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Ovulation stimulating substances in Drosophila biarmipes males: their origin, genetic variation in the response of females, and molecular characterization.

Ovulation responses of Drosophila biarmipes females to an injection of methanolic extract from conspecific males vary with the strains of females. This strain difference seems to be controlled by a small number of autosomal genes, with low responsiveness being recessive. Strangely, all D. biarmipes strains show a high level of ovulation after mating. We pursued the reason for this discrepancy and found that D. biarmipes males produce two different substances with ovulation-inducing activity. One of them is derived from the accessory glands and effective in females of all strains. Another originates in the ejaculatory duct and is inactive in some strains. In an active HPLC fraction of the ejaculatory duct extract, we found a peptide consisting of 32 amino acids. Its C-terminal region has a striking similarity to the sex-peptide of D. melanogaster, but the N-terminal region was entirely different. Evolutionary implications of these findings are discussed.

Amino Acid Sequence↗

Purification and characterization of a juvenile hormone binding protein from the hemolymph of the fourth instar tobacco hornworm, Manduca sexta.

A protein which binds the insect juvenile hormone has been isolated from the hemolymph of the fourth instar tobacco hornworm, Manduca sexta (Lepidoptera). Bioassay and chemical characterization of the bound ligand from the purified binding protein indicates that this molecule is the primary macromolecule responsible for juvenile hormone transport in the hemolymph of this insect. The juvenile hormone binding protein has been purified using gel filtration, ion exchange chromatography and preparative polyacrylamide gel electrophoresis. The protein is a single polypeptide chain of about 28,000 daltons with a sedimentation coefficient of 2.2S and an isoelectric point of 5.0. Binding analysis using a hydroxyapatite batch assay indicates that the juvenile hormone binding protein has one binding site with a Ka of 1.2 times 10(7) M-1 at 4 degrees C.

Animals↗

[Analogs of juvenile hormones in the control of insects of medical or sanitary importance].

The growth and moulting or immature insects is related to three main groups of hormones : cerebral, ecdysonal and juvenile. Juvenile hormone analogues mark a new stage in the control of detrimental insects, and have significant advantages over the conventional insecticides. In order to establish the effectiveness of juvenile hormone analogues a series of biotests were performed : the wax pupal test, injections, systemic and topical applications, the treatment of food, pulverizations and exposure to vapors. In the laboratory, the efficiency of the analogues was tested against the following genera : Anopheles, Aedes, Culex, Culiseta, Musca, Stomoxys and Blattella, obtaining several effects, such as : lengthening of the larval stages, blocking of metamorphosis and ecdysis, blocking of the emergence of adults, morphogenetic ovicide, sterilizing and lethal effects, etc. Exploratory trials were performed in the field with compounds whose effectiveness had been demonstrated, against some Aedes, Musca, Stomoxys, Pediculus and Bovicola species. Laboratory experiments showed, however, that the insects might become resistant to juvenile hormone analogues.

Aedes↗

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta.

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.

Adipose Tissue↗

Discovery of insect anti-juvenile hormones in plants.?2U.

Two simple chromenes with anti-JH activity have been isolated and identified from the bedding plant Ageratum houstoianum. By contact and fumigation these compounds induce precocious metamorphosis and sterilization in several hemipteran species of insects. Certain holometabolous species are sterilized, forced into diapause, or both. Each of these biological actions is equivalent to removal of the corpora allata, which produce the JH's, and is reversible by treatment with exogenous JH. Thus, the action of these compounds is to stop the production or depress the titer of the JH's. To our knowledge, this is the first discovery of anti-JH, and we hope it will guide the way to the emergence of a fourth generation of safe and insect-specific pesticides.

Female↗

Cloning of a putative juvenile hormone-responsive storage protein gene from the tobacco budworm, Heliothis virescens.

A cDNA clone with 78% amino acid identity to a basic juvenile hormone (JH)-suppressible hemolymph protein from the cabbage looper, Trichoplusia ni, was isolated from the tobacco budworm, Heliothis virescens. This clone was obtained upon screening a cDNA library derived from larval fat body of a pesticide resistant strain of H. virescens with a cDNA probe for Drosophila melanogaster glutathione S-transferase. By comparison with other insect storage proteins, this clone was predicted to be part of an approximately 2,300 nucleotide (nt) cDNA, of which 691 nt were isolated and sequenced. The partial cDNA clone hybridizes to a RNA of approximately 2,370 nt in H. virescens. Treatment with a juvenoid (2-[1-methyl-2-(4-phenoxyphenoxy)ethoxy] pyridine; pyriproxifen) leads to a decrease in RNA levels of this putative hemolymph storage protein in early fifth stadium larvae of H. virescens, prior to commitment. In contrast, treatment in late fifth stadium (after commitment to pupal development) leads to an increase in the RNA level of this JH-responsive gene. This is the first report of both induction and suppression of storage protein RNA levels in the same stadium. We have given this gene the designation Hv-SP4 (H. virescens, storage protein 4; accession no. U48594). Genetic segregation analysis of restriction fragment length polymorphisms (RFLPs) defined by Hv-SP4 has shown that it is the product of a single-copy, Mendelian, autosomal gene.

Amino Acid Sequence↗

Insect physiology: the emerging story of ecdysis.

The discovery of a new insect peptide hormone that triggers ecdysis - shedding of an old cuticle - has revealed hidden layers of intricacy about an insect behavior previously thought to be mediated by a single neuropeptide.

Animals↗

Physiological effectors of hyperglycemic neurohormone biosynthesis in an insect.

Biosynthesis of hypertrehalosemic hormone (HTH) by isolated corpora cardiaca (CC) from Blaberus discoidalis cockroaches was measured for the 3-week period following adult ecdysis using an in vitro bioassay based on the incorporation of [3H]tryptophan. Synthetic rates for both males and females were highest following the molt and declined with age. Rates of HTH secretion and levels of stored HTH followed a similar pattern. We found no neural or endocrine brain effects or photophase influence on HTH synthesis. Synthesis was unaffected by treatment with 20-hydroxyecdysone or the juvenile hormone analog methoprene. In starved insects, HTH synthesis doubled and secretion increased 66% relative to fed controls.

Animals↗

Juvenile hormone activity of some glycosidic juvenogens.

In a search for new hormonogen compounds related to insect juvenile hormones we have prepared and bioassayed several beta-D-glucosides of some selected secondary juvenoid alcohols. Due to localization of the activating enzyme beta-D-glucopyranosidase in the epithelium and in the lumen of the digestive tract, the glycosidic juvenogens exhibit juvenile hormone activity in insects only when ingested. They are completely inactive in topical application as well as when injected into the haemolymph. It is suggested that the highly specific and selective action of the hydrophilic, nonvolatile, glycosidic juvenogens could be of practical importance.

Animals↗

Regulation of juvenile hormone esterase gene transcription by juvenile hormone.

Juvenile hormone (JH) is a major hormone regulating insect development. We have obtained a cDNA and a genomic clone for juvenile hormone esterase (JHE), the enzyme that is involved in the degradation of juvenile hormone and which is critical for insect development. Analysis of the regulation of JHE during the final larval stadium in the cabbage looper, Trichoplusia ni, showed that the JHE mRNA levels are maximal on days 2 and 4 of the final stadium. Nuclear run-on analyses demonstrated that changes in JHE mRNA levels are primarily due to changes in the transcription rate of the gene, which may be a single copy in the genome. Treatment with a JH analog resulted in induction of JHE gene transcription, which could be detected within three hours after treatment. Salient features present in the 5' flanking region of this JH-sensitive gene are presented, including the presence of sequences closely resembling binding sites for members of the family of nuclear receptors. This report is the first direct demonstration, by nuclear run-on analysis, of JH induction of gene transcription.

Amino Acid Sequence↗

Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite schistosoma mansoni.

The insect moulting hormones, ecdysone and 20 hydroxyecdysone, were detected by the combined use of radioimmunoassay and high performance liquid chromatography in the human parasite Schistosoma mansoni. On day 11 after infection only the ecdysone form is present, but, on day 40 after infection the ratio between ecdysone and 20 hydroxyecdysone changes with anatomic localization of the adult worms in mammalian host. In the eggs, the ratio of these two hormones is identical to the ratio found in sexually mature worms located in mesenteric veins. These data demonstrate for the first time that S. mansoni synthesizes the steroid hormones ecdysone and 20 OH ecdysone which are potent molecules in stimulating growth and vitello-genesis of this gonochoric trematode.

Aging↗

Isolation, characterization and developmental expression of the ecdysteroid-induced E75 gene of the wax moth Galleria mellonella.

Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone did not encode the N-terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75A and GmE75B, which differed in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA was induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.

Amino Acid Sequence↗

Nucleotide sequence, structure and developmental regulation of LHP82, a juvenile hormone-suppressible hexamerin gene from the waxmoth, Galleria mellonella.

We cloned and sequenced a composite cDNA corresponding to the 2.6 kb last instar-specific, juvenile hormone-suppressible Lhp82 mRNA from Galleria mellonella. The identity of the cDNA was confirmed by N-terminal amino acid sequencing of the purified Lhp82 subunit. In addition, we sequenced all coding regions and most of the intronic DNA as well as 1300 nucleotides of 5' flanking DNA from the Lhp82 gene. The eight exons of the Lhp82 gene specify a pre-protein of 706 residues, including the signal peptide of 18 amino acids. The deduced amino acid sequence of Lhp82 contains four potential N-glycosylation sites, and the calculated isoelectric point and molecular weight of secreted Lhp82 are pI = 6.43 and 79.9 kDa, respectively. Inspection of the 5' flanking and intronic regions of Lhp82 DNA revealed a 301 nucleotide cassette in intron 6 that appears to be a recently inserted repetitive element. We also performed Northern blot and nuclear run-off transcription experiments in order to determine the basis for Lhp82 gene inactivity after day 2 of the pupal stage. The results of these studies indicate that Lhp82 transcription is permanently shut off by the ecdysteroid pulse which occurs in the absence of juvenile hormone beginning 24 h post-pupation.

Amino Acid Sequence↗