20-hydroxyecdysone Concentration Research Articles

ECDYSTEROIDS OF SILENE ITALICA: GLYCOSIDIC AND NONGLYCOSIDIC COMPONENTS AND HPLC-DAD-ESI-MS PROFILE


 Silene italica (L.) Pers. is a species of Caryophyllaceae family which introduced samples contain ecdysteroids that was previously shown (Meng et al., 2001). In this paper, the composition of ecdysteroids of wild-growing S. italica was shown. Using high performance chromatography with diode-array detection and electrospray ionization mass-spectrometry detection (HPLC-DAD-ESI-MS) twenty-two compounds assigned to the ecdysteroid group. The non-glycoside components of S. italica were included twelve compounds and integristerone A, 26-hydroxyintegristerone A, 22-deoxyintegristerone A, 2-deoxyintegristerone A, and 2-deoxypolypodine B were identified for the first time. Ten compounds were characterized as ecdysteroid monoglycosides containing hexose fragment as a carbohydrate part. Trer were derivatives of 20-hydroxyecdysone, polypodine B, ecdysone and 2-deoxypolypodine B. For the first time, the presence of 22-deoxyintegristerone A hexoside non-detected in plant objects was found. Comparative analysis of the ecdysteroids composition of S. italica and well studied species S. italica spp. nemoralis (Báthori et al., 2000, 2002, 2004; Pongrácz et al., 2003; Simon et al., 2002) indicates their proximity. Quantitative analysis of five ecdysteroid content in the organs of S. italica showed that they were unevenly distributed in the plant. The concentration of 20-hydroxyecdysone was 0.10–32.12 mg/g and the total ecdysteroids content was 0.10–40.92 mg/g of dry plant weight. In general, it should be noted that the ability to produce and accumulate ecdysteroids was also observed for the wild samples of S. italica, which cultural samples was noted as ecdysteroid concentrator.

Phytoecdysteroid and Clerodane Content in Three Wild Ajuga Species in Israel

Many species of the genus Ajuga (family Lamiaceae) contain phytoecdysteroids and clerodane diterpenes. Phytoecdysteroids are triterpene-derived analogues of steroid hormones that control molting and metamorphosis in arthropods, whereas clerodanes deter phytophagous insects. We identified and quantified phytoecdysteroid and clerodane contents in three Ajuga plant species in Israel. Leaves and roots of Ajuga iva, Ajuga chamaepitys (Ajuga chia), and Ajuga orientalis were collected from three different populations. Using liquid chromatography–time of flight–mass spectrometry analysis, we identified three phytoecdysteroids: 20-hydroxyecdysone (ecdysterone), makisterone A, and cyasterone and two clerodanes: dihydroajugapitin and columbin. Their contents varied significantly among plant species, organs, and populations. The highest concentrations of 20-hydroxyecdysone, makisterone A, and cyasterone were recorded in leaves and roots of A. iva. Cyasterone content tended to be higher in leaves of A. chamaepitys. Cl...

The retardant effect of 2-Tridecanone, mediated by Cytochrome P450, on the Development of Cotton bollworm, Helicoverpa armigera.

BackgroundPlant allelochemicals act as toxins, inhibitors of digestion, and deterrents that affect the fecundity of insects. These compounds have attracted significant research attention in recent decades, and much is known about the effects of these xenobiotic plant secondary metabolites on insect development. To date, although ecological interactions between xenobiotic plant secondary chemicals that retard insect growth have been observed in many species, it remains unclear how particular allelochemicals influence insect development in a life stage-dependent manner.ResultsWe found that 2-tridecanone can affect insect development; this effect appears similar to the symptoms induced by the physiological imbalance between juvenile and molting hormones in cotton bollworm. We later detected that a decrease in the concentration of 20-hydroxyecdysone occurred alongside the observed symptoms. We next identified the transcriptome of Helicoverpa armigera and eightdigital gene expression libraries for shading light on how 2-tridecanone retarded the development of cotton bollworm. The expression of CYP314A1, CYP315A1, CYP18A1, CYP307A1, and CYP306A1 (unigenes 16487, 15409, 40026, 41217, 35643, 16953, 8199, 13311, and 13036) were found to be induced by 2-tridecanone; these are known to be related to the biosynthesis or metabolism of 20-hydroxyecdysone. Expression analysis and RNA interference studies established that the retardant effect of 2-tridecanone on the development of cotton bollworm is mediated by P450 genes.ConclusionsThe candidate P450 gene approach described and exploited here is useful for identifying potential causal genes for the influence of plant allelochemicals on insect development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3277-y) contains supplementary material, which is available to authorized users.

Tributyltin Synergizes with 20-Hydroxyecdysone to Produce Endocrine Toxicity

One of the great challenges facing modern toxicology is in predicting the hazard associated with chemical mixtures. The development of effective means of predicting the toxicity of chemical mixtures requires an understanding of how chemicals interact to produce nonadditive outcomes (e.g., synergy). We hypothesized that tributyltin would elicit toxicity in daphnids (Daphnia magna) by exaggerating physiological responses to 20-hydroxyecdysone signaling via synergistic activation of the retinoid X receptor (RXR):ecdysteroid receptor (EcR) complex. Using reporter gene assays, we demonstrated that RXR, alone, is activated by a variety of ligands including tributyltin, whereas RXR:EcR heterodimers were not activated by tributyltin. However, tributyltin, in combination with the daphnid EcR ligand 20-hydroxyecdysone, caused concentration-dependent, synergistic activation of the RXR:EcR reporter. Electrophoretic mobility shift assays revealed that tributyltin did not enhance the activity of 20-hydroxyecdysone by increasing binding of the receptor complex to a DR-4 DNA-binding site. Exposure of daphnids to elevated concentrations of 20-hydroxyecdysone caused premature and incomplete ecdysis resulting in death. Tributyltin exaggerated this effect of exogenous 20-hydroxyecdysone. Further, exposure of daphnids to tributyltin enhanced the inductive effects of 20-hydroxyecdysone on expression of the 20-hydroxyecdysone-inducible gene HR3. Continuous, prolonged exposure of maternal daphnids to concentrations of tributyltin resulted in mortality concurrent with molting. Taken together, these results demonstrate that xenobiotics, such as tributyltin, can interact with RXR to influence gene expression regulated by the heterodimeric partner to RXR. The result of such interactions can be toxicity due to inappropriate or exaggerated hormonal signaling. The application of the in vitro/in vivo approach used in this study is discussed in relation to modeling of nonadditive interactions among constituents of chemical mixtures.

Insecticidal activity influence of 20-Hydroxyecdysone on the pathogenicity of Isaria fumosorosea against Plutella xylostella

The bioactivity of the entomopathogenic fungus, Isaria fumosorosea, and the plant secondary chemical, 20-Hydroxyecdysone, against Plutella xylostella were tested. 20-Hydroxyecdysone gave an apparent increase in adjusted larval mortality in a dose-dependent manner, whereas no influence of 20-Hydroxyecdysone was observed on conidial germination, mycelial growth and conidial production of I. fumosorosea. Furthermore, the synergism between 20-Hydroxyecdysone and I. fumosorosea was studied under laboratory and field cage conditions. Percent cumulated adjusted mortality of P. xylostella after the application of different concentrations of 20-Hydroxyecdysone and I. fumosorosea showed that the mortalities were directly related to the concentration of each component in the mixtures and their cumulative effect was evident for an extended period.

Quantitative Cell-Based Reporter Gene Assays Using Droplet-Based Microfluidics

We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data.

Ecdysteroid signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest Spodoptera exigua

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists such as methoxyfenozide (RH-2485) have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, cell lines derived from the polyphagous noctuid pest Spodoptera exigua (Se4 cells) were selected for continuous growth in the presence of high concentrations of 20-hydroxyecdysone (20E) or methoxyfenozide. Here we describe an analysis of ecdysteroid receptor signaling in the ecdysteroid-resistant Se4 cell lines. In contrast to other ecdysteroid-resistant cell lines described in literature, our data support the existence of a normal functioning ecdysteroid receptor complex in the resistant Se4 cell lines: (1) using a recombinant BmNPV baculovirus as a transduction tool, activation of an ecdysone-responsive luciferase cassette was demonstrated; (2) the early gene HR3 is constitutively expressed in the resistant cell lines that are grown in the presence of 20E or methoxyfenozide. Quantitative RT-PCR experiments indicated that expression levels of SeEcR mRNA were comparable among sensitive and resistant cell lines. Sequencing of PCR fragments also revealed the presence of SeEcR mRNA with a wild-type ligand-binding domain in resistant cells. Finally, a possible role for the gene FTZ-F1, whose expression correlates with the absence of circulating ecdysteroids during insect development, in the resistance mechanism was investigated. However, it was observed that FTZ-F1, in contrast to what is observed during insect development, is constitutively expressed in Se4 cells and that its expression is not regulated by the addition of ecdysteroid. It is proposed that the resistance mechanism in Se4 cells resides at the coupling between the conserved hierarchical cascade of early and early-late gene expression and the differentiation program in the Se4 cell line. The use of insect cell lines for the investigation of resistance against dibenzoylhydrazine ecdysone agonists and their relevance for uncovering resistance mechanisms in insects during pest control programs is discussed.

Identification of the molting hormone of the sweet potato ( Bemisia tabaci) and greenhouse ( Trialeurodes vaporariorum) whitefly

In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.

Shell protein characteristics and vitellogenin-like proteins in brine shrimp Artemia franciscana exposed to municipal effluent and 20-hydroxyecdysone

We developed biomarkers to monitor the endocrine-disrupting potential of contaminants and municipal effluents in aquatic arthropods. Artemia fransciscana shrimp were cultured and exposed to increasing concentrations of 20-hydroxyecdysone (20HE) and solid phase (C-8) municipal effluent extract (MEE) for 48 h at 20 °C. The levels of vitellogenin (Vtg)-like proteins, alkali-labile phosphates in total proteins and acetylcholinesterase (AChE) activity were determined in soft tissues. The levels of acid-soluble, alkali-soluble (sclerotin), neutral-soluble (arthropodin) proteins and chitin were determined to characterize the maturation state of shells. Both 20HE and the municipal effluent extract readily increased the total activity of acetylcholinesterase, alkali-labile phosphates in proteins and vitellogenin-like proteins in brine shrimp. In shells, 20HE and the effluent extract increased the proportion of chitin in shells and acid-soluble proteins but were not statistically significant for the latter. The proportion of sclerotin was increased by 20HE but was not changed by the effluent extract. The proportion of arthropodin was decreased by both 20HE and the effluent extract. Correlation analysis revealed that, as expected, the proportion of acid-soluble protein for biomineralisation and chitin levels were positively correlated and the proportion of arthropodin and sclerotin were negatively correlated in shells exposed to 20HE. Principal component analysis revealed that the proportion of chitin, arthropodin, sclerotin and acid-soluble proteins explained 63% of the responses. Shell protein and carbohydrate contents, controlled by the molting hormone 20HE, could be used as efficient markers for ecdysial properties of various contaminants. The municipal effluent appears to contain ecdysial-like chemicals that are capable of producing changes in shell protein composition that are similar to 20HE.

The Effects of Ingested 20-Hydroxyecdysone on the Larvae of Aglais urticae, Inachis io, Cynthia cardui (Lepidoptera: Nymphalidae) and Tyria jacobaeae (Lepidoptera: Arctiidae)

A comparative survey was carried out to investigate the effects, distribution and metabolism of ingested 20-hydroxyecdysone in four species of lepidopteran larvae in relation to the phytoecdysteroid content of the insect’s host plants. Analysis of the leaves of the host plants of each of the species revealed a strong relationship between the levels of phytoecdysteroids and the relative tolerance of the larvae to ingested 20-hydroxyecdysone. Monophagous or oligophagous species (Aglais urticae, Inachis io) feeding on ecdysteroid-negative host plants were either deterred from feeding or showed marked abnormalities in growth and development after incorporation of 20-hydroxyecdysone in their diets. Oligophagous or polyphagous species (Tyria jacobaeae, Cynthia cardui) which feed on host plants from families which are known to contain phytoecdysteroid-positive species, were able to tolerate low levels of 20-hydroxyecdysone in their diets, but exhibited developmental defects at high concentrations. These species were termed semi-tolerant. In each of the species, ingested [3H]20-hydroxyecdysone appeared to follow the same fate as injected [3H]20-hydroxyecdysone. The data are compared to those obtained in previous studies, where truly polyphagous species were shown to tolerate very high concentrations of 20-hydroxyecdysone in their diets by the production of ecdysteroid 22-fatty acyl esters. © 1997 Elsevier Science Ltd. All rights reserved

Developmental synchrony of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) within Aedes albopictus (Diptera: Cuclicidae).

Development of Ascogregarina taiwanensis Lien & Levine was synchronized with its host, the Asian tiger mosquito, Aedes albopictus (Skuse). Trophozoites of the parasite migrate to the Malpighian tubules where the gamonts fuse to form gametocysts and later mature oocysts. In this study, we observed that water temperature, which alters the development time of mosquito larvae, also affected parasite development. A. taiwanensis developed in a shorter time at 29 degrees C, whereas at 14 degrees C, the development was extended to as long as 17 d. Few mature oocysts were produced at 29 degrees C, although gametocysts occasionally were found in the lumen of Malpighian tubules. In contrast, gametocysts did not develop at 14 degrees C unless pupation occurred. Stage transformation of the parasite was determined by the metamorphosis of the mosquito host. Gametocyst formation occurred earlier when infected larvae were injected with an appropriate concentration of 20-hydroxyecdysone, indicating this molting hormone was involved in regulating the synchronous development of A. taiwanensis in its mosquito host.

In vivo muscle protein synthesis rates in the American lobster Homarus americanus during the moult cycle and in response to 20-hydroxyecdysone

Simultaneous measurements of in vivo rates of protein synthesis (Ks) in claw, leg and abdominal muscles were made in the American lobster Homarus americanus at three stages of the moult cycle. Ks values are significantly elevated during the premoult (stage D2-D3) and fall during the intermoult (stage C4) periods in all three muscles. Postmoult (stage A/B) levels are not significantly elevated above intermoult levels. Intermoult levels are between 0.3 and 0.4 % protein synthesized per day. In the premoult animals, the ribosomal activity (milligrams protein synthesized per microgram RNA per day) of the claw, abdominal and leg muscles is elevated three- to fivefold. The claw muscle maintains an elevated ribosomal activity into the postmoult stage whereas, by this stage, that of the other muscle tissues has fallen to intermoult levels. The RNA/protein ratios of the three muscle groups from intermoult, premoult and postmoult animals do not show any significant differences. 18S ribosomal RNA levels fluctuate slightly, with no consistent pattern over the moult cycle. In vivo injection of premoult concentrations of 20-hydroxyecdysone (20-HE) into intermoult lobsters results in elevated Ks values and ribosomal activity for the muscles after 3 days. RNA/protein ratios remain constant in the muscles in response to injections of 20-HE in vivo. In vitro preparations of leg muscle treated with 20-HE did not show similar elevated rates of protein synthesis.

Ecdysone and 20-hydroxyecdysone supplements to the diet affect larval development in the silkworm, Bombyx mori, differently

Ecdysone and 20-hydroxyecdysone were added to artificial diets of the silkworm, Bombyx mori, to evaluate their effects on larval development and the ecdysteroid titer. Ecdysone at more than 50 p.p.m. induced extra larval ecdyses. In contrast, extra larval ecdyses did not occur at any concentration of 20-hydroxyecdysone. 20-Hydroxyecdysone present at a high concentration from hatching onward caused mortality during the second instar. In larvae reared on an ecdysone-supplemented diet the ecdysteroid titer rapidly increased, then declined sharply in each instar. In larvae reared on 20-hydroxyecdysone-supplemented diet, the ecdysteroid titer increased and remained high throughout the second instar. These results suggest that the effect of ecdysone on the mechanism regulating the ecdysteroid titer is qualitatively different from that of 20-hydroxyecdysone.

Mating and 20-hydroxyecdysone cause increased galactogen synthesis in the albumen gland explants of Helix aspersa (mollusca)

1. 1. Mating causes a significant increase in [ 14G]glucose incorporation into newly synthesized galactogen by albumen gland explants of Helix aspersa. 2. 2. 20-Hydroxyecdysone, irrespective of the concentration used did not increase galactogen synthesis in albumen gland explants in virgin snails. 3. 3. In mated snails immediately following egg-laying, 20-hydroxyecdysone at a concentration of 10 −8M caused significant increase in galactogen synthesis in albumen gland explants. 4. 4. In mated snails prior to egg-laying, a 10 −7 M concentration of 20-hydroxyecdysone was required for the maximum increases of [ 14C]glucose uptake into galactogen. 5. 5. These results suggest that 20-hydroxyecdysone plays a hormonal role in controlling galactogen synthesis in the albumen gland of H. aspersa.

INDUCTION OF PRECOCIOUS MOLTING AND CLAW TRANSFORMATION IN ALPHEID SHRIMPS BY EXOGENOUS 20-HYDROXYECDYSONE

Exogenous micromolar concentrations of 20-hydroxyecdysone were used to accelerate the molt cycle in the snapping shrimp, Alpheus heterochelis. Shrimps were exposed to the hormone by adding it to the seawater in which they were cultured. Five days of continuous exposure of an animal to a hormone concentration of 5 µgm/ml shortened the winter molt cycle length by 18 days, or 65%. Claw transformation, a commonly observed phenomenon in alpheid shrimps, also was accelerated at these concentrations, and postmolt animals exhibited appropriate modifications in external claw morphology and, in some cases, restructuring of the claw closer muscle according to schedules identical with those of animals having normal molt cycle lengths. These studies strengthen the hypothesis that the cellular events which underlie claw transformation are modulated by endogenous ecdysteroids.

Regulation of juvenile hormone biosynthesis by 20-hydroxyecdysone during the fourth larval instar of the tobacco hornworm, Manduca sexta

A previous study of the role of 20-hydroxyecdysone in the regulation of juvenile hormone (JH) biosynthesis during pupal commitment in the last (fifth, V) larval instar of Manduca sexta revealed that the increase in the hemolymph edysteroid titer that elicits commitment also stimulates the corpora allata (CA) to synthesize JHs I and III and/or their acids. This stimulation is exerted indirectly via the brain-corpora cardiaca and results in the postcommitment increase in the JH titer that is important for the molt to a pupa. The possibility that a similar form of interendocrine regulation of JH biosynthesis by 20-hydroxyecdysone is involved in the control of larval molting in Manduca was investigated for the molt from the fourth (IV) to the V larval instar. During the IV instar, the increase in the hemolymph ecdysteroid titer and the capacity of the CA to synthesize JHs I and III in vitro correlated temporally in a manner suggestive of CA regulation by 20-hydroxyecdysone. With an in vitro approach, physiological concentrations of 20-hydroxyecdysone (⩾ 1 μg ml −1 hemolymph) were shown to have a dose-dependent trophic effect on CA activity, specifically stimulating JH I synthesis. Since neither basally active nor stimulated CA contained significant amounts of JH I, it appears that 20-hydroxyecdysone acts at the level of biosynthesis rather than at the level of release. The specificity of 20-hydroxyecdysone stimulation of JH I synthesis was demonstrated by the inability of the biologically inactive ecdysteroids 22-isoecdysone and 5α-ecdysone to activate the glands. 20-Hydroxyecdysone's regulation of IV instar larval CA compared with its regulation of the CA during pupal commitment is discussed.

Counteraction by 20-hydroxyecdysone of the effect of juvenile hormone on phosphorylation of ribosomal protein S6

Phosphorylation of ribosomal protein S6, induced when the fat body of Sarcophaga peregrina (flesh-fly) larvae was incubated in vitro in the presence of 32P, was found to be suppressed by juvenile hormone. This suppressive effect of juvenile hormone was counteracted by a physiological concentration of 20-hydroxyecdysone. Since 20-hydroxyecdysone is known to induce phosphorylation of S6 in vivo, this reaction was suggested to be regulated by the balance of the effects of ecdysone and juvenile hormone.

Interendocrine control by 20-hydroxyecdysone of the corpora allata of Manduca sexta

A positive interendocrine control by 20-hydroxyecdysone regulates the corpora allata (CA) and thus the hemolymph titers of the juvenile hormones (JHs) during the fourth and fifth larval stadia of the tobacco hornworm, Manduca sexta. The effect of 20-hydroxyecdysone is exerted via the brain-corpora cardiaca (Br-CC), and the kinetics of stimulation by the hormone suggest that its control of the CA is indirect, possibly involving neural effectors. Co-incubation of precommitment CA with Br-CC resulted in a stimulation of the synthesis of JH I acid equivalent to that achieved by the incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) with 20-hydroxyedcysone, suggesting that a diffusible regulatory factor may be involved in the stimulation. During pharate pupal development, a close temporal relationship exists between the drop in the ecdysteroid titer and declines in CA activity and the JH hemolymph titer, suggesting that 20-hydroxyecdysone may now inhbit the CA. Incubations of day 6 Br-CC-CA with physiological concentrations of 20-hydroxyecdysone resulted in an apparent dose-dependent inhibition of CA activity. Thus in Manduca, 20-hydroxyecdysone may exert varying stage-specific interendocrine effects on the CA which direct the postembryonic development of this species.

Ecdysone metabolism in adult crickets, Gryllus bimaculatus

The fate of injected [ 3H]ecdysone has been investigated in female and male adults of the Mediterranean field cricket, Gryllus binaculatus (de Geer). The metabolism is similar in both sexes and at various stages of adult life. Several classes of apolar metabolites (A1–A5) represent the major compounds. The amount of polar conjugates is low in all tissues, as are the concentrations of 20-hydroxyecdysone. Ovaries are the only organs capable of storing considerable amounts of ecdysteroids. The amount of radiolabelled ecdysteroid activity (mostly [ 3H]ecdysone) excreted during the first 24 hr after injection is high. The chemical identity of the apolar metabolites is not yet known. A2, which is the major apolar compound, has recently been identified as a complex of ecdysone conjugates with abundant long-chain fatty acids (Hoffman et al., 1985 Life Sci. 37, 185–192). Incubations with tissue homogenates in vitro have shown that several organs are capable of converting ecdysone into apolar compounds. Apolar ecdysteroid acyl esters represent a newly identified class of ecdysone conjugates from insects. Their role in regulation of free ecdysteroid titres during the reproductive period in female crickets is discussed.

Ecdysteroid hormone levels in the pupa of the bertha armyworm, Mamestra configurata Wlk., during spermatogenesis

Maturation of the spermatocysts in the testes of post-diapause pupae of M. configurata from the round form to the elongated form began about 5 days (0.15 units) after the initiation of pupal-adult development at 20°C. Spermatocyst maturation coincided with rising levels of 20-hydroxyecdysone in the developing pupa and occurred well after the rise and fall in the level of ecdysone. At the time of peak 20-hydroxyecdysone concentration at day 12 (0.34 units) of pupal-adult development, about 50–60% of the spermatocysts were in the elongated form and by the time (day 16, 0.49 units) 20-hydroxyecdysone had fallen to low levels all of the spermatocysts were elongated. Experimental suppression of ecdysone and 20-hydroxyecdysone to low levels had no effect on spermatocyst maturation. Evidently, high concentrations of these ecdysteroids are not required for spermatogenesis. Further, since male and female pupae show similar patterns of ecdysone and 20-hydroxyecdysone production during pupal-adult development, it seems unlikely that the high concentrations of ecdysteroids are produced solely to regulate spermatogenesis. It is hypothesized that the temporary production of high concentration of 20-hydroxyecdysone during pupal-adult development of M. configurata ultimately serves as an effective feedback mechanism to ensure that production of 20-hydroxyecdysone is terminated in preparation for adult eclosion.