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.     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.     

Fungal ecdysteroid-22-oxidase, a new tool for manipulating ecdysteroid signaling and insect development

Steroid hormones ecdysteroids regulate varieties of developmental processes in insects. Although the ecdysteroid titer can be increased experimentally with ease, its artificial reduction, although desirable, is very difficult to achieve. Here we characterized the ecdysteroid-inactivating enzyme ecdysteroid-22-oxidase (E22O) from the entomopathogenic fungus Nomuraea rileyi and used it to develop methods for reducing ecdysteroid titer and thereby controlling insect development. K(m) and K(cat) values of the purified E22O for oxidizing ecdysone were 4.4 μM and 8.4/s, respectively, indicating that E22O can inactivate ecdysone more efficiently than other ecdysteroid inactivating enzymes characterized so far. The cloned E22O cDNA encoded a FAD-dependent oxidoreductase. Injection of recombinant E22O into the silkworm Bombyx mori interfered with larval molting and metamorphosis. In the hemolymph of E22O-injected pupae, the titer of hormonally active 20-hydroxyecdysone decreased and concomitantly large amounts of inactive 22-dehydroecdysteroids accumulated. E22O injection also prevented molting of various other insects. In the larvae of the crambid moth Haritalodes basipunctalis, E22O injection induced a diapause-like developmental arrest, which, as in normal diapause, was broken by chilling. Transient expression of the E22O gene by in vivo lipofection effectively decreased the 20-hydroxyecdysone titer and blocked molting in B. mori. Transgenic expression of E22O in Drosophila melanogaster caused embryonic morphological defects, phenotypes of which were very similar to those of the ecdysteroid synthesis deficient mutants. Thus, as the first available simple but versatile tool for reducing the internal ecdysteroid titer, E22O could find use in controlling a broad range of ecdysteroid-associated developmental and physiological phenomena.     

Juvenile hormone esterase activity in the pupating and diapausing larvae of Sesamia nonagrioides

The development of the Mediterranean corn borer, Sesamia nonagrioides, under long-day (LD) photoperiod is associated with juvenile hormone (JH) decline and pupation in the 5th or 6th larval instar. The larvae grown under short-day (SD) conditions maintain a moderate JH titer and enter diapause during which they undergo several extra larval molts. Both types of larvae exhibit similar levels of juvenile hormone esterase (JHE) activity that increases in each instar during the period of low ecdysteroid titer and drops when the titer rises to a molt-inducing peak. A suppression of JHE activity within 24h after application of an ecdysteroid agonist suggests that the drop of activity is a rapid and possibly direct response to ecdysteroids or their agonist. Esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP) suppressed more than 98% of the JHE activity without affecting pupation timing and adult development. The data indicate that JHE is not crucial for the switch between larval development, diapause, and metamorphosis in S. nonagrioides.     

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endoparasite, and (2) the low level of ecdysteroids in pseudoparasitized hosts results from the host's intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained.     

Developmental Arrest and Ecdysteroid Deficiency Resulting from Mutations at the Dre4 Locus of Drosophila

Loss-of-function mutations of the dre4 gene of Drosophila melanogaster caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. We postulate that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency.     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.     

Ecdysone 20-hydroxylation and 3-epimerization in larvae of the gypsy moth,Lymantria dispar L.: tissue distribution and developmental changes

The potential for ecdysone metabolism was determined for various larval tissues of the gypsy moth, Lymantria dispar. Homogenates of fat body, midguts, and Malpighian tubules, taken on different days during the second half of the fifth instar, were incubated with [(3)H]ecdysone, and the products were analyzed by reversed-phase and normal-phase HPLC. All tissues showed conversion to 20-hydroxyecdysone, and midguts also produced 3-epiecdysone. Ecdysone 20-monooxygenase (E20MO) activity in the fat body increased from a low level on day 5 to a peak on day 11, coinciding with the peak in the hemolymph ecdysteroid titer on the penultimate day of the instar. Midguts and Malpighian tubules showed E20MO activity only during the last 3 or 4days of the instar, with the highest activity also occurring on the penultimate day. For the midguts, the appearance of the E20MO coincided with the transition from larval to pupal tissue. No activity was detected in larval midguts. 3-Epiecdysone formation, however, was mainly found in larval midguts, with only marginal activity detectable in pupal midguts.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Host regulation effects on Heliothis virescens (F.) larvae inducd by teratocytes of Cardiochiles nigriceps Viereck (Lepidoptera,Noctuidae-Hymenoptera,Braconidae)

Teratocytes deriving from the serosal membrane of Cardiochiles nigriceps Viereck, obtained “in vitro” from embryos hatched on a semidefined medium, were injected at different numbers and in different developmental stages of nonparasitized Heliothis virescens (F.) last instar larvae. Host development was affected by teratocyte injections and the responses registered ranged from normal to complete inhibition of pupation, according to the number of teratocytes injected and the developmental stage of the larva at time of injection. Complete pupation failure was observed when teratocytes derived from 4C nigriceps embryos were injected into 1st day 5th instar (new-slender stage) host larvae. Complete pupation occurred when teratocytes from 2 embryos were injected into 3rd or 4th day 5th instars (burrow-digging or day 1 cell formation stage). Intermediate responses, such as the formation of pupal cuticle without ecdysis or with only partial ecdysis, were obtained with intermediate teratocyte numbers, or host developmental stages. All pupae derived from teratocyte injected larvae failed to develop into adults normally obtained from control injected larvae. The larval weight just before pupation was negatively affected only when teratocyte injections were performed on 1st day 5th instar H. virescens larvae. Teratocyte injections altered the hemolymph protein titer to a level similar to that occurring in parasitized larvae. At the same time the ecdysteroid titer was characterized by a late significant increase, which reached values almost 3 times greater than found in normally parasitized larvae, and also surpassed the highest values registered for nonparasitized larvae. Ligation of parasitized larvae between the meso- and metathorax demonstrated that when the prothoracic glands were excluded, there was almost no ecdysteroid production posterior to the ligation. Ligations performed on parasitized larvae to isolate parasitoid eggs before hatching in the last abdominal segments, demonstrated that only virus and venom determined a reduction of the ecdysteroid titer. On the basis of these results the possible role of teratocytes in affecting the biological activity of ecdysteroids is postulated and discussed in a wider context of host-parasitoid physiological interactions.     

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid – A mechanism that determines the timing of metamorphosis

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.     

Analysis of molting and metamorphosis in the ecdysteroid-deficient mutant L(3)3DTS of Drosophila melanogaster

The dominant temperature-sensitive mutation L(3)3^DTS (DTS-3) in Drosophila melanogaster causes lethality of heterozygotes during the third larval instar at the restrictive temperature (29°C). Temperature-shift experiments revealed two distinct temperature-sensitive periods, with lethal phases during the third larval instar (which may persist for 4 weeks) and during the late pupal stage. At 29°C mutant imaginal discs are unable to evert in situ, but did evert normally if cultured in the presence of exogenous ecdysterone or when implanted into wild-type larval hosts. The only morphologically abnormal tissue present in the lethal larvae is the ring gland, the prothoracic gland being greatly hypertrophied in third instar DTS-3 larvae. Injection of a single wild-type ring gland rescued these mutant larvae, indicating that the mutant gland is functionally, as well as morphologically, abnormal. Finally, the mutant larvae were shown to have less than 10% of the wild-type ecdysteroid levels. These results are all consistent with a proposed lesion in ecdysteroid hormone production in DTS-3 larvae. A comparison with the phenotypes of other “ecdysone-less” mutants is presented.     

Developmental profile of the changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx mori: correlation with ecdysteroid secretion

A very sensitive time-resolved fluoroimmunoassay for the prothoracicotropic hormone (PTTH) of the silkworm Bombyx mori has been established. The lower limit of detection in this assay was 0.1 pg. With this assay method, the amounts of PTTH in the central nervous system and hemolymph were quantified. PTTH was detected only in the brain within the central nervous system, and, in the fifth instar, its content in the brain increased gradually with larval growth and decreased rapidly after the beginning of wandering. A substantial amount of PTTH was also found in the retrocerebral complex of day-3 fifth instar larvae, accounting for 28% of total PTTH. The PTTH titer in hemolymph changed dramatically during Bombyx development, with a small peak in the middle of the fourth instar, medium-sized peaks at the wandering and prepupal stages in the fifth instar, and a large prolonged peak during early pupal-adult development. The changes were overall closely correlated with those in hemolymph ecdysteroid titer. However, some unexpected aspects of PTTH dynamics in hemolymph have also been disclosed. Based on these observations, the significance of PTTH secretion in the control of insect development is discussed.     

Effects of ecdysteroid agonist RH-2485 reveal interactions between ecdysteroids and juvenile hormones in the development of Sesamia nonagrioides

Larvae of Sesamia nonagrioides developing under long day (LD) conditions pupate in the 5th or 6th instar, whereas under the short day (SD) conditions, they undergo several supernumerary larval molts and are regarded as diapausing. The development in early larval instars occurs in the LD larvae at a moderate and in the SD larvae at a high juvenile hormone (JH) titer; ecdysteroid titer cycles similarly under both conditions. The transformation to pupa is initiated by a burst of ecdysteroids at undetectable JH levels, whereas extra larval molts in the diapausing larvae are associated with moderate JH titer and irregular rises of ecdysteroids. Application of 0.2 ppm RH-2485 to the diet of the 6th instar larvae promotes hormonal changes supporting metamorphosis in the LD larvae and slightly accelerates larval molts in the diapausing SD larvae. The 0.5- and 1-ppm doses revert these patterns of endocrine regulations to a mode typical for early larval instars. Particularly dramatic is a JH titer increase provoked within 24 h in the LD larvae. After the treatment, both the LD and SD larvae undergo a series of larval molts, suggesting that hormonal programming of the larval development has been stabilized. A few insects receiving 1 ppm RH-2485, and a high proportion of those fed with 5 ppm RH-2485, deposit two cuticles within a single apolysis and die.