Neuroendocrine regulation of Drosophila metamorphosis requires TGFbeta/Activin signaling.

In insects, initiation of metamorphosis requires a surge in the production of the steroid hormone 20-hydroxyecdysone from the prothoracic gland, the primary endocrine organ of juvenile larvae. Here, we show that blocking TGFß/Activin signaling, specifically in the Drosophila prothoracic gland, results in developmental arrest prior to metamorphosis. The terminal, giant third instar larval phenotype results from a failure to induce the large rise in ecdysteroid titer that triggers metamorphosis. We further demonstrate that activin signaling regulates competence of the prothoracic gland to receive PTTH and insulin signals, and that these two pathways act at the mRNA and post-transcriptional levels, respectively, to control ecdysone biosynthetic enzyme expression. This dual regulatory circuitry may provide a cross-check mechanism to ensure that both developmental and nutritional inputs are synchronized before initiating the final genetic program leading to reproductive adult development. As steroid hormone production in C. elegans and mammals is also influenced by TGFß/Activin signaling, this family of secreted factors may play a general role in regulating developmental transitions across phyla.

Prothoracicotropic hormone regulates developmental timing and body size in Drosophila.

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.

Germline-dependent gene expression in distant non-gonadal somatic tissues of Drosophila.

BACKGROUND: Drosophila females commit tremendous resources to egg production and males produce some of the longest sperm in the animal kingdom. We know little about the coordinated regulation of gene expression patterns in distant somatic tissues that support the developmental cost of gamete production. RESULTS: We determined the non-gonadal gene expression patterns of Drosophila females and males with or without a germline. Our results show that germline-dependent expression in the non-gonadal soma is extensive. Interestingly, gene expression patterns and hormone titers are consistent with a hormone axis between the gonads and non-gonadal soma. CONCLUSIONS: The germline has a long-range influence on gene expression in the Drosophila sexes. We suggest that this is the result of a germline/soma hormonal axis.

Manduca sexta prothoracicotropic hormone: evidence for a role beyond steroidogenesis.

Prothoracicotropic hormone (PTTH) is a homodimeric brain peptide hormone that positively regulates the production of ecdysteroids by the prothoracic gland of Lepidoptera and probably other insects. PTTH was first purified from heads of adult domestic silkworms, Bombyx mori. Prothoracic glands of Bombyx and Manduca sexta undergo apoptosis well before the adult stage is reached, raising the recurring question of PTTH function at these later stages. Because Bombyx has been domesticated for thousands of years, the possibility exists that the presence of PTTH in adult animals is an accidental result of domestication for silk production. In contrast, Manduca has been raised in the laboratory for only five or six decades. The present study found that Manduca brains contain PTTH at all stages examined post-prothoracic gland apoptosis, i.e., pharate adult and adult life, and that PTTH-dependent changes in protein phosphorylation and protein synthesis were observed in several reproductive and reproduction-associated organs. The data indicate that PTTH indeed plays a role in non-steroidogenic tissues and suggest possible future avenues for determining which cellular processes are being so regulated.

Daphnia Halloween genes that encode cytochrome P450s mediating the synthesis of the arthropod molting hormone: evolutionary implications.

BACKGROUND: In crustaceans and insects, development and reproduction are controlled by the steroid hormone, 20-hydroxyecdysone (20E). Like other steroids, 20E, is synthesized from cholesterol through reactions involving cytochrome P450s (CYPs). In insects, the CYP enzymes mediating 20E biosynthesis have been identified, but evidence of their probable presence in crustaceans is indirect, relying solely on the ability of crustaceans to synthesize 20E. RESULTS: To investigate the presence of these genes in crustaceans, the genome of Daphnia pulex was examined for orthologs of these genes, the Halloween genes, encoding those biosynthetic CYP enzymes. Single homologs of spook-CYP307A1, phantom-CYP306A1, disembodied-CYP302A1, shadow-CYP315A1 and shade-CYP314A1 were identified in the Daphnia data base. Phylogenetic analysis indicates an orthologous relationship between the insect and Daphnia genes. Conserved intron/exon structures and microsynteny further support the conclusion that these steroidogenic CYPs have been conserved in insects and crustaceans through some 400 million years of evolution. CONCLUSION: Although these arthropod steroidogenic CYPs are related to steroidogenic CYPs in Caenorhabditis elegans and vertebrates, the data suggest that the arthropod steroidogenic CYPs became functionally specialized in a common ancestor of arthropods and are unique to these animals.

Global identification of bursicon-regulated genes in Drosophila melanogaster.

BACKGROUND: Bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Recent studies indicate that the action of bursicon is mediated by a specific G protein-coupled receptor DLGR2 and the cAMP/PKA signaling pathway. However, little is known regarding the genes that are regulated by bursicon. The identification of bursicon-regulated genes is the focus of this investigation. RESULTS: We used DNA microarray analysis to identify bursicon-regulated genes in neck-ligated flies (Drosophila melanogaster) that received recombinant bursicon (r-bursicon). Fifty four genes were found to be regulated by bursicon 1 h post r-bursicon injection, 52 being up-regulated and 2 down-regulated while 33 genes were influenced by r-bursicon 3 h post-injection (24 up-regulated and 9 down-regulated genes). Analysis of these genes by inference from the fly database http://flybase.bio.indiana.edu revealed that these genes encode proteins with diverse functions, including cell signaling, gene transcription, DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism, cytoskeleton formation, immune response and cell-adhesion. Twenty eight genes randomly selected from the microarray-identified list were verified by real time PCR (qPCR) which supported the microarray data. Temporal response studies of 13 identified and verified genes by qPCR revealed that the temporal expression patterns of these genes are consistent with the microarray data. CONCLUSION: Using r-bursicon, we identified 87 genes that are regulated by bursicon, 30 of which have no previously known function. Most importantly, all genes randomly selected from the microarray-identified list were verified by real time PCR. Temporal analysis of 13 verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. The composite data suggest that these genes play important roles in regulating the cuticle sclerotization and wing expansion processes. The data obtained here will form the basis for future studies aimed at elucidating the exact mechanisms upstream from the secretion of bursicon and its binding to target cells.

Molecular evolution of the insect Halloween family of cytochrome P450s: phylogeny, gene organization and functional conservation.

The insect molting hormone, 20-hydroxyecdysone (20E), is a major modulator of the developmental processes resulting in molting and metamorphosis. During evolution selective forces have preserved the Halloween genes encoding cytochrome P450 (P450) enzymes that mediate the biosynthesis of 20E. In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. The analyzed insect genomes each contains single orthologs of Phantom (CYP306A1), Disembodied (CYP302A1), Shadow (CYP315A1) and Shade (CYP314A1), the terminal hydroxylases. In Drosophila melanogaster, the Halloween gene spook (Cyp307a1) is required for the biosynthesis of 20E, although a function has not yet been identified. Unlike the other Halloween genes, the ancestor of this gene evolved into three paralogs, all in the CYP307 family, through gene duplication. The genomic stability of these paralogs varies among species. Intron-exon structures indicate that D. melanogaster Cyp307a1 is a mRNA-derived paralog of spookier (Cyp307a2), which is the ancestral gene and the closest ortholog of the coleopteran, lepidopteran and mosquito CYP307A subfamily genes. Evolutionary links between the insect Halloween genes and vertebrate steroidogenic P450s suggest that they originated from common ancestors, perhaps destined for steroidogenesis, before the deuterostome-arthropod split. Conservation of putative substrate recognition sites of orthologous Halloween genes indicates selective constraint on these residues to prevent functional divergence. The results suggest that duplications of ancestral P450 genes that acquired novel functions may have been an important mechanism for evolving the ecdysteroidogenic pathway.

Protein kinase C modulates ecdysteroidogenesis in the prothoracic gland of the tobacco hornworm, Manduca sexta.

The prothoracic gland is the primary source of ecdysteroid hormones in the immature insect. Ecdysteroids coordinate gene expression necessary for growth, molting and metamorphosis. Prothoracicotropic hormone (PTTH), a brain neuropeptide, regulates ecdysteroid synthesis in the prothoracic gland. PTTH stimulates ecdysteroid synthesis through a signal transduction cascade that involves at least four protein kinases: protein kinase A (PKA), p70 S6 kinase, an unidentified tyrosine kinase, and the extracellular signal-regulated kinase (ERK). In this report, the participation of protein kinase C (PKC) in PTTH signalling is demonstrated and characterized. PTTH stimulates PKC activity through a PLC and Ca(2+)-dependent pathway that is not cAMP regulated. Inhibition of PKC inhibits PTTH-stimulated ecdysteroidogenesis as well as PTTH-stimulated phosphorylation of ERK and its upstream regulator, MAP/ERK kinase (MEK). These observations reveal that the acute regulation of prothoracic gland steroidogenesis is dependent on a web of interacting kinase pathways, which probably converge on factors that regulate translation.

Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis.

The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active insect molting hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.

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.

Ca2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: evidence for mobilization and entry mechanisms.

Prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.

Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster.

Mutation of members of the Halloween gene family results in embryonic lethality. We have shown that two of these genes code for enzymes responsible for specific steps in the synthesis of ecdysone, a polyhydroxylated sterol that is the precursor of the major molting hormone of all arthropods, 20-hydroxyecdysone. These two mitochondrial P450 enzymes, coded for by disembodied (dib) (CYP302A1) and shadow (sad) (CYP315A1), are the C22 and C2 hydroxylases, respectively, as shown by transfection of the gene into S2 cells and subsequent biochemical analysis. These are the last two enzymes in the ecdysone biosynthetic pathway. A third enzyme, necessary for the critical conversion of ecdysone to 20-hydroxyecdysone, the 20-monooxygenase, is encoded by shade (shd) (CYP314A1). All three enzymes are mitochondrial although shade has motifs suggesting both mitochondrial and microsomal locations. By tagging these enzymes, their subcellular location has been confirmed by confocal microscopy. Shade is present in several tissues as expected while disembodied and shadow are restricted to the ring gland. The paradigm used should allow us to define the enzymes mediating the entire ecdysteroid biosynthetic pathway.

Developmental profiles of ecdysteroids, ecdysteroid receptor mRNAs and DNA binding properties of ecdysteroid receptors in the Ixodid tick Amblyomma americanum (L.).

Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins.

Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis.

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied (dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade (shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom (phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase (Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3beta,5beta-ketodiol) conversion to 2,22-dideoxyecdysone (3beta,5beta-ketotriol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval-larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme.

Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects.

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.

Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish.

BACKGROUND: Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon ß (burs ß), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown until now. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we show in Drosophila melanogaster that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. CONCLUSIONS/SIGNIFICANCE: Our results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data we infer that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle.

MET is required for the maximal action of 20-hydroxyecdysone during Bombyx metamorphosis.

Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, Bombyx mori, participate in 20E signal transduction. Met is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of EcR-USP (the ecdysone receptor genes), RNAi knockdown of Met at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during Bombyx metamorphosis.

Hsc70 binds to ultraspiracle resulting in the upregulation of 20-hydroxyecdsone-responsive genes in Helicoverpa armigera.

To probe the specific functions of the chaperone protein Hsc70 in 20-hydroxyecdysone signaling, we report on the roles of the Hsc70 from Helicoverpa armigera. RT-PCR analysis revealed that the genes for HaEcRB1 and HaUSP1 were upregulated in 5th molting and metamorphic molting larvae, whereas HaHsc70 maintained a constitutive expression level throughout larval development. Silencing HaEcRB1, HaUSP1 or HaHsc70 by RNAi inhibited the expression of a set of 20E-responsive genes. Immunocytochemical assay demonstrated that HaHsc70 is located predominantly in the cytoplasm of unstimulated cells and partially translocated to the nucleus after stimulation by 20E. Knockdown of HaHsc70 by RNAi decreased the amount of both HaEcRB1 and HaUSP1 in the nucleus. HaHsc70 was capable of binding to HaUSP1 in pull-down assays. These data suggest that Hsc70 participates in the 20E signal transduction pathway via binding to USP1 and mediating the expression of EcRB1, USP1 and then a set of 20E-responsive genes.

The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis.

Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal-regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation of the Torso/ERK pathway.

Studies on the Black Box: incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta.

It has long been hypothesized that the oxidation of 7-dehydrocholesterol (7dC), made from dietary cholesterol (C), to 3-oxo-7dC (3-oxo-Delta(5,7)C) immediately precedes the unknown "Black Box" oxidations that lead to the formation of the first up-stream intermediate exhibiting the highly characteristic ecdysteroid structure of the steroid molting hormone of insects, crustaceans and some other arthropods. Perhaps rate-limiting and under the control of the prothoracicotropic hormone (PTTH), the biosynthesis of 3-oxo-7dC and its subsequent oxidative modifications have been difficult to study because of their apparent instability, i.e. no intermediates between 7dC and the diketol (3-oxo-25,22,2-trideoxyecdysone) have ever been observed or identified in insect prothoracic gland incubations with radiolabelled precursors. However, we show that 3-oxo-7dC can be converted into lipophilic, photosensitive, ketone-blocked (PSKB) ketal derivatives which will release 3-oxo-7dC when and where desired following brief irradiation with innocuous long-wave (365 nm) UV-light both in vivo and in vitro. In this manner, 3-oxo-7dC is quickly and efficiently incorporated into ecdysteroids by adult male and female Drosophila raised on a diet containing the PSKB ketals and in prothoracic glands of Manduca sexta incubated with the ketals emulsified into media. The instability of 3-oxo-7dC and its spontaneous transformation into extensively electron-delocalized intermediates will be discussed in relation to a possible mechanism of the Black Box oxidations eventually leading to the production of the active molting hormone 20-hydroxyecdysone (20E).