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Temporal analysis of ecdysteroidogenic activity of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori.

The cellular mechanism underlying ecdysteroidogenesis during the fourth larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid biosynthetic activity of the prothoracic glands, cAMP accumulation of the gland cells, the in vitro release of prothoracicotropic hormone (PTTH), etc. According to the differential responsiveness of prothoracic glands to PTTH, dibutyryl cAMP (dbcAMP), and 1-methyl-3-isobutylxanthine (MIX), the following different stages were classified and changes in PTTH signal transduction were assumed. During the first stage (between days 0 and 1), the glands showed low basal and PTTH-stimulated activities in both cAMP accumulation and ecdysteroidogenesis, and PTTH release in vitro was maintained at low but detectable levels, implying that a low but sustained PTTH signal may be transduced to prothoracic gland cells. On day 1.5, when low basal ecdysteroid production of the prothoracic glands was being maintained, both the responsiveness of glands to the stimulation of PTTH and PTTH release in vitro dramatically increased, indicating greatly increased PTTH transduction. On day 3 (when the basal ecdysteroidogenesis became maximal) and afterwards, high PTTH release in vitro was maintained, but the gland showed no response to PTTH, implying that the refractoriness of gland cells to PTTH may occur at this stage. We assume that the development-specific changes in PTTH signal transduction during the penultimate larval instar may play a critical role in regulating changes in ecdysteroidogenesis of the prothoracic glands.

1-Methyl-3-isobutylxanthine↗

Molecular cloning of three distinct cDNAs, each encoding a different adipokinetic hormone precursor, of the migratory locust, Locusta migratoria. Differential expression of the distinct adipokinetic hormone precursor genes during flight activity.

Three distinct cDNAs encoding the preproadipokinetic hormones I, II, and III (prepro-AKH I, II, and III), respectively, of Locusta migratoria have been isolated and sequenced. The three L. migratoria AKH precursors have an overall architecture similar to that of other precursors of the AKH/red pigment-concentrating hormone (RPCH) family identified so far. The AKH I and II precursors of L. migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least homologous to the Manduca sexta, Drosophila melanogaster, and Carcinus maenas AKH/RPCH precursors, we favor the opinion that the L. migratoria AKH III precursor is evolutionary more related to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors than to the AKH I and II precursors of S. gregaria, S. nitans, or L. migratoria. In situ hybridization showed signals for the different AKH mRNAs to be co-localized in cell bodies of the glandular lobes of the corpora cardiaca. Northern blot analysis revealed the presence of single mRNA species encoding the AKH I precursor (approximately 570 bases), AKH II precursor (approximately 600 bases), and AKH III precursor (approximately 670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (approximately 2.0 times each) and the AKH III mRNA (approximately 4.2 times) in the corpora cardiaca.

Amino Acid Sequence↗

Characterization of the AeaHP gene and its expression in the mosquito Aedes aegypti (Diptera: Culicidae).

The sequence and tissue expression of the gene encoding a peptide hormone Aea-HP-I, known to inhibit host-seeking behavior, has been characterized for the yellowfever mosquito, Aedes aegypti (L.). The open reading frame reveals a prepropeptide that would be processed into three identical peptides. The gene contains four short introns and exists as a single genomic copy. Transcripts of the gene were present in the brain, terminal ganglion, and midgut of adults, and in females, its expression profile differed for each tissue before and during a reproductive cycle. Peptides resulting from this expression were identified in the female tissues by immunoassays. Numerous neurosecretory cells and neurons in the nervous system were immunostained by an Aea-HP-I antiserum. Hundreds of endocrine cells were stained similarly in the midgut, thus contributing to the 10 times greater amount of immunoreactive peptide in an abdomen than in a head, as determined with an Aea-HP-I radioimmunoassay. Based on these results, neurosecretory cells and midgut endocrine cells are likely sources of Aea-HPs shown to reach highest hemolymph titer at the same time as host seeking is inhibited in female Ae. aegypti during a reproductive cycle.

Aedes↗

Neurohormone D induces ionic current changes in cockroach central neurones.

The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), increases the frequency of spontaneous activity in dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana. The increase in spike frequency is accompanied by changes in the shape and the amplitude of the single action potentials, e.g. a more pronounced afterhyperpolarization. Effects of NHD on membrane currents were investigated in these DUM cells with whole-cell voltage-clamp measurements. A voltage-independent Ca2+ current flowing at the resting potential (ICa,R) was found. NHD, at nanomolar concentrations, enhanced this ICa,R in a concentration-dependent manner. 0.1 mM Cd2+ markedly reduced ICa,R and in this case ICa,R was hardly potentiated by NHD. In the presence of NHD a fast activating Ca(2+)-dependent K+ current sensitive to charybdotoxin and to low concentrations of tetraethylammonium was augmented. The enhanced afterhyperpolarization of action potentials can be accounted for by the increase in the Ca(2+)-dependent K+ current. The changes of the membrane currents induced by NHD are discussed with respect to further effects on the spike pattern and in relation to the previously described mode of action of AKH-peptides in other preparations.

Animals↗

Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta.

The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

Animals↗

Primary role of adipokinetic hormone in the formation of low density lipophorin in locusts.

It was demonstrated that the primary action of adipokinetic hormone (AKH) is to stimulate calcium ion uptake into the fat body cell, subsequently causing the formation of diacylglycerol from triacylglycerol. Furthermore, it was also shown that AKH is not directly responsible for increased diacylglycerol uptake by lipophorin from the fat body. The diacylglycerol level of the fat body was found to increase by an average of 2.4-fold after 90 min of incubation in the presence of AKH. Calcium ion was also found to be essential in the action of AKH on the fat body. Supporting this is the observation that calcium ionophore mimics the AKH action in vivo and in vitro; injection of calcium ionophore into adult locusts as well as incubation of hemolymph with fat body and ionophore caused the transformation of high density lipophorin to low density lipophorin. When the fat body, preincubated with or without AKH, was reincubated with hemolymph, diacylglycerol uptake by lipophorin occurred for both incubations. In some sets of experiments, low density lipophorin particles were formed even in the hemolymph that was incubated with fat body preincubated without AKH, indicating that AKH is not directly responsible for its formation. Calcium ion was found not to be necessary for the diacylglycerol uptake process to occur.

Animals↗

Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide.

The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact. This monomeric PTTH has about one-half the biological activity of intact PTTH. Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond. The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH. In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages.

Amino Acid Sequence↗

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm, Manduca sexta.

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.

Aging↗

Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus.

Methyl farnesoate, the crustacean juvenoid, is synthesized and secreted from the mandibular organs of crustaceans under the negative control of the sinus gland-derived mandibular organ-inhibiting hormone (MO-IH). Previously we isolated and sequenced two isoforms, MO-IH-1 and MO-IH-2, differing by just one amino acid, from sinus glands of the edible crab, Cancer pagurus. We now report the isolation of cDNAs encoding MO-IH-1 and MO-IH-2 by a combination of reverse-transcriptase-mediated PCR in conjunction with 5' and 3' rapid amplification of cDNA ends ('RACE'). Full-length clones of MO-IH-1 and MO-IH-2 encoded a 34-residue putative signal peptide and the mature 78-residue MO-IH sequences. Northern blot analysis of various tissues showed that MO-IH expression is confined to the X-organ (a cluster of perikarya within the eye). Southern blot analysis indicated that there are approx. 10 copies of the gene for MO-IH in C. pagurus. Additional Southern blotting experiments detected MO-IH-hybridizing bands in another Cancer species, C. antennarius. In support of this, an HPLC-radioimmunoassay analysis of sinus gland extracts of C. antennarius and C. magister also revealed MO-IH-like immunoreactivity.

Amino Acid Sequence↗

Vitellogenin and ecdysteroid titers in Ixodes scapularis during vitellogenesis.

Ecdysteroids are the only hormones unequivocally identified thus far in ticks. We found a positive correlation between ecdysteroid concentration and vitellogenin synthesis in female Ixodes scapularis. Vitellogenin (Vg) synthetic activity was measured by an in vitro assay for Vg, involving incubations of the fat body with 35S-methionine and immunoprecipitation collected on a solid-phase matrix, protein A. Vitellogenin synthetic activity in the fat body was undetectable in unfed females but was detected after tick attachment to the host. Vitellogenin production in the fat body remained low from attachment until 2 days prior to detachment from the host. Vitellogenin synthesis in the fat body peaked 2 days after detachment and declined to a level 2-3 times above background from 6 days after dropping from the host through oviposition. A peak of ecdysteroids in females 6 days after attachment preceded an increasing rate of Vg synthesis, suggesting a positive correlation between these parameters. Ecdysone and 20-hydroxyecdysone, the 2 major ecdysteroids present during vitellogenesis, could not be detected in females prior to feeding or 2 days after attachment to the host; however, concentrations began to increase 4 days after attachment, peaked during rapid engorgement, and subsequently declined.

Analysis of Variance↗

Role of juvenile hormone and allatotropin on nutrient allocation, ovarian development and survivorship in mosquitoes.

Teneral reserves are utilized to initiate previtellogenic ovarian development in mosquitoes. Females having emerged with low teneral reserves have reduced juvenile hormone (JH) synthesis and previtellogenic development. We investigated what role JH, allatotropin (AT) and other head-factors play in the regulation of previtellogenic ovarian development and adult survivorship. Factors from the head are essential for corpora allata (CA) activation and reproductive maturation. We have shown that decapitation of females within 9-12h after adult ecdysis prevented normal development of the previtellogenic follicles; however maximum previtellogenic ovarian development could be induced in decapitated females by topically applying a JH analog. When females were decapitated 12 or more hours after emergence nutritional resources had been committed to ovarian development and survivorship was significantly reduced. To study if allatotropin levels correlated with teneral reserves, we measured AT titers in the heads of two adult phenotypes (large and small females) generated by raising larvae under different nutritional diets. In large mosquitoes AT levels increased to a maximum of 45 fmol in day 4; in contrast, the levels of allatotropin in the heads of small mosquitoes remained below 9 fmol during the 7 days evaluated. These results suggest that only when nutrients are appropriate, factors released from the brain induce the CA to synthesize enough JH to activate reproductive maturation.

Aedes↗

Hormonal control of the orange coloration of diapause pupae in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae).

Diapause pupae of Papilio xuthus show color polymorphism, represented by diapause-green, orange, and brownish-orange types that are each associated with specific pupation sites. We investigated the role of the site of pupation on the induction of the development of orange types (or brownish-orange types), and the endocrine mechanism underlying the control of color polymorphism in short-day pupae. All short-day larvae of the wandering stage developed into orange or brownish-orange type pupae when they were placed in rough-surfaced containers after gut-purge. Utilizing a pharate pupal ligation between the thorax and abdomen, the endocrine mechanism underlying the control of color polymorphism was shown to involve a head-thorax factor (Orange-Pupa-Inducing Factor: OPIF) that induced orange types in short-day pupae. OPIF was bioassayed using the ligated abdomens of short-day pharate pupae. OPIF was extractable with 2% NaCl solution from 5th-instar larval ganglia complexes following the mesothoracic complex (TG(2,3)-AG(1-7)), but it could not be extracted with either acetone or 80% ethanol solution. OPIF may not exist in the brains of day-0 pupae or in brain-subesophageal ganglion and prothoracic ganglion complexes of 5th-instar larvae. The short-day pharate pupae responded to OPIF in a dose-dependent manner.

Animals↗

Investigation of presumptive mobilization pathways for calcium in the steroidogenic action of big prothoracicotropic hormone.

Ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm Manduca sexta is stimulated by the cerebral neuropeptide prothoracicotropic hormone (PTTH). PTTH-stimulated cAMP synthesis and ecdysone secretion are dependent on the presence of extracellular calcium, suggesting that PTTH enhances calcium entry into the cytosol. Such entry into the cytosol might involve the opening of a plasma membrane calcium channel, or a mechanism dependent upon prior inositol triphosphate (IP3)-mediated release of intracellularly stored calcium. In pupal prothoracic glands, PTTH does not increase IP3 or other inositol phosphates over-times ranging from seconds up to 30 min, even in the presence of lithium. However, the L-type calcium channel antagonist nitrendipine completely prevents PTTH-stimulated ecdysone synthesis. A 41 kDa G-protein in prothoracic glands is ADP-ribosylated by pertussis toxin. However, PTTH-stimulated ecdysone synthesis is unaffected by prior exposure to pertussis toxin, indicating that the 41 kDa protein is not involved in the acute stimulation of steroidogenesis. By contrast, cholera toxin has a stimulatory effect on ecdysone secretion suggesting the involvement of a Gs-like protein. Based on the absence of PTTH-stimulated inositol phosphate formation in pupal prothoracic glands, it is suggested that calcium mobilization may occur through the opening of a calcium channel, possibly regulated by Gs.

Animals↗

Can the "challenge hypothesis" be applied to insects?

The challenge hypothesis, conceived for testosterone and vertebrates, has recently been applied to juvenile hormone (JH) and insects. Scott [Scott, M.P., 2006a. Resource defense and juvenile hormone: the "challenge hypothesis" extended to insects. Horm. Behav. 49, 276-281] found that JH in the burying beetle Nicrophorus orbicollis increased in response to a social challenge in the presence of a breeding resource, while there was no such JH response in the purported brood parasite Nicrophorus pustulatus. Two important implications of the challenge hypothesis that need to be tested are whether JH affects dominance and why there are interspecific differences in the JH response to a social challenge. The effect of JH on dominance in burying beetles was examined by topical application of JH III to one of two competing females at 24 h and again at 1 h prior to presentation of a breeding resource (mouse carcass). JH supplementation had no effect on dominance in intraspecific interactions in N. orbicollis, as measured by possession of the carcass on Day 1, 3 or 7. Similarly, JH did not alter carcass ownership during competition between N. orbicollis and N. pustulatus nor did it affect reproductive success. Irrespective of JH supplementation, N. pustulatus became increasingly dominant as the trials progressed, rarely occupying the carcass on Day 1, but excluding N. orbicollis in nearly half the trials by Day 7. These findings, and a brief review of the burying beetle literature, suggest that the challenge hypothesis, as applied to testosterone and vertebrates, does not yet have an analogous model for JH and insects.

Aggression↗

Allatotropin regulation of juvenile hormone synthesis by the corpora allata from the lubber grasshopper, Romalea microptera.

The in vitro synthesis of juvenile hormone (JH) by corpora allata (CA) from the lubber grasshopper (Romalea microptera) was stimulated by low concentrations of brain extract and this effect was reduced at higher concentrations, suggesting the presence of allatotropin (AT) and allatostatin (AST) factors in the brain. The AT activity of brain extracts caused a rapid and reversible stimulation and appeared to be a peptide(s). Reversed phase (C18) HPLC analysis of brain extracts disclosed two peaks of AT activity but no significant AST activity. Manse-AT, Schgr-NPF, and Locmi-FLRF had no effect on JH synthesis by lubber CA, indicating that the Rommi-AT factors are distinct from these peptides. High concentrations of Dippu-AST-7 and Grybi-AST-1 inhibited JH synthesis, implying that AST factors might be present in lubber grasshoppers. CA response to AT activity of brain extracts varied during the oviposition cycle ( approximately 35 days), with the maximum response occurring on days 16-18. AT activity of brain extracts also varied during the cycle, being highest on day 25. Our data suggest that the lubber CA is largely regulated by AT activity, and that JH synthesis reflects both CA response to AT activity and the level of AT activity in the brain.

Animals↗

Evidence that free fatty acids in trophocytes of Periplaneta americana fat body may be regulated by the activity of phospholipase A2 and cyclooxygenase.

Previous studies have shown that palmitic, stearic, oleic and linoleic acid levels in trophocytes prepared from the fat body of male Periplaneta americana are increased following treatment of the cells with hypertrehalosemic hormone (HTH). Melittin, an activator of phospholipase A2, mimicked the action of HTH by increasing the free fatty acid content in a concentration-dependent manner. The increase caused by HTH could be eliminated by pretreatment of the trophocytes with 1 mM 4'-bromophenacyl bromide (BPB), an inhibitor of phospholipase A2. BPB also decreases the concentration of free fatty acids in trophocytes not treated with HTH but by a smaller margin. Nordihydroguaiaretic acid (NDGA) and indomethacin, inhibitors of lipoxygenase and cyclooxygenase, respectively, eliminated the increase in free fatty acids evoked by HTH. In the absence of HTH both inhibitors increased the free fatty acid content of the trophocytes, an effect consistent with the known mode of action of these agents. None of the inhibitors tested, all of which blocked HTH activated trehalose synthesis, prevented activation of phosphorylase by HTH. This is taken as evidence that other downstream sites are also important in the regulation of trehalose production by the fat body. It is suggested that the increase in free fatty acids evoked by HTH, or metabolites of those fatty acids, may regulate the synthesis and release of trehalose from the trophocytes because of potential effects on trehalose phosphate synthase, trehalose 6-phosphate phosphatase, and the trehalose transport mechanism in the trophocyte membrane.

Acetophenones↗

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris.

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.

Animals↗

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori.

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.

Adenylyl Cyclases↗