Kiyoshi Hiruma

Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in Wings

Using the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA clone, we isolated three genomic clones for EcR from the tobacco hornworm, Manduca sexta. Subsequent isolation and sequencing of several cDNAs yielded a homolog of the B1 isoform with 50, 95 and 70% amino acid identities with DmEcR in the N-terminal A/B, the DNA binding and the ligand binding domains respectively. Unlike Drosophila, an intron occurs between the exons encoding the two zinc fingers of Manduca EcR (MsEcR). A 6.0 kb mRNA encoding MsEcR was found in both larval wing discs and prothoracic glands and in pupal wings. During the final larval instar, the mRNA was maximal in the wing discs at one day after wandering (W1), whereas in the prothoracic gland EcR mRNA increased rapidly to high levels on day 2 and remained high thereafter. During the onset of adult development, two peaks of EcR mRNA were observed in wings from day 3 to 5 and on day 8 after pupal ecdysis. These two peaks correlated with the time of increasing titers of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The EcR mRNA peaks always preceded the large ecdysteroid peak, suggesting that the transcription of the EcR gene is induced by a low concentration of ecdysteroid in vivo.

Juvenile Hormone Is Required to Couple Imaginal Disc Formation with Nutrition in Insects

In starved larvae of the tobacco hornworm moth Manduca sexta, larval and imaginal tissues stop growing, the former because they lack nutrient-dependent signals but the latter because of suppression by juvenile hormone. Without juvenile hormone, imaginal discs form and grow despite severe starvation. This hormone inhibits the intrinsic signaling needed for disc morphogenesis and does so independently of ecdysteroid action. Starvation and juvenile hormone treatments allowed the separation of intrinsic and nutrient-dependent aspects of disc growth and showed that both aspects must occur during the early phases of disc morphogenesis to ensure normal growth leading to typical-sized adults.

Molecular analysis of the mode of action of RH-5992, a lepidopteran-specific, non-steroidal ecdysteroid agonist

Abstract The dibenzoyl hydrazine, RH-5992, induces precocious molting in all lepidopteran larvae tested including the tobacco hornworm, Manduca sexta and the spruce budworm, Choristoneura fumiferana. Synthesis of a new cuticle including formation of a new head capsule that is incompletely sclerotized and lack of ecdysis are some of the major phenotypic effects. Similar to 20E, RH-5992 induces Manduca hormone receptor 3 (MHR3) mRNA and suppresses 14 kDa larval cuticular protein (LCP-14) transcript levels in 4th instar epidermis of Manduca , cultured in vitro . The ED 50 for induction of MHR3 was 1.4 × 10 −7 M, 10 times less than that of 20E. When the epidermis was exposed to 20E for 17 h and then cultured in hormone-free medium for a further 48 h, the LCP-14 mRNA level went up to nearly 60% of the maximal level reached in the untreated control. However, when RH-5992 was used, the level remained low even after 48 h. Dopa decarboxylase (DDC) transcription normally occurs at the end of the molt in preparation for sclerotization and in the epidermis cultured in vitro requires initial exposure to 20E for 17 h followed by its removal. Both in vivo and transient in vitro treatment with RH-5992 prevented DDC expression for up to 48 h after the removal of the compound. Thus, RH-5992 mimics the action of 20E on the expression of these 3 genes, but its effects persist in the tissue much longer than 20E.

Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

Abstract The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.

HORMONAL REGULATION OF EPIDERMAL METAMORPHOSIS IN VITRO : CONTROL OF EXPRESSION OF A LARVAL-SPECIFIC CUTICLE GENE

Fourth (penultimate) instar larval epidermis of the tobacco hornworm, Manduca sexta, was used to develop an in vitro culture system to study the hormonal control of metamorphosis at both the cellular and the molecular level. Immediate exposure to 4 x 10(-6) M 20-hydroxyecdysone (20-HE) for more than 8 hr, followed by hormone-free medium for 24 hr, caused the formation of a new larval cuticle. By contrast, incubation in hormone-free medium for more than 24 hr prior to exposure to 20-HE allowed pupal cuticle synthesis. The cessation of expression of the larval-specific cuticular gene LCP-14 occurred rapidly in response to 20-HE during the larval molt in vitro (half-life: ca. 6 hr), even in the presence of 3 x 10(-8) M JH I. This suppression by 20-HE was prevented by cycloheximide, indicating that 20-HE does not act directly on this gene. Incubation with alpha-amanitin showed that the half-life of LCP-14 was more than 10 hr. Thus, 20-HE must both suppress gene transcription and destabilize the mRNA. LCP-14 mRNA subsequently reappeared 24 hr after exposure to hormone-free medium, indicating that suppression was temporary. By contrast, when JH and its effects were absent after preincubation in hormone-free medium for 48 hr, 20-HE caused permanent suppression of LCP-14 mRNA, since the mRNA did not reappear after removal of 20-HE. Exposure of Day 2 fifth instar larval epidermis to 3 x 10(-7) M 20-HE, which causes pupal commitment in the absence of JH I, also permanently suppressed LCP-14 gene expression.

Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2

ABSTRACT MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5′ flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 μg of 20E per ml inManduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1–USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at −671 and the adjacent GGGTCA being most critical. The EcRE1 at −342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1–USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1–USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1–USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1–USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the sameKd (7 × 10−10 M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.

Granular phenoloxidase involved in cuticular melanization in the tobacco hornworm: regulation of its synthesis in the epidermis by juvenile hormone

The granular phenoloxidase (PO) that is responsible for cuticular melanization in Manduca sexta larva was purified and an antibody was prepared. This granular PO was found to consist of four isozymes of 90 kDa with isoelectric points ranging from 5.7 to 5.85. The enzyme was immunologically and electrophoretically distinct from the cuticular wound PO, a second cuticular PO common to all larval cuticle, and the hemolymph PO. Both [14C]mannose and [14C]sialic acid were incorporated into the granular PO, showing that this granular PO was a glycoprotein whose sugar moiety was a complex oligosaccharide. When no juvenile hormone (JH) was present at the head capsule slippage (HCS) stage, the epidermis began synthesizing PO 6 hr later. This epidermal synthesis was maximal 12 hr after HCS at which time the PO appeared in the cuticle, and then synthesis declined. When synthesis ceased about 23 hr after HCS, no further incorporation into the cuticle was observed. As melanization proceeded, immunologically detectable cuticular PO decreased. Application of 0.1 microgram JH I at the time of HCS inhibited synthesis of PO by the epidermis and thus prevented melanization. JH application after PO synthesis had begun (8 hr after HCS) prevented its subsequent synthesis, causing partial melanization. Thus, the absence of JH is necessary during the period of epidermal synthesis of the granular PO to allow complete melanization.

The molecular mechanisms of cuticular melanization: The ecdysone cascade leading to dopa decarboxylase expression in Manduca sexta

Many insect developmental color changes are known to be regulated by both ecdysone and juvenile hormone. Yet the molecular mechanisms underlying this regulation have not been well understood. This review highlights the hormonal mechanisms involved in the regulation of two key enzymes [dopa decarboxylase (DDC) and phenoloxidase] necessary for insect cuticular melanization, and the molecular action of 20-hydroxyecdysone on various transcription factors leading to DDC expression at the end of a larval molt in Manduca sexta. In addition, the ecdysone cascade found in M. sexta is compared with that of other organisms.

The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release

When the parasitoid Apanteles kariyai laid eggs into host Pseudaletia separata larvae, before prothoracicotropic hormone (PTTH) was released in the last instar preparatory to metamorphosis, the host did not pupate and the larvae of the wasps emerged. The ecdysteroid titer of unparasitized intact larvae increased up to 1 microgram/ml 1 day before pupation, whereas the titer of parasitized larvae was maintained at a low level without the surge. Isolated prothoracic glands from intact larvae synthesized much more ecdysone than those of parasitized larvae both in vivo and in vitro. Administration of exogenous PTTH caused the activation of the prothoracic glands seen during parasitization. Injection of 20-hydroxyecdysone (20-HE) into the parasitized larvae caused by hosts pupation, but did not affect the development of the wasp larvae. However, the sensitivity of the integument to 20-HE was lower in parasitized than in unparasitized larvae. Injection of a mixture of adult wasp calyx and venom fluids into last instar unparasitized larvae delayed their pupation, suggesting that calyx and venom fluids are factors contributing to disturbance of the normal function of brain-prothoracic gland system. These results show that parasitization inhibits secretion and/or synthesis of PTTH and also delays the larval-pupal commitment of the integument by keeping the ecdysteroid level low.

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

SummaryWhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both α-MDH and α-fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 μg/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.