Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta

E75A and E75B, isoforms of the E75 orphan nuclear receptor, are sequentially up-regulated in the abdominal epidermis of the tobacco hornworm Manduca sexta by 20-hydroxyecdysone (20E) during larval and pupal molts, with E75A also increasing at pupal commitment (Zhou et al., Dev. Biol. 193, 127-138, 1998). We have now cloned E75C and show that little is expressed in the epidermis during larval life with trace amounts seen just before ecdysis. Instead, E75C is found in high amounts during the development of the adult wings as the ecdysteroid titer is rising, and this increase was prevented by juvenile hormone (JH) that prevented adult development. By contrast, E75D is expressed transiently during the larval and pupal molts as the ecdysteroid titer begins to decline and again just before ecdysis, but in the developing adult wings is expressed on the rise of 20E. Removal of the source of JH had little effect on either E75C or E75D mRNA expression during the larval and pupal molts. At the time of pupal commitment, in vitro experiments show that 20E up-regulates E75D and JH prevents this increase. Neither E75A nor E75D mRNA was up-regulated by JH alone. Thus, E75C is primarily involved in adult differentiation whereas E75D has roles both during the molt and pupal commitment.     

Insulin/IGF signaling regulates the change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone

At the beginning of the final larval (fifth) instar of Manduca sexta, imaginal precursors including wing discs and eye primordia initiate metamorphic changes, such as pupal commitment, patterning and cell proliferation. Juvenile hormone (JH) prevents these changes in earlier instars and in starved final instar larvae, but nutrient intake overcomes this effect of JH in the latter. In this study, we show that a molecular marker of pupal commitment, broad, is up-regulated in the wing discs by feeding on sucrose or by bovine insulin or Manduca bombyxin in starved final instar larvae. This effect of insulin could not be prevented by JH. In vitro insulin had no effect on broad expression but relieved the suppression of broad expression by JH. This effect of insulin was directly on the disc as shown by its reduction in the presence of insulin receptor dsRNA. In starved penultimate fourth instar larvae, broad expression in the wing disc was not up-regulated by insulin. The discs became responsive to this action of insulin during the molt to the fifth instar together with the ability to become pupally committed in response to 20-hydroxyecdysone. Thus, the Manduca bombyxin acts as a metamorphosis-initiating factor in the imaginal precursors.     

Hormonal Control of Epidermal Cell Development

SYNOPSIS. During larval life the insect epidermis makes a larvalcuticle and certain pigments due to the presence of juvenilehormone (JH) at critical times during the molt cycle. The presenceof JH also permits growth of imaginal discs and maintains strictlylarval epidermis. At metamorphosis the lepidopteran epidermisresponds to a low level of 20- hydroxyecdysone (20HE) in theabsence of JH by becoming pupally committed, then later it formsa pupal cuticle when more 20HE appears, even though JH is present.During the change of commitment, DNA synthesis occurs but isnot essential, whereas both RN A and protein synthesis are.The major changes in the translatable mRNA population at thistime are threefold: a decline in most larval cuticle mRNAs,a transient increase followed by a disappearance of a few larvalcuticle mRNAs, and an appearance of at least one ‘pupalcommitment’ mRNA and two to three mRNAs for small pupalcuticular proteins. Similar changes are seen in the proteinsynthetic patterns. Thus, a pupally committed cell is one whichcan no longer make larval products but which is not yet ableto make most pupal products. Juvenile hormone prevents the changeto pupal commitment by directing some of both the primary andthe secondary actions of 20HE on the genome.     

Commencement of pupal commitment in late penultimate instar and its hormonal control in wing imaginal discs of the silkworm, Bombyx mori

Pupal commitment of the wing imaginal disc of the silkworm, Bombyx mori, is completed shortly after the final (fifth) larval ecdysis. Pupal commitment was induced by in vitro culture with 20-hydroxyecdysone (20E). Shortly after the head capsule slippage (HCS) that occurs approximately 24 h before the final larval ecdysis, the discs become competent to respond to 20E, indicating that the process of pupal commitment begins in the late penultimate (fourth) instar. The simultaneous presence of methoprene (JHA) with 20E suppressed the pupal commitment at 4 ng/ml for the discs at 12 h after HCS and at 240 ng/ml for the discs at the ecdysis. Thus, the discs rapidly lose their sensitivity to JH at the end of the fourth instar. Day 0 fourth wing discs were not pupally committed by 20E when freshly dissected discs were exposed to 20E. By contrast, exposure to 20E after a pre-culture in a hormone free medium induced the pupal commitment. In those discs, the effective JHA concentration to suppress the 20E effects was 0.1 ng/ml. The present data suggest that pupal commitment proceeds through two stages from a reversible state that begins at around HCS to an irreversible state early in the fifth instar. The loss of sensitivity to JH is the primary impetus to begin the process and 20E is the factor that drives the discs to enter the reversible state.     

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=6.3×10^–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 μg/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. Received: 16 July 1998 / Accepted: 5 August 1998     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis

Background

Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometabolous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage.

Methods

Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference approaches.

Results

We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning.

Conclusions

In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and complex functions observed in holometabolous species.

General significance

Our results shed new light to the evolution from hemimetaboly to holometaboly regarding BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from regulating wing development, to determining pupal morphogenesis.     

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

Temporal patterns of protein synthesis inManduca epidermis during the change to pupal commitment in vitro: their modulation by 20-hydroxyecdysone and juvenile hormone

Summary The epidermis of final instar tobacco hornworm larvae,Manduca sexta, becomes committed to pupal differentiation in response to ecdysteroid in the absence of juvenile hormone (JH). Many changes in protein synthetic patterns have been noted during this time (Kiely and Riddiford 1985). To determine which of these changes are caused by ecdysteroid and which are important for the change of commitment, we have incubated larvally-committed epidermis for 24 h with 1 g/ml 20-hydroxyecdysone (20HE) and 3 g/ml epoxygeranylsesamole (EGS) (a JH mimic), with 3 g/ml EGS alone, or in hormone-free medium. Synthesis of larval-specific proteins such as insecticyanin and larval cuticular proteins was reduced to trace amounts or was undetectable after culture with 20HE for 24 h. The larval cuticular proteins that are greatly increasedin vivo on day 3 were not synthesized after exposure to 20HEin vitro. Ecdysteroid increased the synthesis of many of the proteins first seenin vivo on day 3 or during the wandering stage. The synthesis of about half of these latter proteins was inhibited by JH, indicating that they were likely part of the change of commitment. Other proteins that appear at this stagein vivo showed increased synthesis also in hormone-free medium and therefore were independent of the change of commitment.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.