Ecdysone Biosynthesis Research Articles

Identification and expression profile of Halloween genes involved in ecdysteroid biosynthesis in Spodoptera littoralis

20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. The biosynthesis of ecdysteroid is triggered and under the control of the neuropeptide, prothoracicotropic hormone (PTTH). To date, five cytochrome P450 enzymes, namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. These genes are called Halloween genes and mediate the biosynthesis of 20E from cholesterol. In this study, we extended these works to a major pest insect in agriculture, the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes, and the converted amino acid sequences were compared with those of other insects. The phylogenetic analysis clearly showed separated clusters of each gene and the evolutional conservation in insects with a high similarity in Lepidoptera. Spo, phm, dib and sad were predominantly expressed in prothoracic glands, and shd was expressed in fat body and Malpighian tubules at the last instar larvae. Spo expression was kept high level between day 2 and day 4 after ecdysis. The expression of phm and dib peaked at day 2, and sad and shd expressions peaked at day 2 and day 4 after ecdysis. In addition, the hemolymph ecdysteroid titer showed a small peak at day 2 and a large peak at day 4 after ecdysis. These results suggest the importance of Halloween genes in ecdysone biosynthesis by prothoracic glands and conversion of ecdysone into 20E by fat body in larval–pupal metamorphosis.

A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis

In insects, the neuropeptide prothoracicotropic hormone (PTTH) stimulates production of ecdysone (E) in the prothoracic glands (PGs). E is the precursor of the principal steroid hormone, 20-hydroxyecdysone (20E), that is responsible for eliciting molting and metamorphosis. In this study, we used quantitative phosphoproteomics to investigate signal transduction events initiated by PTTH. We identified Spook (CYP307A1), a suspected rate-limiting enzyme for E biosynthesis, and components of the mitogen-activated protein kinase (MAPK) pathway, as major phosphorylation targets of PTTH signaling. Further, proteins not previously linked to PTTH and ecdysone biosynthesis were identified as targets of PTTH signaling. These include proteins involved in signal transduction, endosomal trafficking, constituents of the cytoskeleton and regulators of transcription and translation. Our screen shows that PTTH likely stimulates E production by activation of Spook, an integral enzyme in the E biosynthetic pathway. This directly connects PTTH signaling to the pathway that produces E. A new mechanism for regulation of E biosynthesis in insects is proposed.

New players in the regulation of ecdysone biosynthesis

Insect ecdysone steroid hormone regulates major developmental transitions, such as molting and metamorphosis. The production of ecdysone correlates well with the timing of these transitions. Finding out how the ecdysone biosynthesis is regulated is crucial to fully understand these sophisticated developmental switches. Here we summarized recent findings in the regulation of ecdysone biosynthesis from the aspects of cell signaling, key biosynthetic enzymes and substrate cholesterol trafficking.

Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo

Programmed cell death, or apoptosis, is an essential event in animal development. Spatiotemporal analysis of caspase activation in vivo could provide new insights into programmed cell death occurring during development. Here, using the FRET-based caspase-3 indicator, SCAT3, we report the results of live-imaging analysis of caspase activation in developing Drosophila in vivo. In Drosophila, the salivary gland is sculpted by caspase-mediated programmed cell death initiated by the steroid hormone 20-hydroxyecdysone (ecdysone). Using a SCAT3 probe, we observed that caspase activation in the salivary glands begins in the anterior cells and is then propagated to the posterior cells in vivo. In vitro salivary gland culture experiments indicated that local exposure of ecdysone to the anterior salivary gland reproduces the caspase activation gradient as observed in vivo. In betaFTZ-F1 mutants, caspase activation was delayed and occurred in a random pattern in vivo. In contrast to the in vivo response, the salivary glands from betaFTZ-F1 mutants showed a normal in vitro response to ecdysone, suggesting that betaFTZ-F1 may be involved in ecdysteroid biosynthesis and secretion of ecdysone from the ring gland for local initiation of programmed cell death. These results imply a role of betaFTZ-F1 in coordinating the initiation of salivary gland apoptosis in development.

Host‐induced ecdysteroids in the stop‐and‐go oogenesis in a synovigenic parasitoid wasp

Eupelmus vuilleti (Hymenoptera; Eupelmidae) is a solitary ectoparasitoid producing yolk-rich eggs. The female oviposits mainly on the fourth larval instar of Callosobruchus maculatus (Coleoptera; Bruchidae), which develop within pods and seeds of Vigna unguiculata (Fabacae). Parasitoid females are synovigenic, i.e., they are born with immature eggs and need to feed from the host to sustain egg production during their entire lifetime. However, eggs are rapidly resorbed in unfavourable conditions and an efficient stop-and-go mechanism controls oogenesis in such animals. In this study, the possible involvement of ecdysteroids in the regulation of parasitoid oogenesis is examined. In a first step, the identity and titre of ecdysteroids in reproductively active and inactive female parasitoids were investigated by high performance liquid chromatography followed by enzyme immuno-assay (EIA/HPLC). A larger secretion of ecdysone was found in female during their reproductive period compared with inactive females. In a second step, both the secretion of ecdysteroids into the medium of in vitro incubated ovaries and the ecdysteroid content of females reared with or without host were measured (EIA). The presence of the host, which represents both the oviposition site and the nutritional source, induced an active biosynthesis of ecdysone. This synthesis started at a slow rate after host introduction and reached a maximum after 48 h. When hosts were available, this synthesis was cyclic and continuous during the entire female lifetime. These results showed that host presence triggered ovarian synthesis of ecdysteroids, which are involved in a stop-and-go regulation of egg production linked to host availability.

Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

BackgroundTransposons, i.e. transposable elements (TEs), are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search.ResultsTwelve novel transposons, including LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), MITEs (miniature inverted-repeat transposable elements), one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1) implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1) involved in ecdysone biosynthesis and developmental regulation are free of TE insertions.ConclusionThese results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes.

The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone

The developmental events occurring during moulting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the moulting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20E (20-hydroxyecdysone), are mediated by four cytochrome P450 (P450) enzymes, encoded by genes in the Halloween family. Orthologues of the Drosophila Halloween genes phantom (phm; CYP306A1), disembodied (dib; CYP302A1), shadow (sad; CYP315A1) and shade (shd; CYP314A1) were obtained from the endocrinological model insect, the tobacco hornworm Manduca sexta. Expression of these genes was studied and compared with changes in the ecdysteroid titre that controls transition from the larval to pupal stage. phm, dib and sad, which encode P450s that mediate the final hydroxylations in the biosynthesis of ecdysone, were selectively expressed in the prothoracic gland, the primary source of ecdysone during larval and pupal development. Changes in their expression correlate with the haemolymph ecdysteroid titre during the fifth (final) larval instar. Shd, the 20-hydroxylase, which converts ecdysone into the more active 20E, is expressed in tissues peripheral to the prothoracic glands during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The results indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses triggering moulting.

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.

A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.

Molting defective is required for ecdysone biosynthesis

20-hydroxyecdysone was discovered as the major biologically active insect steroid hormone half a century ago, yet much remains to be learned about its biosynthesis and its activities. 20-hydroxyecdysone controls many biological processes, including progression between larval stages, entry to pupariation and metamorphosis. A number of genes required for 20-hydroxyecdysone production have been identified, including those encoding enzymes that mediate four of the late steps of biosynthesis. A second smaller group of low ecdysone mutants do not encode enzymes. Here, we report identification of one such gene, which we call molting defective, on the basis of its lethal phenotype. molting defective encodes a nuclear zinc finger protein required for ecdysone biosynthesis.

Physiological and molecular interaction in the host–parasitoid system Heliothis virescens–Toxoneuron nigriceps: current status and future perspectives

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the tobacco budworm Heliothis virescens (F.) (Lepidoptera, Noctuidae). Parasitized H. virescens larvae are developmentally arrested and show a complex array of pathological symptoms ranging from the suppression of the immune response to an alteration of ecdysone biosynthesis and metabolism. Most of these pathological syndromes are induced by the polydnavirus associated with T. nigriceps (TnBV). An overview of our recent research work on this system is described herein. The mechanisms involved in the disruption of the host hormonal balance have been further investigated, allowing to better define the physiological model previously proposed. A functional genomic approach has been undertaken to identify TnBV genes expressed in the host and to assess their role in the major parasitoid-induced pathologies. Some TnBV genes cloned so far are novel and do not show any similarity with genes already available in genomic databases, while others code for proteins having conserved domains, such as aspartic proteases and tyrosine phosphatases. Sequencing of the entire TnBV genome is in progress and will considerably contribute to the understanding of the molecular bases of parasitoid-induced host alterations.

Steroid control of longevity in Drosophila melanogaster.

Ecdysone, the major steroid hormone of Drosophila melanogaster, is known for its role in development and reproduction. Flies that are heterozygous for mutations of the ecdysone receptor exhibit increases in life-span and resistance to various stresses, with no apparent deficit in fertility or activity. A mutant involved in the biosynthesis of ecdysone displays similar effects, which are suppressed by feeding ecdysone to the flies. These observations demonstrate the importance of the ecdysone hormonal pathway, a new player in regulating longevity.

Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster.

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C22- and C2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [3H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast, dib (CYP302A1) is responsible for the conversion of the [3H]ketotriol to [3H]2dE. When cells are transfected with both dib and sad, they metabolize the [3H]ketotriol to [3H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

In vitro degradation of the Neb-Trypsin Modulating Oostatic Factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.

Effect of 1-(4-phenoxyphenoxypropyl)imidazole (KS-175) on larval growth in the silkworm Bombyx mori

1-(4-Phenoxyphenoxypropyl)imidazole (KS-175), which has two types of characteristic moieties of insect growth regulators (IGRs), the phenoxyphenoxyalkyl group of juvenile hormone analogs (JHAs) and imidazole of 1,5-disubstituted imidazole such as KK-42, was tested for its biological activity on the silkworm, Bombyx mori. Penultimate (4th) instar larvae topically treated with KS-175 did not molt for more than 20 days. This activity was different from that reported for any IGRs. After the treatment, ecdysteroid levels in the hemolymph did not increase and the cells of the prothoracic gland had shrunk. When the treated penultimate larvae were fed an artificial diet supplemented with 20 ppm of 20-hydroxyecdysone, the larvae molted to the ultimate (5th) instar with a timing similar to that of control larvae fed a diet with or without 20-hydroxyecdysone. These results suggest that topical application of KS-175 irreversibly damages ecdysone biosynthesis in the prothoracic glands.

Insect trypsin modulating oostatic factor (Neb-TMOF) and its analogs: Preliminary structure/biological function relationship studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH2 (III), NO2 (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH2)6], and [Phe(NO2)6]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)6]- and [D-His(Bzl)6]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH2)6-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.

Multiple control of ecdysone biosynthesis in blowfly larvae: Interaction of ecdysiotropins and ecdysiostatins

Multiple control of ecdysone biosynthesis in blowfly larvae: Interaction of ecdysiotropins and ecdysiostatins

Multiple control of ecdysone biosynthesis in blowfly larvae: Interaction of ecdysiotropins and ecdysiostatins

Multiple control of ecdysone biosynthesis in blowfly larvae: Interaction of ecdysiotropins and ecdysiostatins

Effects of juvenile hormone analogues and ecdysteroid biosynthesis effectors on egg production in crickets,Gryllus bimaculatusde Geer (Ensifera, Gryllidae)

Summary Allatectomy experiments revealed that the corpora allata, and thus juvenile hormone (JH), do not have an all-encompassing control on egg production in Gryllus bimaculatus, though they do exert a marked quantitative effect on oviposition rate. Rate of egg laying and number of eggs produced by allatectomized females reared at 27°C were much reduced in comparison with sham-operated controls. Topical application of the JH analogues methoprene, hydroprene and fenoxycarb onto allatectomized females restored egg production. This rise in fecundity was high in the case of methoprene treatment, but low in the case of hydroprene application. Females reared at the suboptimal temperature of 19°C also produced only a small number of eggs. Administration of the JH analogues to 19°C females again resulted in a distinct increase in egg production. Since we believe that ecdysteroids may account for that portion of eggs which is produced and layed by allatectomized females, intact animals reared at the optimal temperature of 27°C were treated with three potential ecdysone biosynthesis inhibitors, the imidazole compounds KK42 and ketoconazole and the benzoyl phenyl urea derivative diflubenzuron. Treatment of females with DFB and ketoconazole significantly reduced total fecundity, whereas KK42 showed only a moderate effect. Injections of 20-hydroxyecdysone into allatectomized 27°C females did not affect egg laying, whereas a combined treatment of hydroprene + 20-hydroxyecdysone restored egg production to a more or less normal rate.

Sterols that modify moulting in insects

The development of insects is under the control of a steroid hormone, ecdysone. This paper reviews suicide substrate type inactivators designed to inhibit the biosynthesis of ecdysone. Several series of acetylenic derivatives of cholesterol were synthesized and their biological effects on the prothoracic glands of the locust were investigated in vitro, enabling structure-activity relationships to be studied. Information on the ability of brassinosteroids, a series of plant growth regulators, to inhibit ecdysteroid activities of insects is discussed as a precursor to a study of brassinosteroid ecdysone mimics.