ecdysterone and pyriproxyfen

Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action in Aedes aegypti mosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fed Ae. aegypti females but blocked the development of primary follicles at Christopher's Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Krüppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, including HR3 and E75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism in Ae. aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF.

Topics: Aedes; Animals; Culicidae; Ecdysterone; Fat Body; Female; Glycogen; Insect Proteins; Insecticides; Juvenile Hormones; Methoprene; Ovary; Pyridines; RNA Interference; Sterilization; Triglycerides

A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis.

Topics: Amino Acid Sequence; Amino Acid Transport Systems; Amino Acids, Neutral; Animals; Coleoptera; Ecdysterone; Feces; Gene Expression Regulation, Developmental; Insect Proteins; Juvenile Hormones; Molecular Sequence Data; Phylogeny; Pupa; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sequence Homology, Amino Acid

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

In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome.

Topics: Animals; Bees; Blotting, Northern; Blotting, Southern; Blotting, Western; Ecdysterone; Female; Gene Dosage; Juvenile Hormones; Male; Ovary; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; Vitellogenins

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