Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity

The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: effects of the ecdysone agonist RH 5849 on ecdysone 20-monooxygenase activity

The non-steroidal ecdysone agonist RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) was found to inhibit in a dose-response and apparently competitive fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity in the midgut of wandering stage last instar larvae of the tobacco hornworn, Manduca sexta. More effectively on a per molar basis than the naturally occurring molting hormones ecdysone and 20-hydroxyecdysone, RH 5849 was also found to elicit the dramatic 50-fold increase in midgut steroid hydroxylase activity (which normally occurs with the onset of the wandering stage) when injected into competent head or thoracic ligated pre-wandering last instar larvae. These data support and extend the potential usefulness of RH 5849 as a pharmacological probe for further investigating the actions of ecdysteroids and their role(s) in the regulation of ecdysteroid monooxygenases.     

Ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta (L.) Midgut: Is the dual localization real?

The dual localization of ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta larval midgut was investigated. Cosubstrate requirements and response to osmolarity of the microsomal ecdysone 20-monooxygenase system were found to be different from those previously reported for the mitochondrial enzyme system. The microsomal monooxygenase utilized NADPH and, less efficiently, NADH as cosubstrates. NADPH and NADH effects were neither additive nor synergistic. NADPH yielded identical activities in isotonic and hypotonic incubations. Mitochondria and microsomes showed no synergistic interaction for ecdysone 20-hydroxylation. After washing of the mitochondria, a large proportion of their ecdysone 20-monooxygenase activity was lost. The extent of the loss was inversely correlated to the concentration of mitochondria in the incubation mixture. The addition of bovine serum albumin to the incubations (2 mg/ml) largely restored the original activities. The microsomal contamination in mitochondrial pellets after each of three successive washings was determined by measuring the activity of a microsomal marker enzyme, NADPH-cytochrome c reductase. At each step of the purification, the ecdysone 20-monooxgenase activity of the mitochondrial preparations far exceeded the activity attributable to the microsomal contamination. These results confirm the existence of two independent ecdysone 20-monooxygenase systems in the midgut of M. sexta larvae.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: evidence for the involvement of a neuroendocrine-endocrine axis during larval-pupal development

Ecdysone 20-monooxygenase activity and the factors which may regulate this steroid hydroxylase were examined in the midgut of the tobacco hornworm, Manduca sexta, during the last larval stadium. Radioassay experiments revealed that midgut ecdysone 20-monooxygenase undergoes a single 50-fold increase in activity temporally coincident with the onset of the wandering stage. The increase in midgut monooxygenase activity was prevented by actinomycin D and cycloheximide, and could be elicited in head (but not thoracic) ligated animals by a brain-retrocerebral complex factor(s) released at the same time as prothoracicotropic hormone. In contrast, ecdysone or 20-hydroxyecdysone could elicit the increase in enzyme activity in both head and thoracic ligated animals. These data suggest the operation of a neuroendocrine-endocrine axis in the regulation of midgut ecdysone 20-monooxygenase activity.     

Midgut and fatbody mitochondrial transhydrogenase activities during larval-pupal development of the tobacco hornworm, Manduca sexta

Midgut and fatbody mitochondria from fifth larval instar Manduca sexta display a membrane-associated transhydrogenase that catalyzes a reversible hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming activity occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg⁺⁺-dependent ATPase. During the ten-day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the fifty-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations were made apparent. Moreover, that the increases in all transhydrogenase activities resulted from de novo enzyme synthesis were indicated by the cycloheximide-dependent reductions in these activities.     

Regulation of ecdysone hydroxylation in Locusta migratoria: Rôle of the moulting hormone level

After repetitive injections of moderate doses of ecdysone, ecdysterone or phenobarbital to young Vth (last) instar larvae of Locusta migratoria, the conversion rate of ecdysone to ecdysterone in vivo is significantly higher than in control insects. Similarly, 5 hr after injection of a low dose of ecdysone or ecdysterone, a strong ‘induction’ of ecdysone 20-monooxygenase activity occurs. This ‘inductive’ effect is blocked by cycloheximide.Simultaneous injections of ecdysone and ecdysterone show that hydroxylation of ecdysone is inhibited by the product of the reaction, ecdysterone. Removal of the prothoracic glands and X-ray treatment of the hemocytopoietic tissue do not affect ecdysone hydroxylation. The mechanism of induction and inhibition of ecdysone 20-monooxygenase shown in this study is probably responsible for the important variations of this key enzyme which have been reported from several insect species.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Characterization of ecdysone 20-monooxygenase activity in wandering stage larvae of Drosophilamelanogaster: Evidence for mitochondrial and microsomal cytochrome P-450 dependent systems

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone to 20-hydroxyecdysone, was characterized in wandering stage larvae of Drosophila melanogaster using an in vitro radioassay in conjunction with analytical thin layer chromatography. 20-Hydroxyecdysone was confirmed to be the product of the enzyme radioassay system by high pressure liquid chromatography. The 20-monooxygenase was found to be most active in a 0.10 M phosphate buffer, pH 7.5, was inhibited by Ca²⁺, Mg²⁺ and Se⁴⁺ and exhibited a temperature optimum at 35°C. Differential centrifugation, sucrose step gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with both the mitochondrial and microsomal fractions. Substrate kinetics experiments indicated that the mitochondrial and microsomal monooxygenase systems exhibit apparent K_ms for ecdysone of 6.4 × 10⁻⁸ and 9.9 × 10⁻⁸ M, respectively, with apparent V_maxs of 4.1 and 10.2 pg 20-hydroxyecdysone formed/min per mg tissue equiv., respectively. Both monooxygenase systems were inhibited by their product 20-hydroxyecdysone. The cytochrome P-450 nature of these insect steroid hydoxylases was initially suggested by their requirement for NADPH, NADH was approximately half as effective in supporting the mitochrondrial monooxygenase activity. In addition, both monooxygenase systems were inhibited by carbon monoxide, ellipticine, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Photochemical action spectra of ecdysone 20-monooxygenase activity confirmed the cytochrome P-450 dependency of both the mitochondrial and microsomal ecdysone 20-hydroxylase systems.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae)

A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x 10(-10) and 1.6 x 10(-10) M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6 x 10(-7) M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced approximately 56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was approximately 1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Ecdysterone biosynthesis: a microsomal cytochrome-P-450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust.

Ecdysone 20-monooxygenase, an enzyme which converts ecdysone to ecdysterone (the major moulting hormone of insects) has been characterized in cell-free preparations of tissues from African migratory locust. The product of the reaction has been identified as ecdysterone on the basis of several microchemical derivatization and chromatographic methods. Ecdysone 20-monooxygenase activity is located primarily in the microsomal fraction which also carries NADPH cytochrome c reductase and cytochrome P-450, as shown by sucrose density gradient centrifugation. Optimal conditions for the ecdysone 20-monooxygenase assay have been determined. The enzyme has a Km for ecdysone of 2.7 x 10(-7) M and is competitvely inhibited by ecdysterone (Ki = 7.5 x 10(-7) M). Ecdysone 20-monooxygenase is a typical cytochrome P-450 linked monooxygenase: the reaction requires O2 and is inhibited by CO, an effect partially reversed by white light. The enzyme is effectively inhibited by several specific monooxygenase inhibitors and by sulfhydryl reagents, but not by cyanide ions. Ecdysone elicits a type I difference spectrum when added to oxidized microsomes. NADPH acts as preferential electron donor. The transfer of reducing equivalents proceeds through NADPH cytochrome c (P-450) reductase: ecdysone 20-monooxygenase is inhibited by cytochrome c. Both NADPH cytochrome c reductase and ecdysone 20-monooxygenase are inhibited by NADP+ and show a similar Km for NADPH. The Malpighian tubules have the highest specific activity of ecdysone 20-monooxygenase, while fat body contain most of the cytochrome P-450 and NADPH cytochrome c reductase.     

Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.     

Effects of a fat body extract on larval midgut cells and growth of lepidoptera

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentration-dependent manner. This effect was compared with those of insect molting hormones, ecdysone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.     

Ecdysone 20-monooxygenase in a cricket,Gryllus bimaculatus (Ensifera,Gryllidae)—characterization of the microsomal midgut steroid hydroxylase in adult females

Summary Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml^–1. K⁺ and Na⁺ (10^–3–10^–1 M), Ca²⁺ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg²⁺ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent K_m for ecdysone of 3.7·10^–7 M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10^–6 M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I₅₀ of 8·10^–4 M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein^–1 was calculated for midgut microsomes of 4-day-old females.Abbreviations GO carbon monoxide - EDTA ethylenediamine tetraacetic acid - HPLC high performance liquid chromatography - I ₅₀ concentration for 50% inhibition - KCN potassium cyanide - K ₁ inhibition constant - K _m Michaelis-Menten constant - MOPS 3-morpholinopropanesulfonic acid - NADH/NAD ⁺ nicotinamide adenine dinucleotide reduced/oxidized - NADPH/NADP ⁺ nicotinamide adenine dinucleotide phosphate reduced/oxidized - Na ₂ S ₂ O ₄ sodium dithionite - SEM Standard error of mean - TLC thin-layer chromatography - TRIS 2-amino 2-hydroxymethyl-1,3-propanediol (trishydroxymethyl aminomethane) - V _max maximal reaction velocity     

The effect of the juvenile hormone analog, fenoxycarb, on ecdysone receptor B1 expression in the midgut of Bombyx mori during larval-pupal metamorphosis

The Bombyx mori (Lepidoptera: Bombycidae) midgut undergoes remodeling during the larval-pupal metamorphosis. All metamorphic events in insects are controlled by mainly two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate, has been shown to be one of the most potent juvenile hormone analogs against a variety of insect species. In this study, the effect of fenoxycarb on EcR-B1 protein expression in the midgut of Bombyx mori during the remodeling processwas investigated. Fenoxycarb was topically treated to the beginning of the fifth instar Bombyx larvae. Its application prolonged the last instar and prevented metamorphic events. Analyses were performed from day 6 of the fifth instar to 24 hr after pupation in controls and to day 14 of the fifth instar in the fenoxycarb treated group. According to our results, the presence of EcR-B1 in the midguts of the fenoxycarb treated group during the feeding period suggested that EcR-B1 was involved in the functioning of larval cells and during this period fenoxycarb did not affect EcR-B1 status. Immediately after termination of the feeding stage, the amount of EcR-B1 protein increased, which indicated that it may strengthen the ecdysone signal for commitment of remodeling process. In the fenoxycarb treated group, its upregulation was delayed, which may be related to the inhibition of ecdysone secretion from the prothoracic gland.     

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.