Action Of 20-hydroxyecdysone Research Articles

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin has been used to control agricultural pests for a long time; however, the molecular mechanism of azadirachtin on lepidopterans is still not clear. In this study, the fourth instar larvae of fall armyworm were fed with azadirachtin, and then the ecdysis was blocked in the fourth instar larval stage (L4). The prothoracic glands (PGs) of the treated larvae were dissected for RNA sequencing to determine the effect of azadirachtin on ecdysis inhibition. Interestingly, one of the PG-enriched genes, the nuclear hormone receptor 3 (HR3), was decreased after azadirachtin treatment, which plays a critical role in the 20-hydroxyecdysone action during ecdysis. To deepen the understanding of azadirachtin on ecdysis, the HR3 was knocked out by using the CRISPR/Cas9 system, while the HR3 mutants displayed embryonic lethal phenotype; thus, the stage-specific function of HR3 during larval molting was not enabled to unfold. Hence, the siRNA was injected into the 24 h L4 larvae to knock down HR3. After 96 h, the injected larvae were blocked in the old cuticle during ecdysis which is consistent with the azadirachtin-treated larvae. Taken together, we envisioned that the inhibition of ecdysis in the fall armyworm after the azadirachtin treatment is due to an interference with the expression of HR3 in PG, resulting in larval mortality. The results in this study specified the understanding of azadirachtin on insect ecdysis and the function of HR3 in lepidopteran in vivo.

Stage-dependent effects of 20-hydroxyecdysone on DNA synthesis of corpus allatum cells in the silkworm, Bombyx mori.

DNA synthesis in cells of the corpus allata (CA) of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU); developmental changes during the 3rd, 4th, and last larval instars and effects of 20-hydroxyecdysone treatment were examined. During both the 3rd and 4th larval instars, the number of DNA-synthesizing cells fluctuated, and relatively low levels were observed during the middle stages. On day 0 of the last larval instar, the number of DNA-synthesizing cells per gland was 9.2, which then increased on day 1 and remained at levels ranging from 12.9 and 16.9 cells per gland. A major peak level (28 BrdU-labeled cells per gland) occurred on day 8, two days after larvae entered the wandering stage. When last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 0 or 1, the number of DNA-synthesizing cells dramatically decreased to very low levels and these low levels were maintained throughout the remainder of the instar. However, no effect was observed when last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 3, indicating the stage-specific action of 20-hydroxyecdysone. The mechanism by which 20-hydroxyecdysone treatment inhibits DNA synthesis of CA cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. It was found that the decrease in responsiveness of DNA synthesis of CA cells of 20-hydroxyecdysone-treated larvae to stimulation by growth factors from hemolymph may have been, at least in part, responsible for the indirect inhibitory effects of 20-hydroxyecdysone.

Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast.

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported. This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes.

Phytoecdysteroids in seeds of Lloydia serotina (Liliaceae)

Seeds of a number of species in the Liliaceae (sensu Brummitt, 1992) were examined for the presence of ecdysteroid agonist and antagonist activities. No species were antagonistic to 20-hydroxyecdysone action on the ecdysteroid-responsive Drosophila melanogaster B(II) cell line and only one extract, that of Lloydia serotina, was agonistic. This activity is attributable to the presence of phytoecdysteroids as detected by ecdysteroid-specific radioimmunoassay and the agonist version of the B(II) bioassay. HPLC in conjunction with radioimmunoassay and bioassay have been used to determine the ecdysteroid profile. The major ecdysteroids present are identified as 20-hydroxyecdysone and polypodine B (5beta,20-dihydroxyecdysone).

Control of activity and regression of the silk glands in the last-larval instar of Galleria mellonella

The weight increase of silk glands in isolated larval abdomens is enhanced by brain implants which stimulate RNA and protein synthesis. It is proposed that, in intact last-instar larvae, a blood-borne factor from the brain promotes silk gland activity during the facultative feeding period. In the post-feeding larvae, some starving larvae and in isolated larval abdomens the silk glands regress. The regression is accelerated and the glands degenerate under the action of 20-hydroxyecdysone. This effect is not associated with an increase in protease and RN-ase activities. In the normal larvae, the protease activity increases gradually during the last instar to a maximum at the time of regression and drops in the degenerating glands. RN-ase activity is maintained at a high level in the fully active, regressing and degenerating glands The results indicate that regression and degeneration of the silk glands are caused by structural cell reorganization rather than by an appearance of cytolytic enzymes.

The role of central aminergic neurons in the action of 20-hydroxyecdysone on neurosecretory cells of Rhodnius prolixus

The enhancement of electrical activity of the neurosecretory cells in the brain and corpus cardiacum of Rhodnius prolixus induced by 20-hydroxyecdysone has been used as a means of examining the role of aminergic neurons in this reflex. The response of the brain and corpus cardiacum from mated ovariectomized females to 20-hydroxyecdysone was blocked by phentolamine and phenoxybenzamine (α-aminergic receptor antagonists) but not by propranolol (a β-aminergic receptor antagonist). Preparations taken from ‘reserpinzed’ females failed to respond to 20-hydroxyecdysone. Dopamine at 10 −7 M was capable of mimicking 20-hydroxyecdysone in activating the neurosecretory system from mated ovariectomised females as well as from ‘reserpinized’ mated ovariectomised females. The response to dopamine was blocked by phentolamine. The neurosecretory system from virgin ovariectomized females failed to respond to 10 −7 or 10 −6 M dopamine, but was activated by 10 −5 M dopamine. It is concluded that the action of 20-hydroxyecdysone onto the neurosecretory cells is indirect and involves aminergic interneurons. The results also suggest that the mating stimuli may function by enhancing the response of neurons to amines.