methyl-farnesoate and pyriproxyfen

Analysis of the disruptive effects of chemicals on lipids in invertebrates is limited by our poor knowledge of the lipid metabolic pathways and the complete lipidome. Recent studies shown that juvenoids and bisphenol A disrupted the dynamics of lipid droplets in the crustacean Daphnia magna. This study used ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) to study how juvenoids (pyriproxyfen and methyl farnesoate) and bisphenol A disrupt the dynamics of glycerophospholipids and glycerolipids in Daphnia adults and their allocation to eggs. Lipidomic analysis identified 234 individual lipids corresponding to three classes of glycerolipids, seven of glycerophospholipids, and one of sphingolipids, of which 194 changed according to the chemical treatments and time. Adult females in the control and bisphenol A treatment groups had low levels of triacylglycerols but high levels of glycerophospholipids, whereas those in the juvenoid treatment groups had high levels of triacylglycerols and low levels of glycerophospholipids. The opposite trend was observed for the lipid contents in the eggs produced. Because the juvenoids reduced reproduction dramatically, the females allocated less triacylglycerols to their eggs than the controls did. Interestingly, females exposed to bisphenol A allocated less triacylglycerols to their eggs despite producing a similar number of eggs as that of the controls. Thin-layer chromatography analyses confirmed the UHPLC/TOFMS results and allowed qualitative determination of cholesterol, which was also accumulated in females exposed to the juvenoids.

Topics: Animals; Benzhydryl Compounds; Chromatography, High Pressure Liquid; Daphnia; Fatty Acids, Unsaturated; Female; Glycerophospholipids; Juvenile Hormones; Lipid Droplets; Mass Spectrometry; Ovum; Phenols; Pyridines; Reproduction; Resource Allocation; Water Pollutants, Chemical

The analysis of lipid disruptive effects in invertebrates is limited by our poor knowledge of the lipid metabolic pathways. A recent study showed that tributyltin activated the ecdysteroid, juvenile hormone and retinoic X receptor signaling pathways, and disrupted the dynamics of neutral lipids in the crustacean Daphnia magna impairing the transfer of triacylglycerols to eggs and hence promoting their accumulation in post-spawning females. Tributyltin disruptive effects correlated with lower fitness for offspring and adults. The present study aims to addresses effects of existing compounds on storage lipids in post-spawning females and their health effects. D. magna individuals were exposed 12 chemicals that included vertebrate obesogens (tributyltin, triphenyltin, bisphenol A, nonylphenol, di-2-ethylhexyl phthalate), other contaminants known to affect arthropods (pyriproxyfen, fenarimol, methoprene, emamectin benzoate and fluoxetine), as well as the natural hormones methyl farnesoate and 20-hydroxyecdysone. Reproductive effects were also assessed. Quantitative changes in storage lipids accumulated in lipid droplets were studied using Nile red staining, which showed a close relationship with whole organism levels of triacylglycerols. Ten compounds altered storage lipids in a concentration related manner enhancing (tributyltin, bisphenol A, methyl farnesoate, pyriproxyfen and 20-hydroxyecdysone) or decreasing (nonylphenol, fenarimol, emamectin benzoate, methoprene and fluoxetine) their levels in post-spawning females. Eight compounds that altered lipid levels also had detrimental effects on growth and/or reproduction.

Topics: Animals; Daphnia; Fatty Acids, Unsaturated; Female; Juvenile Hormones; Phenols; Pyridines; Reproduction; Trialkyltin Compounds; Water Pollutants, Chemical

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.. 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.. 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.

Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Crustacea; Daphnia; Endocrine System; Fatty Acids, Unsaturated; Female; Fertility; Genes, Reporter; Life Cycle Stages; Ligands; Luciferases; Male; Molecular Sequence Data; Population Density; Pyridines; Sex Determination Processes; Sex Ratio; Signal Transduction; Transcription Factors

The effect of the insecticide, pyriproxyfen on early ovary synthesis was examined in the Gecarcinid land crab, Gecarcoidea natalis. Crabs were fed a mixture of either leaf litter and bait containing 0.5% (wt/wt) pyriproxyfen (experimental groups), or a mixture of leaf litter and a control bait containing no pyriproxyfen (control groups), at simulated baiting doses of 2 kg ha(-1) and 4 kg ha(-1), during the period in which G. natalis synthesises its ovaries. A third group of crabs were fed ad libitum either the bait containing 0.5% Pypriproxyfen or the control bait. Pyriproxyfen affected early ovary development in G. natalis. The ovaries from crabs in the experimental groups at all baiting levels had a higher total nitrogen content and dry mass than the ovaries from crabs in the control groups. Pyriproxyfen affected the histology of the ovaries. Ovaries from animals in the experimental groups were more mature, containing more previtellogenic and early vitellogenic oocytes, of a larger diameter, than the ovaries from crabs in the control groups. Significant amounts of pyriproxyfen accumulated within the midgut gland and ovary, the hypothesised target tissues, while minor amounts of pyriproxyfen was accumulated in the muscle, a hypothesised non target tissue. Pyriproxyfen may have stimulated early ovary development and induced synthesis of yolk protein by mimicking methyl farnesoate and thus causing endocrine disruption. Given this, pyriproxyfen should not be used to control invasive insects in environments where gecarcinid and other land crab species are present.

Topics: Animals; Brachyura; Endocrine Disruptors; Fatty Acids, Unsaturated; Female; Ovary; Pyridines

It was reported that males daphnid Daphnia magna that have been induced by methyl farnesoate exposure exhibit higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than females. Male neonates are also known to be induced by exposure to juvenile hormone analogs, such as fenoxycarb and pyriproxyfen. If these analogs can be used to produce male progeny, the biological and physiological studies of daphnid male would be progressed since the effects of these analogs were several hundred times higher than that of methyl farnesoate. Therefore, in the present study, it was investigated that the chemical sensitivity of male neonates induced by exposure to juvenile hormone (methyl farnesoate) and its analogs. The minimum concentrations of methyl farnesoate, fenoxycarb and pyriproxyfen to induce 100% male-reproduction were 200nM (50microg/l), 0.23nM (70ng/l) and 0.31nM (100ng/l), respectively. In addition, no reduction of relative reproduction was observed at the juvenoid concentrations in 24h exposure producing 100% male progeny. The median effective concentrations (EC50) of potassium dichromate for immobility of male neonates, established by a standardized method for investigating sensitivity to chemicals, were significantly higher (12-29%) than that of females at least after 24h exposure in all the male neonates induced by juvenoids used in this study (P<0.05). This study demonstrated that the male daphnids induced by exposure to juvenile hormone and its analogs exhibit similar chemical tolerance.

Topics: Animals; Animals, Newborn; Daphnia; Fatty Acids, Unsaturated; Juvenile Hormones; Male; Phenylcarbamates; Potassium Dichromate; Pyridines; Reproduction; Sex Differentiation

Terpenoid hormones function as morphogens throughout the animal kingdom and many of these activities are mediated through members of the retinoid X group of nuclear receptors (RXR; NR2B). In the present study, RXR was cloned from the water flea Daphnia magna, a primitive crustacean of the class Branchiopoda, and characterized with respect to phylogeny, developmental expression, and hormonal regulation. The full length daphnid RXR cDNA was cloned by initial PCR amplification of a cDNA fragment from the highly conserved DNA-binding domain followed by extension of the fragment using RACE PCR. The full length cDNA was 1888 base pairs in length and coded for a 400 amino acid protein that exhibited the five-domain structure of a nuclear receptor superfamily member. The RXR protein shared significant identity with other NR2B group members. Phylogenetic analyses of the ligand-binding domain of the receptor revealed that daphnid RXR clustered with RXR from decapod crustaceans on a branch of the phylogenetic tree that was distinct from RXRs known to bind retinoic acids and juvenile hormones. Daphnid RXR mRNA levels were greatest in embryos that were early in development and progressively declined through the initial five stages of embryo development. Adult females expressed higher levels of RXR mRNA than did males and exposure of females to the terpenoid mimic pyriproxyfen reduced RXR mRNA to levels approaching levels in males. RXR mRNA levels in males were refractory to pyriproxyfen. These results show that branchiopod crustaceans dynamically express RXR which should be evaluated as a candidate receptor for the terpenoid hormone methyl farnesoate which functions as a sex determinant in these organisms.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Daphnia; DNA, Complementary; Ecdysterone; Fatty Acids, Unsaturated; Female; Gene Expression Regulation, Developmental; Male; Molecular Sequence Data; Phylogeny; Pyridines; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Alignment

Hemoglobin levels are significantly elevated in the crustacean Daphnia magna by juvenoid hormones. The present study was undertaken to identify the specific globin (hb) genes that are induced by juvenoids and to identify putative juvenoid response elements (JREs) that may mediate this induction. Gene product of globin 2 (hb2), but not globin 1 and globin 3, was robustly elevated following juvenoid treatment of daphnids. A candidate JRE, located in the promoter of hb2, bound activated factor(s) in response to juvenoid treatment of daphnids. This hormone-induced protein:JRE interaction was robust when daphnids were reared at high oxygen tension but was inhibited when daphnids were reared under low pO2, implying that hypoxia might act to disrupt juvenoid-mediated endocrine signaling. The candidate JRE consists of a steroid/retinoid-response element-like core adjacent to a 5' AT-rich extension and thus bears resemblance to response elements that bind monomeric nuclear receptors. The induction of hb2 mRNA levels by juvenoid treatment occurred rapidly (within 4 h of exposure) and was not attenuated by treatment of daphnids with cycloheximide. In contrast, cycloheximide treatment did block hormone-mediated elevations in hemoglobin protein levels. Thus, induction of hb2 by juvenoids was not dependent upon the synthesis of secondary transcription factors that bound the JRE but was likely due to activation of the gene directly by the juvenoid-receptor complex. Affinity pull-down experiments with nuclear proteins extracted from juvenoid-treated daphnids using the JRE as bait yielded a 52kDa candidate for a monomeric nuclear receptor in D. magna that may mediate the regulatory activity of juvenoids.

Topics: Animals; Base Sequence; Cycloheximide; Daphnia; Fatty Acids, Unsaturated; Hemoglobins; Juvenile Hormones; Molecular Sequence Data; Promoter Regions, Genetic; Pyridines; Receptors, Cytoplasmic and Nuclear; Response Elements; RNA, Messenger; Signal Transduction

Commensurate with the decline in many crustacean populations has been an accumulation in reports of sexually ambiguous individuals within these populations. The cause of gynandromorphism or intersex among crustaceans is unknown. We show that gynandromorphism in the branchiopod crustacean Daphnia magna is initiated by the sex-determining hormone methyl farnesoate when levels of the hormone are intermediate between low levels that stimulate the production of broods containing all female offspring and high levels that stimulate the production of broods of all male offspring. The incidence of hormonally-induced gynandromorphism was low (0.14% at the maximum stimulatory hormone concentrations) but was significantly increased (46-fold) when the animals were hormone-treated at 30 degrees C. Some environmental chemicals also can stimulate the gynandromorphic phenotype as we demonstrated with the insecticide pyriproxyfen. Gynandromorphism occurs due to inadequate signaling of male-sex determination since: a) gynandromorphs did not occur in a population that was producing only female offspring; and, b) conditions that stimulated gynandromorphism also reduced the incidence of male offspring. We suggest that male sex determination normally occurs prior to the first embryonic cleavage. Elevated temperature may alter the timing of sex determination such that methyl farnesoate signaling occurs after the first embryonic cleavage and bilateral gynandromorphism occurs as a consequence of signaling to only one of the daughter cells. These results demonstrate that environmental factors can cause aberrant sex determination via perturbations in methyl farnesoate signaling.

Topics: Animals; Daphnia; Disorders of Sex Development; Environment; Fatty Acids, Unsaturated; Female; Incidence; Male; Phenotype; Pyridines; Sex Determination Processes; Temperature

U.S. Environmental Protection Agency is charged with developing a screening and testing paradigm for detecting endocrine toxicity of chemicals that are subject to regulation under the Food Quality Protection and the Safe Drinking Water Acts. In this study, we developed and evaluated a screening assay that could be employed to detect juvenoid-related endocrine-modulating activity in an invertebrate species. Juvenoid activity, anti-juvenoid activity, and juvenoid potentiator activity of chemicals was assessed using the water flea Daphnia magna. Male sex determination is under the regulatory control of juvenoid hormone, presumably methyl farnesoate, and this endpoint was used to detect juvenoid modulating activity of chemicals. Eighteen chemicals were evaluated for juvenoid agonist activity. Positive responses were detected with the juvenoid hormones methyl farnesoate and juvenile hormone III along with the insect growth regulating insecticides pyriproxyfen, fenoxycarb, and methoprene. Weak juvenoid activity also was detected with the cyclodiene insecticide dieldrin. Assays performed repetitively with compounds that gave either strong positive, weak positive, or negative response were 100% consistent indicating that the assay is not prone to false positive or negative responses. Five candidate chemicals were evaluated for anti-juvenoid activity and none registered positive. Four chemicals (all trans-retinoic acid, methoprene, kinoprene, bisphenol A) also were evaluated for their ability to potentiate the activity of methyl farnesoate. All registered positive. Results demonstrate that an in vivo assay with a crustacean species customarily employed in toxicity testing can be used to effectively screen chemicals for juvenoid-modulating activity.

Topics: Animals; Benzhydryl Compounds; Daphnia; Ecdysteroids; Endocrine System; Endpoint Determination; Fatty Acids, Unsaturated; Female; Juvenile Hormones; Male; Methoprene; Pesticides; Phenols; Phytol; Pyridines; Reproducibility of Results; Sex Differentiation; Toxicity Tests; Tretinoin; United States; United States Environmental Protection Agency

Methyl farnesoate is a juvenoid hormone that regulates a variety of processes in crustaceans including male sex determination among daphnids (Branchiopoda, Cladocera). The synthetic juvenoids pyriproxyfen and fenoxycarb mimic the action of methyl farnesoate in daphnids. In the present study we tested the hypothesis that juvenoids also can regulate ecdysteroid activity in a crustacean (Daphnia magna). Methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. Exposure of ecdysteroid-responsive cells to 20-hydroxyecdysone reduced cell proliferation and increased mRNA levels of the ecdysone receptor and its partner protein ultraspiracle. Co-treatment of cells with the juvenoid pyriproxyfen attenuated all of these ecdysteroid mediated responses. While juvenoids functioned as anti-ecdysteroids in both intact embryos and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. The combined effects of pyroproxyfen with the ecdysteroid synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone were evaluated in an effort to discern whether the action of the juvenoids were additive with those of know anti-ecdysteroids. The anti-ecdysteroid effects of pyriproxyfen were non-additive with those of either anti-ecdysteroid. Rather, joint effects conformed to a model of synergy. These results demonstrated that juvenoids elicit anti-ecdysteroidal activity in a crustacean through a unique mechanism of action. A model involving receptor partner deprivation is proposed that explains the synergistic interactions observed.

Topics: Animals; Cell Proliferation; Cells, Cultured; Daphnia; DNA Primers; Drosophila; Ecdysteroids; Ecdysterone; Embryo, Nonmammalian; Fatty Acids, Unsaturated; Gene Expression Regulation; Juvenile Hormones; Models, Biological; Phenylcarbamates; Pyridines; Pyrimidines; Receptors, Steroid; RNA, Messenger; Sex Determination Processes; Signal Transduction; Testosterone