Male-biased Sex Ratio Research Articles

Sex-different response in growth traits to resource heterogeneity explains male-biased sex ratio

In dioecious plants, differences in growth traits between sexes in a response to micro-environmental heterogeneity may affect sex ratio bias and spatial distributions. Here, we examined sex ratios, stem growth traits and spatial distribution patterns in the dioecious clonal shrub Aucuba japonica var. borealis, in stands with varying light intensities. We found that male stems were significantly more decumbent (lower height/length ratio) but female stems were upright (higher height/length ratio). Moreover, we found sex-different response in stem density (no. of stems per unit area) along a light intensity gradient; in males the stem density increased with increases in canopy openness, but not in females. The higher sensitivity of males in increasing stem density to light intensity correlated with male-biased sex ratio; fine-scale sex ratio was strongly male-biased as canopy openness increased. There were also differences between sexes in spatial distributions of stems. Spatial segregation of sexes and male patches occupying larger areas than female patches might result from vigorous growth of males under well-lit environments. In summary, females and males showed different growth responses to environmental variation, and this seemed to be one of possible causes for the sex-differential spatial distributions and locally biased sex ratios.

Male-biased sex ratio does not promote increased sperm competitiveness in the seed beetle, Callosobruchus maculatus.

Sperm competition risk and intensity can select for adaptations that increase male fertilisation success. Evolutionary responses are examined typically by generating increased strength of sexual selection via direct manipulation of female mating rates (by enforcing monandry or polyandry) or by alteration of adult sex ratios. Despite being a model species for sexual selection research, the effect of sexual selection intensity via adult sex-ratio manipulation on male investment strategies has not been investigated in the seed beetle, Callosobruchus maculatus. We imposed 32 generations of experimental evolution on 10 populations of beetles by manipulating adult sex ratio. Contrary to predictions, males evolving in male-biased populations did not increase their testes and accessory gland size. This absence of divergence in ejaculate investment was also reflected in the fact that males from male-biased populations were not more successful in either preventing females from remating, or in competing directly for fertilisations. These populations already demonstrate divergence in mating behaviour and immunity, suggesting sufficient generations have passed to allow divergence in physiological and behavioural traits. We propose several explanations for the absence of divergence in sperm competitiveness among our populations and the pitfalls of using sex ratio manipulation to assess evolutionary responses to sexual selection intensity.

Simple settlement decisions explain common dispersal patterns in territorial species.

Dispersal is one of the least-understood aspects of animal behaviour. For example, little is known of the mechanisms that determine how individuals express different dispersal behaviours in response to different circumstances. Uncovering these mechanisms is important for our understanding of spatial population dynamics. Using agent-based simulations, we examine how simple decision rules generate individual-level dispersal plasticity, and how this can influence population-scale dispersal dynamics. We model a territorial, monogamous population inhabiting a completely homogeneous environment. Dispersal variability therefore emerges solely as a result of between-individual interactions (competition, settlement, reproduction), which are governed by simple decision-making algorithms. We show that complex dispersal dynamics, including sex biases and strong density dependence, emerge naturally from simple rule-based behaviours. Dispersal is particularly sensitive to the inclusion of mate availability as a criterion for settlement: if neither sex evaluates mate availability, dispersal distances tend to decline at low densities, leading to a strong Allee effect from reduced pairing success. If one sex evaluates mate availability (females), Allee effects are largely avoided, but female-biased dispersal generates increasingly male-biased adult sex ratios at low densities. Sex biases are eliminated if both sexes evaluate mate availability, but population growth rates tend to be reduced due to survival costs and reduced pairing success. Our models suggest that simple decision mechanisms can explain several dynamic patterns that are commonly observed among territorial species. Importantly, these patterns emerge in the absence of environmental heterogeneity or between-individual variation in dispersal phenotypes, two conditions that are often invoked to explain dispersal heterogeneity in nature. This has implications for studies seeking to examine the causes of dispersal variability in wild populations, suggesting that observed patterns could be largely driven by the social and demographic conditions experienced by sampled individuals. Further insights could be gained by examining how selection operates on decision rules in different life-history and environmental circumstances, and how this might interact with selection on other demographic traits. Uncovering the decision rules used during settlement should be a priority for those wishing to understand and predict dispersal patterns in nature.

Regional warming and the thermal regimes of American crocodile nests in the Tempisque Basin, Costa Rica

Spatial variation in global climate change makes population-specific responses to this enigmatic threat pertinent on a regional scale. Organisms with temperature-dependent sex determination (TSD) potentially possess a unique physiological susceptibility that threatens population viability if rapid environmental effects on sex ratios render populations non-viable. A heavily male-biased sex ratio for hatchling American crocodiles of the Tempisque Basin, Costa Rica requires assessment of how nest temperature affects sex determination at this site, how females might compensate for these effects when creating nests, and how current patterns of climate change might alter future sex ratios and survival in hatchling cohorts. We demonstrate high within-nest variation in temperature but predict a female bias at hatching based on nest temperatures quantified here. Further, our data suggest that egg size and metabolic heating associated with this factor outweighs microhabitat parameters and depth in influencing nest thermal regimes. Finally, we document regional warming in the Tempisque Basin over the last 15 years and project that further heating over the next 15 years will not yield hatchling sex ratios as male biased as those currently found at this site. Thus, we find no support for nest temperature or climate change as likely explanations for male-biased American crocodile (Crocodylus acutus) sex ratios in the Tempisque Basin.

Effects of thyroid endocrine manipulation on sex-related gene expression and population sex ratios in Zebrafish

Thyroid hormone reportedly induces masculinization of genetic females and goitrogen treatment delays testicular differentiation (ovary-to-testis transformation) in genetic males of Zebrafish. This study explored potential molecular mechanisms of these phenomena. Zebrafish were treated with thyroxine (T4, 2nM), goitrogen [methimazole (MZ), 0.15mM], MZ (0.15mM) and T4 (2nM) (rescue treatment), or reconstituted water (control) from 3 to 33days postfertilization (dpf) and maintained in control water until 45dpf. Whole fish were collected during early (25dpf) and late (45dpf) testicular differentiation for transcript abundance analysis of selected male (dmrt1, amh, ar) and female (cyp19a1a, esr1, esr2a, esr2b) sex-related genes by quantitative RT-PCR, and fold-changes relative to control values were determined. Additional fish were sampled at 45dpf for histological assessment of gonadal sex. The T4 and rescue treatments caused male-biased populations, and T4 alone induced precocious puberty in ∼50% of males. Male-biased sex ratios were accompanied by increased expression of amh and ar and reduced expression of cyp19a1a, esr1, esr2a, and esr2b at 25 and 45dpf and, unexpectedly, reduced expression of dmrt1 at 45dpf. Goitrogen exposure increased the proportion of individuals with ovaries (per previous studies interpreted as delay in testicular differentiation of genetic males), and at 25 and 45dpf reduced the expression of amh and ar and increased the expression of esr1 (only at 25dpf), esr2a, and esr2b. Notably, cyp19a1a transcript was reduced but via non-thyroidal pathways (not restored by rescue treatment). In conclusion, the masculinizing activity of T4 at the population level may be due to its ability to inhibit female and stimulate male sex-related genes in larvae, while the inability of MZ to induce cyp19a1a, which is necessary for ovarian differentiation, may explain why its “feminizing” activity on gonadal sex is not permanent.

Cryptic male choice: experimental evidence of sperm sex ratio and seminal fluid adjustment in relation to coital rate.

The differential allocation hypothesis suggests that a mother should adjust the sex of offspring in relation to her mate's attractiveness, thereby increasing future reproductive fitness when her sons inherit the attractive traits. More attractive males have been shown to sire more sons, but it is possible that the sex ratio skew could be a result of paternal rather than maternal manipulation, which would be a more parsimonious explanation. We manipulated coital rate (an indicator of attractiveness) in laboratory mice and showed that males that mate more often have higher levels of glucose in their semen despite lower blood glucose levels. Since peri-conceptual glucose levels in utero increase male conceptus survival, this could result in male-biased sex ratios. The males that mated most also had more remaining X-chromosome-bearing-spermatozoa, suggesting depletion of Y-chromosome-bearing-spermatozoa during mating. We hypothesise that males may alter both seminal fluids and X:Y ratios in an ejaculate to influence subsequent sex ratios. Our results further support a paternal role in sex allocation.

Sex allocation and secondary sex ratio in Cuban boa (Chilabothrus angulifer): mother’s body size affects the ratio between sons and daughters

Secondary sex ratios of animals with genetically determined sex may considerably deviate from equality. These deviations may be attributed to several proximate and ultimate factors. Sex ratio theory explains some of them as strategic decisions of mothers improving their fitness by selective investment in sons or daughters, e.g. local resource competition hypothesis (LRC) suggests that philopatric females tend to produce litters with male-biased sex ratios to avoid future competition with their daughters. Until now, only little attention has been paid to examine predictions of sex ratio theory in snakes possessing genetic sex determination and exhibiting large variance in allocation of maternal investment. Cuban boa is an endemic viviparous snake producing large-bodied newborns (∼200g). Extremely high maternal investment in each offspring increases importance of sex allocation. In a captive colony, we collected breeding records of 42 mothers, 62 litters and 306 newborns and examined secondary sex ratios (SR) and sexual size dimorphism (SSD) of newborns. None of the examined morphometric traits of neonates appeared sexually dimorphic. The sex ratio was slightly male biased (174 males versus 132 females) and litter sex ratio significantly decreased with female snout-vent length. We interpret this relationship as an additional support for LRC as competition between mothers and daughters increases with similarity of body sizes between competing snakes.

Maladaptive Sex Ratio Adjustment in the Invasive Brine Shrimp Artemia franciscana

Sex allocation theory is often hailed as the most successful area of evolutionary theory due to its striking success as a predictor of empirical observations [1]. Most naturally occurring sex ratios can be explained by the principle of equal investment in the sexes [2-4] or by cases of "extraordinary" sex allocation [5]. Deviations from the expected sex ratio are often correlated with weak selection or low environmental predictability (e.g., [6, 7]); true cases of aberrant sex allocation are surprisingly rare [8]. Here, we present a case of long-lasting maladaptive sex allocation, which we discovered in invasive populations of theexclusively sexual brine shrimp Artemia franciscana. A.franciscana was introduced to Southern Franceroughly 500 generations ago [9]; since then, it hascoexisted with the native asexual species Artemiaparthenogenetica [10]. Although we expect A.franciscana to produce balanced offspring sex ratios, we regularly observed extremely male-biased sex ratios in invasive A.franciscana, which were significantly correlated to the proportion of asexuals in the overall population. We experimentally proved that both invasive- and native-range A.franciscanaoverproduced sons when exposed to excess females,without distinguishing between conspecificand asexual females. We conclude that A.franciscana adjust their offspring sex ratio in function of the adult sex ratio but are information limited inthe presence of asexual females. Their facultative adjustment trait, which is presumably adaptive in theirnative range, has thus become maladaptive in the invasive range where asexuals occur. Despite this, ithas persisted unchanged for hundreds of generations.

King Penguins in Zoos: Relating Breeding Success to Husbandry Practices

As global climate change warms the Southern Ocean, wild king penguins ( Aptenodytes patagonicus ) possibly face drastic population declines in the future. Moreover, the captive breeding program in Europe is currently unsustainable and it is thus imperative to determine what is required for successfully breeding king penguins. A questionnaire was used to assess the husbandry practices and breeding success of 12 American and European zoos. Overall, a positive relationship was found between yearly egg productivity and density of king penguins held in enclosures in different zoos. All zoos examined showed male-biased adult sex ratios and yearly egg productivity was found to be negatively related to the deviation from an even sex ratio. Mean hatching success was greater in zoos performing outside walks with king penguins than in those that did not. Based on records over 40 years, the population of the sustainably breeding Basel Zoo in Switzerland was modelled using stage-based matrix models to get insight into its breeding success. Years of medium colony size (density of 0.25 to 0.38 individuals per m 2 ) displayed a larger growth rate than years with high or low colony size and years with equal sex ratios showed a higher growth rate than those with male-biased or female-biased sex ratios. The results suggest zoological institutions should focus on increasing density and striving toward equal sex ratios within king penguin populations to maximize breeding success. Where possible, adding outside walks should also be taken into consideration.

Gonadal differentiation and temperature effects on sex determination in the freshwater pike silverside Chirostoma estor Jordan 1880

The gonadal description of the freshwater atherinopsid pike silverside Chirostoma estor suggests that the gonads differentiate as ovaries or testes by 8 weeks after hatching when raised at 21 °C. Thermal treatments at 14 °C, 21 °C and 29 °C applied from fertilisation, clearly affected phenotypic sex ratios, suggesting that the thermolabile window of sex determination occurred early in development. In this study, exposure to the highest temperature led to male-biased sex ratios in this species. However, the effects of the lower and medium temperatures on the sex ratios were less clear, suggesting the presence of a mixture of genotypic and temperature-dependent sex determination (TSD) mechanisms in C. estor, similar to other atherinopsids. This work further enhances our knowledge regarding the diversity and plasticity of TSD mechanisms in atherinopsid and teleost fish.

Sex ratios at birth after induced abortion.

Skewed male:female ratios at birth have been observed among certain immigrant groups. Data on abortion practices that might help to explain these findings are lacking. We examined 1 220 933 births to women with up to 3 consecutive singleton live births between 1993 and 2012 in Ontario. Records of live births, and induced and spontaneous abortions were linked to Canadian immigration records. We determined associations of male:female infant ratios with maternal birthplace, sex of the previous living sibling(s) and prior spontaneous or induced abortions. Male:female infant ratios did not appreciably depart from the normal range among Canadian-born women and most women born outside of Canada, irrespective of the sex of previous children or the characteristics of prior abortions. However, among infants of women who immigrated from India and had previously given birth to 2 girls, the overall male:female ratio was 1.96 (95% confidence interval [CI] 1.75-2.21) for the third live birth. The male:female infant ratio after 2 girls was 1.77 (95% CI 1.26-2.47) times higher if the current birth was preceded by 1 induced abortion, 2.38 (95% CI 1.44-3.94) times higher if preceded by 2 or more induced abortions and 3.88 (95% CI 2.02-7.50) times higher if the induced abortion was performed at 15 weeks or more gestation relative to no preceding abortion. Spontaneous abortions were not associated with male-biased sex ratios in subsequent births. High male:female ratios observed among infants born to women who immigrated from India are associated with induced abortions, especially in the second trimester of pregnancy.

Male-biased infant sex ratios and patterns of induced abortion.

Two linked research papers by Urquia and colleagues examine infant sex selection, a long-standing and controversial topic.[1][1],[2][2] In their first study, the authors investigate birth certificate data to examine skewed infant sex ratios among mothers born in India compared with mothers born in

Redescription and biology of Diopatra neapolitana (Annelida: Onuphidae), a protandric hermaphrodite with external spermaducal papillae

A one-year study of the reproductive biology of a population of Diopatra neapolitana at Villaviciosa estuary, northern Spain, was undertaken. Field observations together with a histological study of monthly collected individuals revealed that the population was iteroparous, had a discontinuous reproductive season with a resting period during August and September and a spawning season from March to July. The study showed that D. neapolitana was not dioecious as previously suggested but consisted of protandric sequential hermaphrodites, pure males and pure females with a male biased sex ratio of 3:1. During the peak reproductive period from May to August we observed simultaneous hermaphrodites with two dorsal papillae per segment in the branchial region. Histological studies demonstrated that the papillae were acting as seminal vesicles, storing own sperm, and also as sperm ducts, providing an exit route; hence we termed them ‘spermaducal papillae’. The papillae are not the only sperm repositories as the coelom of males and simultaneous hermaphrodites in smaller size classes is also filled with sperm. The worms are broadcast spawners with a brief pelagic larval stage as previously reported but the finer points of this unusual fertilisation system need still to be elaborated. Diopatra cryptornata was recently described as a new species, supposedly differing from D. neapolitana in chaetal detail and the possession of the papillae. We have shown conclusively with morphological and genetical studies that the former species is a junior synonym of the latter. In the absence of type material we are here designating a neotype from recently collected material from Naples.

The ‘extreme female brain’: increased cognitive empathy as a dimension of psychopathology

Baron-Cohen's ‛extreme male brain’ theory postulates that autism involves exaggerated male-typical psychology, with reduced empathizing (considered here as social–emotional interest, motivation and abilities) and increased systemizing (non-social, physical-world and rule-based interest, motivation and abilities), in association with its male-biased sex ratio. The concept of an ‘extreme female brain’, involving some combination of increased empathizing and reduced systemizing, and its possible role in psychiatric conditions, has been considerably less well investigated. Female-biased sex ratios have been described in two conditions, depression and borderline personality disorder (BPD), that also show evidence of increases in aspects of empathy in some studies. We evaluated the hypothesis that BPD and depression can be conceptualized in the context of the ‘extreme female brain’ by: (1) describing previous conceptualizations of the extreme female brain model, (2) reviewing evidence of female-biased sex ratios in BPD and depression, (3) conducting meta-analyses of performance on the Reading the Mind in the Eyes test (RMET) among individuals with BPD, clinical or sub-clinical depression, and other psychiatric conditions involving altered social cognition and mood (schizophrenia, bipolar disorder, eating disorders, and autism), in relation to disorder sex ratios, and (4) evaluating previous evidence of increased empathic performance in these, and related, psychiatric conditions, and (5) synthesizing these lines of evidence into models for causes and effects of an ‘extreme female brain’. Our primary empirical results are that RMET performance is enhanced in sub-clinical depression, preserved in borderline personality disorder, and reduced in other disorders (by meta-analyses), and that across disorders, more male-biased patient sex ratios are strongly associated with worse RMET performance of patients relative to controls. Our findings, in conjunction with previous work, suggest that increased cognitive empathizing mediates risk and expression of some psychiatric conditions with evidence of female biases, especially sub-clinical depression and borderline personality disorder, in association with increased attention to social stimuli, higher levels of social and emotional sensitivity, negative emotion biases, and over-developed mentalist thought. These results link evolved human sex differences with psychiatric vulnerabilities and symptoms, and lead to specific suggestions for future work.

Production of germ cell‐deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor-derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell-deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell-deficient rainbow trout did not show a male-biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell-deficient and control recipients. The donor-derived germ cells showed significantly higher proliferation in the gonads of germ cell-deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell-deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell-deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end-knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia.

The effects of quantitative fecundity in the haploid stage on reproductive success and diploid fitness in the aquatic peat moss Sphagnum macrophyllum.

A major question in evolutionary biology is how mating patterns affect the fitness of offspring. However, in animals and seed plants it is virtually impossible to investigate the effects of specific gamete genotypes. In bryophytes, haploid gametophytes grow via clonal propagation and produce millions of genetically identical gametes throughout a population. The main goal of this research was to test whether gamete identity has an effect on the fitness of their diploid offspring in a population of the aquatic peat moss Sphagnum macrophyllum. We observed a heavily male-biased sex ratio in gametophyte plants (ramets) and in multilocus microsatellite genotypes (genets). There was a steeper relationship between mating success (number of different haploid mates) and fecundity (number of diploid offspring) for male genets compared with female genets. At the sporophyte level, we observed a weak effect of inbreeding on offspring fitness, but no effect of brood size (number of sporophytes per maternal ramet). Instead, the identities of the haploid male and haploid female parents were significant contributors to variance in fitness of sporophyte offspring in the population. Our results suggest that intrasexual gametophyte/gamete competition may play a role in determining mating success in this population.

Imagination in human social cognition, autism, and psychotic-affective conditions

Complex human social cognition has evolved in concert with risks for psychiatric disorders. Recently, autism and psychotic-affective conditions (mainly schizophrenia, bipolar disorder, and depression) have been posited as psychological ‘opposites’ with regard to social-cognitive phenotypes. Imagination, considered as ‘forming new ideas, mental images, or concepts’, represents a central facet of human social evolution and cognition. Previous studies have documented reduced imagination in autism, and increased imagination in association with psychotic-affective conditions, yet these sets of findings have yet to be considered together, or evaluated in the context of the diametric model. We first review studies of the components, manifestations, and neural correlates of imagination in autism and psychotic-affective conditions. Next, we use data on dimensional autism in healthy populations to test the hypotheses that: (1) imagination represents the facet of autism that best accounts for its strongly male-biased sex ratio, and (2) higher genetic risk of schizophrenia is associated with higher imagination, in accordance with the predictions of the diametric model. The first hypothesis was supported by a systematic review and meta-analysis showing that Imagination exhibits the strongest male bias of all Autism Quotient (AQ) subscales, in non-clinical populations. The second hypothesis was supported, for males, by associations between schizophrenia genetic risk scores, derived from a set of single-nucleotide polymorphisms, and the AQ Imagination subscale. Considered together, these findings indicate that imagination, especially social imagination as embodied in the default mode human brain network, mediates risk and diametric dimensional phenotypes of autism and psychotic-affective conditions.

Cold Tolerance and Sex-Dependent Hypothermia May Explain Winter Sexual Segregation in a Farmland Bird.

Migration is an important event in the life cycle of many organisms, but considerable intraspecific variation may occur in its timing and/or destination, resulting in sexual segregation during wintering periods. In this study, we tested the body size hypothesis, or cold tolerance hypothesis, which predicts that body size dimorphism modulates metabolic costs associated with cold climate. Using the Eurasian skylark, we first investigated whether this species showed sexual differential migration. Then we explored the body size hypothesis by experimentally testing the effect of low ambient temperature (Ta) on both metabolic rate (MR) and body temperature (Tb). We tested for sex-related differences in metabolism and in energy-saving mechanism (hypothermia). We found clear differential migration by sex in skylark wintering populations, with a male-biased sex ratio decreasing toward southern latitudes. Measurements on captive birds at 20°, 6°, and -5°C demonstrated a significant increase in MR when Ta decreased, but there is no difference between sexes. While both males and females reduced their Tb overnight, Tb reduction was more pronounced in females exposed to the coldest temperature treatment. In addition, we found that individuals with the most reduced Tb lost less body weight during the night, suggesting that Tb reduction may help minimize energy expenditure when conditions become constraining. Our study suggests that functional mechanisms may be involved in latitudinal segregation between sexes and supports the hypothesis that sex-specific physiological strategies and thermal tolerance may explain segregation between sexes.

Genetic and morphological sex identification methods reveal a male-biased sex ratio in the Ivory Gull Pagophila eburnea

Sex identification of birds is relevant to studies of evolutionary biology and ecology and is often a central issue for the management and conservation of populations. The Ivory Gull Pagophila eburnea (Phipps, 1774) is a rare high-Arctic species whose main habitat is sea ice throughout the year. This species is currently listed Near Threatened by the IUCN, because populations have drastically declined in part of the species distribution in the recent past. Here we tested molecular sexing methods with different types of samples. Molecular sexing appeared to be very efficient with DNA extracted from muscle, blood, and buccal swabs, both for adults and young chicks. We also performed morphological analyses to characterize sexual size dimorphism in Ivory Gulls sampled in three distinct regions: Greenland, Svalbard, and Russia. Males were larger than females for all morphometric measurements, with little overlap between sexes. Discriminant analysis based on six morphometric variables correctly classified ~95 % of the individuals, even when using two variables only, i.e., gonys height and skull length. Therefore, both molecular and biometric methods are useful for sexing Ivory Gulls. Interestingly, our results indicate a male-biased sex ratio across all Ivory Gull populations studied, including two samples of offspring (67.8 % males).

Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17α-ethinylestradiol and 17β-trenbolone.

Environmental estrogens and androgens can be present simultaneously in aquatic environments and thereby interact to disturb multiple physiological systems in organisms. Studies on interaction effects in fish of androgenic and estrogenic chemicals are limited. Therefore, the aim of the present study was to evaluate feminization and masculinization effects in zebrafish (Danio rerio) exposed to combinations of two synthetic steroid hormones detected in environmental waters: the androgen 17β-trenbolone (Tb) and the oestrogen 17α-ethinylestradiol (EE2). Juvenile zebrafish were exposed between days 20 and 60 post-hatch to different binary mixtures of Tb (1, 10, and 50 ng/L) and EE2 (2 and 5 ng/L). The endpoints studied were whole-body homogenate vitellogenin concentration at 40 days post-hatch, and sex ratio including gonad maturation at 60 days post-hatch. The feminizing potency of 5 ng/L of EE2, alone as well as in combination with Tb, was clear in the present study, with exposures resulting in almost all-female populations and females being sexually immature. Masculinization effects with male-biased sex ratios were observed when fish were exposed to 2 ng/L of EE2 in combination with Tb concentrations. Intersex fish were observed after exposure to mixtures of 2 ng/L EE2 with 50 ng/L Tb. Sexual maturity generally increased among males at increasing concentrations of Tb. The results of the present study show that exposure to environmentally relevant mixtures of an oestrogen and androgen affects the process of gonad differentiation in zebrafish and lead to sexual disruption.