Annotation, phylogenetics, and expression of the nuclear receptors in Daphnia pulex.

BACKGROUND: The nuclear receptor superfamily currently consists of seven gene subfamilies that encompass over 80 distinct receptor proteins. These transcription factors typically share a common five-domain structure with a highly conserved DNA-binding domain. Some nuclear receptors are ubiquitous among the metazoans, while others are unique to specific phylogenetic groups. Crustaceans represent the second largest group of arthropods with insects being the largest. However, relative to insects, little is known about the nuclear receptors of crustaceans. The aim of this study was to identify putative nuclear receptors from the first assembled genome of a crustacean Daphnia pulex http://wFleaBase.org. Nuclear receptor expression was evaluated and receptors were subjected to phylogenetic analyses to gain insight into evolution and function. RESULTS: Twenty-five putative nuclear receptors were identified in D. pulex based on the presence of a conserved DNA-binding domain. All of the nuclear receptor protein sequences contain a highly homologous DNA-binding domain and a less conserved ligand-binding domain with the exception of the NR0A group. These receptors lack a ligand-binding domain. Phylogenetic analysis revealed the presence of all seven receptor subfamilies. The D. pulex genome contains several nuclear receptors that have vertebrate orthologs. However, several nuclear receptor members that are represented in vertebrates are absent from D. pulex. Notable absences include receptors of the 1C group (peroxisome proliferators-activated receptors), the 3A group (estrogen receptor), and the 3C group (androgen, progestogen, mineralcorticoid, and glucocorticoid receptors). The D. pulex genome also contains nuclear receptor orthologs that are present in insects and nematodes but not vertebrates, including putative nuclear receptors within the NR0A group. A novel group of receptors, designated HR97, was identified in D. pulex that groups with the HR96/CeNHR8/48/DAF12 clade, but forms its own sub-clade. Gene products were detected in adult female D. pulex for 21 of the 25 receptors. CONCLUSION: Nuclear receptors are ancient proteins with highly conserved DNA-binding domains. The DNA-binding domains of the nuclear receptors of D. pulex contain the same degree of conservation that is typically found within nuclear receptors of other species. Most of the receptors identified in D. pulex have orthologs within the vertebrate and invertebrate lineages examined with the exception of the novel HR97 group and the Dappu-HR10 and potentially the Dappu-HR11 receptors found in D. pulex. These groups of receptors may harbour functions that are intrinsic to crustacean physiology.

A transgenerational endocrine signaling pathway in Crustacea.

BACKGROUND: Environmental signals to maternal organisms can result in developmental alterations in progeny. One such example is environmental sex determination in Branchiopod crustaceans. We previously demonstrated that the hormone methyl farnesoate could orchestrate environmental sex determination in the early embryo to the male phenotype. Presently, we identify a transcription factor that is activated by methyl farnesoate and explore the extent and significance of this transgenerational signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Several candidate transcription factors were cloned from the water flea Daphnia pulex and evaluated for activation by methyl farnesoate. One of the factors evaluated, the complex of two bHLH-PAS proteins, dappuMet and SRC, activated a reporter gene in response to methyl farnesoate. Several juvenoid compounds were definitively evaluated for their ability to activate this receptor complex (methyl farnesoate receptor, MfR) in vitro and stimulate male sex determination in vivo. Potency to activate the MfR correlated to potency to stimulate male sex determination of offspring (pyriproxyfen>methyl farnesoate>methoprene, kinoprene). Daphnids were exposed to concentrations of pyriproxyfen and physiologic responses determined over multiple generations. Survivial, growth, and sex of maternal organisms were not affected by pyriproxyfen exposure. Sex ratio among offspring (generation 2) were increasingly skewed in favor of males with increasing pyriproxyfen concentration; while, the number of offspring per brood was progressively reduced. Female generation 2 daphnids were reared to reproductive maturity in the absence of pyriproxyfen. Sex ratios of offspring (generation 3) were not affected in this pyriproxyfen lineage, however, the number of offspring per brood, again, was significantly reduced. CONCLUSIONS: Results reveal likely components to a hormone/receptor signaling pathway in a crustacean that orchestrates transgenerational modifications to important population metrics (sex ratios, fecundity of females). A model is provided that describes how these signaling processes can facilitate population sustainability under normal conditions or threaten sustainability when perturbed by environmental chemicals.

Tributyltin synergizes with 20-hydroxyecdysone to produce endocrine toxicity.

One of the great challenges facing modern toxicology is in predicting the hazard associated with chemical mixtures. The development of effective means of predicting the toxicity of chemical mixtures requires an understanding of how chemicals interact to produce nonadditive outcomes (e.g., synergy). We hypothesized that tributyltin would elicit toxicity in daphnids (Daphnia magna) by exaggerating physiological responses to 20-hydroxyecdysone signaling via synergistic activation of the retinoid X receptor (RXR):ecdysteroid receptor (EcR) complex. Using reporter gene assays, we demonstrated that RXR, alone, is activated by a variety of ligands including tributyltin, whereas RXR:EcR heterodimers were not activated by tributyltin. However, tributyltin, in combination with the daphnid EcR ligand 20-hydroxyecdysone, caused concentration-dependent, synergistic activation of the RXR:EcR reporter. Electrophoretic mobility shift assays revealed that tributyltin did not enhance the activity of 20-hydroxyecdysone by increasing binding of the receptor complex to a DR-4 DNA-binding site. Exposure of daphnids to elevated concentrations of 20-hydroxyecdysone caused premature and incomplete ecdysis resulting in death. Tributyltin exaggerated this effect of exogenous 20-hydroxyecdysone. Further, exposure of daphnids to tributyltin enhanced the inductive effects of 20-hydroxyecdysone on expression of the 20-hydroxyecdysone-inducible gene HR3. Continuous, prolonged exposure of maternal daphnids to concentrations of tributyltin resulted in mortality concurrent with molting. Taken together, these results demonstrate that xenobiotics, such as tributyltin, can interact with RXR to influence gene expression regulated by the heterodimeric partner to RXR. The result of such interactions can be toxicity due to inappropriate or exaggerated hormonal signaling. The application of the in vitro/in vivo approach used in this study is discussed in relation to modeling of nonadditive interactions among constituents of chemical mixtures.

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna).

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.