Daphnids (Daphnia magna) utilize cyclic parthenogenesis as a reproductive strategy. During periods of abundant resources, these organisms reproduce asexually. In response to environmental cues that signal the onset of environmental adversity, daphnids produce males and reproduce sexually. The environmental cues that stimulate the sexual reproductive phase are well known; however, the endocrine signals that transduce these environmental cues remain unknown. The present study was undertaken to test the hypothesis that the crustacean juvenoid hormone, methyl farnesoate, is a male sex determinant in this species. Continuous exposure to aqueous concentrations of methyl farnesoate greater than approximately 30 nM stimulated a concentration-dependent production of male-containing broods of organisms. Short-term exposures to methyl farnesoate during periods of egg and embryo maturation revealed that male sex determination occurred during a specific 12-hour period of ovarian egg development. Exposure of eggs to 400 nM methyl farnesoate during this sensitive developmental period resulted in the production of all-male broods of offspring, while exposure to concentrations as low as 52 nM produced mixed broods of males and females. This active concentration range of methyl farnesoate is consistent with levels measured in the hemolymph of some decapod crustaceans. These results demonstrate that methyl farnesoate is capable of programming daphnid embryos to develop into males and is likely the endocrine factor responsible for initiating the sexual reproductive phase in these organisms.

Daphnid culture. Daphnids (Daphnia magna)were cultured in incubators at a density of 40 adults in 1 L of medium at a temperature and photoperiod of 20°C and 16 hr light. Algae (Selenastrum capricornutum), cultured in Bold's basal medium, was used as a food source for daphnids during culturing and

Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis' sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis' sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology.

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Synthetic glucocorticoids are pharmaceutical compounds prescribed in human and veterinary medicine as anti-inflammatory agents and have the potential to contaminate natural watersheds via inputs from wastewater treatment facilities and confined animal-feeding operations. Despite this, few studies have examined the effects of this class of chemicals on aquatic vertebrates. To generate data to assess potential risk to the aquatic environment, we used fathead minnow 21-d reproduction and 29-d embryo-larvae assays to determine reproductive toxicity and early-life-stage effects of dexamethasone. Exposure to 500 µg dexamethasone/L in the 21-d test caused reductions in fathead minnow fecundity and female plasma estradiol concentrations and increased the occurrence of abnormally hatched fry. Female fish exposed to 500 µg dexamethasone/L also displayed a significant increase in plasma vitellogenin protein levels, possibly because of decreased spawning. A decrease in vitellogenin messenger ribonucleic acid (mRNA) expression in liver tissue from females exposed to the high dexamethasone concentration lends support to this hypothesis. Histological results indicate that a 29-d embryo-larval exposure to 500 µg dexamethasone/L caused a significant increase in deformed gill opercula. Fry exposed to 500 µg dexamethasone/L for 29 d also exhibited a significant reduction in weight and length compared with control fry. Taken together, these results indicate that nonlethal concentrations of a model glucocorticoid receptor agonist can impair fish reproduction, growth, and development.

In this study, 9 chemicals were chosen from a recent report on surface water concentrations of a variety of xenobiotics to test the hypothesis that the toxicity of chemical mixtures could be estimated using a model based on the toxicity of the individual chemicals. Concentration-response curves for the endpoints of lifespan, growth rate, and fecundity were generated for each chemical experimentally using the crustacean, Daphnia magna. From this data, a mathematical model for the combined toxicity of these chemicals was generated that merged the concepts of concentration addition and independent joint action. Toxicity of a mixture was modeled at various levels at which the ratio of the chemicals within the mixture was maintained at that reported for median detected environmental levels. Toxicity of the mixture was then determined experimentally and compared to model predictions. The model accurately predicted the most sensitive endpoint, as well as the lowest toxic effect level of the mixture. Results demonstrated that, for this mixture of chemicals, toxicity was not influenced significantly by interactions among the chemicals and a single constituent dominated toxicity. According to model predictions and experimental results, the median detected environmental concentrations of chemicals constituting this mixture provided no margin of safety.

Environmental signals can activate neuro-endocrine cascades that regulate various physiological processes. In the present study, we demonstrate that two responses to environmental stress signaling in the crustacean Daphnia magna -hemoglobin accumulation and male offspring production -are co-elevated by the crustacean terpenoid hormone methyl farnesoate and several synthetic analogs. Potency of the hormones with respect to the induction of both hemoglobin and male offspring was highly correlated, suggesting that both processes are regulated by the same terpenoid signaling pathway. Six clones of the D. pulex/pulicaria species complex that were previously characterized as unable to produce male offspring and five clones that were capable of producing males were evaluated for both hemoglobin induction and male offspring production in response to methyl farnesoate. Four of the five male-producing clones produced both hemoglobin and male offspring in response to the hormone. Five of the six non-male-producing clones produced neither hemoglobin nor males in response to the hormone. These results provide additional evidence that both physiological processes are regulated by the same signaling pathway. Furthermore, the results indicate that the non-male-producing clones are largely defective in some methyl farnesoate signaling component, downstream from methyl farnesoate synthesis but upstream from the genes regulated by the hormone. A likely candidate for the site of the defect is the methyl farnesoate receptor. As a consequence of this defect, non-male-producing clones have lost their responsiveness to environmental signals that are transduced by this endocrine pathway. This defect in signaling would be likely to enhance population growth in stable environments due to the elimination of males from the population, assuming that other processes critical to population growth are not also compromised by this defect.

Abstract-Standard reproductive assays with daphnids involve parthenogenetically reproducing females and exclude the assessment of effects on sexual reproduction. The goals of this study were to characterize sexual differentiation of male and female daphnids (Daphnia magna) and to evaluate whether exposure to putative endocrine-disrupting chemicals may perturb the development of sex characteristics. Anatomical sex differences that developed during maturation in males included elongated first antennae and morphologic alterations in the head capsule and carapace edge. Reproductive maturation in females was associated with the development of a brood chamber and abdominal process. Alterations in the growth rates of the first antennae of males and the abdominal process of females were used to evaluate the effects of chemical exposure on the development of these sex characteristics during maturation. Exposure of female daphnids to the nonsteroidal vertebrate estrogen diethylstilbesterol and the insect juvenile hormone analog methoprene at concentrations as low as 3.0 and 0.080 M, respectively, stimulated development of the abdominal process. Exposure of males to the steroidal vertebrate androgen androstenedione (Ն6.0 M) stimulated development of the first antennae. These results demonstrate that the development of secondary sex characteristics in daphnids can be altered by chemical exposure.