Body size determination in insects: a review and synthesis of size‐ and brain‐dependent and independent mechanisms

Body size determination requires a mechanism for sensing size and a mechanism for linking size information to the termination of growth. Although the hormonal mechanisms that terminate growth are well elucidated, the mechanisms by which a body senses its own size are only partially understood; most of this understanding has come from the study of the mechanisms that control insect moulting and metamorphosis. We first review and discuss advances in our understanding of the physiological mechanisms by which insect larvae sense their size. Second, we present new findings on how larvae in which the size‐sensing mechanism has been disrupted eventually terminate growth (in a size‐independent manner). We synthesize recent insights into the genetic and molecular mechanisms of ecdysteroid regulation in Drosophila melanogaster with developmental physiology findings in Manduca sexta, paving the way for an integrated understanding of the mechanisms of body size regulation.     

SILKWORM 30K PROTEIN INHIBITS ECDYSONE‐INDUCED APOPTOSIS BY BLOCKING THE BINDING OF ULTRASPIRACLE TO ECDYSONE RECEPTOR‐B1 IN CULTURED Bm5 CELLS

This study investigates the mechanism through which increased 30K protein inhibits ecdysone‐induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20‐hydroxyecdysone (20E) after transfection with the pIZT/V5‐His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5‐His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti‐30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5‐His‐transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor‐B1 (EcR‐B1). Anti‐30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5‐His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR‐B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR‐B1‐containing complexes from Bm5 cells transfected with control pIZT/V5‐His vector and treated with 20E showed that EcR‐B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5‐His‐transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR‐B1 through formation of a 30K/EcR‐B1 complex, resulting in inhibition of 20E‐induced Bm5 cell apoptosis.     

MicroRNA Let‐7 targets the ecdysone signaling pathway E75 gene to control larval–pupal development in Bactrocera dorsalis

MicroRNAs (miRNAs) regulate various biological processes during insect developme nt;however, their role in larval-pupal development in oriental fruit fly, Bactrocera dorsalis (Hendel) remains unknown. In the current study, we address the biological function of a conserved miRNA, Bdo-Let-7 in the regulation of BdE75 gene, which belongs to the ecdysone signaling pathway and participates in the larval-pupal development in B. dorsalis. Using dual luciferase reporter assay in HEK293T cells we show that Bdo-Let-7 miRNA interacts with the 3' untranslated region of BdE75 gene and suppresses its expression. The Bdo-Let?7 and BdE75 are also co-expressed in the larval-pupal stages and in different tissues of B. dorsalis .In in vivo experiments, the injectio n of Bdo-Let-7 agomir and antagomir in third instar larvae down- and up-regulated the expression of BdE75、 respectively. The 20-hydroxyecdysone (20E) injection assay shows that 20E up-regulated the expression of Bdo-Let-7 on the 5th day of the larvae. Moreover, abnormal pupation and eclosion were observed after larval Bdo-Let-7 antagomir injection. Based on these results, we show that Bdo-Let-7 regulates the ecdysone signaling pathway through the exact dose of BdE75 gene, and is indispensable for normal larval-pupal development in B. dorsalis.     

昆虫成虫蜕皮激素研究进展

绝大多数成体昆虫羽化后,幼虫期间负责蜕皮激素合成的前胸腺即发生退化,但在一些内部生理及外部环境因子的调控下,某些成体组织(如生殖腺)可扮演类似前胸腺的角色合成与分泌蜕皮激素。蜕皮激素的功能发挥是经受体介导的,包括核受体(如EcR/USP)和膜受体(如DopEcR),它们广泛表达于成体许多组织,参与成虫行为、生殖、寿命、滞育及免疫应答等众多方面的调节,对维持基本的生理功能具有重要作用。就成虫蜕皮激素的产生组织及影响其滴度的因素、成虫蜕皮激素受体概述与组织分布、成虫蜕皮激素信号通路的功能发挥等研究进展方面加以综述。     

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.     

Control of ecdysteroidogenesis: Activation and inhibition of prothoracic gland activity

The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insectManduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca²⁺ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a β tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the gland's potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.     

The metabolism of ingested and injected [3H]ecdysone by final instar larvae of Heliothis armigera

ABSTRACT. When larvae of Heliothis armigera (Hüber) are fed on a diet containing [³H]ecdysone they produce large quantities of ecdysone 22-palmitate, probably in the gut. The radiolabel is excreted in the faeces chiefly as ecdysone 22-palmitate, but appreciable quantities of unchanged ecdysone are also found together with traces of ecdysone 2-and 3-acetates. No polar metabolites are found with ingested ecdysone, and the ecdysone probably did not penetrate the gut wall, since on injection into the body, ecdysone was appreciably converted into polar metabolites.     

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.     

BhSGAMP‐1, a gene that encodes an antimicrobial peptide,is developmentally regulated by the direct action of 20‐OH ecdysone in the salivary gland of Bradysia hygida (Diptera,Sciaridae)

Recently we have shown that BhSGAMP‐1 is a developmentally regulated reiterated gene that encodes an antimicrobial peptide (AMP) and is expressed exclusively in the salivary glands, at the end of the larval stage. We show, for the first time, that a gene for an AMP is directly activated by 20‐OH ecdysone. This control probably involves the participation of short‐lived repressor(s). We also found that the promoter of BhSGAMP‐1 is not equipped with elements that respond to infection, provoked by the injection of microorganisms, in the salivary glands or in the fat body. We produced polyclonal antibodies against the synthetic peptide and found that the BhSGAMP‐1 peptide is secreted in the saliva. The BhSGAMP‐1 gene was also activated during the third larval molt. These facts confirm our hypothesis that this preventive system of defense was selected to produce an environment free of harmful microorganisms in the insect's immediate vicinity, during molts. genesis 47:847–857, 2009. © 2009 Wiley‐Liss, Inc.     

Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change. i.e., cell aggregation, in response to treatment with 1 microM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20-30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.     

Haemolymph ecdysone concentrations in Hyalophora cecropia pupae,dauer pupae and adults

Haemolymph ecdysone concentrations were determined by radioimmunoassay in diapausing pupae, pharate adults, adults, and chilled dauer pupae. The concentration in diapausing pupae after 6 months chilling (5.35 pg/μl) increased dramatically after 3 days at 27°C (>200 pg/μl) and then decreased to low levels in adult females (1.63 pg/μl). In adult males ecdysone was undetectable in all except one animal. Dauer pupae showed a decrease from 6.1 to 1.7 pg/μl 1 day after being transferred from 6 to 27°C. Over a 3-day period the value increased to 3.19 pg/μl and remained constant for more than a year. These results suggest that diapausing pupae with and without brain neurosecretory cells maintain a low concentration of ecdysone in the haemolymph.     

Metabolism of [3H]-ecdysone in Schistocerca gregaria; formation of ecdysteroid acids together with free and phosphorylated ecdysteroid acetates

The metabolism of [³H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid tit re, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetate, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were identified as metabolites in both the free form and as polar conjugates. Comparison of the intact polar conjugates of the ecdysteroid acetates on two HPLC systems with the corresponding authentic compounds indicated that they were 3-acetylecdysone-2-phosphate and 3-acetyl-20-hydroxyecdysone-2-phosphate. Other major polar metabolites were identified as ecdysonoic acid and 20-hydroxyecdysonoic acid. Ecdysone metabolism in fifth-instar S. gregaria is apparently an age-dependent process. Early in the instar, excretion of both free and conjugated ecdysteroids, as well as ecdysteroid 26-acids, occurs. At this stage the level of ecdysteroid acetates in the conjugated (phosphate) form is high, in contrast to the free ecdysteroids, where ecdysone predominates. When the endogenous hormone titre is high, the formation of ecdysteroid acetates is less, the major excreted matabolites at that stage being conjugated 20-hydroxyecdysone together with ecdysteroid-26-acids, but little free ecdysteroids. Acetylation of ecdysone occurs primarily in the gastric caecae. Ecdysone-3-acetate (mainly as polar conjugate) is also a major product of ingested ecdysone in early fifth-instar Locusta migratoria.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Developmental regulation of granule size and numbers in larval salivary glands of drosophila by steroid hormone ecdysone

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, approximately 3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5x10(-6) m ecdysone.     

Juvenile hormone and moulting hormone titres in diapause- and non-diapause destined flesh flies

Failure of the brain to stimulate the prothoracic gland to release ecdysone has been widely regarded as the basis for diapause in insect pupae. In diapause-destined flesh flies, the absence of a peak of moulting hormone around the time of pupal head eversion supports this contention, but in addition, major pulses of juvenile hormone (JH) activity with a rhythmicity of 24 hr are unique to flesh flies destined for pupal diapause. JH activity persists during diapause, and a pulse of JH precedes the rise of moulting hormone that initiates adult development.     

The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.