Ecdysteroid levels throughout the life cycle of the desert locust, Schistocerca gregaria

Moulting hormone levels for all stages of the life cycle of the desert locust, Schistocerca gregaria, have been determined using gas chromatography with electron capture detection of the trimethylsilylated hormones. During larval development, the major hormone detected is 20-hydroxyecdysone with smaller quantities of ecdysone present. In mature adult females the major ecdysteroid observed is a polar conjugate of ecdysone, with smaller quantities of conjugated 20-hydroxyecdysone also present. During embryonic development the pattern changes from a high proportion of conjugated ecdysone in the early stages to give more free hormone and a higher proportion of 20-hydroxyecdysone in later stages. The highest titre of 20-hydroxyecdysone found in this insect is during the 5th larval instar. Maximal levels of ecdysteroid per insect are found in mature females just before oviposition, while the highest level of ecdysteroid per g of tissue is found in the eggs.     

Expression,subcellular localization and protein‑protein interaction of four isoforms of EcR/USP in the common cutworm

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval?pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real‐time quantitative polymerase chain reaction in different tissues during the larval–pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein?protein interaction were explored by transient expression and far‐western blotting, respectively. All the four genes were significantly up‐regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20‐hydroxyecdysone (20E) induction except for USP2, and USP1 could be up‐regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein?protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval?pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.     

Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).     

家蚕蜕皮与变态的内分泌调控

家蚕的蜕皮与变态是由前胸腺分泌的脱皮素（ｍｏｌｔｉｎｇ ｈｏｒｍｏｎｅ或 ｅｃｄｙｓｔｅｒｏｉｄ简称 ＭＨ）及由咽侧体分泌的保幼激素（ｊｕｖｅｎｉｌｅ ｈｏｒｍｏｎｅ）控制的,而促有前胸腺激素（ｐｒｏｔｈｏｒａｃｉｃｏｔｒｏｐｉｃ ｈｏｒｍｏｎｅ,以下简称ＰＴＴＨ）的功能为刺激前胸腺分泌蜕皮素。笔者近１０年来从家蚕内分泌体系的一系列研究中发现,蜕皮素浓度的变化可以通过控制咽侧体的保幼激素的生物合成来影响幼虫发育,而ＰＴＴＨ的信息传递可通过调控前胸腺的功能,进而影响血淋巴中蜕皮素浓度。     

hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals. This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.     

An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone

It has been suggested that Pentatomomorpha utilise the C₂₈ ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C₂₇ hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His₆-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His₆ tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [³H]ponasterone A (K_d = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula.     

Spiny lobster development: mechanisms inducing metamorphosis to the puerulus: a review

This review outlines current knowledge of mechanisms effecting metamorphosis in decapod crustaceans and insects. The comparative approach demonstrates some of the complexities that need resolving to find an answer to the question raised frequently by ecologists: “What triggers metamorphosis in spiny lobsters?” It is evident that crustacean moulting and metamorphosis are genetically controlled through endocrine systems that mediate gene expression. The molecular mechanisms underlying these developmental processes have been studied intensively in insects, particularly in the fruitfly, Drosophila melanogaster (Diptera), and some lepidopteran species. Comparatively, there is minimal information available for a few decapod crustacean species, but none for spiny lobsters (Palinuridae). Nothing was known of hormone signalling transduction pathways, via nuclear receptors (NRs) and gene activation during larval moults in palinurids—until a recent, ground-breaking study of early phyllosomal development of Panulirus ornatus by Wilson et al. (Rock Lobster Enhancement and Aquaculture Subprogram. FRDC Project 2000/263, Australian Govt, Fisheries Research and Development Corporation and Australian Institute of Marine Science, Nov 2005). Their study not only identified homologues of five hormone NRs of D. melanogaster, but also patterns of gene regulation showing strong similarities to those of gene expression found in insect larval development. Their results indicated that control of moulting and metamorphosis in palinurids closely parallels that in insects, suggesting that insects can serve as model systems for elucidating molecular mechanisms in larval decapods. In insects and crustaceans, the steroid hormone, ecdysone, (20E) initiates moulting. In insects, juvenile hormone (JH) mediates the type of larval moult that occurs, either anamorphic or metamorphic. The latter results when the level of JH in the haemolymph drops in the final larval instar. High levels of JH inhibit the metamorphic moult during insect larval development. The interaction of 20E and JH is not fully understood, and the operative molecular mechanisms are still being elucidated. No nuclear receptor for JH has been identified, and alternative JH signalling pathways await identification. In decapod crustaceans, methyl farnesoate (MF), a precursor of JH, replaces the latter in other functions mediated by JH in insects; but there is little evidence indicating that MF plays a similar ‘antimetamorphic’ role in decapod larval moults.     

The POU Factor Ventral Veins Lacking/Drifter Directs the Timing of Metamorphosis through Ecdysteroid and Juvenile Hormone Signaling

Although endocrine changes are known to modulate the timing of major developmental transitions, the genetic mechanisms underlying these changes remain poorly understood. In insects, two developmental hormones, juvenile hormone (JH) and ecdysteroids, are coordinated with each other to induce developmental changes associated with metamorphosis. However, the regulation underlying the coordination of JH and ecdysteroid synthesis remains elusive. Here, we examined the function of a homolog of the vertebrate POU domain protein, Ventral veins lacking (Vvl)/Drifter, in regulating both of these hormonal pathways in the red flour beetle, Tribolium castaneum (Tenebrionidae). RNA interference-mediated silencing of vvl expression led to both precocious metamorphosis and inhibition of molting in the larva. Ectopic application of a JH analog on vvl knockdown larvae delayed the onset of metamorphosis and led to a prolonged larval stage, indicating that Vvl acts upstream of JH signaling. Accordingly, vvl knockdown also reduced the expression of a JH biosynthesis gene, JH acid methyltransferase 3 (jhamt3). In addition, ecdysone titer and the expression of the ecdysone response gene, hormone receptor 3 (HR3), were reduced in vvl knockdown larvae. The expression of the ecdysone biosynthesis gene phantom (phm) and spook (spo) were reduced in vvl knockdown larvae in the anterior and posterior halves, respectively, indicating that Vvl might influence ecdysone biosynthesis in both the prothoracic gland and additional endocrine sources. Injection of 20-hydroxyecdysone (20E) into vvl knockdown larvae could restore the expression of HR3 although molting was never restored. These findings suggest that Vvl coordinates both JH and ecdysteroid biosynthesis as well as molting behavior to influence molting and the timing of metamorphosis. Thus, in both vertebrates and insects, POU factors modulate the production of major neuroendocrine regulators during sexual maturation.     

Physiological aspects of nm-g mutant: An ecdysteroid-deficient mutant of the silkworm,Bombyx mori

We examined physiological and endocrinological properties of the nm-g mutant of the silkworm, whose development was arrested at 1st or 2nd larval instar. The larva of the nm-g homozygote continued to feed and grow past the stage when a normal larva moulted to the next instar. The nm-g larva attained more than twice the body weight of a normal moulting larva, but remained in the same instar with no sign of the moulting until it finally died. The ecdysteroid concentration in the haemolymph of the nm-g larva was reduced and an increase in the titre, which occurred before moulting in the normal larva, was not observed. Injection of 20-hydroxyecdysone induced the nm-g larva to moult and ecdyse to the next larval instar. From these results, we conclude that nm-g is an ecdysteroid-deficient mutant.     

Induction of supernumerary larval moulting in the tobacco hornworm Manduca sexta: interaction of bisacylhydrazine ecdysteroid agonists with endogenous Juvenile Hormone

Abstract When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH‐5849 or tebufenozide (RH‐5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth‐stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH‐5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult‐promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone‐agonist properties. Sixth‐stage larvae resulting from exposure to critical dietary concentrations of RH‐5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth‐stage larval cuticle.     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.     

Developmental profiles of ecdysteroids, ecdysteroid receptor mRNAs and DNA binding properties of ecdysteroid receptors in the Ixodid tick Amblyomma americanum (L.)

Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins.     

Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle.

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.