Analysis of ecdysteroidogenic activity of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The cellular mechanism underlying ecdysteroidogenesis throughout the last larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid secretory activity of the prothoracic glands and cAMP accumulation of gland cells, as well as changes in responsiveness to stimulation by prothoracicotropic hormone (PTTH) and 1-methyl-3-isobutylxanthine (MIX). It was found that the prothoracic glands during the first 3 days of the last instar cannot produce detectable ecdysteroid and showed no response to stimulation by PTTH or 1-methyl-3-isobutylxanthine (MIX). However, artificial elevation of cellular cAMP levels by in vitro dibutyryl cAMP treatment stimulated the glands to secrete detectable ecdysteroid, implying the presence of a cAMP-dependent ecdysteroidogenic apparatus during this stage. From days 3 to 8, basal gland activities fluctuated, but the glands showed activation responses to PTTH and to the chemicals that increase cellular cAMP levels. After the occurrence of the peak in basal gland activity on day 9, glands on day 10 showed no response to PTTH, implying a refractory state of the glands to PTTH stimulation. For cAMP accumulation, it was found that glands on day 2 began to show increased cAMP accumulation to PTTH, implying that the acquisition of gland competency for elevation of cAMP levels after stimulation by PTTH precedes that of ecdysteroid production. Moreover, during most parts of the last larval instar (between days 3 and 8) and at the pupation stage, greatly increased cAMP accumulation upon stimulation by PTTH was observed only in the presence of MIX, indicating that cAMP phosphodiesterase levels may be high during these stages. From these results, we concluded that development-specific PTTH signal transduction during the last larval instar, which shows a different pattern from that of the penultimate larval instar, may play an important role in regulating changes in prothoracic gland activity and in leading to larval-pupal metamorphosis.     

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

Effects of RH-5992 on ecdysteroidogenesis of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori

Stage-dependent effects of RH-5992 on ecdysteroidogenesis of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx mori, were studied in the present report. When larvae were treated with RH-5992 during the early stages of the fourth larval instar (between day 0 and day 1), initially ecdysteroid levels in the hemolymph were inhibited. However, 24 h after RH-5992 application, ecdysteroid levels were greatly increased as compared with those treated with acetone. The examination of the in vitro prothoracic gland activity upon RH-5992 application during the early stages of the fourth larval instar confirmed a short-term inhibitory effect. When RH-5992 was applied to the later stages of the fourth larval instar, no effects on both hemolymph ecdysteroid levels and prothoracic gland activity were observed. Addition of RH-5992 to incubation medium strongly inhibited ecdysteroid secretion by the prothoracic glands from the early fourth instar, indicating direct action of RH-5992 on ecdysteroidogenesis by prothoracic glands. Four hours after application with RH-5992 on day 1.5, prothoracic glands still showed an activated response to PTTH in both PTTH-cAMP signaling and the extracellular signal-regulated kinase (ERK) signaling. Moreover, addition of RH-5992 to incubation medium did not interfere with the stimulatory effect of the glands to PTTH in ecdysteroidogenesis. These results indicated that both PTTH-cAMP signaling and PTTH-ERK signaling may not be involved in short-term inhibitory regulation by RH-5992.     

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.     

In vitro and in vivo stimulation of extracellular signal-regulated kinase (ERK) by the prothoracicotropic hormone in prothoracic gland cells and its developmental regulation in the silkworm, Bombyx mori

In this study, we investigated activation of the extracellular signal-regulated kinase (ERK) by the prothoracicotropic hormone (PTTH) in prothoracic gland cells of the silkworm, Bombyx mori. The results showed that the PTTH stimulated ERK phosphorylation as this depends on time and dose and ecdysteroidogenic activity. The ERK phosphorylation inhibitors, PD 98059 and U0126, blocked both basal and PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis. In addition, activation of glandular ERK phosphorylation by the PTTH appeared to be developmentally regulated with the refractoriness of gland cells to the PTTH occurring during the latter stages of both the fourth and last larval instars. Moreover, in vitro activation of ERK phosphorylation of prothoracic glands by the PTTH was also verified by in vivo experiments: injection of the PTTH into day 6 last instar larvae greatly increased the activity of glandular ERK phosphorylation and ecdysteroidogenesis. These results suggest that development-specific changes in ERK phosphorylation may play a role in PTTH stimulation of ecdysteroidogenesis.     

Involvement of PI3K/Akt signaling in PTTH-stimulated ecdysteroidogenesis by prothoracic glands of the silkworm, Bombyx mori

The prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by prothoracic gland in larval insects. Previous studies showed that Ca²⁺, cAMP, extracellular signal-regulated kinase (ERK), and tyrosine kinase are involved in PTTH-stimulated ecdysteroidogenesis by the prothoracic glands of both Bombyx mori and Manduca sexta. In the present study, the involvement of phosphoinositide 3-kinase (PI3K)/Akt signaling in PTTH-stimulated ecdysteroidogenesis by B. mori prothoracic glands was further investigated. The results showed that PTTH-stimulated ecdysteroidogenesis was partially blocked by LY294002 and wortmannin, indicating that PI3K is involved in PTTH-stimulated ecdysteroidogenesis. Akt phosphorylation in the prothoracic glands appeared to be moderately stimulated by PTTH in vitro. PTTH-stimulated Akt phosphorylation was inhibited by LY294002. An in vivo PTTH injection into day 6 last instar larvae also increased Akt phosphorylation of the prothoracic glands. In addition, PTTH-stimulated ERK phosphorylation of the prothoracic glands was not inhibited by either LY294002 or wortmannin, indicating that PI3K is not involved in PTTH-stimulated ERK signaling. A23187 and thapsigargin, which stimulated B. mori prothoracic gland ERK phosphorylation and ecdysteroidogenesis, could not activate Akt phosphorylation. PTTH-stimulated ecdysteroidogenesis was not further activated by insulin, indicating the absence of an additive action of insulin and PTTH on the prothoracic glands. The present study, together with the previous demonstration that insulin stimulates B. mori ecdysteroidogenesis through PI3K/Akt signaling, suggests that crosstalk exists in B. mori prothoracic glands between insulin and PTTH signaling, which may play a critical role in precisely regulated ecdysteroidogenesis during development.     

Daily changes in neuroendocrine control of moulting hormone secretion in the prothoracic gland of the cockroach Periplaneta americana (L.)

Time of day related changes in ecdysteroid secretion by the prothoracic gland of last instar nymphs were studied using in vitro coincubations of prothoracic glands and brains under a 12-h light:12-h dark cycle. The experiments reveal that the cells of the prothoracic gland of the cockroach nymphs do not have an endogeneous circadian oscillator determining rhythmicity of ecdysteroid secretion. PTTH release in the scotophase is responsible for the peak of ecdysteroid production during the photophase.     

Insulin stimulates ecdysteroidogenesis by prothoracic glands in the silkworm,Bombyx mori

It is generally accepted that the prothoracicotropic hormone (PTTH) is the stimulator of ecdysteroidogenesis by prothoracic glands in larval insects. In the present study, we investigated activation of ecdysteroidogenesis by bovine insulin in prothoracic glands of the silkworm, Bombyx mori. The results showed that the insulin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. In addition, insulin also stimulated both DNA synthesis and viability of prothoracic glands. Insulin-stimulated ecdysteroidogenesis was blocked by either LY294002 or wortmannin, indicating involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Activation of ecdysteroidogenesis by insulin appeared to be developmentally regulated. Moreover, in vitro activation of ecdysteroidogenesis of prothoracic glands by insulin was also verified by in vivo experiments: injection of insulin into day 6 last instar larvae greatly increased both hemolymph ecdysteroid levels and ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of Akt and the insulin receptor was stimulated by insulin, and stimulation of Akt phosphorylation appeared to be PI3K-dependent and developmentally regulated. Insulin did not stimulate extracellular signal-regulated kinase (ERK) signaling of the prothoracic glands. These results suggest that in silkworm prothoracic glands, in addition to the PTTH and an autocrine factor, ecdysteroidogenesis is also stimulated by insulin during development.     

Regulation of prothoracic gland ecdysteroidogenic activity leading to pupal metamorphosis

The prothoracic glands of early last (fifth) instar larvae of the silkworm are inactive with regard to ecdysteroidogenesis and unresponsive to prothoracicotropic hormone (PTTH) [J. Insect Physiol. 31 (1985) 455]. In an attempt to elucidate the hormonal mechanisms that cause the inactivity, we compared the effects of PTTH, dibutyryl cyclic AMP (dbcAMP), a cAMP phosphodiesterase inhibitor (IBMX), juvenile hormone analogue (JHA) and 20-hydroxyecdysone (20E) on secretory activity of the third, fourth and fifth instar glands. Among the factors examined, feedback inhibition by 20E was indicated to be the most likely factor. Inhibition was moderate in the third and early fourth instars while 20E strongly inhibited the glands of middle fourth instar larvae. The inhibitory effect of 20E was reduced by removal of the brain and corpora allata. Once the glands were suppressed by 20E to the degree of exhibiting neither secretory activity nor responsiveness to PTTH, dbcAMP or IBMX did not elicit ecdysone secretion at all. Thus the feedback inhibition may shut down ecdysteroidogenesis although it is obscure whether it affects the intracellular transductory cascade from the PTTH receptor through cAMP. Taken together, this evidence suggests that inactivity of the gland in the early fifth instar is brought about by feedback inhibition of the glands by 20E occurring in the late fourth instar, and that this inactivity is maintained by the juvenile hormone found in the early fifth instar.     

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.     

S6 phosphorylation results from prothoracicotropic hormone stimulation of insect prothoracic glands: A role for S6 kinase

The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). Previous work from this laboratory revealed that PTTH acts via a cascade including Ca²⁺/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the 56 protein of the 40S ribosomal subunit. The jmmunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. We demonstrate here that rapamycin inhibited PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there was a 75% inhibition of ecdysteroid biosynthesis. Similar results, were observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis was accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays revealed that injection of rapamycin into day 6 fifth instar larvae resulted in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamor-phosis. When S6 kinase (S6K) activity was examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the date revealed that rapamycin inhibited S6K activity. The composite data suggest that rapamycin inhibits a signal transduction element leading to p34 phosphorylation that is necessary for PTTH-stimulated ecdysteroidogenesis in this insect endocrine gland, and lend further support to the concept that p34 is S6. © 1994 Wiley-Liss, Inc.     

Prothoracic gland inactivation in Heliothis virescens (F.) (Lepidoptera:Noctuidae) larvae parasitized by Cardiochiles nigriceps Viereck (Hymenoptera:Braconidae)

Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. nigriceps genome, which, in contrast, are undetectable in H. virescens.     

Temporal changes in DNA synthesis of prothoracic gland cells during larval development and their correlation with ecdysteroidogenic activity in the silkworm, Bombyx mori

DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the 3rd, 4th, and last larval instars were examined. During the early stages of both the 3rd and 4th larval instars, a dramatic increase in the number of DNA-synthesizing cells of the prothoracic glands was detected. However, during the latter stages of each instar, the number of DNA-synthesizing cells greatly decreased. The determination of glandular protein content showed that dramatic increases occurred during the latter stages of each larval instar. Comparison of changes in prothoracic gland cell DNA synthesis with ecdysteroidogenic activity showed that the increase in DNA synthesis precedes ecdysteroidogenesis. The cellular mechanism underlying changes in prothoracic gland cell DNA synthesis during the last two larval instars was further analyzed by determining the in vitro DNA synthesis of the glands, their responsiveness to hemolymph growth factors, and changes in the growth-promoting activity of hemolymph during development. It was found that both growth factors and the responsiveness of the prothoracic gland cells to growth factors from hemolymph may play roles in regulating DNA synthesis of gland cells.     

家蚕蜕皮与变态的内分泌调控

家蚕的蜕皮与变态是由前胸腺分泌的脱皮素（ｍｏｌｔｉｎｇ ｈｏｒｍｏｎｅ或 ｅｃｄｙｓｔｅｒｏｉｄ简称 ＭＨ）及由咽侧体分泌的保幼激素（ｊｕｖｅｎｉｌｅ ｈｏｒｍｏｎｅ）控制的,而促有前胸腺激素（ｐｒｏｔｈｏｒａｃｉｃｏｔｒｏｐｉｃ ｈｏｒｍｏｎｅ,以下简称ＰＴＴＨ）的功能为刺激前胸腺分泌蜕皮素。笔者近１０年来从家蚕内分泌体系的一系列研究中发现,蜕皮素浓度的变化可以通过控制咽侧体的保幼激素的生物合成来影响幼虫发育,而ＰＴＴＨ的信息传递可通过调控前胸腺的功能,进而影响血淋巴中蜕皮素浓度。     

The ecdysoneless (ecd1ts) mutation disrupts ecdysteroid synthesis autonomously in the ring gland of Drosophila melanogaster

Ring glands dissected from homozygous l(3)ecd1ts wandering larvae and upshifted in vitro to the restrictive temperature, 29 degrees C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29 degrees C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29 degrees C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29 degrees C autonomously and may also interfere with PTTH release.     

Developmental changes in hemolymph ecdysteroid level and prothoracicotropic hormone activity during the fifth larval instar of the Eri silkworm, Samia cynthia ricini

Developmental changes in hemolymph ecdysteroid level, ecdysteroid synthesis by prothoracic glands （PGs） in vitro, prothoracicotropic hormone （PTTH） activity in brain extracts, and PTTH activity in the hemolymph were measured during the fifth larval instar of the Eri silkworm, Samia cynthia ricini. The changing patterns of hemolymph ecdysteroid level and ecdysteroid synthesis by laGs in vitro are similar to each other, with maximums on day 9. However, on this day, hemolymph ecdysteroid level was substantially higher than ecdysteroid synthesis by PGs in vitro suggesting a high PTTH activity in the hemolymph on day 9. Moreover, the changing pattern of PTTH activity in brain extracts is also similar to that of PTTH activity in the hemolymph, both peaking on day 9. However, on this day, activity in brain extracts was much smaller than PTTH activity in the hemolymph implying that most PTTH synthesized by the brain is secreted to the hemolymph and the brain stores a very little amount of PTTH. This study provides unique insights onto the hormonal regulation of ecdysteroid synthesis in the Eri silkworm and is useful for our future studies on signal transduction of insect neurolaelatides.     

Bombyx mori prothoracicostatic peptide inhibits ecdysteroidogenesis in vivo

Bombyx prothoracicostatic peptide (Bom-PTSP) is a brain neuropeptide that has recently been reported to have in vitro inhibitory activity to prothoracicotropic hormone (PTTH)-stimulated ecdysteroid biosynthesis in the prothoracic gland of the silkworm, Bombyx mori. In the present report, Bom-PTSP has been shown to significantly decrease hemolymph ecdysteroid titer in the fifth instar larvae when Bom-PTSP was injected into the fifth instar day 8 silkworm larvae, resulting in significant delay in spinning behavior. This is the first evidence that Bom-PTSP inhibits in vivo ecdysteroidogenesis in the silkworm.     

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.