Redundant ecdysis regulatory functions of three nuclear receptor HR3 isoforms in the direct-developing insect Blattella germanica

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B(1) and BgHR3-B(2). Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.     

Cell culture techniques for studying insect cuticle

Evidence that biosynthetic pathways critical to the formation of insect cuticle are retained in continuous insect cell lines opens new possibilities for research on the cuticle system. Recent findings indicate that chitin, molting hormone, and catecholamines are all produced by a vesicle cell line derived from embryos of the cockroach Blattella germanica. The chitin that is formed by this cell line is particulate and does not show the characteristic featherlike crystalline structure found in mature cuticle. The molting hormone is produced as ecdysone and is released into the culture medium. The addition of 20-hydroxyecdysone to the cultures increases the production of chitin fourfold. These responses are similar to those found in insect organ cultures.     

Antioxidant effect of 20-hydroxyecdysone in a model system

Changes in the level of lipid free-radical oxidation in mitochondrial fraction at the presence of 20-hydroxyecdysone in 1, 2, 4, 6, 8 microM concentration with the help of a chemiluminescence (ChL) method were investigated in vitro. Statistically authentic reduction of ChL kinetic parameters--I(s) and tg alpha, at 20-hydroxyecdysone presence in concentration of 2 microM was found. 20-hydroxyecdysone administration in concentration 4 microM and more leads to the statistically authentic change of all four ChL parameters. The antioxidizing effect of 20-hydroxyecdysone was compared with action of such antioxidant as a hydroquinone. The higher activity of 20-hydroxyecdysone as an antioxidant in comparison with the hydroquinone was shown. For changes of all four measured kinetic parameters of ChL, concentration of the hydroquinone as much 2-fold than for 20-hydroxyecdysone is necessary. On the basis of our researches in the model system a conclusion was made that 20-hydroxyecdysone has an antioxidizing action on lipid free radical oxidation in mitochondrial fraction in dependents on concentration. 20-Hydroxyecdysone has antioxidizing properties directly, in these conditions in vitro, when its metabolites formation does not occur yet.     

RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica

Ecdysteroids play a major role during developmental growth in insects. The more active form of these hormones, 20-hydroxyecdysone (20E), acts upon binding to its heterodimeric receptor, formed by the two nuclear receptors, EcR and RXR/USP. Functional characterization of USP has been exclusively conducted on the holometabolous insect Drosophila melanogaster. However, it has been impossible to extend such analysis to primitive-hemimetabolous insects since species of this group are not amenable to genetic analysis. The development of methodologies based on gene silencing using RNA interference (RNAi) after treatment with double-stranded RNA (dsRNA) in vivo has resolved such limitations. In this paper, we show that injection of dsRNA into the haemocoel of nymphs and adults of the cockroach Blattella germanica can be used to silence gene function in vivo. In our initial attempt to test RNAi techniques, we halted the expression of the adult-specific vitellogenin gene. We then used the same technique to silence the expression of the B. germanica RXR/USP (BgRXR) gene in vivo during the last nymphal instar. BgRXR knockdown nymphs progressed through the instar correctly but they arrested development at the end of the stage and were unable to molt into adults. The results described herein suggest that RXR/USP function, in relation to molting, is conserved across the insect Class.     

Reproductive effects in German cockroaches by ecdysteroid agonist RH-0345, juvenile hormone analogue methoprene and carbamate benfuracarb

Blatta germanica is the more prevalent cockroach species in Algeria. In the present study, we tested the effect on reproduction in B. germanica of two insect growth regulators, RH-0345, a benzoylhydrazine analogue that mimics the action of 20-hydroxyecdysone, and methoprene, one of the most commercially important juvenile hormone analogues, and a novel carbamate insecticide, benfuracarb. The compounds were applied topically (10 and 20 microg/insect for RH-0345, and 1 and 10 microg/insect for methoprene) or orally administrated (at 2% for benfuracarb) on newly emerged females and evaluated on reproductive events during the adult life (2, 4 and 6 days). Treatment with RH-0345 and benfuracarb reduced significantly the number of oocytes, the size and the volume of the basal oocyte during the experimental period. Methoprene distorted the ovarian development since it caused a significant reduction in the number of oocytes at 2, 4 and 6 days for the two tested doses, and an increase in oocyte size at 2, 4 and 6 days with 1 microg and a decrease with 10 microg. In a second series of experiments, the effects of these compounds were assayed on the ovarian proteins. Data from biochemical analysis revealed that RH-0345 and benfuracarb reduced the ovarian amounts of proteins, while treatment with methoprene increased it during the sexual maturation.     

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. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

Response of cultured Aedes aegypti fat bodies to 20-hydroxyecdysone

Fat bodies from non-blood-fed Aedes aegypti, stimulated in vitro by 10⁻⁴ M and 10⁻⁶ M of 20-hydroxyecdysone, were found to synthesize and release vitellogenin into the culture medium. Vitellogenin-specific monoclonal antibodies were utilized in an enzyme-linked immunosorbent assay procedure for quantification of vitellogenin in small aliquots of medium taken periodically from the culture. A minimal exposure of 5 h to 20-hydroxyecdysone was shown to be needed before the fat bodies would respond. Time-course of vitellogenin production in vitro was found to be identical to that observed in vivo. Vitellogenin-titre profiles were also investigated in cultured fat bodies from blood-fed A. aegypti. In all cases, response patterns were not affected by the presence or absence of 20-hydroxyecdysone after the fat bodies had been stimulated by blood meal to produce vitellogenin. We suggest here that initiation and control of vitellogenin synthesis is a programmed response to 20-hydroxyecdysone.     

The effects of 20-hydroxyecdysone on the differentiationin vitro of cells from the eye imaginal disc fromDrosophila melanogaster

We have examined the effects of the insect ecdysteroid, 20-hydroxyecdysone, on the differentiation of neuronal and non-neuronal elements in the developing adult visual system, usingin vitro methods inDrosophila. We examined the differentiation of early neuronal markers in the presence and absence of 1 μg/ml 20-hydroxyecdysone. Immunoreactivity to 22C10, a marker of an early neuronal antigen, as well as to the photoreceptor-specific antibody 24B10, suggests that differentiation of neuronal and photoreceptor antigens does not require 20-hydroxyecdysone. In eye-discs cultured from animals 5 hours after the white prepupa (P+5), ommochrome pigmentation first appeared after 2 days in 1 μg/ml 20-hydroxyecdysone, but cultures lacked pigmentation without 20-hydroxyecdysone. Our culture conditions failed to support the formation of the second screening pigment, drosopterins, even with 20-hydroxyecdysone. Eye discs from P+5 also formed lenses and interommatidial bristles in culture when 20-hydroxyecdysone was added but not in cultures devoid of the hormone. The differentiation of synaptotagmin and the elongation of extending photoreceptor neurites from eye disc fragments both occur in the absence of 20-hydroxyecdysone in cultures, but adding the hormone increased average neurite length. The threshold for enhanced neurite length was less than 125 ng/ml 20-hydroxyecdysone. Eye-disc cultures also developed immunoreactivity to histamine, the photoreceptor transmitter, from synthesis not re-uptake, in both the presence and in the absence of 20-hydroxyecdysone. These findings suggest that photoreceptor axons may be able to release transmitterin vivo both when they grow into the optic lobe and during the subsequent events in synapse formation.     

Investigation of ecdysteroid content and composition of Silene repens indigenous in Mongolia and introduced into western Siberia

The ecdysteroid profile was investigated by HPLC, and it has been found that 2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, polypodine B, 20-hydroxyecdysone, and integristerone A are synthesized in the aerial part. The composition of biologically active substances (BAS) has been investigated, and the presence of triterpene glycosides, alkaloids and flavonoids has been confirmed; tannins have been detected for the first time. It has been shown that the ecological conditions, such as altitude above sea level and humidity, influence the ecdysteroid composition and BAS levels.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

Ecdysteroid levels during post-diapause development and 20-hydroxyecdysone-induced development in male pupae of Mamestra configurata Wlk.

Post-diapause development in male pupae of Mamestra configurata Wlk. was characterized by the appearance of large, transitory peaks of ecdysone (2.8 μg/g live wt) at day 8 and 20-hydroxyecdysone (2.2 μg/g) at day 12 which declined to low levels prior to adult eclosion at day 28.Treatment of diapausing pupae with 20-hydroxyecdysone elicited a progression of dose-dependent physiological and pathological effects, including termination of diapause, development, accelerated development, and accelerated development leading to malformation and death. At a dose of 7.5 μg 20-hydroxyecdysone/g, all treated pupae terminated diapause, developed with little mortality and produced a high proportion of morphologically perfect adults. However, there were no large peaks of ecdysone or 20-hydroxyecdysone in treated pupae, possibly due to feedback inhibition by 20-hydroxyecdysone.At doses greater than 7.5 μg/g, development was accelerated markedly, survival decreased precipitously (0% at 15 μg/g) and the proportion of malformed adults increased sharply. Pupae that received a lethal dose of 20-hydroxyecdysone died almost synchronously after undergoing accelerated development for 18–20 days, indicating that they encounter a common, hormone-induced developmental block. Pupae receiving 15 μg/g also showed no edcysone or 20-hydroxyecdysone peaks, but had a prolonged period of hyperecdysonism which likely caused their accelerated development and death.     

Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner

The imaginal discs of Sarcophaga were found not to develop normally in the presence of galactose, a hapten sugar of Sarcophaga lectin, or anti-Sarcophaga lectin antibody. Wing and leg discs cultured with these substances became morphologically abnormal and no imaginal discs reached the stage of terminal differentiation, even in the presence of 20-hydroxyecdysone. The development of the imaginal discs was shown to be autonomously regulated in an autocrine manner by Sarcophaga lectin; namely Sarcophaga lectin was secreted by the imaginal discs in the presence of 20-hydroxyecdysone, and the stimulus of self-induced Sarcophaga lectin seemed to be indispensable for further development of the imaginal discs. Sarcophaga lectin was originally found as a defense protein, but these results show that it plays independent roles in both defense and development.     

Regulation of antimicrobial peptide synthesis in larvae of <Emphasis Type="Italic">Calliphora vicina</Emphasis> (Diptera,Calliphoridae): a dose-dependent effect of ecdysteroids

Ecdysteroids are multifunctional hormones regulating virtually all morphogenetic events in insects. Their role in stress and immune response regulation is not well researched. Here we study the effect of 20-hydroxyecdysone on antimicrobial peptide synthesis in the larvae of Calliphora vicina. An inverse correlation was observed between the 20-hydroxyecdysone titer and antimicrobial peptide concentration in the hemolymph of naïve and bacteria challenged maggots. High and low doses of 20-hydroxyecdysone, injected simultaneously with bacterial cells, had an opposite effect on antimicrobial peptide synthesis in the diapausing larvae. Morphogenetically effective doses of 20-hydroxyecdysone demonstrated immunosuppressive activity. A low dose of 20-hydroxyecdysone, on the contrary, moderately stimulated antimicrobial peptide synthesis. The data suggest that ecdysteroids are directly involved in the regulation of the immune system activity and that the final effect is dose-dependant.     

Ecdysone 20-monooxygenase activity in Drosophila virilis strains varying in ecdysteroid response to heat stress

The effect of various duration of heat stress (38 degrees C) on the activity of ecdysone 20-monooxygenase converting ecdysone into 20-hydroxyecdysone has been studied in D. virilis of wild type and mutant strain females, which differ by the mode of heat stress response of ecdysone and 20-hydroxyecdysone. We are the first to show that heat stress induces activity of ecdysone 20-monooxygenase in Drosophila females and enzyme activity correlates with the level of 20-hydroxyecdysone.     

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.     

20-Hydroxyecdysone-induced differentiation and apoptosis in the Drosophila cell line, l(2)mbn

20-Hydroxyecdysone has an inhibitory effect on the proliferation of l(2)mbn cells, causes vacuolization and fragmentation of cells, and promotes a strong phagocytotic activity. From several lines of evidence, it can be concluded that 20-hydroxyecdysone induces apoptosis. Long-term video observations following the fate of individual cells, scanning and transmission electron microscopy reveal the typical characteristics of apoptosis: sequestration of small cellular protuberances or larger parts of the cell with nuclear fragments (apoptotic bodies), chromatin condensation, condensation and vesiculation of cytoplasm, whereas the mitochondria retain their normal appearance. The induction of apoptosis by 20-hydroxyecdysone was confirmed by the TUNEL reaction and quantitatively determined by a method based on this reaction. Onset of apoptosis precedes phagocytotic activity. JH III alone has no clear-cut effect on l(2)mbn cells. In double treatments, the inhibitory effect of 20-hydroxyecdysone on cell proliferation is significantly reduced by the addition of JH III. Whether or not JH III also reduces apoptotic activity is not yet clear. It is shown that the l(2)mbn cell line is an advantageous model system for the exploration of steroid-induced apoptosis.     

Isolation and identification of three ecdysteroid conjugates with a C-20 hydroxy group in eggs of locusta migratoria

Three ecdysteroids conjugates with a hydroxy group at C-20 were isolated from developing eggs of Locusta migratoria and identified as 22-phosphate conjugates of 2-deoxy-20-hydroxyecdysone, 20-hydroxyecdysone and 20-hydroxyecdysone acetate.     

Characterization of ecdysteroid 26-hydroxylase: an enzyme involved in molting hormone inactivation

Insect molting hormone (ecdysteroid) inactivation occurs by several routes, including 26-hydroxylation and further oxidation to the 26-oic acids. Thus, the ecdysteroid 26-hydroxylase is a critical enzyme involved in precise regulation of ecdysteroid titers during insect development. Administration of the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hydroxylase were investigated. The mitochondrial enzyme was found to have optimal activity at a pH of 7. 5 in a Hepes or sodium phosphate buffer at 30-37 degrees C. The apparent K(m) of the microsomal 26-hydroxylase for 20-hydroxyecdysone substrate was lower than that of the mitochondrial enzyme for either 20-hydroxyecdysone or ecdysone substrate. The V(max) of the 26-hydroxylase in both subcellular fractions was slightly higher using 20-hydroxyecdysone as substrate compared to ecdysone. Demonstration that activity of the mitochondrial 26-hydroxylase was inhibited by incubation in a CO (or N(2)) atmosphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided strong evidence that the hydroxylase is cytochrome P450-dependent. Indirect evidence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxylase(s) could exist in a less active dephosphorylated state or more active phosphorylated state. Using Escherichia coli alkaline phosphatase to remove covalently bound phosphate groups, the activity of the 26-hydroxylase was decreased and, conversely, activity was enhanced using a cAMP-dependent protein kinase with appropriate cofactors. In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-treated fractions.     

Role of low ecdysteroid titre in acquisition of competence for pupal transformation in Bombyx mori

Penultimate-instar larvae of Bombyx mori were neck-ligated or ligated posterior to the prothoracic glands. Repetitive injections of 20-hydroxyecdysone every 3 or more hours elicited the gut purge in thorax-ligated animals. Single injections of 20-hydroxyecdysone up to 40 μg failed to induce the gut purge. However, a single injection of 20-hydroxyecdysone together with juvenile hormone analogue, resulted in larval moulting of thorax-ligated animals. Once the thorax-ligated larvae showed the gut purge, a single injection of 20-hydroxyecdysone was enough to provoke pupation. The change in ecdysteroid titre in those animals receiving repeated injections was compared with that observed in neck-ligated larvae that spontaneously underwent the gut purge followed by precocious pupation. These data indicate that the very low ecdysteroid titre found before the gut purge is important for the acquisition of competence to undergo the gut purge in response to a small ecdysteroid surge just before the gut purge.