The complex metabolism of poststerone in male rats.

Ecdysteroids are not endogenous to mammals, but are normal components of the food intake of many mammalian species consuming phytoecdysteroid-containing plants. The most frequently encountered phytoecdysteroid is 20-hydroxyecdysone (20E). Several pharmaceutical effects have been observed after ecdysteroid injection or ingestion, but it is not clear to what extent metabolites generated in the mammalian body contribute to these effects. The C21-ecdysteroid poststerone (Post) is a metabolite of 20E in rodents. Post analogues are key intermediates in the metabolism of exogenous ecdysteroids possessing a C20/22-diol. The pharmacokinetics, bioavailability and metabolism of Post have been assessed in male rats after ingestion and injection. The bioavailability of Post is significantly greater than that of 20E and the presence of an efficient entero-hepatic cycle allows Post to be effectively metabolised to a wide range of metabolites which are excreted mainly in the faeces, but also to some extent in the urine. Several of the major metabolites in the bile have been identified unambiguously as 3-epi-poststerone, 16α-hydroxypoststerone, 21-hydroxypoststerone and 3-epi-21-hydroxypoststerone. Conjugates are also present. Parallels are drawn to the metabolism of endogenous vertebrate steroid hormones, to which Post bears more similarity than 20E.

Ecdysteroid metabolism in mammals: The fate of ingested 20-hydroxyecdysone in mice and rats.

Phytoecdysteroids are molecules derived from sterol metabolism and found in many plants. They display a wide array of pharmacological effects on mammals (e.g. anabolic, anti-diabetic). Although these effects have been long established, the molecular targets involved remain to be identified. Like endogenous steroid hormones and bile acids, which are biochemically related, ingested or injected phytoecdysteroids undergo a set of reactions in mammals leading to the formation of numerous metabolites, only some of which have been so far identified, and it is presently unknown whether they represent active metabolites or inactivation products. In the large intestine, ecdysteroids undergo efficient 14-dehydroxylation. Other changes (reductions, epimerization, side-chain cleavage) are also observed, but whether these occur in the liver and/or large intestine is not known. The purpose of this study was to investigate the pharmacokinetics of 20-hydroxyecdysone (20E), the most common phytoecdysteroid, when administered to mice and rats, using, when required, tritium-labelled molecules to permit metabolic tracking. Bioavailability, the distribution of radioactivity and the kinetics of formation of metabolites were followed for 24-48 hours after ingestion and qualitative and quantitative analyses of circulating and excreted compounds were performed. In mice, the digestive tract always contains the majority of the ingested 20E. Within 30 min after ingestion, 20E reaches the large intestine, where microorganisms firstly remove the 14-hydroxyl group and reduce the 6-one. Then a very complex set of metabolites (not all of which have yet been identified) appears, which correspond to poststerone derivatives formed in the liver. We have observed that these compounds (like bile acids) undergo an entero-hepatic cycle, involving glucuronide conjugation in the liver and subsequent deconjugation in the intestine. Despite the very short half-life of ecdysteroids in mammals, this entero-hepatic cycle helps to maintain their plasma levels at values which, albeit low (≤0.2 µM), would be sufficient to evoke several pharmacological effects. Similar 20E metabolites were observed in mice and rats; they include in particular 14-deoxy-20E, poststerone and 14-deoxypoststerone and their diverse reduction products; the major products of this metabolism have been unambiguously identified. The major sites of metabolism of exogenous ecdysteroids in mammals are the large intestine and the liver. The entero-hepatic cycle contributes to the metabolism and to maintaining a low, but pharmacologically significant, concentration of ecdysteroids in the blood for ca. 24 h after ingestion. These data, together with parallel in vitro experiments provide a basis for the identification of 20E metabolite(s) possibly involved in the physiological effects associated with ecdysteroids in mammals.

Detection and quantitation of ecdysterone in human serum by liquid chromatography coupled to tandem mass spectrometry.

The polyhydroxylated phytosteroid ecdysterone is present in various plants (e.g. spinach). It is widely marketed as the active component of dietary supplements, due to its reported health and performance promoting effects. For evaluation of its actual bioavailability, a fast and sensitive method was developed, optimized and validated for human serum. Instrumental analysis was performed utilizing liquid chromatography-tandem mass spectrometry with positive electrospray ionization and acquisition in multiple reaction mode. Solid phase extraction and dilute-and-inject (following protein precipitation) were tested to isolate ecdysterone from human serum. Both methods were compared in the light of the preset analytical target profile. The limit of detection (LOD) and quantitation (LOQ) for ecdysterone in human serum after SPE extraction corresponded to 0.06 ng/mL and 0.14 ng/mL, respectively, meeting the requested sensitivity of the method. The assay was linear over the range of 0.10 ng/mL to 20.83 ng/mL. As expected, the sensitivity of the SPE method was better than that of the dilute-and-inject procedure, which did not allow for quantitation of all post administration serum samples. Accuracy (relative error; %) and precision (coefficient of variation; %), were both within acceptance criteria (<15%). The developed method was successfully applied to a ten week intervention study conducted in young men performing regular resistance training. Different doses of supplements containing ecdysterone from spinach extract have been administered during the study and the quantitation of ecdysterone in serum samples has been successfully conducted. Ecdysterone could be quantified in all post-administration samples using solid phase extraction (SPE) for sample pretreatment.

Ecdysteroids are present in the blood of wild passerine birds.

Ecdysteroids (arthropod molting hormones) play an important role in the development and sexual maturation of arthropods, and they have been shown to have anabolic and "energizing" effect in higher vertebrates. The aim of this study was to assess ecdysteroid diversity, levels according to bird species and months, as well as to observe the molting status of hard ticks (Acari: Ixodidae) infesting the birds. Therefore, blood samples and ticks were collected from 245 birds (244 songbirds and a quail). Mass spectrometric analyses showed that 15 ecdysteroids were regularly present in the blood samples. Molting hormones biologically most active in insects (including 20-hydroxyecdysone [20E], 2deoxy-20E, ajugasterone C and dacryhainansterone) reached different levels of concentration according to bird species and season. Similarly to ecdysteroids, the seasonal presence of affected, apolytic ticks peaked in July and August. In conclusion, this study demonstrates the presence of a broad range and high concentrations of ecdysteroids in the blood stream of wild-living passerine birds. These biologically active, anabolic compounds might possibly contribute to the known high metabolic rate of songbirds.

Larvicidal efficacy of Adiantobischrysene from Adiantum latifolium against Oryctes rhinoceros through disrupting metamorphosis and impeding microbial mediated digestion.

BACKGROUND: Oryctes rhinoceros Linn. (Coleoptera: Scarabaeidae) is a serious pest of coconuts and other palms. Symbiotic gut bacteria play significant roles in the digestion of cellulosic materials as well as in some other physiological processes essential for the existence of O. rhinoceros larvae. The study was undertaken to isolate a compound with antibacterial and larvicidal activities from the leaves of Adiantum latifolium Lam. following a bioassay-guided method. RESULTS: Methanol extract (ME) of dry leaf powder of A. latifolium showed larvicidal activity against third-instar O. rhinoceros (LD50 , 5018 mg/kg) with antibacterial activity on its gut microbiota. An in vitro study showed the bacteria Bacillus cereus, Micrococcus lylae, Stenotrophomonas maltophilia, Kocuria rosea, Burkholderia mallei, Staphylococcus epidermidis, S. arlettae and Corynebacterium afermentans identified from the larval gut were sensitive to ME. Bioactivity-guided isolation of the compound by liquid-liquid extraction and column chromatography resulted in Adiantobischrysene which showed antibacterial and larvicidal activity (LD50 , 8.4 mg/kg) and led to weight loss and precocious metamorphosis in larvae. An enzyme immunoassay showed a large peak in 20-hydroxyecdysone that commits larvae to precocious metamorphosis. CONCLUSION: This study demonstrated that the antibacterial and metamorphosis disrupting activity of Adiantobischrysene make it a natural pesticidal compound against O. rhinoceros. © 2018 Society of Chemical Industry.

Modification of hormonal balance in larvae of the corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae) due to sublethal Bacillus thuringiensis protein ingestion.

Bacillus thuringiensis (Bt) corn, Zea mays L., is highly efficient against the corn borer Sesamia nonagrioides (Lefèbvre) (Lepidoptera: Noctuidae) when the larvae feed only on the transgenic plants. However, when they feed on Bt leaves during only part of their development, thus ingesting sublethal amounts of Bt toxins, some larvae survive. A previous study reported a prolonged development and precocious diapause induction in larvae fed on a diet with sublethal amounts of Cry1Ab protein. To determine whether these effects were accompanied by a modification of the hormonal balance, S. nonagrioides larvae were fed on sublethal amounts of Bt protein provided in Bt leaves or in the diet. The larvae that survived had higher levels of juvenile hormone (JH), whereas their level of ecdysteroids did not increase sufficiently to allow pupation, leading to a longer larval development and more larval molts. This response may be considered a defense mechanism that allows some larvae to survive toxin ingestion; it is similar the response to insecticidal toxins or viruses observed in other larvae. Changes in the hormone levels in diapausing larvae were undetectable, probably because these changes were masked by the higher level of JH in the hemolymph of diapausing larvae and because of lack of ecdysteroid titer increase, a phenomenon that is usually observed a few days before pupation in nondiapausing larvae. These results should be taken into account in the establishment of non-Bt refuges to prevent development of Bt-resistance in S. non-agrioides populations.

Metabolic effects of 20-OH-ecdysone in ovariectomized rats.

Postmenopausal women develop often obesity which may be prevented by 20-OH-Ecdysone (Ecd). This was investigated in ovariectomized (ovx) rats. They were orally treated with 3 doses of Ecd (18, 56 or 116 mg/day/animal). Positive controls received 159 microg estradiol (E2). Quantitative computer tomography at the level of the abdomen and the metaphysis of the tibia allowed estimation of surface, fat depots and muscles. The highest dose of Ecd resulted in serum concentrations of 0.4 x 10(-6)M. Serum E2 concentrations in the positive controls were 73.3+/-24.41 pg/ml. E2 but not Ecd stimulated uterine weights. Under Ecd ovx animals gained less fat but had more muscle mass. Serum TSH, T4 and T3 levels remained unaffected while E2 treatment increases T4 but decreases T3 levels. Ecd at the lowest dose lowered serum LDL and did not result in increased serum triglycerides, an effect seen in the E2 treated rats. At the Ecd highest dose serum HDL was higher than in the controls. In conclusion Ecd has beneficial effects on fat and muscle tissue and may be able to prevent the metabolic syndrome and sarcopenia by a non-estrogenic mechanism.

Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulation.

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210-330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20-OH ecdysone (20-OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20-OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity.

The ectoparasitic wasp Eulophus pennicornis (Hymenoptera: Eulophidae) uses instar-specific endocrine disruption strategies to suppress the development of its host Lacanobia oleracea (Lepidoptera: Noctuidae).

To successfully complete its development, the gregarious ectoparasitoid Eulophus pennicornis must inhibit the moult of its host, Lacanobia oleracea. In the present study, we examined the possibility that moult- and metamorphosis-associated endocrine events may be disrupted in caterpillars parasitized as newly moulted last (sixth) instars. Juvenile hormone (JH) titres on days 2 and 5 of the final stadium were significantly higher (> 100 fold) in parasitized than in non-parasitized hosts, in which JH was essentially absent. Elevated JH levels were associated with reduced haemolymph JH esterase (JHE) activity (down by 99.8%) and enhanced in vitro JH biosynthesis by the corpora allata (CA) (up to 4.5 fold). Wasp adults and/or larvae, in which we measured high levels of JH III (up to 2.7 ng/g), but little or no JH I or JH II, were not seen as likely sources of JH in parasitized hosts, in which we found mostly JH I and JH II. In addition, removal of parasitoid eggs or larvae after oviposition did not prevent the rise in JH titres seen in parasitoid-laden hosts, suggesting that wasp venom may be responsible for the observed hormonal dysfunction. Host haemolymph 20-hydroxyecdysone (20-E) levels were largely unaffected by parasitism during the final stadium although they were observed to increase earlier and decrease more rapidly in parasitized insects. We compare these results with those reported earlier for L. oleracea larvae parasitized by E. pennicornis as penultimate (fifth) instars, which display significantly depressed 20-E titres relative to control larvae. We conclude that E. pennicornis employs host endocrine-disruption strategies that differ according to whether the host is parasitized as a penultimate or final-stadium larva.

Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta.

During metamorphosis, the insect nervous system undergoes considerable remodeling: new neurons are integrated while larval neurons are remodeled or eliminated. To understand further the mechanisms involved in transforming larval to adult tissue we have mapped the metamorphic changes in a particularly well established brain area, the antennal lobe of the sphinx moth Manduca sexta, using an antiserum recognizing RFamide-related neuropeptides. Five types of RFamide-immunoreactive (ir) neurons could be distinguished in the antennal lobe, based on morphology and developmental appearance. Four cell types (types II-V, each consisting of one or two cells) showed RFamide immunostaining in the larva that persisted into metamorphosis. By contrast, the most prominent group (type I), a mixed population of local and projection neurons consisting of about 60 neurons in the adult antennal lobe, acquired immunostaining in a two-step process during metamorphosis. In a first step, from 5 to 7 days after pupal ecdysis, the number of labeled neurons reached about 25. In a second step, starting about 4 days later, the number of RFamide-ir neurons increased within 6 days to about 60. This two-step process parallels the rise and fall of the developmental hormone 20-hydroxyecdysone (20E) in the hemolymph. Artificially shifting the 20E peak to an earlier developmental time point resulted in the precocious appearance of RFamide immunostaining and led to premature formation of glomeruli. Prolonging high 20E concentrations to stages when the hormone titer starts to decline had no effect on the second increase of immunostained cell numbers. These results support the idea that the rise in 20E, which occurs after pupal ecdysis, plays a role in the first phase of RFamide expression and in glomeruli formation in the developing antennal lobes. The role of 20E in the second phase of RFamide expression is less clear, but increased cell numbers showing RFamide-ir do not appear to be a consequence of the declining levels in 20E that occur during adult development.

Ecdysteroid control of cell proliferation during optic lobe neurogenesis in the moth Manduca sexta.

Cell proliferation within the optic lobe anlagen is dependent on ecdysteroids during metamorphosis of the moth Manduca sexta. We use cultured tissues to show that ecdysteroids must be maintained above a sharp threshold concentration to sustain proliferation. Proliferation can be turned on and off repeatedly simply by shifting the ecdysteroid concentration to above or below this threshold. In subthreshold hormone, cells arrest in the G2 phase of the cell cycle. Ecdysteroid control of proliferation is distinguished from differentiative and maturational responses to ecdysteroids by requiring tonic exposure to the hormone and lower levels of 20-hydroxyecdysone, and by being sensitive to either 20-hydroxyecdysone or its precursor, ecdysone. These characteristics allow optic lobe development to be divided into two ecdysteroid-dependent phases. Initially, moderate levels of ecdysteroid stimulate proliferation. Later, high levels of 20-hydroxyecdysone trigger a wave of apoptosis within the anlage that marks completion of its proliferative phase.

[Interaction of C27-sterols with erythrocyte membranes]. / Issledovanie vzaimodeistviia C27-sterinov s membranami éritrotsitov.

It is established in the in vitro experiments that the interaction of natural C27-sterols: cholesterol, 7-dehydrocholesterol, cholecalciferol (vitamin D3), calcitriol (1 alpha, 25-dihydroxyvitamin D3), ecdysterone (20-hydroxyecdysone) and 2-deoxyecdysterone modulate both lipid and protein phases of human erythrocyte membrane at the concentration range 10(-6)-10(-12) M. These effects depend on the sterols concentration and on the number and position of hydroxy groups in the sterol molecules.

Insect hormones in vertebrates: anabolic effects of 20-hydroxyecdysone in Japanese quail.

Ecdysteroids are hormones controlling cell proliferation, growth and the developmental cycles of insects and other invertebrates. They are occasionally present in various unrelated plants for no apparent reason; no phytohormonal function has yet been identified. In certain cases, ecdysteroids are accumulated to high levels in leaves, roots or seeds. Some ecdysteroid-containing plants have been known as medicinal plants for centuries. One of them, Leuzea carthamoides Iljin (Asteraceae), growing in Central Asia, contains 0.4% ecdysteroid in dry roots and 2% in seeds. A pharmacological preparation from this plant, "Ecdisten', is already available as a commercial preparation for its anabolic, tonic and other effects, for medical use (review). It remained problematic, however, whether ecdysteroids were truly responsible for these effects, because Leuzea contains a number of other biologically active compounds in addition to ecdysteroids. We extracted and purified ecdysteroids from the seeds of Leuzea. With 6 g of 96% 20-hydroxyecdysone (20E), we made a large-scale feeding assay with Japanese quail to find out whether ecdysteroid alone could duplicate the anabolic effects of the seeds. We found that the 96% ecdysteroid increased the mass of the developing quails in a dose-dependent manner, with the rate of increase proportional to the ecdysteroid content in the seeds; there was a 115% increase in living mass with 100 mg kg-1 of pure 20E compared with 109.5% increase with 100-180 mg kg-1 20E equivalents in the seeds. We conclude that the plethora of growth-promoting, vitamin-like effects induced in vertebrates by Leuzea is mediated by ecdysteroids.

Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. I. Hemolymph titers and metabolites.

Hemolymph ecdysteroid (Ecd) titers were measured using radioimmunoassay (RIA) during the molt cycle of the American lobster, Homarus americanus. Individual animals showed small, transitory rises of Ecds which increased in magnitude with the onset of premolt and culminated in a large premolt peak at morphological stages D2(2)-D3(1). Male lobsters had significant postmolt peaks and late premolt titers that remained high until ecdysis. In females, postmolt peaks were absent and late premolt titers reached basal levels before ecdysis. At least seven different Ecd metabolites were identified by high-performance liquid chromatography-RIA analyses. High polarity products (HP) were the most abundant metabolites in virtually every molt stage. Titers of HP were significantly higher in males during late postmolt-early intermolt and in late premolt. Levels of 20-hydroxyecdysone (20E) were equivalent in both sexes and correlated with the morphological changes associated with premolt. Evidence was also obtained for the presence of ecdysone, ponasterone A, and other as yet unidentified metabolites. The pattern of Ecd metabolites in the hemolymph supports other data indicative of 20E as the major molting hormone. Metabolism of 20E is primarily toward more polar compounds, including conjugates.

Effect of 20-hydroxyecdysone on the salivary glands of the male tick, Amblyomma hebraeum.

Female ticks (Acari: Ixodidae) feed only once in the adult stage, dying after laying a large batch of eggs. During the early post-engorgement stage, haemolymph ecdysteroid titre rises, which is probably responsible for autolysis of the salivary glands that takes place at this time. Males, on the other hand, can re-attach and feed numerous times during the adult stage. Males were fed on rabbits for either 7 or 14 days. Haemolymph was collected either the day of removal from the host or 4 days later, and ecdysteroid titre was measured by radioimmunoassay. The approximate titre in all 4 groups was 20 ng of 20-hydroxyecdysone (20-OHE) equivalents/ml haemolymph. Fluid secretory competence in vitro can be used as an index of salivary-gland degeneration. The glands dissected from fed males which had been left off the host for 4 days lost 62% of their fluid secretory competence compared to glands dissected shortly after the males were removed. This loss in fluid secretory competence was reversed by allowing ticks left off the host of 4 days to resume feeding. Male salivary glands lost fluid secretory competence when exposed for 4 days in organ culture to 20-OHE; the effect was maximal at the lowest concentration tested (20 ng/ml). Thus, although male salivary glands were highly sensitive to 20-OHE, it is still not clear whether this hormone causes the tissue to degenerate.

Ecdysteroid-like compounds in the serum and urine of African patients infected with Loa loa and Mansonella perstans microfilariae.

Ecdysteroids are compounds related to 20-hydroxyecdysone, the insect moulting hormone. Surprisingly, they have been found in serum and urine of patients infected with helminths. In these cases, the substances are assumed to be produced by the parasites and, therefore, might be used as a marker of parasitic infection. Thus, we need to know exactly which species, at which developmental stage, can release ecdysteroids in such large quantities that they could be detected in the biological fluids of the host. Large-scale investigations must, accordingly, be devoted to the major species of helminths. In the present study, we examined 100 African patients with Loa loa and/or Mansonella perstans microfilaraemia. About 70 of them had high levels of ecdysteroid-like materials in serum or urine. In contrast, uninfected patients and European controls had much lower concentrations. However, the ecdysteroid titres did not reflect the concentration of microfilariae actually present in the blood, and some heavily infected patients were even negative. Nevertheless, the most important point was that high ecdysteroid levels in man were always associated with a pathological condition. The precise significance of the phenomenon should be determined.

Cytophysiological correlations between prothoracic gland activity and hemolymph ecdysteroid concentrations in Rhodnius prolixus during the fifth larval instar: further studies in normal and decapitated larvae.

Hemolymph ecdysteroid titers in fifth instar larvae of Rhodnius prolixus were determined by radioimmunoassay, and their prothoracic glands were excised and examined by electron microscopy. During the last larval instar, the titer of ecdysteroid increased between the head-critical period until Day 13, at which time the peak titer was 3100 pg 20-hydroxyecdysone equivalents/microliter. The activation of secretory cells at the time of the second period of prothoracicotropic hormone release was correlated with the development of major cellular organelles. The smooth endoplasmic reticulum first appeared at the head-critical period and then proliferated in close relation to the increase in ecdysteroid titer until Day 13, after which time it disappeared. Mitochondria expand and develop tubular cristae. They are closely associated with the smooth endoplasmic reticulum. When insects were decapitated, hemolymph ecdysteroid titer remained below 10 pg/microliter and the prothoracic gland cells failed to develop smooth endoplasmic reticulum. We conclude that in the prothoracic gland cells as well as other steroidogenic tissues the smooth endoplasmic reticulum in association with mitochondria is involved in ecdysone biosynthesis.

Patterns of serum ecdysteroids during induced and uninduced proecdysis in the fiddler crab, Uca pugilator.

Eyestalk-intact and eyestalkless fiddler crabs, Uca pugilator, have similar temporal patterns of circulating serum ecdysteroids during proecdysis. Both groups of animals showed two distinct transient peaks of radioimmunoassay (RIA)-active ecdysteroids. Peak 1 occurred 3 weeks prior to ecdysis and preceded the onset of rapid proecdysial limb bud growth. Peak 2 was a larger peak that occurred a few days prior to ecdysis. Thin-layer chromatography profiles of the two peaks showed at least seven RIA-active compounds common to both peaks. The relative abundance of these compounds differed between the two peaks. The role of the eyestalks in control of circulating ecdysteroids was limited to maintenance of intermolt conditions. During proecdysis, the control of circulating ecdysteroid levels was located outside of the eyestalks. There was no correlation between limb bud growth rates and serum ecdysteroid levels during proecdysis.

Development of microsomal cytochrome P-450 monooxygenases during the last larval instar of the locust, Locusta migratoria: correlation with the hemolymph 20-hydroxyecdysone titer.

Several enzyme activities were measured in microsomes from Malpighian tubules and from fat body of the locust, Locusta migratoria, during the last larval instar and the 20-hydroxyecdysone titer was determined in the hemolymph. Ecdysone 20-monooxygenase, the cytochrome P-450 dependent monooxygenase which converts ecdysone to the active molting hormone 20-hydroxyecdysone had a low activity in the beginning of the instar, but showed a peak in both Malpighian tubules and fat body which coincided with the peak of 20-hydroxyecdysone in the hemolymph. The varying ratios of ecdysone and 20-hydroxyecdysone in L. migratoria hemolymph may therefore be accounted for by these changes in ecdysone 20-monooxygenase activity. The amounts of cytochrome P-450 and the activity of NADPH-cytochrome c reductase also showed a peak on day 5 of the instar, as did the activity of cytochrome P-450 linked lauric acid omega-hydroxylase in fat body microsomes. In larvae experimentally deprived of molting hormone, the activities of the cytochrome P-450 monooxygenases were low. The possible role of ecdysteroids in the control of developmental changes of cytochrome P-450 monooxygenases is discussed.