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Flies on steroids--Drosophila metamorphosis and the mechanisms of steroid hormone action.

Recent studies have provided new insights into the molecular mechanisms by which the steroid hormone ecdysone triggers the larval-to-adult metamorphosis of Drosophila. Ecdysone-induced transcription factors activate large sets of secondary-response genes and provide the competence for subsequent regulatory responses to the hormone. It seems likely that similar hormone-triggered regulatory hierarchies exist in other higher organisms and that Drosophila is providing our first glimpses of the complexities of these gene networks.

Animals↗

Locust adipokinetic hormone stimulates lipid mobilization in Manduca sexta.

Adipokinetic hormone, a decapeptide isolated from the locust, stimulates mobilization of diacylglycerols from the locust fat body and loading of the lipid transport protein, lipophorin. Injection of the synthetic locust adipokinetic hormone into a sphinx moth, Manduca sexta, causes lipid loading of lipophorin. The lipophorin decreases in density from 1.11 to 1.06 g/ml, and a soluble protein from the hemolymph (apolipophorin III) associates with the lipophorin particle. Administration of intermediate doses of hormone indicates that lipophorin is converted directly to the low density form; no appreciable amounts of intermediate density particles are formed.

Adipose Tissue↗

Identification and characterization of adipokinetic hormone (Locusta migratoria)-like immunoreactivity in the human cerebrospinal fluid.

Using an antiserum raised against locust adipokinetic hormone I, considerable quantity of adipokinetic hormone-like immunoreactivity was demonstrated in the human cerebrospinal fluid. The immunoreactivity was characterized by gel permeation and high performance liquid chromatography. The main immunoreactive component in the cerebrospinal fluid coeluted with adipokinetic hormone I. These results suggest that adipokinetic hormone may contribute to the neuronal function in the human central nervous system.

Chromatography, Gel↗

A possible role of SchistoFLRFamide in inhibition of adipokinetic hormone release from locust corpora cardiaca.

The distribution and actions of FMRFamide-related peptides (FaRPs) in the corpora cardiaca of the locust Locusta migratoria were studied. Antisera to FMRFamide and SchistoFLRFamide (PDVDHVFLRFamide) label neuronal processes that impinge on glandular cells in the glandular lobe of the corpora cardiaca known to produce adipokinetic hormones. Electron microscopic immunocytochemistry revealed that these FaRP-containing processes form synaptoid contacts with the glandular cells. Approximately 12% of the axon profiles present in the glandular part of the corpus cardiacum contained SchistoFLRFamide-immunoreactive material. Retrograde tracing of the axons in the nervus corporis cardiaci II with Lucifer yellow revealed 25-30 labelled neuronal cell bodies in each lateral part of the protocerebrum. About five of these in each hemisphere reacted with the SchistoFLRFamide-antiserum. Double-labelling immunocytochemistry showed that the FaRP-containing processes in the glandular lobe of the corpora cardiaca are distinct from neuronal processes, reacting with an antiserum to the neuropeptide locustatachykinin. The effect of the decapeptide SchistoFLRFamide and the tetrapeptide FMRFamide on the release of adipokinetic hormone I (AKH I) from the cells in the glandular part of the corpus cardiacum was studied in vitro. Neither the deca- nor the tetrapeptide had any effect on the spontaneous release of AKH I. Release of AKH I induced by the phosphodiesterase inhibitor IBMX, however, was reduced significantly by both peptides. These results point to an involvement of FaRPs as inhibitory modulators in the regulation of the release of adipokinetic hormone from the glandular cells.

1-Methyl-3-isobutylxanthine↗

Immunohistochemical localisation of the hypertrehalosaemic hormone II (Cam-HrTH-II) and related peptides in the nervous system of Carausius morosus and Sarcophaga bullata.

A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the Lom-AKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.

Amino Acid Sequence↗

Induction of vitellogenin gene transcription in vitro by juvenile hormone in Blattella germanica.

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10(-7) or 10(-8) M), partially inhibited the response to JH III.

Animals↗

Heat stress affects oogenesis differently in wild-type Drosophila virilis and a mutant with altered juvenile hormone and 20-hydroxyecdysone levels.

The link between reproduction and environmental signals is poorly understood at the physiological, genetic and molecular levels. We describe a mutant strain of Drosophila virilis that has altered responses to heat stress. Heat stress in wild-type females results in oocyte maturation delays, degradation of early vitellogenic egg chambers, inhibition of yolk protein gene expression in follicle cells and accumulation of mature oocytes. The mutant females have increased levels of ecdysteroids and decreased juvenile hormone degradation, and show all of the heat-stress-induced reproductive effects observed in wild-type flies, without exposure to heat stress. During oogenesis in mutant females following heat stress there is an increase in early vitellogenic oocyte degradation and some degradation of late egg chambers. 20-Hydroxyecdysone levels, but not juvenile hormone degradation, change following heat stress in mutant females.

Animals↗

Immunocytochemical distribution of pigment-dispersing hormone in the cephalic ganglia of polyneopteran insects.

Material detectable with antisera to the pigment-dispersing hormone (PDH) is regarded as a component of the circadian clock residing in some insects in the optic lobe. This paper demonstrates that the position of the PDH-positive neurones and the course of their processes are similar in all representatives of the insect cohort Polyneoptera. A basic morphological pattern, which includes the proximal frontoventral (Pfv), distal posteriodorsal (Dpd) and posterioventral (Dpv) clusters of PDH-positive neurones, was found in the examined species of locusts, crickets, walking sticks, cockroaches, earwigs and termites. The Pfv cluster is located close to the accessory medulla and usually consists of a set of smaller and a set of larger perikarya. The Dpd and Dpv clusters occupy a dorsal and a ventral position, respectively, at the distal edge of the medulla. These clusters are lacking in stonefly and praying mantid species. The fan-like arrangement of PDH-positive fibres within the frontal medulla face (the locusts and the praying mantid have an additional, smaller fan on the posterior medulla face) is another characteristic feature of Polyneoptera. One (two in the locusts and the praying mantid) nerve bundle runs from the optic lobe to the lateral protocerebrum where it ramifies. One branch gives rise to a fibre network frontally encircling brain neuropile in the area of mushroom bodies. One thin fibre in the crickets and the earwig, and several thicker and anastomosing fibres in the other insects, connect the brain hemispheres. The arrangement of other PDH-positive structures specifies taxa within Polyneoptera. Specific features comprise the presence of PDH-positive perikarya in protocerebrum (walking stick and termite), deutocerebrum (cricket, walking stick, and one cockroach species), tritocerebrum (another cockroach species), and the suboesophageal ganglion (cricket, walking stick and termite). In the walking stick and the termite, PDH-positive fibres pass from the cephalic to the frontal ganglion and from there via the recurrent nerve to the corpora cardiaca where they make varicosities indicative of peptide release into the haemolymph.

Animals↗

Cloning and characterization of ecdysone receptor and ultraspiracle cDNAs from Spodoptera litura.

The insect molting hormone, ecdysone, induces a series of events critical for ecdysis, beginning with the formation of a complex between ecdysteroid receptor (EcR) and its partner protein, ultraspiracle (Usp). In the present study, we cloned cDNAs encoding EcR and Usp by RT-PCR and 5'-/3'-RACE from the common cutworm, Spodoptera litura, a destructive agricultural pest insect in East Asia. Hexahistidine-tagged recombinant EcR and Usp of S. litura were expressed in E. coli, and the proteins, after refolding, exhibited the specific binding to an ecdysone analogue in vitro.

Animals↗

Allatotropin released by the brain controls larval molting in Galleria mellonella by affecting juvenile hormone synthesis.

Monoclonal antibodies were raised against a protein fraction which was purified from larval brains of Galleria mellonella and which was shown to stimulate the corpora allata to synthesize juvenile hormone in vivo as well as in vitro. Immunoblot analysis revealed that one polypeptide band of 20 kDa was specifically recognized. Two pairs of median neurosecretory cells of the brain and the cells of the corpus cardiacum were demonstrated to be immunoreactive to the antibodies. Our results sustain the hypothesis that the larval brain directly governs insect larval molting by controlling JH synthesis via an allatrophic factor.

Animals↗

Neuroendocrinological and molecular aspects of insect reproduction.

This review summarizes recent advances and novel concepts in the area of insect reproductive neuroendocrinology. The role of 'classic' hormones, such as ecdysteroids and juvenoids, to control reproduction is well documented in a large variety of insect species. In adult gonads, ecdysteroids appear to induce a cascade of transcription factors, many of which also occur during the larval molting response. Recent molecular and functional data have created opportunities to study an additional level of regulation, that of neuropeptides, growth factors and their respective receptors. As a result, many homologs of factors playing a role in vertebrate reproductive physiology have been discovered in insects. This review highlights several neuropeptides controlling the biosynthesis and release of the 'classic' insect hormones, as well as various peptides and biogenic amines that regulate behavioural aspects of the reproduction process. In addition, hormone metabolizing enzymes and second messenger pathways are discussed with respect to their role in reproductive tissues. Finally, we speculate on future prospects for insect neuroendocrinological research as a consequence of the recent 'Genomics Revolution'.

Animals↗

Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene.

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5'-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding beta-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EcRE) was localized at position -75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.

Animals↗

The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical.

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high performance liquid chromatography from the corpus cardiacum of the Colorado potato beetle, Leptinotarsa decemlineata. The sequences of both peptides, designated Led-CC-I and Led-CC-II, were determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. The C-terminal of both peptides were blocked and neither molecule was cleaved by carboxypeptidase. Both peptides were found to be octapeptides; Led-CC-I has the primary structure pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, and Led-CC-II has the primary sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2. These structures are identical to the two hypertrehalosaemic hormones from the American cockroach. Preliminary experiments show that the synthetic peptides are apparently involved in the control of amino acid metabolism during flight of the potato beetle.

Amino Acid Sequence↗

Potential role for a FTZ-F1 steroid receptor superfamily member in the control of Drosophila metamorphosis.

FTZ-F1, a member of the steroid receptor superfamily, has been implicated in the activation of the homeobox segmentation gene fushi tarazu early in Drosophila embryogenesis. We have cloned a developmental isoform of FTZ-F1 and found that it is expressed as a product of the previously identified, midprepupal chromosome puff at 75CD. The 75CD puff occurs in the midst of a period of intense puffing activity that is triggered in response to the steroid hormone ecdysone at the onset of metamorphosis. Indirect immunofluorescent staining for FTZ-F1 on Drosophila polytene chromosomes reveals binding to over 150 chromosomal targets, which include 75CD itself and prominent late prepupal puffs that are predicted to be regulated by midprepupal puff proteins. These results suggest a role for FTZ-F1 as a regulator of insect metamorphosis and underscore the repeated utilization of a regulatory protein for widely separate developmental pathways.

Amino Acid Sequence↗

Identification of a glycogenolysis-inhibiting peptide from the corpora cardiaca of locusts.

A mass spectrometric study of the peptidome of the neurohemal part of the corpora cardiaca of Locusta migratoria and Schistocerca gregaria shows that it contains several unknown peptides. We were able to identify the sequence of one of these peptides as pQSDLFLLSPK. This sequence is identical to the part of the Locusta insulin-related peptide (IRP) precursor that is situated between the signal peptide and the B-chain. We designated this peptide as IRP copeptide. This IRP copeptide is also present in the pars intercerebralis, which is likely to be the site of synthesis. It is identical in both L. migratoria and S. gregaria. It shows no effect on the hemolymph lipid concentration in vivo or muscle contraction in vitro. The IRP copeptide is able to cause a decreased phosphorylase activity in locust fat body in vitro, opposite to the effect of the adipokinetic hormones and therefore possibly represents a glycogenolysis-inhibiting peptide.

Amino Acid Sequence↗

The chemical and biological properties of methoxyfenozide, a new insecticidal ecdysteroid agonist.

Methoxyfenozide [N-tert-butyl-N'-(3-methoxy-o-toluoyl)-3,5-xylohydrazide; RH-2485] is the newest diacylhydrazine insecticide to reach the marketplace. It binds with very high affinity to the ecdysone receptor complex (EcR:USP) in lepidopteran insects [Kd = 0.5 nM (Plodia)], where it functions as a potent agonist, or mimic, of the insect molting hormone, 20-hydroxyecdysone (20E). Methoxyfenozide exhibits high insecticidal efficacy against a wide range of important caterpillar pests, including many members of the family Pyralidae, Pieridae, Tortricidae and Noctuidae. It is most effective when ingested by the target caterpillar, but it also has some topical and ovicidal properties. It is modestly root systemic, but not significantly leaf-systemic. Evidence collected to date indicates that methoxyfenozide has an excellent margin of safety to non-target organisms, including a wide range of non-target and beneficial insects.

Administration, Oral↗