Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth,Lymantria dispar (L.) following studies on identification,titers and synthesis of ecdysteroids in last-instar females

Summary Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.Abbreviations A _r activation ratio - HPLC high performance liquid chromatography - HPSEC high performance size-exclusion chromatography - PG prothoracic gland - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locusta migratoria L.

The kinetics of secretion of ecdysone by the prothoracic glands of Locusta migratoria were studied during the last larval instar. Three stages of intense production of ecdysone (α-ecdysone) were monitored during this developmental period: they correspond to three peaks of moulting hormone concentration in the blood, which indicates that the main regulation of the moulting hormone titre is achieved through variations in prothoracic gland activity. In the haemolymph the ratio of ecdysone to ecdysterone (20-hydroxy-ecdysone) is in favour of ecdysone during the two first moulting hormone peaks ecdysterone being by far predominant over ecdysone at the time of the third (major) peak; these results support previous studies on the metabolic fate of injected labelled ecdysone during the same developmental period in Locusta migratoria.     

Molting hormone titer changes and their significance during development of the colorado beetle, Leptinotarsa decemlineata

Molting hormone (MH) titer in whole animal extracts of Leptinotarsa decemlineata was determined by chemical extraction and the Musca test (1 MU = 3.5 ng ecdysterone) during the developmental span from newly-ecdysed fourth instar larva to an adult 3 days after eclosion. Within the 17-day period, 21 age groups were chosen to estimate the MH titer. Two peaks of MH titer were detected, one in the post-feeding larval stage and the other during the pupal and pharate adult stage. MH activity was first detected in 2-day-old post-feeding larvae, and reached a maximum of 23.5 MU/g tissue on the third day. It began to decline on 3.5 days, and fell to 5.5 MU/g tissue on 4.5 days, the time of larval-pupal ecdysis. In the pupal and pharate adult stage MH rose after the first day and increased to a maximum of 91.5 MU/g tissue on the third day. The titer again declined on the fourth day, and became undetectable one day before adult emergence and in adults 3 days after emergence. MH was demonstrated to be produced by isolated larval abdomens. A peak of 11.5 MU/g tissue was detected in 7-day post-ligation preparations. The titer decreased to 6.9 MU/g tissue in 10-day post-ligation preparations, which was the time of the ecdysis. The finding raises questions concerning the rôle of MH synthesis by other tissues in relation to the function of the prothoracic glands during insect development.     

Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: Evidence for a fat body stimulatory factor

Juvenile hormone or ZR512 applied topically to day-5, fifth-instar, neck-ligated Manduca sexta larvae results in the acceleration of pharate pupal development when compared to neck-ligated, untreated larvae. This occurs as a result of an increase in the haemolymph ecdysteroid titre. Juvenile hormone, therefore, appears to stimulate ecdysone synthesis by the prothoracic glands of these animals, but not directly as shown by in vitro analysis. When ecdysone synthesis by the prothoracic glands of these ZR512- or juvenile hormone-treated animals was analyzed in vitro, increased gland activity was demonstrated but this did not occur until at least 2 days after treatment. This time lag in response supports the concept of an indirect stimulation of the prothoracic glands. Incubation of fat body from these ZR512- or juvenile hormone-treated, neck-ligated, larvae in 19AB culture medium revealed that the resulting pre-conditioned medium was capable of stimulating prothoracic glands in vitro up to 9-fold in a dose-dependent manner. A developmental profile was generated of the amount of this stimulatory factor released into the medium by fat body of untreated larvae representing each day of the last instar, and revealed that maximal release occurred with fat body from day-9 animals. The alterations in the amount of factor release by the fat body during larval-pupal development roughly correlated with the juvenile hormone titre and suggested a possible role for this factor in the regulation of the ecdysteroid titre. In contrast to the prothoracicotropic hormone, the fat body stimulatory factor is heat labile and has an apparent mol. wt in the 30,000 Dalton range. These data, particularly the kinetics of prothoracic gland stimulation, suggest that the factor may be a protein transporting a substrate for ecdysone biosynthesis to the prothoracic glands.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

Juvenile hormone coordinates the regulation of the hemolymph ecdysteroid titer during pupal commitment in Manduca sexta

The timing and magnitude of the pupal commitment peak in the hemolymph ecdysteroid titer of fifth instar Manduca sexta larvae are controlled by the combined effects of prothoracicotropic hormone (PTTH), a prothoracic gland-stimulating factor present in the hemolymph, and the biosynthetic competence of the prothoracic glands themselves. The present data indicate those individual effects are coordinated by juvenile hormone (JH): (1) Treatment of larvae with the JH analog (7S)-hydroprene prevents the normal precommitment drop in the titer of the stimulatory factor; (2) treatment of larvae with (7S)-hydroprene suppresses in a dose- and time-dependent manner the biosynthetic competence of the prothoracic glands; and (3) (7S)-hydroprene acts directly on the brain to inhibit the release of PTTH in vitro. Thus, during Manduca development, a drop in the JH titer early in the fifth instar results in a rapid drop in the titer of the stimulatory factor, the gradual acquisition by prothoracic glands of biosynthetic competence, and lastly, the gated release of PTTH into the hemolymph. The resulting increase in ecdysone synthesis by the prothoracic glands gives rise to the small peak in the ecdysteroid titer that drives pupal commitment.     

Changes in ecdysone titre during pupal-adult development in the silkworm, Bombyx mori

Changes in the ecdysone titre of the silkworm, Bombyx mori, during pupal-adult development were estimated. The average value of the maximum titre, which was observed on the second day after pupal ecdysis, was about 0·8 μg equivalent of β-ecdysone/g of live weight in both sexes.

There is a distinct sexual dimorphism in the pattern of the ecdysone titre. The male exhibited a single sharp peak on the second day whereas the female showed the second peak on the fifth day. When the female was ovariectomized, the ‘female type’ ecdysone pattern was converted to the ‘male type’. In the female pharate adult 7 days after pupal ecdysis, ecdysone activity accumulated in the ovaries.

The relationship between the ecdysone titre and adult differentiation, especially during ovarian development, is discussed.     

Regulation of insect prothoracic glands: Existence of a haemolymph stimulatory factor in Manduca sexta

The primary regulator of ecdysone biosynthesis by insect prothoracic glands is the prothoracicotropic hormone. However, it now appears that other factors, secondary regulators, may modulate prothoracic gland activity. One such factor has been isolated from the haemolymph of Manduca larvae. This haemolymph factor stimulates in vitro ecdysone synthesis by larval and pupal prothoracic glands by approx. 5-fold. It has an apparent mol. wt of ～330 kD, is protease-sensitive and is heat labile, the latter clearly distinguishing it from the prothoracicotropic hormone. Further, its steroidogenic effects and those of prothoracicotropic hormone are additive. Treatment of larval or pupal prothoracic glands with both moieties simultaneously effects an approx. 10-fold increase in ecdysone synthesis. The haemolymph titre of the stimulatory factor is low at commitment of the last-larval instar, then increases by approx. 3-fold later in the instar during pharate-pupal development. This increase in the titre is sufficient to effect a significant increase in prothoracic gland activity that could be physiologically important. Thus, it appears that the fluctuating level of this haemolymph stimulatory factor may act in conjunction with prothoracicotropic hormone to regulate the haemolymph ecdysteroid titre by modulating the ecdysone biosynthetic activity of the prothoracic glands.     

Alterations in DNA-dependent RNA polymerase activity during the development of the tobacco hornworm, Manduca sexta.

RNA polymerases I, II and III have been detected in the extracts of fat body and integument of the tobacco hornworm, and their activity during larval-pupal-adult metamorphosis has been measured. Total RNA polymerase activity of both tissues reaches a peak just prior to the wandering stage of the fifth instar larva. The enzyme activity of the integument declines thereafter while, in the fat body, a change in the cellular compartmentalization of enzyme activity occurs during development. This is indicated by the observations that RNA polymerase activity, which was predominantly in the soluble fraction up until the onset of the wandering stage, declines rapidly during the wandering stage while RNA polymerase activity in the insoluble-pellet fraction increases. A steady-state level is reached just prior to pupation, and the enzyme activity remains at that level during pharate adult development. The α-amanitin-sensitive enzyme appears to be responsible for most of the RNA polymerase activity during larval life. The findings that the peak of RNA polymerase activity in both tissues and the subsequent changes in compartmentalization in the fat body occur coincidentally with the first surge of α-ecdysone release by the prothoracic glands raises the possibility that control of RNA polymerase activity may be humorally mediated.     

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.     

The secretion and metabolism of alpha-ecdysone by cockroach (Leucophaea maderae) tissues in vitro

Hemolymph β-ecdysone levels are high (～1.6 μg/ml) in late last instar cockroach ($\text{Leucophaea}$$\text{maderae}$) nymphs; the level of α-ecdysone (～0.1 μg/ml) is evidently subphysiological. Cultured leg regenerates, target organs of ecdysone, are capable of slowly converting α- to β-ecdysone. Cultured prothoracic glands secrete α-ecdysone, which was identified by complete mass spectrometry. These results are consistent with the view that α-ecdysone, secreted by the prothoracic gland, functions as a prohormone which is converted into the active moulting hormone, β-ecdysone, in other tissues.     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm,Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.     

BIOSYNTHESIS OF ECDYSONE IN THE ISOLATED ABDOMEN OF THE SILKWORM,BOMBYX MORI*

During pupal-adult development of the silkworm, Bombyx mori, the ecdysone titer changes, exhibiting two maxima in the females: one on the second day of pupal development and the other just before adult emergence. During the second maximum, ecdysone accumulates in the ovaries. It also accumulates in isolated abdomens, which were prepared just after pupal ecdysis and induced to initiate adult development by injection with β-ecdysone. Several lines of evidence suggest that ecdysone is synthesized in the ovary itself.     

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.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

Metabolism of 3H-α-ecdysone in Gryllus bimaculatus during the eighth larval instar

During the 8th larval instar of Gryllus bimaculatus the metabolites of exogenous tritiumlabelled α-ecdysone were isolated from animals and from excrement, qualified by TLC and quantified in the scintillation counter. Besides β-ecdysone, there were 3 other compounds more polar than α-ecdysone (compounds I, II, and III), in addition to two less polar ones: compounds IV and V.The pattern of hormone changes characteristically during the period of development investigated. Thus on the 3rd day compound IV, on the 4th day α-ecdysone, and on the 5th day, at the moment of the hormone titre maximum, β-ecdysone dominates.Whether all ecdysone metabolites represent products of inactivation will be discussed in this article.     

An in vitro study on regulation of prothoracic gland activity in the early last-larval instar of the silkworm Bombyx mori

The endocrine mechanisms that regulate prothoracic gland (PG) activity in early stages of final larval instar of the silkworm Bombyx mori were investigated using a newly developed long-term cultivation system of the gland. The PGs dissected from day-0 fifth instar larvae did not secrete detectable amounts of ecdysone for the first 24 h in culture but started secretion within the next 2 days. The amount of secreted ecdysone increased day by day. When day-0 PGs were co-cultivated with corpora allata, however, they remained inactive for at least 8 days. PGs dissected from 1-day younger larvae (day-3 fourth instar larvae) secreted ecdysone for the first 24 h but stopped secretion for the next 24 h, followed by recovery of ecdysone secretory activity. By contrast, PGs from day-1 fourth instar larvae remained active throughout a cultivation period without any sign of inactivation. However, when the same glands were exposed to a high titer of 20-hydroxyecdysone for the second 24h in culture, they gradually lost their activity. These results indicate that PGs of fourth instar larvae are inactivated by ecdysteroid through a negative feedback mechanism and that thus inactivated PGs spontaneously recover ecdysone secretory activity in the early fifth instar unless inhibited by juvenile hormone.     

Steroidal regulation of hydrolyzing activity of the dietary carbohydrates in the silkworm, Bombyx mori

Blood sugar is an essential energy source for growth and development and is maintained at a constant level through precise regulation of formation and utilization. Sugars are produced from dietary carbohydrates by enzymatic hydrolysis in the digestive tract, which are under the homeostatic control of paracrine and prandial mechanisms in mammals. Here, we show that dietary carbohydrates hydrolyzing activity of the digestive tract is developmentally regulated by the steroid hormone ecdysone in the silkworm, Bombyx mori. The dietary carbohydrates hydrolyzing activity remained high throughout the last larval period and then decreased to negligible levels until the pupal period. However, dietary carbohydrates digestive activities were constitutively high when the steroidogenic organ, prothoracic glands were ablated. The prothoracic glands produced and released a large amount of ecdysone at the end of the larval period, suggesting that ecdysone is responsible for the decrease in dietary carbohydrates hydrolyzing activity. In fact, ecdysone decreased the activity to negligible levels in silkworms lacking the prothoracic glands. The present results indicate that the dietary carbohydrates hydrolyzing activity is regulated by ecdysone and that an increase in ecdysone titer decreases that activity at the end of the larval period, suggesting that ecdysone is essential for metabolic coordination during development.