Release Of Ecdysone Research Articles

Growth Regulating Effects of Natural Pesticide, Azadirachtin from Neem Tree (Azadirachta indica A. Juss) on Insects: A Review

Azadirachtin is a tetranortriterpenoid derived from the neem tree Azadirachta indica A. juss. Azadirachtin has attracted worldwide attention as promising feeding deterrent, insect growth disruptor and sterilent. Azadirachtin based pesticides become very much popular because of its biodegradability and least toxicity to non- target organisms. It has now been promoted as an alternative insecticide in integrated pest management programmes. Azadirachtin has an ability to modify or suppress haemolymph ecdysteroid and juvenile hormone titres through inhibition of the secretion of brain peptides prothoracicotropic hormone and allatotropic hormone, which are responsible for inducing the synthesis and release of ecdysone from the prothoracic gland and juvenile hormone from corpora allata, respectively. Blocking the synthesis and release of ecdysone and juvenile hormone leading to disruption of coordination of moulting process. Furthermore, this compound is known to cause degenerative changes in the cells of insect endocrine glands. This review focuses on growth regulating effects of natural pesticide, azadirachtin from neem tree (Azadirachta indica A. Juss) on insects.

Expression profiles and possible functions of the ecdysone-related genes in the midgut stem cells of the silkworm, Bombyx mori L., 1758 (Lepidoptera: Bombycidae)

The insect midgut has remarkable similarities with the vertebrate intestine especially concerning controlling cell regulation by the stem cells. While the formation of the pupal midgut from stem cells is regulated by ecdysone, it is inhibited by juvenile hormone via suppression of ecdysone release. This study investigated the possible functions of ecdysone-related genes in the stem cells of Bombyx mori L., 1758 (Lepidoptera: Bombycidae) during larval-pupal metamorphosis. The study was conducted in the Ege University silkworm culture laboratory and insect physiology research laboratory during the years between 2014-2018. Juvenile hormone analogue, fenoxycarb was applied to the fifth instar Bombyx larvae to delayed or inhibit the formation of the pupal midgut. Morphologic observations were performed by hematoxylin plus eosin staining; the proliferation rate of stem cells was analyzed by bromodeoxyuridine cell proliferation assay and expression patterns of ecdysone-related genes were detected by quantitative real-time reverse transcriptase-polymerase chain reaction. Expression states of genes and developmental events of the midgut were differently affected by fenoxycarb treatment in an application time-dependent manner. According to results, genes were firstly classified based on their sensitivity of fenoxycarb, then grouped according to their expression profile in connection with morphological evaluations of stem cells.

Larval feeding status regulates the transcript levels of genes encoding PTTH and allatoregulatory peptides in silkworm Bombyx mori.

In Bombyx mori, two dorsolateral neurosecretory cells (NSCs) in each of the two brain lobes have been identified as prothoracicotropic hormone (PTTH) producing cells. This neuropeptide in insects stimulates the prothoracic gland for the synthesis and release of ecdysone, responsible for the molting events. Allatotropin (AT) and allatostatin (AST) are allatoregulatory neuropeptides that regulate juvenile hormone biosynthesis. Here, by using RT-qPCR, we showed that in B. mori, nutritional stress modulates the mRNA expression of AT and AST-C (allatostain type C) in the central nervous system consisting of the brain lobes and all the associated ganglia. Using whole-mount in situ hybridization, we showed that the feeding status of Bombyx larvae also influences the expression of PTTH in the NSCs of the brain. Food deprivation significantly decreased the mRNA expression levels of PTTH in larvae at active or terminal growth period. Further, we showed that insulin modulates the expression level of PTTH. However, its action was dependent on the feeding status of the larvae. At feeding, the insulin decreased the PTTH expression level, while at food deprivation, the insulin increased the PTTH expression level. The data thus indicates that larval feeding status plays an important role in altering the mRNA expression levels of allatoregulatory peptide genes and PTTH.

The Effects of Juvenile Hormone Analogue, Fenoxycarb on the Last Instar Testes of Bombyx mori

The development and metamorphosis of insects is regulated by juvenile hormone (JH) and ecdysone hormone, which are worked antagonist. Fenoxycarb is an effective juvenile hormone analogue on many insects. In this study, the effect of fenoxycarb on the last larval stage Bombyx mori testes was investigated. Male larvae on the 6th day of the last instar are divided into 2 groups. 1 ng of fenoxycarb dissolved in acetone was topically applied to larvae in the treatment group and the remaining 18 larvae were used as the control group. Control and treated groups were followed to until pupation from day when they showed cocoon spinning behavior. After dissection, several of testes were entreated routine histological processes and photographed. In treatment group, it was seen that fenoxycarb reduced spermatogonium formation by suppressing ecdysone release and caused a decrease in mature sperm. Remaining testes are used to the analysis of total carbohydrate and glycogen content. In conclusion, fenoxycarb has the reducing effect on spermatogenesis and it can cause to decrease of total carbohydrate content because of stress in insects. These histological and biochemical results may be in support of the use of fenoxycarb as an insect growth regulator (IGR) for harmful insects.

Structural Basis of Neurohormone Perception by the Receptor Tyrosine Kinase Torso

In insects, brain-derived Prothoracicotropic hormone (PTTH) activates the receptor tyrosine kinase (RTK) Torso to initiate metamorphosis through the release of ecdysone. We have determined the crystal structure of silkworm PTTH in complex with the ligand-binding region of Torso. Here we show that ligand-induced Torso dimerization results from the sequential and negatively cooperative formation of asymmetric heterotetramers. Mathematical modeling of receptor activation based upon our biophysical studies shows that ligand pulses are "buffered" at low receptor levels, leading to a sustained signal. By contrast, high levels of Torso develop the signal intensity and duration of a noncooperative system. We propose that this may allow Torso to coordinate widely different functions from a single ligand by tuning receptor levels. Phylogenic analysis indicates that Torso is found outside arthropods, including human parasitic roundworms. Together, our findings provide mechanistic insight into how this receptor system, with roles in embryonic and adult development, is regulated.

Fleeing heat and light

Fleeing heat and light

Bantam miRNA Promotes Systemic Growth by Connecting Insulin Signaling and Ecdysone Production

During the development of multicellular organisms, body growth is controlled at the scale of the organism by the activity of long-range signaling molecules, mostly hormones. These systemic factors coordinate growth between developing tissues and act as relays to adjust body growth in response to environmental changes [1]. In target organs, long-range signals act in concert with tissue-autonomous ones to regulate the final size of a given tissue. In Drosophila, the steroid hormone ecdysone plays a dual role: peaks of secretion promote developmental transitions and maturation, while basal production negatively controls the speed of growth. The antagonistic action of ecdysone and the conserved insulin/insulin growth factor (IGF) signaling pathway regulate systemic growth and modulate final body size [2, 3]. Here we unravel an unexpected role of bantam microRNA in controlling body size in Drosophila. Our data unveil that, in addition to its well-characterized function in autonomously inducing tissue growth [4-9], bantam activity in ecdysone-producing cells promotes systemic growth by repressing ecdysone release. We also provide evidence that the regulation of ecdysone production by insulin signaling relies on the repression of bantam activity. These results identify a molecular mechanism that underlies the crosstalk between these two hormones and add a new layer of complexity to the well-characterized role of bantam in growth control.

Ecdysone Control of Developmental Transitions: Lessons from Drosophila Research

The steroid hormone ecdysone is the central regulator of insect developmental transitions. Recent new advances in our understanding of ecdysone action have relied heavily on the application of Drosophila melanogaster molecular genetic tools to study insect metamorphosis. In this review, we focus on three major aspects of Drosophila ecdysone biology: (a) factors that regulate the timing of ecdysone release, (b) molecular basis of stage- and tissue-specific responses to ecdysone, and (c) feedback regulation and coordination of ecdysone signaling.

Nuclear Receptor DHR4 Controls the Timing of Steroid Hormone Pulses During Drosophila Development

Author SummarySteroid hormones play fundamental roles in development and disease. They are often released as pulses, thereby orchestrating multiple physiological and developmental changes throughout the body. Hormone pulses must be regulated in a way so that they have a defined beginning, peak, and end. In Drosophila, pulses of the steroid hormone ecdysone govern all major developmental transitions, such as the molts or the transformation of a larva to a pupa. While we have a relatively good understanding of how an ecdysone pulse is initiated, little is known about how hormone production is turned off. In this study, we identify a critical regulator of this process, the nuclear receptor DHR4. When we interfere with the function of DHR4 specifically in the ecdysone-producing gland, we find that larvae develop much faster than normal, and that this is caused by the inability to turn off ecdysone production. We show that DHR4 oscillates between cytoplasm and nucleus of ecdysone-producing cells under the control of a neuropeptide that regulates ecdysone production. When the neuropeptide pathway is inactive, DHR4 enters the nucleus and represses another gene, Cyp6t3, for which we show a novel role in the production of ecdysone.

Expressions of the cytochrome P450 monooxygenase gene Cyp4g1 and its homolog in the prothoracic glands of the fruit fly Drosophila melanogaster (Diptera: Drosophilidae) and the silkworm Bombyx mori (Lepidoptera: Bombycidae)

Here we describe the expression profiles of the cytochrome P450 monooxygenase gene Cyp4g1 in the fruit fly, Drosophila melanogaster Meigen, and its homolog in the silkworm, Bombyx mori L. We identified Cyp4g1 by a microarray analysis to examine the expression levels of 86 predicted D. melanogaster P450 genes in the ring gland that contains the prothoracic gland (PG), an endocrine organ responsible for synthesizing ecdysteroids. B. moriCyp4g25 is a closely related homolog of D. melanogaster Cyp4g1 and is also expressed in the PG. A developmental expression pattern of Cyp4g25 in the PG is positively correlated with a fluctuation in hemolymph ecdysteroid titer in the late stage of the final instar. Moreover, the expression of Cyp4g25 in cultured PGs is significantly induced by the addition of prothoracicotropic hormone (PTTH), a neuropeptide hormone that stimulates the synthesis and release of ecdysone. We propose that Cyp4g1 and Cyp4g25 are the candidates that play a role in regulating PG function and control ecdysteroid production and/or metabolism during insect development.

Ecdysone regulates size-dependent development

Ecdysone regulates size-dependent development

Encapsulated ecdysone by internal gelation of alginate microspheres for controlling its release and photostability

Alginate microspheres prepared by internal gelation were chosen as carrier for a model insect hormone, ecdysone, owing non-toxicity of the polymers and mild conditions of the method. The coated colloids were characterized using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The in vitro release of ecdysone from microspheres was studied in simulated insect midgut conditions. The photostabilization of unencapsulated and encapsulated ecdysone exposed to ultraviolet light was evaluated. The effect of the ecdysone-loaded alginate microspheres on the biology and mortality of Martianus dermestoides Chevrolat larvae was investigated. The results showed that ecdysone-loaded alginate microspheres which were obtained by association had a mean diameter of 52.8 ± 9.8 μm. The encapsulation efficiency and the encapsulation yield of the ecdysone-loaded alginate microspheres were about 80.50 ± 3.6% and 86.55 ± 5.3%, respectively. Swelling studies were conducted in phosphate buffered saline solution and diffusion coefficients ( D) and diffusional exponents ( n) for water transport were determined using the empirical equation. Alginate microspheres swelled 1.85-fold of their original size. The in vitro release experiments revealed that the release rate of ecdysone from the alginate microspheres was proportional to the release time. The results were analyzed by using a semi-empirical equation and it was found that the drug release mechanism was “anomalous transport”. The photolysis experiments demonstrated that the UV light-induced decomposition of ecdysone was markedly decreased by encapsulation into microspheres. Therefore, it could be considered as an effective system to enhance the photostability of ecdysone. Toxicity of the novel ecdysone-loaded alginate microsphere insecticide was higher to the larvae of M. dermestoides Chevrolat compared to the unencapsulated ecdysone as indicated by the lower EC 50 value.

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

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

Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.

Body-Size Regulation: Combining Genetics and Physiology

New research has revealed that the activity of the insulin-signaling pathway in the prothoracic gland of Drosophila modulates ecdysone release and thereby influences both the duration and rate of larval growth.

Ras Activity in the Drosophila Prothoracic Gland Regulates Body Size and Developmental Rate via Ecdysone Release

In Drosophila, each of the three larval instars ends with a molt, triggered by release of steroid molting hormone ecdysone from the prothoracic gland (PG). Because all growth occurs during the larval stages, final body size depends on both the larval growth rate and the duration of each larval stage, which in turn might be regulated by the timing of ecdysone release. Here, we show that the expression of activated Ras, PI3 kinase (PI3K), or Raf specifically in the PG reduces body size, whereas activated Ras or PI3K, but not Raf, increases PG cell size. In contrast, expression of either dominant-negative (dn) Ras, Raf, or PI3K increases body size and prolongs the larval stages, leading to delayed pupariation, whereas expression of dn-PI3K, but not of dn-Raf or dn-Ras, reduces PG cell size. To test the possibility that altered ecdysone release is responsible for these phenotypes, we measured larval ecdysone levels indirectly, via the transcriptional activation of two ecdysone targets, E74A and E74B. We found that the activation of Ras within the PG induces precocious ecdysone release, whereas expression of either dn-PI3K or dn-Raf in the PG greatly attenuates the [ecdysone] increase that causes growth cessation and pupariation onset. We conclude that Ras activity in the PG regulates body size and the duration of each larval stage by regulating ecdysone release. We also suggest that ecdysone release is regulated in two ways: a PI3K-dependent growth-promoting effect on PG cells, and a Raf-dependent step that may involve the transcriptional regulation of ecdysone biosynthetic genes.

Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM), and produced large vacuoles and small electrondense granules; insects fed on Ringer's saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringer's saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringer's saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringer's saline meal with ecdysone; Ringer's saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.

Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon

The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.

Dual requirement for the EcR/USP nuclear receptor and the dGATAb factor in an ecdysone response in Drosophila melanogaster.

The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit.

Control of prothoracic gland activity by juvenile hormone in fourth instar Manduca sexta larvae

In Manduca sexta, larval-larval molting of the IVth instar ensues in response to a large pulse of ecdysteroid. Ecdysone release by the prothoracic glands (PG), in turn, is stimulated by prothoracicotropic hormone (PTTH) from the brain. We show that juvenile hormone (JH) is necessary and its titer need not fall for PTTH release to occur. Application of the JH analog methoprene to larvae did not shift the hemolymph ecdysteroid titer profile nor did it affect PG activity in vitro. On the other hand, allatectomy abolished the ecdysteroid pulse which could be restored by application of methoprene. Neck-ligation and debraining experiments show that this effect of methoprene is mediated by the brain. In neck-ligated larvae, i.e. when PTTH is eliminated, the ecdysteroid hemolymph titer rises gradually and leads to a larval-pupal molt. In this situation, methoprene suppressed the ecdysteroid titer completely, presumably by direct inhibition of the PG. Possible mechanisms of ecdysteroid regulation in intact and neck-ligated IVth instar larvae are discussed.