Genetic tools to study juvenile hormone action in Drosophila.

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Met and gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

Elucidation of the regulation of an adult cuticle gene Acp65A by the transcription factor Broad.

Broad (BR), an ecdysone-inducible transcription factor, is a major determinant of the pupal stage. The misexpression of BR-Z1 isoform (BR-Z1) during adult development of Drosophila melanogaster prevents the expression of the adult cuticle protein 65A gene (Acp65A). We found that the proximal 237 bp of the 5' flanking region of Acp65A were sufficient to mediate this suppression. A targeted point mutation of a putative BR-Z1 response element (BRE) within this region showed that it was not involved. Drosophila hormone receptor-like 38 (DHR38) is required for Acp65A expression. We found that BR-Z1 repressed DHR38 expression and that BR's inhibition of Acp65A expression was rescued by exogenous expression of DHR38. Thus, BR-Z1 suppresses Acp65A expression by preventing the normal up-regulation of DHR38 at the time of adult cuticle formation.

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.

Development and survival of Anopheles gambiae eggs in drying soil: influence of the rate of drying, egg age, and soil type.

Little is known about the contribution made by the egg stage of African malaria vectors to the rapid rise in adult populations following the onset of seasonal rains. To examine this issue, we evaluated the viability of Anopheles gambiae eggs in drying soil in the laboratory. Survival data were collected from field-caught mosquitoes kept in sandy loam soil and laboratory-reared colonies kept in sandy loam soil and black cotton soil. Under high, medium, and low soil-moisture regimes, egg viability declined sharply with increased duration of drying. Eggs remained viable in drying sandy loam soil for 1, 5, and 10 days, but not after 15 or 20 days. The most dramatic decline in hatching success occurred between drying days 1 (78-83% hatch) and 5 (20-23% hatch). In contrast, eggs reared in high-moisture black cotton soil remained viable for up to 15 days. Furthermore, after 5 drying days, high-, medium-, and low-moisture soils averaged 59, 47, and 31% hatching success, respectively. We recovered unhatched eggs from sandy loam soils to examine the developmental status of the embryos. A majority of the unhatched eggs that were recovered from days 15 and 20 in sandy loam soils contained fully developed late-stage embryos. Thus, unhatched eggs completed embryonic development but probably died before receiving an appropriate hatching stimulus. Our results suggest that the absolute moisture content of the soil does not alone determine hatching success of anopheline eggs. Rather, soil moisture, together with the rate of drying, physiological factors associated with the age of the egg, and the type of soil in which the egg rests likely influence survival.

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta.

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.

Regulation of transcription factors MHR4 and betaFTZ-F1 by 20-hydroxyecdysone during a larval molt in the tobacco hornworm, Manduca sexta.

During the last larval molt in Manduca sexta, a number of transcription factors are sequentially expressed. Unlike E75A and MHR3, whose mRNAs are induced when the ecdysteroid titer increases, the expression of MHR4 mRNA occurs transiently at the onset of the decline of ecdysteroid titer followed by betaFTZ-F1 mRNA expression when the ecdysteroid titer becomes low. When day 2 fourth epidermis was exposed to 20-hydroxyecdysone (20E) in vitro, MHR4 mRNA appeared between 12 and 21 h, peaked at 24 h, and then declined. Using the protein synthesis inhibitors cycloheximide and anisomycin both in vivo and in vitro, we found that the MHR4 transcript was directly induced by 20E and required the presence of 20E for its expression. The accumulation of MHR4 mRNA, however, did not occur until a 20E-induced inhibitory protein(s) disappeared. This control of MHR4 expression is unique among the ecdysone-induced transcription factors. When the epidermis was cultured with 20E, betaFTZ-F1 mRNA was not induced until after the removal of 20E as previously found for Drosophila and the silkworm Bombyx mori. The presence of juvenile hormone had no effect on accumulation of either transcript.

Hormonal regulation and patterning of the broad-complex in the epidermis and wing discs of the tobacco hornworm, Manduca sexta.

Expression of Manduca Broad-Complex (BR-C) mRNA in the larval epidermis is under the dual control of ecdysone and juvenile hormone (JH). Immunocytochemistry with antibodies that recognize the core, Z2, and Z4 domains of Manduca BR-C proteins showed that BR-C appearance not only temporally correlates with pupal commitment of the epidermis on day 3 of the fifth (final) larval instar, but also occurs in a strict spatial pattern within the abdominal segment similar to that seen for the loss of sensitivity to JH. Levels of Z2 and Z4 BR-C proteins shift with Z2 predominating at pupal commitment and Z4 dominant during early pupal cuticle synthesis. Both induction of BR-C mRNA in the epidermis by 20-hydroxyecdysone (20E) and its suppression by JH were shown to be independent of new protein synthesis. For suppression JH must be present during the initial exposure to 20E. When JH was given 6 h after 20E, suppression was only seen in those regions that had not yet expressed BR-C. In the wing discs BR-C was first detected earlier 1.5 days after ecdysis, coincident with the pupal commitment of the wing. Our findings suggest that BR-C expression is one of the first molecular events underlying pupal commitment of both epidermis and wing discs.

Different mechanisms underlie phenotypic plasticity and interspecific variation for a reproductive character in drosophilids (Insecta: Diptera).

The insect ovary is a modular structure, the functional unit of which is the ovariole. Ovariole number is positively correlated with potential reproductive output. Among drosophilids (Insecta: Diptera), ovariole number shows both phenotypic plasticity and substantial interspecific and interpopulational variation. Here we examine the mechanistic connection between phenotypic plasticity and genetically fixed variation in ovariole number within the melanogaster species group. When a laboratory population of Drosophila melanogaster was reared under reduced food conditions, differences in ovariole number were entirely due to alterations in cell differentiation during the wandering stage at the very end of larval development. Cell growth and cell death were not affected. When these same flies were reared under a variety of temperatures, ovariole number differences arose during the latter half of the third (final) larval instar. Cell differentiation was affected, although cell number was not, and ovariole number differences were established before metamorphosis. In contrast, genetically fixed, interspecific and interpopulational variability in ovariole number was caused by alterations in the dynamics of cell differentiation and by cell number differences. Furthermore, the stages affected were different in different species and populations in the melanogaster species group, ranging from the first (D. sechellia) through the middle of the third (D. simulans and D. mauritiana) larval stage. Therefore, the mechanistic bases for plasticity-based variability are largely distinct from the mechanistic bases for interspecific and interpopulational variability. Our results suggest that phenotypic plasticity indicates evolutionary flexibility in underlying ontogenetic processes.

Patterns of MHR3 expression in the epidermis during a larval molt of the tobacco hornworm Manduca sexta.

MHR3, an ecdysone-induced transcription factor, was shown to appear in the abdominal epidermis of the tobacco hornworm Manduca sexta in a pattern-specific manner as the 20-hydroxyecdysone (20E) titer rises for the larval molt. The crochet epidermis that forms the hooked setae on the proleg is first to show MHR3 mRNA and protein followed sequentially by the spiracle, the dorsal intrasegmental annuli, the interannular regions, and finally the trichogen and tormogen cells. The protein appears in the nuclei about 8 h before the onset of cuticle formation, is present during the outgrowth of the setae, and disappears after epicuticle formation. In vitro studies showed that MHR3 mRNA induction in the crochet epidermis by 20E was more sensitive (EC(50) = 10(-6) M; 50% induction by 2 h exposure to 4 x 10(-6) M 20E) and did not require protein synthesis for maximal accumulation compared to the dorsal epidermis. The ecdysone receptor complex is present in both tissues at the outset of the molt and therefore is not a determining factor in these responses. Thus, in addition to the ecdysone receptor complex, region-specific factors govern both sensitivity and timing of responsiveness of MHR3 to 20E to ensure that this transcription factor will be present when needed for its differentiative role.

Parallel alterations in the timing of ovarian ecdysone receptor and ultraspiracle expression characterize the independent evolution of larval reproduction in two species of gall midges (Diptera: Cecidomyiidae).

Although most insects reproduce in the adult stage, facultative larval or pupal reproduction (paedogenesis) has evolved at least six times independently in insects, twice in gall midges of the family Cecidomyiidae (Diptera). Paedogenesis in gall midges involves the precocious growth and differentiation of the ovary in an otherwise larval form. We have previously shown that the timing of expression of the Ecdysone Receptor (EcR) and Ultraspiracle (USP), the two proteins that constitute the functional receptor for the steroid hormone 20-hydroxyecdysone, regulates the timing and progression of ovarian differentiation in Drosophila melanogaster (Diptera: Drosophilidae). Here we test the hypothesis that precocious activation of EcR and USP in the ovaries of paedogenetic gall midges allows for precocious ovarian differentiation. Using monoclonal antibodies directed against insect EcR and USP proteins, we first show that when these gall midges are reared under conditions that promote typical, metamorphic development, up- regulation of EcR and USP occurs in the final larval stage. By contrast, in the paedogenetic life cycle, EcR and USP are up-regulated early in the first larval stage. A similar pattern is seen for two independently-evolved paedogenetic gall midges, Heteropeza pygmaea and Mycophila speyeri. We discuss our results in the context of developmental constraints on the evolution of paedogenesis in dipteran insects.

The origins of insect metamorphosis.

Insect metamorphosis is a fascinating and highly successful biological adaptation, but there is much uncertainty as to how it evolved. Ancestral insect species did not undergo metamorphosis and there are still some existing species that lack metamorphosis or undergo only partial metamorphosis. Based on endocrine studies and morphological comparisons of the development of insect species with and without metamorphosis, a novel hypothesis for the evolution of metamorphosis is proposed. Changes in the endocrinology of development are central to this hypothesis. The three stages of the ancestral insect species-pronymph, nymph and adult-are proposed to be equivalent to the larva, pupa and adult stages of insects with complete metamorphosis. This proposal has general implications for insect developmental biology.

The sequence of Locusta RXR, homologous to Drosophila Ultraspiracle, and its evolutionary implications.

The cellular response to steroid hormones is mediated by nuclear receptors which act by regulating transcription. In Drosophila melanogaster, the receptor for the insect molting hormone, 20-hydroxyecdysone, is a heterodimer composed of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The DNA binding domains of arthropod USPs and their vertebrate homologs, the retinoid X receptor (RXR) family, are highly conserved. The ligand binding domain sequences, however, divide into two distinct groups. One group consists of sequences from members of the holometabolous higher insect orders Diptera and Lepidoptera, the other of sequences from vertebrates, a crab and a tick. We here report the sequence of an RXR/USP from the hemimetabolous orthopteran, Locusta migratoria. The locust RXR/USP ligand binding domain clearly falls in the vertebrate-crab-tick rather than the dipteran-lepidopteran group. The reason for the evolutionarily abrupt divergence of the dipteran and lepidopteran sequences is unknown, but it could be a change in the type of ligand bound or the loss of ligand altogether.

Activation of a delayed-early gene encoding MHR3 by the ecdysone receptor heterodimer EcR-B1-USP-1 but not by EcR-B1-USP-2.

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 micrograms of 20E per ml in Manduca 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 same Kd (7 x 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.

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta.

Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10(-5) M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC50=6.3x10(-7) M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 microgram/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC50 as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected.

Juvenile hormone prevents ecdysteroid-induced expression of broad complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta.

A cDNA homolog of the Drosophila melanogaster Broad Complex (BRC) gene was isolated from the tobacco hornworm, Manduca sexta, which shows a predicted 88% amino acid identity with Drosophila BRC in the N-terminal BTB domain. Three zinc finger domains encoding homologs of the Drosophila Z2, Z3, and Z4 domains (93, 100, and 85% identity, respectively) were obtained by RT-PCR. In Manduca dorsal abdominal epidermis, BRC RNAs were not observed during the larval molt. Three BRC transcripts-6.0, 7.0, and 9.0 kb-first appeared at the end of the feeding stage of the fifth (final) instar when the epidermis is exposed to ecdysteroids in the absence of juvenile hormone (JH) and becomes committed to pupal differentiation. These RNAs were induced in day 2 fifth larval epidermis in vitro by 20-hydroxyecdysone (20E) in the absence of JH with dose-response and time courses similar to the induction of pupal commitment. This induction by 20E in vitro was prevented by the presence of JH I at levels seen in vivo during the larval molt. In the wing discs, the BRC RNAs appeared shortly after ecdysis to the fifth instar and coincided with the onset of metamorphic competence of these discs. Application of a JH analogue pyriproxifen during the fourth instar molt delayed and reduced the levels of BRC mRNAs seen in the wing discs in the early fifth instar, but did not completely prevent their appearance in this tissue that first differentiates at metamorphosis. The expression of the BRC transcription factors thus appears to be one of the first molecular indications of the genetic reprogramming of the epidermis necessary for insect metamorphosis. How JH prevents BRC expression in this epidermis may provide the key to understanding how this hormone controls metamorphosis.

The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian morphogenesis during Drosophila metamorphosis.

Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the ecdysone receptor (EcR) and its partner ultraspiracle (USP). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of USP and the EcR-A isoform in all but one of eight mesoderm-derived somatic cell types. The one exception is the larval terminal filament (TF) cells, in which only USP is detectable during cell differentiation. In cells destined to form the basal stalks and anterior oviduct, USP colocalizes with what appears to be the EcR-B2 isoform. Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. Our results demonstrate that proper expression of the ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects.

Identification of proteins and developmental expression of RNAs encoded by the 65A cuticle protein gene cluster in Drosophila melanogaster.

Proteins of the third instar larval cuticle of Drosophila melanogaster, LCP5-LCP9, were purified and their N-terminal sequences determined. Three of these proteins (LCP5, 6, and 8) were found to be encoded by two multicopy genes previously mapped to the gene cluster at 65A 5-6 on the left arm of the third chromosome. The analysis of the patterns of developmental expression of the 8 distinct genes at this site showed that all but two were expressed during larval life. The patterns fell into three groups: one where expression was all through larval life, one where expression was primarily in the third instar, and one only during the production of the adult cuticle. One duplicated gene was not expressed in the Canton S strain at any time from the embryo to adult ecdysis. These findings indicate that there is not a unique set of cuticle proteins in the third versus the first and second instar larval cuticles and indicates that overlapping gene sets in several different gene clusters encode the proteins of the cuticle of different developmental stages.

Steroid and neuronal regulation of ecdysone receptor expression during metamorphosis of muscle in the moth, Manduca sexta.

Ecdysteroids regulate the remodeling of the dorsal external oblique 1 (DEO1) muscle during metamorphosis in Manduca sexta (). We show that the temporal and spatial patterning of the A and B1 isoforms of the ecdysone receptor (EcR) within muscle DEO1 corresponds with the developmental fates of the fibers. Using antibodies directed to specific isoforms of EcR, we show that the expression of various EcR isoforms in myonuclei differ among the five fibers of DEO1 and correspond with the developmental response of the muscle to the changing steroid titers and to the pattern of innervation. Muscle degeneration and apoptosis of myonuclei in all fibers are correlated with the expression of only EcR-A just before pupal ecdysis and then with the expression of low levels of both EcR-A and EcR-B1 shortly after pupation. Only the first fiber of muscle DEO1 participates in the regrowth of the adult muscle, and only this fiber shows an upregulation of EcR-B1 that is evident at 3 d after pupal ecdysis. Denervation of the muscle prevents both the upregulation of EcR-B1 and myoblast proliferation. We conclude that the developmental fate of muscle DEO1 during metamorphosis is orchestrated by interactions between rising and falling ecdysteroid titers, the pattern of expression of EcR isoforms by the muscle, and interactions with other cells in the local environment.

Regulation of the transcription factor E75 by 20-hydroxyecdysone and juvenile hormone in the epidermis of the tobacco hornworm, Manduca sexta, during larval molting and metamorphosis.

The homolog of the ecdysteroid-induced transcription factor E75A in Drosophila melanogaster was cloned from the tobacco hornworm, Manduca sexta, and its developmental expression and hormonal regulation were analyzed. Both E75A and E75B mRNAs were found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20E in vitro caused the rapid, transient induction of E75A RNA with a peak at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) showed a 10-fold higher sensitivity to 20E (EC50 = 2 x 10(-8) M for fourth instar and 2 x 10(-7) M for fifth instar epidermis). The presence of the protein synthesis inhibitor anisomycin had no effect on the induction but prevented the decline, indicating that E75A RNA was directly induced by 20E, but its down-regulation depended on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, caused an increased accumulation of E75A RNA throughout the culture period although the temporal pattern was unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis.