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.
- cytochrome P450
- Halloween gene
- prothoracic gland
Insect moulting hormones (ecdysteroids) control and co-ordinate the periodic moults of growing immature insects and the metamorphic differentiation into pupae and adult. The insect Halloween genes encode the terminal cytochrome P450 (P450) hydroxylases mediating the biosynthesis of ecdysteroids . Mutations disrupting these specific P450 steroid hydroxylases in Drosophila result in morphogenetic abnormalities such as failure of head involution and cuticle formation, leading to embryonic death [2–6]. This emphasizes the physiological importance of the Halloween P450 enzymes, in contrast with many catalytically versatile P450 enzymes that metabolize xenobiotics, and which can be knocked out without obvious change in phenotype . Despite considerable effort, the identification of the P450 enzymes involved in ecdysteroid biosynthesis remained elusive for more than five decades. The breakthrough came from the discovery that the embryonic lethal phenotypes of the Drosophila melanogaster Halloween family of mutants presumably were caused by a low ecdysteroid titre . P450 enzymes encoded by these Halloween genes were identified from their known cytological position and the catalytic competences were demonstrated by expression in Drosophila S2 cells . It was found that Drosophila Phantom (Phm; CYP306A1), Disembodied (Dib; CYP302A1), Shadow (Sad; CYP315A1) and Shade (Shd; CYP314A1) catalyse the final four hydroxylations, yielding 20E (20-hydroxyecdysone) the principal insect moulting hormone [3–6].
phm, dib and sad are expressed in the prothoracic gland cells of the ring gland, the principal source of ecdysteroids during larval development, where they mediate the last three steps in the formation of ecdysone from dietary cholesterol [4,5]. In many lepidopteran insects, ecdysone is, however, not the primary product of the prothoracic glands . Instead, 3-dehydroecdysone is produced and secreted, by what is believed to be an essentially similar biosynthetic pathway [8–10], and this prohormone is immediately converted into ecdysone in the haemolymph by a reductase . Ecdysone is then converted into 20E in peripheral tissues such as the fat body, midgut and Malpighian tubules by a hydroxylation at C-20 catalysed by Shd, the E20MO (ecdysone 20-mono-oxygenase) [3,12]. These Halloween genes are expressed also in the ovaries, consistent with the importance of 20E for normal oogenesis .
Here, we provide a short review of recent advances in unravelling the roles of the steroidogenic insect P450s with emphasis on their developmental regulation in the tobacco hornworm, Manduca sexta, during the final (fifth) larval instar.
There has been major progress in this field over the last 5 years with the molecular characterization of terminal enzymatic steps . Despite this great achievement, resulting from the utilization of molecular genetics, details of earlier steps in the biosynthetic pathway remain to be elucidated .
Early steps: sterol precursors to the ketodiol (2,22,25-trideoxyecdysone)
Insects, which are sterol heterotrophs, obtain mainly cholesterol or phytosterols depending on their dietary habits. Phytophagous insects primarily ingest phytosterols that are first dealkylated to cholesterol, the immediate sterol precursor of ecdysteroids. In the prothoracic gland cells, cholesterol is first converted into 7dC (7-dehydrocholesterol) by the action of the 7,8-dehydrogenase. Both dealkylation and the cholesterol to 7dC conversion are believed to involve P450-catalysed reactions by yet unidentified enzymes . However, a recent study indicates that the 7,8-dehydrogenation may be carried out by Neverland, a Rieske-domain protein .
The product of the 7,8-dehydrogenation, 7dC, is subjected to a unique and mysterious transformation to 5β[H]-3β, 14α-dihydroxy-cholesta-7-ene-6-one (ketodiol), the first recognizable ecdysteroid-like molecule (extensively reviewed in [1,11,14]). No intermediates have been characterized between 7dC and the ketodiol and even the subcellular site of this biochemical transformation remains conjectural. The nature of this so-called ‘Black Box’ reaction has eluded molecular and even biochemical characterization despite a great deal of investigation. Various biochemical scenarios have been proposed for this transformation that may well include P450-catalysed reactions . So far, the P450 enzymes involved in ecdysteroid biosynthesis (i.e. the Halloween P450s) have all been identified from ‘low ecdysteroid’ mutants of Drosophila. The only ‘orphan’ P450 enzyme in the Halloween family of low ecdysteroid mutants is Spook (Spo; CYP307A1). The experimental paradigm that determined the function of the other Halloween P450s has failed to assign a specific enzymatic role to Spo [15,16]. The rescue of homozygous spo mutants provided an artificial pulse of ecdysteroid intermediates, which indicates that Spo probably acts upstream of Phm [i.e. upstream of the ketodiol (2,22,25-trideoxyecdysone)]. However, 7dC does not rescue spo mutants as it does with the woc , dnpc1a  and neverland mutants , indicating that Spo is not the 7,8-dehydrogenase. Several functions have been proposed for Spo including the possibility that Spo is either involved in the Black Box reaction or synthesizes a novel signal molecule required for ecdysteroid biosynthesis [15,16].
Ecdysteroidogenic Halloween P450s: M. sexta
Because all insects utilize the same active ecdysteroid, namely 20E, synthesized from cholesterol through the same series of sterol modifications, the Halloween P450 genes may have been structurally and functionally conserved through some 400 million years of insect evolution. This is substantiated by the orthologous relationship of Drosophila, Manduca and silkmoth (Bombyx mori) Halloween genes shown in Figure 1. The conserved primary structure of these genes allowed for the identification of the Manduca orthologues using RT (reverse transcriptase)–PCR [12,16,20]. Unequivocal evidence that these genes are the Manduca orthologues of Drosophila Phm, Dib, Sad and Shd came from functional characterization by expression of the genes in Drosophila S2 cell lines. When expressed in S2 cells with specific ecdysteroid substrates, Manduca Phm is the C25-hydroxylase, Dib the C22-hydroxylase, Sad the C2-hydroxylase and Shd the C20-hydroxylase (Figure 2) [12,20]. While Phm is a microsomal P450, Dib and Sad are mitochondrial. Thus ecdysteroid intermediates are shuttled between intracellular compartments during biosynthesis. In Manduca, E20MO activity is observed in both mitochondrial and microsomal fractions  and it is possible the Shd may reside in either of these locations [3,12] as demonstrated for some mammalian P450s [22,23].
Expression of the Manduca Halloween genes
Throughout embryonic, larval and pupal development of insects, changes in the ecdysteroid titre promote transition from one developmental stage to the next . We carried out an extensive expression analysis of the Manduca Halloween genes to investigate developmentally related changes in their expression [12,16,20] in this established model insect for endocrinological studies.
Consistent with previous studies showing that the Drosophila phm, dib and sad are predominantly expressed in the prothoracic gland cells [4,5], and the classical dogma that these glands are the principal source of ecdysone , the Manduca orthologues of these genes are selectively expressed in the prothoracic glands during the fifth larval instar .
The haemolymph ecdysteroid titre is for a large part directly a result of the production by the prothoracic glands , thus depending on the activity of the steroidogenic enzymes including Phm, Dib and Sad. To examine whether changes in the production of ecdysteroids by the prothoracic glands are associated with transcriptional regulation of the Halloween genes, we carried out detailed analyses of their expression during the fifth instar and during the beginning of pupal–adult development . The final larval instar of Manduca is characterized by two pulses of ecdysteroids; the first small peak around days 3–4 (commitment peak) reprograms the animal to undergo a metamorphic moult in response to the second and large peak (moulting peak) at day 7 that elicits moulting to the pupa . Figure 2 shows that increases in the prothoracic glands' expression of phm, dib and sad were temporally coincident with the two haemolymph ecdysteroid pulses during the fifth larval instar.
During the beginning of pupal–adult development, the prothoracic glands are believed to support the rise in the haemolymph ecdysteroid titre consisting mainly of ecdysone . Six days after pupation, these glands begin to undergo programmed cell death . Although the expression of phm, dib and sad does not closely parallel the ecdysteroid titre during the beginning of pupal–adult development, increases are observed in this period (Figure 2). This agrees with previous reports indicating a role of the prothoracic glands in the production of ecdysone necessary for normal pupal–adult development.
The expression pattern of sad, catalysing the final step in the formation of ecdysone, is reminiscent of the 2-hydroxylase, dib, catalysing the preceding step (Figure 2). It is intriguing that spo and phm exhibit very similar patterns of expression given evidence that Spo may act as an integral protein in the biosynthetic pathway upstream of Phm [15,16].
In Drosophila, spo is not expressed in the ring gland, a composite organ housing the prothoracic gland cells, during larval development but only in the embryo prior to the formation of this gland [15,16]. This indicates that Spo is not required for ecdysteroid production during larval development, even though ecdysteroid production by the prothoracic glands must take place for moulting to occur at these stages. The fact that Manduca spo was expressed in the prothoracic glands during larval development perplexed but encouraged us to re-examine the Drosophila genome. It turned out that Cyp307a2p, believed to be a truncated pseudogene, encoded a complete P450 enzyme. Expression analysis revealed that this closely related paralogue of spo, dubbed spookier (spok; Cyp307a2), is expressed in the embryonic and larval ring gland . Thus it seems that Drosophila (dipteran), in contrast with Manduca and Bombyx (lepidopterans), has two proteins, Spo and Spok, that provide Spo-like activity at different life stages . Distinct spatiotemporal expression of duplicated steroidogenic P450s, like spo and spok, is also known with paralogues of the aromatase (CYP19) in teleosts .
Overall, our expression data indicate that transcriptional up-regulation of phm, dib and sad may occur to support the zenith of ecdysteroid production by the prothoracic glands during the fifth larval instar of Manduca. If Spo is directly involved in the biosynthesis of ecdysone, regulation of its expression probably serves the same purpose, that is, to support the prothoracic glands' changing requirements for synthesizing ecdysteroids.
The E20MO is responsible for the conversion of ecdysone into 20E and thus is of critical importance to normal insect development. Biochemical characterization of the E20MO as being a typical P450 enzyme was done more than 20 years ago  and Smith et al.  described the in vitro activity of this enzyme in the fat body and midgut of Manduca. In these tissues, the activity fluctuates dramatically during the fifth instar. Analysis of Manduca shd mRNA levels reveals the preponderance of expression in the fat body, midgut and Malpighian tubules , which are classical tissues for E20MO activity . There is a remarkable correlation between changes in the activity [29,30] and the levels of shd mRNA in the fat body and midgut during the fifth instar and the first 6 days of pupal–adult development (Figure 2). This suggests that the activity of the Manduca E20MO is strictly dependent on the transcriptional activity, although post-transcriptional control may also occur. In addition to these dramatic changes in the fat body and especially the midgut, significant changes in shd expression were observed in the Malpighian tubules during the fifth instar. These results reveal three overlapping and successive peaks in shd expression, and thereby E20MO activity, reaching a peak in the fat body at days 4–5, in the midgut at days 5–6 and in the Malpighian tubules at days 6–7 of the fifth instar. Therefore it seems possible that the fat body converts ecdysone into 20E during the ‘commitment peak’, whereas the Malpighian tubules, and perhaps to a less degree the midgut, are responsible for this conversion at the time of the moulting peak. During the first 6 days of pupal–adult development when the ecdysteroid titre consists mainly of ecdysone , significant expression of Manduca shd was not observed.
While characterization of the Halloween gene family of P450s has brought conclusive molecular characterization of the terminal hydroxylations in the biosynthetic scheme, attempts to identify enzymes acting prior to the formation of the ketodiol (the substrate of Phm) have been unsuccessful. Identification and characterization of the enzymes involved in these early steps are the final frontiers in the elucidation of the insect enzymes involved in ecdysteroidogenesis.
The apparent transcriptional control of the Halloween genes, including the prominent and rapid changes of shd expression, merits further investigation as knowledge of regulation of these genes is important for understanding modulation of ecdysteroidogenesis. In Drosophila, it was recently demonstrated that βFTZ-F1 (β fushi tarazu factor 1), a homologue of SF1 (steroidogenic factor 1) controlling the expression of vertebrate steroidogenic P450s, is involved in regulation of Phm and Dib . This indicates fundamental conservation of the transcriptional machinery controlling vertebrate and insect steroidogenic P450s.
Uncovering the complete biosynthetic pathway of ecdysteroids and the mechanisms regulating the ecdysteroid titre are important for our understanding of moulting and metamorphosis, which are fundamental processes in the lives of arthropods that have contributed to the indisputable success of this group of animals. Such knowledge may also provide the basis for the development of target-specific agents for pest control.
We are grateful to our collaborators, Dr Michael B. O'Connor and his colleagues at the University of Minnesota (Minneapolis, MN, U.S.A.) and Dr Chantal Dauphin-Villemant and her colleagues at Université Pierre et Marie Curie (Paris, France). This research was supported by grants from the NSF (National Science Foundation; IBN0130825; L.I.G. and J.T.W.) and National Institutes of Health (Bethesda, MD, U.S.A.; GM63198-01; R.R.).
8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology: Independent Meeting held at Swansea Medical School, Swansea, Wales, U.K., 23–27 July 2006. Organized and Edited by D. Kelly, D. Lamb and S. Kelly (Swansea, U.K.).
Abbreviations: 7dC, 7-dehydrocholesterol; Dib, Disembodied; 20E, 20-hydroxyecdysone; E20MO, ecdysone 20-mono-oxygenase; ketodiol, 2,22,25-trideoxyecdysone; P450, cytochrome P450; Phm, Phantom; Sad, Shadow; Shd, Shade; Spo, Spook; Spok, Spookier
- © 2006 The Biochemical Society