier work can be attributed to small subpopulation sizes leading to local mate competition, a condition unlikely to be met in most ESD systems. Simulations revealed that consistent male biases are expected over a wide range of population structures, environmental conditions, and genetic architectures of sex determination, with male excesses as large as 30 percent under some conditions. Given the ubiquity of genetic structure in natural populations, we predict that modest, enduring male biased allocation should be common in ESD species, a pattern consistent with reviews of ESD sex ratios.     

High Temperature Increases the Masculinization Rate of the All-Female (XX) Rainbow Trout “Mal” Population

Salmonids are generally considered to have a robust genetic sex determination system with a simple male heterogamety (XX/XY). However, spontaneous masculinization of XX females has been found in a rainbow trout population of gynogenetic doubled haploid individuals. The analysis of this masculinization phenotype transmission supported the hypothesis of the involvement of a recessive mutation (termed mal). As temperature effect on sex differentiation has been reported in some salmonid species, in this study we investigated in detail the potential implication of temperature on masculinization in this XX mal-carrying population. Seven families issued from XX mal-carrying parents were exposed from the time of hatching to different rearing water temperatures ((8, 12 and 18°C), and the resulting sex-ratios were confirmed by histological analysis of both gonads. Our results demonstrate that masculinization rates are strongly increased (up to nearly two fold) at the highest temperature treatment (18°C). Interestingly, we also found clear differences between temperatures on the masculinization of the left versus the right gonads with the right gonad consistently more often masculinized than the left one at lower temperatures (8 and 12°C). However, the masculinization rate is also strongly dependent on the genetic background of the XX mal-carrying families. Thus, masculinization in XX mal-carrying rainbow trout is potentially triggered by an interaction between the temperature treatment and a complex genetic background potentially involving some part of the genetic sex differentiation regulatory cascade along with some minor sex-influencing loci. These results indicate that despite its rather strict genetic sex determinism system, rainbow trout sex differentiation can be modulated by temperature, as described in many other fish species.     

Environmental condition related reproductive strategies and sex ratio in hydras

Kaliszewicz, A. and Lipińska, A. 2011. Environmental condition related reproductive strategies and sex ratio in hydras. —Acta Zoologica (Stockholm) 00 :1–7. Temperature and food supply appeared to affect sex ratio, sex composition and percentage of sexual individuals in three Hydra species: Hydra vulgaris, Hydra circumcincta and Hydra viridissima. We found three sexes present: females, males and hermaphrodites depending on environmental conditions. Hydra vulgaris appeared to be a species with a temperature‐dependent sex determination (TSD). The males and hermaphrodites were present only under rising temperatures, whereas females were observed exclusively at lowering temperatures. Hydras reproduced asexually at constant room temperature. Unlimited food affected sex ratios and induced the presence of males in H. circumcincta at lowering temperatures. Thus, H. circumcincta may be recognised as another Hydra species in which sex is determined by environmental factors (ESD). Under rising temperatures, the number of hermaphroditic individuals was higher when food supply was unlimited in all three species, indicating that hermaphrodites may need more energy to produce both male and female gonads. Both temperature changes and food supply positively affected asexual reproductive strategies in hydras, especially budding rates. Hydra circumcincta appeared to be less agile than other hydras and able to self‐fertilise. It is likely that self‐fertilisation is an adaptation to the low probability of meeting a mate belonging to the other clone.     

SEX RATIOS AND CONDITIONS REQUIRED FOR ENVIRONMENTAL SEX DETERMINATION IN ANIMALS

Environmental sex determination (ESD) is a system of sexual determination that is influenced by a variable environment. Once sex is determined it is then fixed for life. The model of Charnov & Bull (1977) proposes that ESD is favoured by natural selection when an individual's fitness as a male or female is strongly influenced by environmental conditions and when the individual has little control over which environment it will experience. Adaptive sex ratio variation is considerably easier for organisms with ESD, and this feature is the ultimate cause for the evolution and maintenance of ESD. ESD is taxonomically widely expressed, and more cases are likely to be discovered. Both environmental and genotypic sex determination mechanisms are found in closely related species. Evidence of geographical variation in the degree and in the critical environmental values of ESD within the same species has also been discovered, e.g. in the fish Menidia menidia and in the crustacean Gammarus duebeni. The factors causing sex determination in invertebrates include temperature, daylength, nutrition, density, humidity, ionic composition of the environment, pH, carbon dioxide, UV light, metabolic products, parasites, exposure to the opposite sex of the same species, and in parasitoids also host size, age and type. In vertebrates temperature is the dominant factor causing sex determination, though in fish also pH, salinity, light, water quality and nutrition, and in turtles water potential of the substrate have some effect on the sex expression. Most of these factors influence growth through resource availability or developmental speed. In most cases of ESD in invertebrates and fish, the environmental factor has a gradual effect on the sex expression, in contrast to the typical steep threshold mode found in reptiles. These differences might be due to the fact that invertebrates exhibiting ESD are commonly parasitic or confined to aquatic environments, where less spatial microhabitat differentiation exists. Sex ratio data available from nature for animals with ESD are quite limited, except for reptiles. In the laboratory sex ratios can be varied more widely than what is observed in nature. There are a number of characteristic features some of which are found in each species exhibiting ESD: (1) Patchy environments, (2) variable sex ratios, (3) parthenogenesis in addition to bisexuality, (4) parasitism, (5) aquatic habitats, (6) sexual dimorphism, (7) females larger than males, and (8) local mate competition.     

Sexual differentiation in the spiny softshell turtle (Apalone spinifera), a species with genetic sex determination

It is hypothesized on the basis of sex determination theory that species exhibiting genetic sex determination (GSD) may undergo sexual differentiation earlier in development than species with environmental sex determination (ESD). Most turtle species exhibit a form of ESD known as temperature-dependent sex determination (TSD), and in such species the chronology of sex differentiation is well studied. Apalone spinifera is a species of softshell turtle (Trionychidae) that exhibits GSD. We studied sexual differentiation in this species in order to facilitate comparison to TSD species. Eggs were incubated at two different temperatures and embryos were harvested at various stages of mid to late development. Gonad length was measured with image analysis software, then prepared histologically. Indifferent gonads have differentiated in stage 19 embryos. Histological details of gonadogenesis follow the same pattern as described for other reptiles. Regression of the male paramesonephric duct closely follows testicular differentiation. Gonad lengths are longer at the warmer incubation temperature, and ovaries are generally longer than testes at each stage and for each temperature. Although sexual differentiation takes place at about the same stage as in other turtles with TSD (18-20), in A. spinifera this differentiation is irreversible at this stage, while in some of the TSD species sex is reversible until about stage 22. This immutable, definitive sexual differentiation may support the hypothesis of an accelerated chronology of sex differentiation for this species. We also note that sexual dichromatism at hatching is known in this species and may provide additional evidence of early differentiation. J. Exp. Zool. 290:190-200, 2001.     

Ovotestes suggest cryptic genetic influence in a reptile model for temperature-dependent sex determination

Sex determination and differentiation in reptiles is complex. Temperature-dependent sex determination (TSD), genetic sex determination (GSD) and the interaction of both environmental and genetic cues (sex reversal) can drive the development of sexual phenotypes. The jacky dragon (Amphibolurus muricatus) is an attractive model species for the study of gene–environment interactions because it displays a form of Type II TSD, where female-biased sex ratios are observed at extreme incubation temperatures and approximately 50 : 50 sex ratios occur at intermediate temperatures. This response to temperature has been proposed to occur due to underlying sex determining loci, the influence of which is overridden at extreme temperatures. Thus, sex reversal at extreme temperatures is predicted to produce the female-biased sex ratios observed in A. muricatus. The occurrence of ovotestes during development is a cellular marker of temperature sex reversal in a closely related species Pogona vitticeps. Here, we present the first developmental data for A. muricatus, and show that ovotestes occur at frequencies consistent with a mode of sex determination that is intermediate between GSD and TSD. This is the first evidence suggestive of underlying unidentified sex determining loci in a species that has long been used as a model for TSD.     

Adaptive significance of environmental sex determination in an amphipod

Environmental sex determination (ESD) permits adaptive sex choice under patchy environmental conditions, where the environment affects sex-specific fitness and where offspring can predict their likely adult status by monitoring an appropriate environmental cue. For Gammarus duebeni, an amphipod with ESD, it has been proposed that this flexible sex determination system is adaptive because males gain more from large size. Under ESD, young which are born earlier in the season become mostly males and, experiencing longer to grow, are therefore larger at breeding than females which are born later in the season. In order to test the hypothesis that ESD is adaptive for this species we investigated the relationship between size and fitness for both males and females, in a population of G. duebeni known to have ESD. We measured size related pairing success and fecundity, and used these two measures to calculate the relative fitness gains achieved through an increase in size for either sex. The fitness of both males and females increased with size, but males gained more from an increase in size than did females, throughout the breeding season. The data support the adaptive explanation for the evolution and maintenance of ESD in this species.     

Climate change,sex reversal and lability of sex‐determining systems

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.     

Sex determination in annual fishes: Searching for the master sex-determining gene in Austrolebias charrua (Cyprinodontiformes,Rivulidae)

Evolution of sex determination and differentiation in fishes involves a broad range of sex strategies (hermaphroditism, gonochorism, unisexuality, environmental and genetic sex determination). Annual fishes inhabit temporary ponds that dry out during the dry season when adults die. The embryos exhibit an atypical developmental pattern and remain buried in the bottom mud until the next rainy season. To elucidate genomic factors involved in the sex determination in annual fish, we explored the presence of a candidate sex-specific gene related to the cascade network in Austrolebias charrua. All phylogenetic analyses showed a high posterior probability of occurrence for a clade integrated by nuclear sequences (aprox. 900 bp) from both adults (male and female), with partial cDNA fragments of A. charrua from juveniles (male) and the dsx D. melanogaster gene. The expressed fragment was detected from blastula to adulthood stages showing a sexually dimorphic expression pattern. The isolated cDNA sequence is clearly related to dsx D. melanogaster gene and might be located near the top of the sex determination cascade in this species.     

Nest site choice compensates for climate effects on sex ratios in a lizard with environmental sex determination

Theoretical models suggest that in changing environments natural selection on two traits, maternal nesting behaviour and pivotal temperatures (those that divide the sexes) is important for maintaining viable offspring sex ratios in species with environmental sex determination (ESD). Empirical evidence, however, is lacking. In this paper, we provide such evidence from a study of clinal variation in four sex-determining traits (maternal nesting behaviour, pivotal temperatures, nesting phenology, and nest depth) in Physignathus lesueurii, a wide-ranging ESD lizard inhabiting eastern Australia. Despite marked differences in air and soil temperatures across our five study sites spanning 19° latitude and 1200 m in elevation, nest temperatures did not differ significantly among sites. Lizards compensated for climatic differences chiefly by selecting more open nest sites with higher incident radiation at cooler sites. Clinal variation in the onset of nesting also compensated for climatic differences, but to a lesser extent. There was no evidence of compensation through pivotal temperatures or nest depth. More broadly, our results extend to the egg stage the life history prediction that behaviour is the chief compensatory mechanism for climatic differences experienced by species spanning environmental extremes. Furthermore, our study was unique in revealing that nest site choice influenced mainly the daily range in nest temperatures, rather than mean temperatures, in a shallow-nesting reptile. Finally, indirect evidence suggests that the cue used by nesting lizards was radiation or temperature (through basking or assessing substrate temperatures), not visual detection of canopy openness. We conclude that maternal nesting behaviour and nesting phenology are traits subject to sex ratio selection in P. lesueurii, and thus, must be considered among the repertoire of ESD species for responding to climate change.     

两栖动物性别决定相关基因的研究进展

两栖动物的性别决定机制主要包括遗传性别决定(genetic sex determination,GSD)和环境性别决定(environmental sex determination,ESD).近年来,在两栖动物性别决定和性腺分化机制的研究中,运用分子生物学技术探讨性别决定相关基因及其相互关系方面的研究已获得新的成果....     

How rapidly can maternal behavior affecting primary sex ratio evolve in a reptile with environmental sex determination?

Theoretical models identify maternal behavior as critical for the maintenance and evolution of sex ratios in organisms with environmental sex determination (ESD). Maternal choice of nest site is generally thought to respond more rapidly to sex ratio selection than environmental sensitivity of offspring sex (threshold temperatures) in reptiles with temperature-dependent sex determination (TSD, a form of ESD). However, knowledge of the evolutionary potential for either of these traits in a field setting is limited. I developed a simulation model using local climate data and observed levels of phenotypic variation for nest-site choice and threshold temperatures in painted turtles (Chrysemys picta) with TSD. Both nest-site choice and threshold temperatures, and hence sex ratios, evolved slowly to simulated climate change scenarios. In contrast to expectations from previous models, nest-site choice evolved more slowly than threshold temperatures because of large climatic effects on nest temperatures and indirect selection on maternally expressed traits. A variant of the model, assuming inheritance of nest-site choice through natal imprinting, demonstrated that natal imprinting inhibited adaptive responses in female nest-site choice to climate change. These results predict that females have relatively low potential to adaptively adjust sex ratios through nest-site choice.     

A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus)

Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map.     

Embryonic gonadal and sexual organ development in a small viviparous skink, Niveoscincus ocellatus

The majority of research into the timing of gonad differentiation (and sex determination) in reptiles has focused on oviparous species. This is largely because: (1) most reptiles are oviparous; (2) it is easier to manipulate embryonic developmental conditions (e.g., temperature) of eggs than oviductal embryos and (3) modes of sex determination in oviparous taxa were thought to be more diverse since viviparity and environmental sex determination (ESD)/temperature-dependent sex determination (TSD) were considered incompatible. However, recent evidence suggests the two may well be compatible biological attributes, opening potential new lines of enquiry into the evolution and maintenance of sex determination. Unfortunately, the baseline information on embryonic development in viviparous species is lacking and information on gonad differentiation and sexual organ development is almost non-existent. Here we present an embryonic morphological development table (10 stages), the sequence of gonad differentiation and sexual organ development for the viviparous spotted snow skink (Niveoscincus ocellatus). Gonad differentiation in this species is similar to other reptilian species. Initially, the gonads are indifferent and both male and female accessory ducts are present. During stage 2, in the middle third of development, differentiation begins as the inner medulla regresses and the cortex thickens signaling ovary development, while the opposite occurs in testis formation. At this point, the Müllerian (female reproductive) duct regresses in males until it is lost (stage 6), while females retain both ducts until after birth. In the later stages of testis development, interstitial tissue forms in the medulla corresponding to maximum development of the hemipenes in males and the corresponding regression in the females.     

Increase of cortisol levels after temperature stress activates dmrt1a causing female‐to‐male sex reversal and reduced germ cell number in medaka

In vertebrates, there is accumulating evidence that environmental factors as triggers for sex determination and genetic sex determination are not two opposing alternatives but that a continuum of mechanisms bridge those extremes. One prominent example is the model fish species Oryzias latipes which has a stable XX/XY genetic sex determination system, but still responds to environmental cues, where high temperatures lead to female‐to‐male sex reversal. However, the mechanisms behind are still unknown. We show that high temperatures increase primordial germ cells (PGC) numbers before they reach the genital ridge, which, in turn, regulates the germ cell proliferation. Complete ablation of PGCs led to XX males with germ cell less testis, whereas experimentally increased PGC numbers did not reverse XY genotypes to female. For the underlying molecular mechanism, we provide support for the explanation that activation of the dmrt1a gene by cortisol during early development of XX embryos enables this autosomal gene to take over the role of the male determining Y‐chromosomal dmrt1bY.