methyl-farnesoate and farnesoic-acid

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again "competent" to synthesize JH, which plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or -24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the -24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the -24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.

Topics: Aedes; Animals; Corpora Allata; Ecdysterone; Fatty Acids, Unsaturated; Juvenile Hormones; Metamorphosis, Biological; Methyltransferases; Molting; Pupa

Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Fatty Acids, Unsaturated; Female; Molecular Sequence Data; Nephropidae; Ovary; Reproduction; Retinoid X Receptors; RNA, Messenger; Sequence Alignment; Sequence Analysis, Protein

MicroRNAs (miRNAs) play essential roles in plant and animal development, but the cause and effect of miRNA expression divergence between closely related species and in interspecific hybrids or allopolyploids are unknown. Here, we show differential regulation of a miR163-mediated pathway in allotetraploids and their progenitors, Arabidopsis thaliana and Arabidopsis arenosa. miR163 is a recently evolved miRNA in A. thaliana and highly expressed in A. thaliana, but its expression was undetectable in A. arenosa and repressed in resynthesized allotetraploids. Repression of A. arenosa MIR163 (Aa MIR163) is caused by a weak cis-acting promoter and putative trans-acting repressor(s) present in A. arenosa and allotetraploids. Moreover, ectopic Aa MIR163 precursors were processed more efficiently in A. thaliana than in resynthesized allotetraploids, suggesting a role of posttranscriptional regulation in mature miR163 abundance. Target genes of miR163 encode a family of small molecule methyltransferases involved in secondary metabolite biosynthetic pathways that are inducible by a fungal elicitor, alamethicin. Loss of miR163 or overexpression of miR163 in mir163 mutant plants alters target transcript and secondary metabolite profiles. We suggest that cis- and trans-regulation of miRNA and other genes provides a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses in Arabidopsis-related species and allopolyploids.

Topics: Alamethicin; Arabidopsis; Arabidopsis Proteins; DNA, Complementary; Epigenesis, Genetic; Fatty Acids, Unsaturated; Gene Expression Regulation, Plant; Gene Transfer, Horizontal; Genes, Plant; Genome, Plant; Methyltransferases; MicroRNAs; Mutagenesis, Insertional; Plant Leaves; Polyploidy; Promoter Regions, Genetic; RNA, Plant; Sequence Analysis, DNA; Species Specificity; Stress, Physiological

We report on juvenile hormone (JH) biosynthesis from long-chain intermediates by specific reproductive tissues and the corpora allata (CA) prepared from adult longhorned beetles, Apriona germari. The testes, male accessory glands (MAGs), ovaries, and CA contained the long-chain intermediates in the JH biosynthetic pathway, farnesoic acid (FA), methyl farnesoate (MF), and JH III. The testes and ovaries, but not CA, produced radioactive JH III after the addition of (3)H-methionine and, separately, unlabeled methionine, to the incubation medium. We inferred that endogenous FA is methylated to MF in the testes and ovaries. Addition of farnesol led to increased amounts of FA in the testes, MAGs, ovaries, and CA, indicating oxidation of farnesol to FA. Addition of FA to incubation medium yielded increased JH III, again indicating methylation of FA to MF in the testes, MAGs, ovaries, but not CA. Addition of MF to incubation medium also led to JH III, from which we inferred the epoxidation of MF to JH III. JH biosynthesis from farnesol in the testes, MAGs, and ovaries of A. germari proceeds via oxidation to FA, methylation to MF, and epoxidation to JH III. This is a well-known pathway to JH III, described here for the first time in reproductive tissues of longhorned beetles. © 2010 Wiley Periodicals, Inc.

Topics: Animals; Coleoptera; Corpora Allata; Fatty Acids, Unsaturated; Female; Gonads; Juvenile Hormones; Male

Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. Farnesoic acid O-methyltransferase (FAMeT) is thought to be an important enzyme in the JH biosynthetic pathway, catalyzing methylation of farnesoic acid (FA) to methyl farnesoate (MF). Previous evidence in other insects suggested that FAMeT is rate limiting and regulated by a neuropeptide family, the allatostatins. A full-length cDNA encoding a 296 amino acid putative FAMeT has been isolated. A recombinant (r)FAMeT was cloned, expressed and a specific antiserum generated. rFAMeT was assayed for enzymatic activity using a radiochemical assay. In this assay, no activity was detected either with rFAMeT alone or when added to a corpus allatum CA extract. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of Drosophila melanogaster ring gland. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on sesquiterpenoid biosynthesis. Drome AST appears to have a select effect on JH bisepoxide biosynthesis and not MF or JHIII. Additional analysis of MF, JHIII and JHB3 release in strains with a deficiency or decrease of FAMeT compared to wild type shows no significant decrease in MF, JHIII or JH bisepoxide synthesis. Deficiency strains that reduce the level of FAMeT showed reduced longevity relative to wildtype but this result may be due to other genetic influences.

Topics: Amino Acid Sequence; Animals; Corpora Allata; Drosophila melanogaster; Drosophila Proteins; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Female; Gene Deletion; Juvenile Hormones; Larva; Longevity; Male; Methyltransferases; Molecular Sequence Data; Neuropeptides; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid

The in vivo ligand-binding function and ligand-binding activity of the Drosophila melanogaster retinoid-X receptor (RXR) ortholog, ultraspiracle, toward natural farnesoid products of the ring gland were assessed. Using an equilibrium fluorescence-binding assay, farnesoid products in the juvenile hormone (JH) biosynthesis pathway, and their epoxy derivatives, were measured for their affinity constant for ultraspiracle (USP). Farnesol, farnesal, farnesoic acid and juvenile hormone III exhibited high nanomolar to low micromolar affinity, which in each case decreased upon addition of an epoxide across a double bond of the basic farnesyl structure. Similar analysis of the substitution on C1 of methyl ether, alcohol, aldehyde, and carboxylic acid showed that each conferred weaker affinity than that provided by the methyl ester. Attention was thus focused for a ring-gland farnesoid product that possesses the features of methyl ester and lack of an epoxide. A secreted product of the ring gland, methyl farnesoate, was identified possessing these features and exhibited an affinity for ultraspiracle (K(d) = 40 nm) of similar strength to that of RXR for 9-cis retinoic acid. Mutational analysis of amino acid residues with side chains extending into the ligand-binding pocket cavity (and not interacting with secondary receptor structures or extending to the receptor surface to interact with coactivators, corepressors or receptor dimer partners) showed that the mutation C472A/H475L strongly reduced USP binding to this ring gland product and to JH III, with less effect on other ring-gland farnesoids and little effect on binding by (the unnatural to Drosophila) JH I. Along with the ecdysone receptor, USP is now the second arthropod nuclear hormone receptor for which a secreted product of an endocrine gland that binds the receptor with nanomolar affinity has been identified.

Topics: Animals; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Farnesol; Fatty Acids, Unsaturated; Ligands; Models, Molecular; Mutation; Protein Binding; Recombinant Proteins; Retinoid X Receptors; Sesquiterpenes; Transcription Factors

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.

Topics: Animals; Astacoidea; Eye; Fatty Acids, Unsaturated; Gene Expression Regulation; Hemolymph; Immunohistochemistry; Male; Neuropeptides; Oligopeptides

Previous studies have demonstrated that the synthesis of juvenile hormone (JH) by the isolated corpora allata (CA) complex in vitro as well as the JH titer in the yellow fever mosquito Aedes aegypti are elevated before feeding and low after a blood meal. In the present study, we used an in vitro radiochemical assay to analyze the effect of farnesoic acid (FA) and Aedes allatotropin (Aedes-AT) on the biosynthesis of JH and methyl farnesoate (MF) by the isolated CA complex of A. aegypti adult female. CA complex from day-0 females (0-1 h after emergence) exhibited a low basal juvenile hormone III (JH III) biosynthetic activity and did not respond to either allatotropic or FA stimulation. However, incubation of CA complexes from newly emerged females with Aedes-AT plus FA resulted in very high production of JH III. This is the first report suggesting that allatotropin makes corpora allata in newly emerged females capable for JH biosynthesis. When we studied CA complexes dissected from females 1 day after emergence, the stimulatory action of Aedes-AT was strong and dose-dependent, with maximum stimulation in the range of 10(-8)-10(-9) mol l(-1), suggesting that Aedes-AT is indeed a true allatotropin (a molecule with allatotropic activity) in A. aegypti. The addition to the culture medium of 40 micro mol l(-1) FA, a JH precursor, resulted in a 9-fold increase in JH III biosynthesis in 2-, 4- and 6-day-old sugar-fed females. The two major labeled products synthesized by the stimulated CA complex were identified as JH III and MF by RP-HPLC and GC-MS. Treatment of CA complexes with FA, but not Aedes-AT, resulted in an increase in MF. Application of both Aedes-AT and FA to the CA complexes of 2-, 4- and 6-day-old females resulted in the same effects as FA alone. These data suggest that in sugar-fed females, FA and Aedes-AT exert different effects on the terminal steps in JH biosynthesis.

Topics: Aedes; Age Factors; Analysis of Variance; Animals; Corpora Allata; Fatty Acids, Unsaturated; Female; In Vitro Techniques; Insect Hormones; Juvenile Hormones; Mass Spectrometry; Neuropeptides

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.

Topics: Aedes; Animal Nutritional Physiological Phenomena; Animals; Blood; Corpora Allata; Fatty Acids, Unsaturated; Female; Imidazoles; Insect Hormones; Juvenile Hormones; Neuropeptides; Sesquiterpenes; Sexual Behavior, Animal; Time Factors

A series of farnesoic acid derivatives was prepared and their morphogenic regulatory activities were evaluated. Their inhibitory activities against yeast cell growth and yeast-to-hypha transition examined in Candida albicans cells are dependent upon the chain length as well as the substitution patterns on the isoprenoid template. The preliminary structure-activity relationship of these compounds is described to elucidate the essential structural requirements.

Topics: Candida albicans; Cell Division; Fatty Acids, Unsaturated; Hyphae; Inhibitory Concentration 50; Structure-Activity Relationship; Yeasts

The neuropeptide mandibular organ (MO)-inhibiting hormone (MO-IH), synthesized and secreted from the X-organ-sinus-gland complex of the eyestalk, regulates the biosynthesis of the putative crustacean juvenile hormone, methyl farnesoate (MF). Using radiolabelled acetate as a precursor for isoprenoid biosynthesis, farnesoic acid (FA), farnesol, farnesal, MF and geranyl geraniol were detected in MOs cultured for 24 h. Treatment of MOs with extract of sinus gland inhibited the final step of biosynthesis of MF, catalysed by FA O-methyltransferase. Additionally, treatment of MOs with purified MO-IH exhibited a dose-dependent inhibition of this final step of MF synthesis. The extent of this inhibition was dependent on the ovary stage of the MO-donor animal, being maximal in MOs from animals in the early stages of ovarian development. Assay of FA O-methyltransferase activity, using [3H]FA in the presence of S-adenosyl-l-methionine, demonstrated that the enzyme was located in the cytosolic fraction of MOs and was inhibited by incubation of MOs with MO-IH prior to preparation of subcellular fractions. For cytosolic preparations taken from vitellogenic animals, both Vmax and Km were appreciably lower than for those taken from non-vitellogenic animals. Conversely, eyestalk ablation of early-vitellogenic animals, which removes the source of MO-IH in vivo, resulted in enhancement of the cytosolic FA O-methyltransferase activity. Although both Vmax and Km show an appreciable increase upon eyestalk ablation, the increased enzyme activity is probably reflected by the fact that Vmax/Km (an approximate indication of kcat) has increased 5-fold. The combined evidence demonstrates that MO-IH inhibits FA O-methyltransferase, the enzyme which catalyses the final step of MF biosynthesis in MOs.

Topics: Animals; Brachyura; Fatty Acids, Unsaturated; Female; In Vitro Techniques; Juvenile Hormones; Kinetics; Methyltransferases; Neuropeptides; Polyisoprenyl Phosphates; Subcellular Fractions