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.     

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.     

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.     

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.     

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.     

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.     

Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae)

During the last larval instar, the wing imaginal disks of Precis coenia grow continuously. The rate of disk growth is not disk-autonomous but closely matches the rate of somatic growth of the larva, so that the size of the disks is a function of the size of the body, irrespective of the growth rate of the larva. When larvae are starved, their wing disks cease growth within 4 h, which indicates the existence of an efficient coupling mechanism between the growth of the soma and growth of the imaginal disks. Disk growth is inhibited by juvenile hormone in a dose-dependent manner. In the presence of the hormone the wing disks stop growing even while the larva continues to grow normally. During the last larval instar the wing imaginal disks also undergo a complex differentiation, consisting of the development of the lacunae and tracheation that define the future adult wing venation system. In normally growing larvae, differentiation of the wing disk is tightly correlated with wing size. Differentiation and size can be dissociated by starvation. If larvae are starved at any time after differentiation has begun, differentiation continues at a normal rate, even though the wing disk does not grow. Differentiation does not begin spontaneously in larvae that are starved before differentiation has begun. These findings indicate that the initiation of differentiation and its continuation are controlled independently. Juvenile hormone inhibits differentiation in a dose-dependent manner. Upon treatment with juvenile hormone, the stage of differentiation becomes fixed. These findings indicate that continued differentiation of the wing disk can only occur in the absence of juvenile hormone. Although the circulating level of juvenile hormone may be elevated during starvation, it is unlikely that this elevation is responsible for the observed effect of starvation on growth and differentiation of the disk.     

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.     

Macromolecular synthesis in wing discs of Spodoptera mauritia Boisd: effects of a juvenile hormone analogue

Last instar larvae of S. mauritia treated topically on day 0, day 1, day 2 and day 3 with a daily (dose of 25 microg juvenile hormone analogue (JHA) moulted into supernumerary larvae. The imaginal discs of the supernumerary larvae especially those of mouthparts and thoracic appendages showed pupal characteristics. However the wing discs, which showed only partial differentiation, were uneverted and highly tanned. In an effort to provide an explanation to this anomaly the RNA, DNA and protein profile in the wing discs of supernumerary larvae were studied. Quantitative analysis of DNA, RNA and protein showed a considerable increase in the amount of DNA and protein and a decline in RNA level. SDS-PAGE analysis of wing disc proteins of JHA treated larvae showed a reduction in the expression of many major proteins that were predominant in the wing discs of control larvae. The results suggest that JHA induced inactivation of genes involved in the synthesis of proteins needed for evagination process may be responsible for the formation of uneverted, partially differentiated pupal wing discs in supernumerary larvae.     

The role of juvenile hormone during larval-pupal transformation of Spodoptera littoralis: Switchover in the sensitivity of the prothoracic gland to juvenile hormone

Application of JHA to the final instar of Spodoptera littoralis larvae before they attain maximal body weight causes delay to or completely blocks the onset of metamorphosis, as indicated by the dorsal vessel exposure. When the same dose of JHA is administered to the larvae just after their attaining maximal body weight, at the time of the onset of gut-emptying, metamorphosis is substantially accelerated.By means of ligature experiments, it is shown that JHA has a direct effect on the prothoracic glands. It inhibits them at the beginning of the final instar and then stimulates them shortly before pupation. It seems that under normal conditions JH is necessary for pupal cuticle formation; those larvae which pupate without JH show adult characteristics.     

Brain-independent development in the moth Sesamia nonagrioides

The caterpillars of Sesamia nonagrioides developing under long-day (LD) photoperiod pupate in the 5th or 6th instar whereas under short day (SD) conditions they enter diapause and undergo several extra larval molts. The diapause is terminated within 1-3 instars upon transfer of SD larvae to the LD conditions. Brain removal from the 6th instar larvae promotes pupation followed by imaginal development; however, one third of the SD larvae and 12% of the LD larvae debrained at the start of the instar first undergo 1-2 larval molts. The incidence of larval molts is enhanced by the brain implants. Exclusively pupal molts occur in the LD larvae debrained late in the 6th instar. Decapitation elicits pupation in both LD and SD larvae, except for some of the 4th and 5th and rarely 6th instar that are induced to a fast larval molt. The pupation of decapitated larvae is reverted to a larval molt by application of a juvenile hormone (JH) agonist. No molts occur in abdomens isolated from the head and thorax prior to the wandering stage. Abdomens isolated later undergo a larval (SD insects) or a pupal (LD insects) molt. Taken together the data reveal that in S. nonagrioides (1) several larval molts followed by a pupal and imaginal molt can occur without brain; (2) an unknown head factor outside the brain is needed for the pupal-adult molt; (3) brain exerts both stimulatory and inhibitory effect on the corpora allata (CA); (4) larval molts induced in CA absence suggest considerable JH persistence.     

Time-dosage studies of juvenile hormone action on the development of Plodia interpunctella

The degree of inhibition of larval-pupal ecdysis of Indian meal moths, Plodia interpunctella, by juvenile hormone (JH) treatment depended upon the dosage of hormone and time of treatment. During the last larval instar, the timing aspect operated independently of dosage and had two essential components for effectiveness, (a) early initiation of exposure and (b) maintenance of exposure. The effects of JH treatments could be reversed by removing the insects from the JH diet. In vitro tests with wing disks indicated that JH reversibly inhibited disk development only during the early part of the last larval instar, a time when disks are insensitive to β-ecdysone. After disks acquire full sensitivity to β-ecdysone, they lose their ability to respond to JH.     

Effects of a juvenile hormone analogue on the duration of the fifth instar in the milkweed bug Oncopeltus fasciatus

Topical application of JHA to fifth instar nymphs of Oncopeltus fasciatus, immediately following ecdysis from the fourth instar, decreases the duration of the fifth instar by approximately 36 hr in addition to inducing a supernumerary larval moult. JHA appears to accelerate the time of subsequent ecdysis in two ways: first, the onset of ecdysone secretion is accelerated, and is accompanied by a similarly premature initiation of mitotic activity in epidermal cells. This is the classical prothoracicotropic action of JH. Second, the period between the onset of mitotic activity and the time of ecdysis itself is shortened. That is, once cellular activities associated with the moulting cycle are triggered by ecdysone, such activities are completed more rapidly in the presence of JHA. It appears that the larval-larval moult induced by JHA requires intrinsically less time to accomplish than a normal metamorphic moult.     

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.     

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

The morphostatic actions of juvenile hormone

The maintenance of "status quo" in larvae by juvenile hormone (JH) involves both the programming of ecdysteroid-dependent synthesis during the molt and the suppression of morphogenetic growth during the intermolt. The latter morphostatic action does not require ecdysteroids, and has been studied in the formation of imaginal discs in Manduca sexta. Preultimate larval instars have both invaginated discs and imaginal primordia, both of which grow isomorphically with the larva. In the last instar, the young discs/primordia initiate the morphogenesis and patterning that results in a mature disc. JH suppresses both the initiation and progression of the signaling that transforms immature discs or primordia into a fully patterned imaginal disc. This transformation normally occurs in the context of the rapid growth of the last larval stage, and nutrient-dependent factors appear to be able to override the JH suppression. The morphostatic action of JH may have been important for the evolution of the larval stage. Studies on embryos of basal, hemimetabolous insects show that their premature exposure to JH can truncate patterning programs and cause precocious tissue maturation, factors essential for organizing a novel larval form. This suppression of embryonic patterning then results in embryonic fields that remain dormant as long as JH is present. These are the primordia that can transform into imaginal discs once JH disappears in preparation for metamorphosis.