Ecdysis triggering hormone is essential for larva-pupa-adult transformation in Leptinotarsa decemlineata.

In Drosophila melanogaster, ecdysis triggering hormone (ETH) is the key factor triggering ecdysis behaviour and promoting trachea clearance. However, whether ETH plays the dual roles in non-dipteran insects is unknown. In this survey, we found that Ldeth mRNA levels were positively correlated with circulating 20-hydroxyecdysone (20E) titers in Leptinotarsa decemlineata. Ingestion of an ecdysteroid agonist halofenozide or 20E stimulated the transcription of Ldeth, whereas RNA interference (RNAi) of ecdysteroidogenesis (LdPTTH or LdSHD) or 20E signalling (LdEcR, LdUSP or LdFTZ-F1) genes inhibited the expression, indicating ETH acts downstream of 20E. RNAi of Ldeth at the final instar stage impaired pupation. More than 80% of the Ldeth-depleted beetles remained as prepupae, completely wrapped in the old larval cuticles. These prepupae became withered, dried and darkened gradually, and finally died in soil. The remaining Ldeth hypomorphs pupated and emerged as abnormal adults, bearing smaller and wrinkle elytrum and hindwing. Moreover, the tracheae in the Ldeth hypomorphs were full of liquid. We accordingly proposed that the failure of trachea clearance disenabled air-swallowing after pupa-adult ecdysis and impacted wing expansion. Our results suggest that ETH plays the dual roles, initiation of ecdysis and motivation of trachea clearance, in a coleopteran.

Cucurbitacin B acts a potential insect growth regulator by antagonizing 20-hydroxyecdysone activity.

BACKGROUND: 20-Hydroxyecdysone (20E), a crucial insect steroid hormone, can bind to its cognate nuclear receptor composed of ecdysone receptor (EcR) and ultraspiracle (USP) to activate expression of 20E-response genes, enabling subsequent metamorphosis. In this study, we tried to find out which steroid-like compounds can block insect metamorphosis effectively and provide useful information for biopesticide study. For this purpose, we screened 126 steroid-like compounds for possible 20E antagonists using a dual-luciferase reporter assay with Drosophila melanogaster Kc and Bombyx mori Bm12 cells. RESULTS: Among 126 steroid-like compounds, three cucurbitacins (CucB, D and E) were identified as 20E antagonists in both Kc and Bm12 cells. Notably, CucB caused significant molting defects and mortality in both B. mori and D. melanogaster larvae, and dramatically hindered larval growth of Helicoverpa armigera by its anti-feeding activity. CONCLUSION: In vivo and in vitro experiments demonstrate that CucB acts as a potential insect growth regulator by antagonizing 20E activity and thus blocking molting and metamorphosis induced by 20E signaling. © 2017 Society of Chemical Industry.

Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid--a mechanism that determines the timing of metamorphosis.

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.

Steroid hormone signaling is involved in the age-dependent behavioral response to sex pheromone in the adult male moth Agrotis ipsilon.

In most animals, including insects, male reproduction depends on the detection and processing of female-produced sex pheromones. In the male moth, Agrotis ipsilon, both behavioral response and neuronal sensitivity in the primary olfactory center, the antennal lobe (AL), to female sex pheromone are age- and hormone-dependent. In many animal species, steroids are known to act at the brain level to modulate the responsiveness to sexually relevant chemical cues. We aimed to address the hypothesis that the steroidal system and in particular 20-hydroxyecdysone (20E), the main insect steroid hormone, might also be involved in this olfactory plasticity. Therefore, we first cloned the nuclear ecdysteroid receptor EcR (AipsEcR) and its partner Ultraspiracle (AipsUSP) of A. ipsilon, the expression of which increased concomitantly with age in ALs. Injection of 20E into young sexually immature males led to an increase in both responsiveness to sex pheromone and amount of AipsEcR and AipsUSP in their ALs. Conversely, the behavioral response decreased in older, sexually mature males after injection of cucurbitacin B (CurB), an antagonist of the 20E/EcR/USP complex. Also, the amount of AipsEcR and AipsUSP significantly declined after treatment with CurB. These results suggest that 20E is involved in the expression of sexual behavior via the EcR/USP signaling pathway, probably acting on central pheromone processing in A. ipsilon.

Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila.

Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. We show that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce ßFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying ßFTZ-F1 rescues Cyp18a1-deficient prepupae. Because Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to ßFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process.

Identification and expression profile of a putative basement membrane protein gene in the midgut of Helicoverpa armigera.

BACKGROUND: The midgut undergoes histolysis and remodeling during the larval to adult transition in holometabolous insects, but the molecular mechanisms underlying this process are not well understood. RESULTS: Using Suppression Subtractive Hybridization (SSH), we identified a 531 bp cDNA predicted to encode a 176 amino acid protein, which we call hmg176. Northern and western blot analysis suggested that high levels of hmg176 are expressed in the midgut during molting, but not during metamorphosis. HMG176 protein was detected by immunofluorescence within the membrane of fat bodies and the basement membrane of the midgut of both molting and feeding larvae, but not in metamorphically committed larvae. In situ hybridization revealed that hmg176 transcripts mainly localized to the columnar cells of the midgut. Interestingly, a non-steroidal ecdysone agonist, RH-2485, significantly upregulated expression of hmg176. CONCLUSION: These observations suggest that hmg176 encodes a larval-specific protein that may participate in sustaining larval midgut during larval development, possibly in response to ecdysteroid in vivo. This study will enlighten our understanding of the molecular mechanisms of tissue histolysis during metamorphosis.

Brominated flame retardants: activities in a crustacean development test and in an ecdysteroid screening assay.

Brominated flame retardants (BFRs) were investigated for toxic effects both in vivo and in vitro in two invertebrate bioassays. Subchronic effects of tetrabromobisphenol A (TBBPA), tribromophenol (TBP), and four polybrominated diphenyl ethers ([PBDEs]; BDE-28, BDE-47, BDE-99, and BDE-100) on larval development of the marine copepod Acartia tonsa were studied. For TBBPA and TBP 5-d effective median concentration (EC50) values for inhibition of the larval development rate were 125 and 810 microg/L, respectively, whereas the PBDEs were much more potent with 5-d EC50 in the low microg/L range (1.2 microg/L for BDE-100; 4.2 microg/L for BDE-99; 13 microg/L for BDE-28; and 13 microg/L for BDE-47). These concentrations were up to two orders of magnitude below the 48-h LC50 for acute adult toxicity (108 microg/L for BDE-28; 400 microg/L for TBBPA; 520 microg/L for BDE-100; 705 microg/L for BDE-99; 1,500 microg/L for TBP; and 2,370 microg/L for BDE-47). To distinguish between general toxicological and endocrine-mediated toxic effects, the BFRs were assessed in vitro for ecdysteroid agonistic/antagonistic activity with the ecdysteroid-responsive Drosophila melanogaster B(II)-cell line. The pentabrominated diphenyl ethers BDE-99 and BDE-100 showed weak ecdysteroid antagonistic activity. Thus, these PBDEs may be regarded as potential endocrine disrupters in invertebrates. The combination of in vitro assays and subchronic biotests with ecologically important crustacean species is a rapid and cost-effective tool when screening for sublethal effects of BFRs and other chemicals.

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity.

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.

Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori.

Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 m NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol.mg(-1) protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events.

A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds.

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system.

Insect development is guided by the combined actions of ecdysteroids and juvenile hormones (JHs). The transcriptional effects of ecdysteroids are mediated by a protein complex consisting of the ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), but a corresponding JH receptor has not been defined conclusively. Given that the EcR ligand binding domain (LBD) is similar to that of the JH-responsive rat farnesoid-X-activated receptor (FXR), we sought to define experimental conditions under which EcR-dependent transcription could be promoted by JH. Chinese hamster ovary (CHO) cells were transfected with a plasmid carrying an ecdysteroid-inducible reporter gene, a second plasmid expressing one of the three amino-terminal variants of Drosophila EcR or an EcR chimera, and a third plasmid expressing either the mouse retinoid X receptor (RXR), or its insect orthologue, USP. Each of the EcR variants responded to the synthetic ecdysteroid, muristerone A (murA), but a maximal response to 20-hydroxyecdysone (20E) was achieved only for specific EcR combinations with its heterodimeric partner. Notably, the Drosophila EcR isoforms were responsive to 20E only when paired with USP, and only EcRB2 activity was further potentiated by JHIII in the presence of 20E. EcR chimeras that fuse the activator domains from VP16 or the glucocorticoid receptor to the Drosophila EcR DNA-binding and ligand-binding domains were responsive to ecdysteroids. Again, the effects of JHIII and 20E were associated with specific partners of the chimeric EcRs. In all experiments, the LBD of EcR proved to be the prerequisite component for potentiation by JHIII, and in this conformation may resemble the FXR LBD. Our results indicate that EcR responsiveness is influenced by the heterodimeric partner and that both the N-terminal domain of EcR and the particular ecdysteroid affect JHIII potentiation.

Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a fleshfly, Sarcophaga peregrina.

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.

Potent and selective partial ecdysone agonist activity of chromafenozide in Sf9 cells.

Chromafenozide (ANS-118) is a non-steroidal ecdysone mimic and its insecticidal effect is highly specific to lepidoptera. In order to evaluate the transcription-inducing activity via nuclear ecdysone receptor (EcR) and the mode of action of chromafenozide, ecdysone-responsive reporter gene assay systems were developed in Sf9 and Kc cells. Ponasterone A, a full EcR agonist, induced reporter transcription in a dose-dependent manner in both Sf9 and Kc cells. In contrast, chromafenozide activated reporter transcription with comparable potency to ponasterone A only in Sf9 cells, although its maximum activity was 4-fold lower than that of ponasterone A. When chromafenozide was applied together with ponasterone A to Sf9 cells, it antagonized ponasterone A at nanomolar concentrations. These results suggest that chromafenozide is a potent partial EcR agonist specific to lepidoptera; it appears to bind lepidopteran EcR with comparable affinity to ponasterone A, but may activate the EcR in a different manner.

Identification and ecdysteroid antagonist activity of three oligostilbenes from the seeds of Carex pendula (Cyperaceae).

Methanolic extracts of seeds of several (Carex species were found to antagonise the action of 20-hydroxyecdysone in the Drosophila melanogaster microplate-based B(II) cell bioassay. Bioassay-guided HPLC analysis of seeds of Carex pendula (drooping sedge) provided one previously unknown tetrastilbene (cis-miyabenol A) and two known oligostilbenes (kobophenol B and cis-miyabenol C) as the biologically active compounds (EC50 values were 31, 37 and 19 microM, respectively, vs. 5 x 10(-8) M 20-hydroxyecdysone). The structures and relative stereochemistries of these compounds were deduced by comprehensive ID- and 2D-NMR experiments. These compounds are isolated from Carex pendula for the first time. In vitro experiments with dipteran and lepidopteran ecdysteroid receptor proteins demonstrate that the oligostilbenes are able to compete with radiolabelled ecdysteroid ([3H]ponasterone A) for occupancy of the ligand binding site. IC50/Ki values are similar to the EC50 values obtained in the B(II) bioassay.

Inhibition of [(3)H]ponasterone a binding by ecdysone agonists in the intact Sf-9 cell line.

Ecdysone agonists, including dibenzoylhydrazines, significantly inhibited the binding of [(3)H]ponasterone A ([(3)H]PoA) in intact Sf-9 cells (Spodoptera frugiperda). The amount of [(3)H]PoA binding varied in a concentration-dependent manner. According to the IC(50), concentration at which there is 50% inhibition, the order of potency of typical ecdysone agonists is tebufenozide (RH-5992) > methoxyfenozide (RH-2485) > PoA > 20-hydroxyecdysone > cyasterone > RH-5849, makisterone A > or = inokosterone > ecdysone. The ranking is consistent with that obtained from a cultured integument system of the rice stem borer Chilo suppressalis except for methoxyfenozide. Other compounds whose modes of action are different from that of ecdysteroids, for example respiration inhibitors, plant steroid hormones, and chitin synthesis inhibitors, did not inhibit the binding of [(3)H]PoA significantly. The mammalian hormones estradiol and diethylstilbestrol, and a secondary bile acid, lithocholic acid, significantly inhibited the binding of [(3)H]PoA at 25 microM. However, their binding activity in terms of pIC(50) was either very low or not evaluated.

Resveratrol-type oligostilbenes from Iris clarkei antagonize 20-hydroxyecdysone action in the Drosophila melanogaster B(II) cell line.

Bioassay-guided high-performance liquid chromatography analysis of a MeOH extract of Iris clarkei seeds yielded the resveratrol-type oligomeric stilbenes, ampelopsin B and alpha-viniferin, which antagonize the action of 20-hydroxyecdysone; with a 20-hydroxyecdysone concentration of 50 nM, the ED50 values were 33 microM and 10 microM, respectively. The structures of these compounds were determined by spectroscopic analysis, notably ultraviolet, liquid secondary ion mass spectrometry and modern one- and two-dimensional nuclear magnetic resonance techniques.

Antiepileptic effects of 20-hydroxyecdysone on convulsive seizures in spontaneously epileptic rats.

We examined the effect of 20-hydroxyecdysone (20-HE), a neurosteroid found in insects that is involved in their developmental process, on both tonic convulsion and absence-like seizure in spontaneously epileptic rat (SER). When 20-HE was given orally to SER at 25-200 mg/kg, significant decreases of the tonic convulsion were observed with 100 and 200 mg/kg. Pretreatment of the animal with bicuculline (1 mg/kg, i.p.) antagonized the inhibitory effects of 20-HE. However, absence-like seizures were not affected by 20-HE. These findings indicate that 20-HE produces antiepileptic effects on tonic convulsion by acting on the modulatory site of GABA(A) receptors.

Two limonoids from Turraea obtusifolia (Meliaceae), prieurianin and rohitukin, antagonise 20-hydroxyecdysone action in a Drosophila cell line.

Bioassay-assisted HPLC analyses yielded two prieurianin-type limonoids, prieurianin and rohitukin, from the seeds of Turraea obtusifolia, which act as antagonists of 20-hydroxyecdysone action in the Drosophila melanogaster BII cell line. With a 20-hydroxyecdysone concentration of 5 x 10(-8) M, the ED50 values for prieurianin and rohitukin are 10(-5) M and 1.25 x 10(-4) M, respectively.

Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor.

Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.