methyl-6-7-10-11-bis(epoxy)-3-7-11-trimethyl-2-dodecenoate and farnesoic-acid

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.. A sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).. The method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10-20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.. The proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.

Topics: Aedes; Animals; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Clinical Chemistry Tests; Drosophila melanogaster; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Female; Fluorescent Dyes; Limit of Detection; Oxadiazoles; Sesquiterpenes; Spectrometry, Fluorescence

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 corpus allatum (CA) of adult female Ceratitis capitata produces methyl palmitate (MP) in vitro, in addition to JHB(3) and JH III. Biosynthesized MP migrates on TLC and co-elutes from RP-18 HPLC with synthetic MP. Its identity is verified herein by GCMS. MP production is up-regulated twofold by mevastatin, an inhibitor of mevalonic acid-dependent isoprene biosynthesis. Fosmidomycin, an inhibitor of mevalonic acid-independent isoprene synthesis in graminaceous plants, up-regulates MP synthesis by about fourfold. However, it does not depress JHB(3) biosynthesis concurrently. This suggests that the initial enzyme(s) in the conversion of 1-deoxy-xylulose 5-phosphate to isoprene is presumably present in C. capitata, but is inhibited by fosmidomycin, and this inhibition diverts precursors to MP synthesis. Phytol, an acyclic diterpene, might be suppressing isoprene biosynthesis by CA, thereby resulting in a fourfold increase in the MP biosynthesis. Linolenic acid is an end-product and its presence in incubation media up-regulates MP biosynthesis by twofold, presumably due to the feedback diversion to biosynthesis of C(16:0) and its methyl ester. Biosynthesis of MP is markedly depressed after mating, while otherwise maintained at significantly higher levels in virgin females. MP biosynthesis is significantly reduced in virgin females by direct axonal control but is less consistent after mating.

Topics: Animals; Ceratitis capitata; Chromatography, Thin Layer; Corpora Allata; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Female; Fosfomycin; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation; Linoleic Acid; Lovastatin; Palmitates; Phytol; Sesquiterpenes; Sexual Behavior, Animal; Tritium

Adult female Drosophila melanogaster corpus allatum (CA) synthesize JHB3 from endogenous and exogenous precursors in vitro. We present evidence supporting the thesis that biosynthesis proceeds from precursor FA via initial epoxidation and terminal methylation on the basis of the following: (1) Methyl farnesoate is not epoxidized to JHIII or JHB3; (2) Authentic JHIII is not epoxidized to JHB3; and (3) FABE is markedly metabolized to JHB3. Cerebral allatostatic factors act at some stage subsequent to FA and this precursor is not normally rate-limiting. Additionally, neural inhibition from the brain acts at some biosynthetic step prior to FA.

Topics: Animals; Corpora Allata; Drosophila melanogaster; Epoxy Compounds; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Female; Juvenile Hormones; Male; Methylation

Juvenile hormone (JH) is a major regulator of insect development and reproduction and its titer is determined largely by central nervous system regulation of JH synthesis by the corpora allata. To establish the basis for a molecular genetic dissection of the neuroendocrine system responsible for modulating JH titer, a radiochemical assay was utilized to examine JH synthesis in vitro by the isolated corpus allatum as well as the regulation of this synthesis by brain extracts of wild-type and apterous mutant Drosophila melanogaster females during reproductive maturation. JH production by glands of wild-type females increases in parallel with the progress of ovarian maturation, the major product of the adult corpus allatum being juvenile hormone 3 bis-epoxide (JHB3). Gland activity appears to be regulated by both the availability of JH precursors and the level of terminal oxidase(s) in the JH biosynthetic pathway. The brain contains an allatostatic factor, that is transmitted to the glands via nervous connections. Allatostatin production in the brain appears to be positively regulated by JHB3. Adult corpora allata from the mutants ap4 and ap56f synthesize very low levels of JH; additionally, brains of ap56f homozygotes lack allatostatic activity.

Topics: Animals; Biological Assay; Brain; Corpora Allata; Drosophila melanogaster; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Female; Gene Expression Regulation; Infertility, Female; Juvenile Hormones; Mutation; Neurosecretory Systems; Vitellogenesis