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It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signaling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signaling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis. This article is protected by copyright. All rights reserved.

Metabolism, growth, and development are intrinsically linked, and their coordination is dependent upon inter-organ communication mediated by anabolic, catabolic, and steroid hormones. InDrosophila melanogaster, the corpora cardiaca (CC) influences metabolic homeostasis through adipokinetic hormone (AKH) signaling. AKH has glucagon-like properties and is evolutionarily conserved in mammals as the gonadotropin-releasing hormone, but its role in insect development is unknown. Here we report that AKH signaling alters larval development in a nutrient stress-dependent manner. This activity is regulated by the locusdg2, which encodes a cGMP-dependent protein kinase (PKG). CC-specific downregulation ofdg2expression delayed the developmental transition from larval to pupal life, and altered adult metabolism and behavior. These developmental effects were AKH-dependent, and were observed only in flies that experienced low nutrient stress during larval development. Calcium-mediated vesicle exocytosis regulates ecdysteroid secretion from the prothoracic gland (PG), and we found that AKH signaling increased cytosolic free calcium levels in the PG. We identified a novel pathway through which PKG acts in the CC to communicate metabolic information to the PG via AKH signaling. AKH signaling provides a means whereby larval nutrient stress can alter developmental trajectories into adulthood.

In the present study, the roles of a major serine/threonine protein phosphatase 2A (PP2A) in prothoracicotropic hormone (PTTH)-stimulated prothoracic glands (PGs) of Bombyx mori were evaluated. Immunoblotting analysis showed that Bombyx PGs contained a structural A subunit (A), a regulatory B subunit (B), and a catalytic C subunit (C), with each subunit undergoing development-specific changes. The protein levels of each subunit were not affected by PTTH treatment. However, the highly conserved tyrosine dephosphorylation of PP2A C subunit (PP2A c ), which appears to be related to activity, was increased by PTTH treatment in a time-dependent manner. We further demonstrated that phospholipase C (PLC), Ca2+, and reactive oxygen species (ROS) are upstream signaling for the PTTH-stimulated dephosphorylation of PP2A c . The determination of PP2A enzymatic activity showed that PP2A enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Okadaic acid (OA), a specific PP2A inhibitor, prevented the PTTH-stimulated dephosphorylation of PP2A c and reduced both basal and PTTH-stimulated PP2A enzymatic activity. The determination of ecdysteroid secretion showed that treatment with OA did not affect basal ecdysteroid secretion but did significantly inhibit PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PP2A activity is involved in ecdysteroidogenesis. Treatment with OA stimulated the basal phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) without affecting PTTH-stimulated ERK and 4E-BP phosphorylation. From these results, we hypothesize that PTTH-regulated PP2A signaling is a necessary component for the stimulation of ecdysteroidogenesis, potentially by mediating the link between ERK and TOR signaling pathways.

Insect molting and metamorphosis are controlled by the molt stimulating hormone ecdysone. A recent study suggests that reduced tissue oxygenation correlates with the size-sensing mechanism responsible for triggering molting. When reared in hypoxia, larvae of Manduca sexta and Drosophila melanogaster initiate molting at lower weights than do larvae reared in normoxia. Furthermore, in Drosophila, the signaling gas nitric oxide (NO) appears to be required for normal developmental timing. As observed in Drosophila, NO signaling targets the nuclear hormone receptor beta fushi tarazu transcription factor 1 (βFTZ-F1) through activation of Drosophila hormone receptor 3 (DHR3), two key regulators of ecdysone production and metamorphic tissue progression. We set out to directly examine the effects of hypoxia and NO on ecdysone secretion using prothoracic glands from feeding fifth (last) larval stage M. sexta. Our results indicate that in vitro treatment of prothoracic glands with hypoxia (2% oxygen) or the NO donor DETA-NONOate significantly inhibit ecdysone secretion. Protein markers of glandular activity were also in keeping with an initial inhibition, measured a decrease in phosphorylated ERK (extracellular signal regulated kinase) and an increase in non-phosphorylated 4EBP (eukaryotic initiation factor 4E binding protein). Additionally, gene expression levels of Manduca hormone receptor 3 (mhr3), βftz-f1, nitric oxide synthase (nos), and the PTTH receptor torso, were quantified using real-time PCR. NO treatment increased mhr3 expression and decreased nos expression. Hypoxia increased mhr3 transcription after 2 hr, but decreased transcription after 12 hr, with no effect on nos expression. Both NO and hypoxia had small effects on βftz-f1 expression, yet strongly increased torso transcription. Our results demonstrate that, in isolated prothoracic glands, hypoxia and NO signaling directly inhibit ecdysteroid secretion, but at the same time alter aspects of prothoracic gland function that may enhance secretory response.

Diapause, a programmed developmental arrest, is common in insects, enabling them to survive adverse seasons. It is well established that pupal diapause is regulated by ecdysteroids secreted by the prothoracic glands (PGs), with cessation of ecdysteroid secretion after pupal ecdysis leading to pupal diapause. A major factor regulating the gland activity is prothoracicotropic hormone (PTTH) secreted from the brain. In our previous study, we demonstrated that the cessation of PTTH release after pupal ecdysis resulted in the inactivation of the PGs, leading to pupal diapause in the cabbage army moth Mamestra brassicae. Here we show that a neuropeptide myosuppressin also contributes to the inactivation of PGs at the initiation of diapause. Myosuppressin suppresses PTTH-stimulated activation of the PGs in vitro. Concentrations of myosuppressin in the hemolymph after pupal ecdysis are higher in diapause pupae than in nondiapause pupae.

Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031bp), Akt (855bp), and S6K (918bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas.

Molting in decapod crustaceans is controlled by molt-inhibiting hormone (MIH), an eyestalk neuropeptide that suppresses production of ecdysteroids by a pair of molting glands (Y-organs or YOs). Eyestalk ablation (ESA) activates the YOs, which hypertrophy and increase ecdysteroid secretion. At mid premolt, which occurs 7-14days post-ESA, the YO transitions to the committed state; hemolymph ecdysteroid titers increase further and the animal reaches ecdysis ~3weeks post-ESA. Two conserved signaling pathways, mechanistic target of rapamycin (mTOR) and transforming growth factor-β (TGF-β), are expressed in the Gecarcinus lateralis YO. Rapamycin, an mTOR antagonist, inhibits YO ecdysteroidogenesis in vitro. In this study, rapamycin lowered hemolymph ecdysteroid titer in ESA G. lateralis in vivo; levels were significantly lower than in control animals at all intervals (1-14days post-ESA). Injection of SB431542, an activin TGF-β receptor antagonist, lowered hemolymph ecdysteroid titers 7 and 14days post-ESA, but had no effect on ecdysteroid titers at 1 and 3days post-ESA. mRNA levels of mTOR signaling genes Gl-mTOR, Gl-Akt, and Gl-S6k were increased by 3days post-ESA; the increases in Gl-mTOR and Gl-Akt mRNA levels were blocked by SB431542. Gl-elongation factor 2 and Gl-Rheb mRNA levels were not affected by ESA, but SB431542 lowered mRNA levels at Days 3 and 7 post-ESA. The mRNA level of an activin TGF-β peptide, Gl-myostatin-like factor (Mstn), increased 5.5-fold from 0 to 3days post-ESA, followed by a 50-fold decrease from 3 to 7days post-ESA. These data suggest that (1) YO activation involves an up regulation of the mTOR signaling pathway; (2) mTOR is required for YO commitment; and (3) a Mstn-like factor mediates the transition of the YO from the activated to the committed state.

SUMMARY Two eyestalk (ES) neuropeptides, molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), increase intracellular cGMP levels in target tissues. Both MIH and CHH inhibit ecdysteroid secretion by the molting gland or Y-organ (YO), but apparently through different guanylyl cyclase(GC)-dependent pathways. MIH signaling may be mediated by nitric oxide synthase (NOS) and NO-sensitive GC. CHH binds to a membrane receptor GC. As molting affects neuropeptide signaling, the effects of ecdysteroid on the expression of the land crab Gecarcinus lateralis β subunit of a NO-sensitive GC (Gl-GC-Iβ), a membrane receptor GC (Gl-GC-II) and a NO-insensitive soluble GC (Gl-GC-III) were determined. Gl-GC-Iβ isoforms differing in the absence or presence of an N-terminal 32-amino acid sequence and Gl-GC-III were expressed at higher mRNA levels in ES ganglia, gill,hepatopancreas, ovary and testis, and at lower levels in YO, heart and skeletal muscle. Three Gl-GC-II isoforms, which vary in the length of insertions (+18, +9 and +0 amino acids) within the N-terminal ligand-binding domain, differed in tissue distribution. Gl-GC-II(+18) was expressed highly in striated muscle (skeletal and cardiac muscles); Gl-GC-II(+9) was expressed in all tissues examined (ES ganglia, YO, gill, hepatopancreas, striated muscles and gonads); and Gl-GC-II(+0) was expressed in most tissues and was the dominant isoform in ES and thoracic ganglia. ES ablation, which increased hemolymph ecdysteroid, increased Gl-GC-II(+18) mRNA level in claw muscle. Using real-time RT-PCR, ES ablation increased Gl-GC-Iβ, Gl-GC-III and ecdysone receptor mRNA levels in the YOs ∼ten-, ∼four- and∼twofold, respectively, whereas Gl-GC-II mRNA level was unchanged. A single injection of 20-hydroxyecdysone into intact animals transiently lowered Gl-GC-Iβ in hepatopancreas, testis and skeletal muscle, and certain Gl-GC-II isoforms in some of the tissues. These data suggest that YO and other tissues can modulate responses to neuropeptides by altering GC expression.

Diapause, a programmed developmental arrest at a specific stage, is common in insects and is regulated by hormones. It is well established that in pupal diapause, cessation of ecdysteroid secretion from the prothoracic glands (PGs) after pupal ecdysis leads to diapause initiation, while resumption of its secretion induces post-diapause development. However, what regulates the activity of the glands is poorly understood, especially for the glands of diapause-terminated pupae. In the present study, we investigate the mechanisms by which post-diapause development is regulated in the cabbage armyworm Mamestra brassicae. We demonstrate that the brain is necessary for the initiation of post-diapause development and that the factor in the brain responsible for the activation of the PGs is the prothoracicotropic hormone (PTTH). Further, through measuring the hemolymph PTTH titers by time-resolved fluoroimmunoassay, we show that PTTH is actually released into the hemolymph prior to the activation of the PGs. Although its peak titer is much lower than expected, this low concentration of PTTH is most likely still effective to activate the PGs of post-diapause pupae, because the responsiveness to PTTH of the glands at this stage is very high compared to that of nondiapause pupal PGs. These results strongly suggest that in M. brassicae, PTTH serves as a trigger to initiate pupa-adult development after diapause termination by stimulating the PGs to secrete ecdysteroid.

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.

Secretion of ecdysteroids by the prothoracic glands of the migratory locust, Locusta migratoria L., is enhanced in vitro by tris(hydroxymethyl)aminomethane (tris) in a dose-dependent manner. Glands from larvae on the second day of the penultimate stadium are most sensitive. This action of tris depends on the uptake of calcium; increased production of ecdysteroid does not occur in the presence of cadmium, verapamil or TMB-8, or in calcium-free media. The concentration of unbound Ca(2+), [Ca(2+)]i, in the cytoplasm is measured with the aid of FURA 2/AM. Tris causes a rise of [Ca(2+)]i that is fully suppressed by lanthanum and partially by nitrendipine. Two antagonists of IP3 receptors elicit mutually opposite effects: heparin blocks, whereas 2-APB accelerates, the rise in [Ca(2+)]i. Ryanodine exerts only a slight effect. It is proposed that tris activates locust prothoracic glands in a Ca(2+)-dependent manner that exhibits many similarities to the transduction pathway of prothoracicotropic hormone.

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.

In crustaceans, molt-inhibiting hormone (MIH) has been proposed to regulate molting by inhibiting the secretion of ecdysteroids from the Y-organ. Thus, MIH titer in the hemolymph should be inversely related to ecdysteroid titers during the molt cycle. However, it has not been demonstrated whether the MIH titer in the hemolymph changes during the molt cycle. The purpose of this study was to determine the changes in the MIH titers in the hemolymph during the molt cycle of the American crayfish, Procambarus clarkii, and to discuss the role of MIH in regulation of molting. As predicted by the hypothesis, the hemolymph MIH titer was high at the intermolt stage when the hemolymph ecdysteroid titer was low, and the MIH titer decreased to a basal level at the early premolt stage when the hemolymph ecdysteroid titer began to increase slightly. At the middle premolt stage when the hemolymph ecdysteroid titer increased, the MIH titer was restored to a level as high as that during the intermolt stage. This is in contradiction to the hypothesis. However, the Y-organs at this stage scarcely responded to MIH both in vitro and in vivo. The present findings suggest that ecdysteroid secretion from the Y-organ may be regulated not only by changes in the hemolymph MIH titer, but also by changes in the responsiveness of the Y-organ to MIH.

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.

Molting and metamorphosis are stimulated by the secretion of ecdysteroid hormones from the prothoracic glands. Insulin-like hormones have been found to enhance prothoracic gland activity, providing a mechanism to link molting to nutritional state. In silk moths (Bombyx mori), the prothoracic glands are directly stimulated by insulin and the insulin-like hormone bombyxin. Further, in Bombyx, the neuropeptide prothoracicotropic hormone (PTTH) appears to act at least in part through the insulin-signaling pathway. In the prothoracic glands of Manduca sexta, while insulin stimulates the phosphorylation of the insulin receptor and Akt, neither insulin nor bombyxin II stimulate ecdysone secretion. Involvement of the insulin-signaling pathway in Manduca prothoracic glands was explored using two inhibitors of phosphatidylinositol-3-kinase (PI3K), LY294002 and wortmannin. PI3K inhibitors block the phosphorylation of Akt and 4EBP but have no effect on ecdysone secretion, or on the phosphorylation of the MAPkinase, ERK. Inhibitors that block phosphorylation of ERK, including the MEK inhibitor U0126, and high doses of the RSK inhibitor SL0101, effectively inhibit ecdysone secretion. The results highlight differences between the two lepidopteran insects most commonly used to directly study ecdysteroid secretion. In Bombyx, the PTTH and insulin-signaling pathways intersect; both insulin and PTTH enhance the phosphorylation of Akt and stimulate ecdysteroid secretion, and inhibition of PI3K reduces ecdysteroid secretion. By contrast, in Manduca, the action of PTTH is distinct from insulin. The results highlight species differences in the roles of translational regulators such as 4EBP, and members of the MAPkinase pathway such as ERK and RSK, in the regulation of insect ecdysone secretion, and in the impact of nutritionally-sensitive hormones such as insulin in the control of ecdysone secretion and molting.

Male dung beetles (Onthophagus taurus) facultatively produce a pair of horns that extend from the base of the head: males larger than a threshold body size develop long horns, whereas males that do not achieve this size develop only rudimentary horns or no horns at all. Using topical applications of methoprene, we identified a sensitive period during the feeding stage of third (final) instar larvae when application of methoprene shifted the threshold body size for horn expression. Male larvae that received methoprene at this time delayed horn production until they attained a larger threshold body size than acetone-treated control larvae. This new sensitive period occurs earlier than a sensitive period previously reported for male horn regulation, and it coincides with a morph-specific pulse of ecdysteroid secretion described for this species. It appears that male horn expression is influenced by endocrine events at two different periods of larval development. We incorporate these results into an expanded model for the endocrine regulation of male horn expression.

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.

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.

The molt-inhibiting hormone of the American crayfish Procambarus clarkii (Prc-MIH), a 75-residue polypeptide containing three disulfide bridges, was synthesized by chemical ligation of two peptides, i.e., synthetic Prc-MIH(1–39) and Prc-MIH(40–75)-NH 2 , and by subsequent folding to form the native disulfide-containing peptide molecule. The synthetic peptide was comparable to the natural Prc-MIH in inhibiting ecdysteroid secretion by in vitro bioassay and shared features with the natural Prc-MIH in some biochemical analyses. These results indicate that the chemical ligation method can be used for the synthesis of Prc-MIH. Furthermore, it was demonstrated that synthetic Prc-MIH has hyperglycemic activity, although the activity was weaker than that of the authentic crustacean hyperglycemic hormone in the American crayfish. To examine the structural requirement of the Prc-MIH for eliciting biological activity, an antibody raised against the C-terminal region (residues 55–75) and two synthetic peptides, i.e., a core region (residues 1–54) containing three disulfide bridges and the C-terminal region, were utilized. It is suggested that Prc-MIH exerts its activities through coordination between the core region and the C-terminal region.

The interaction between Ca 2+ and cAMP in the mediation of ecdysteroid secretion from prothoracic glands (PGs) of Bombyx mori was investigated in vitro. Omission of Ca 2+ from the PGs’ incubation medium decreased basal ecdysteroid secretion from day 3 until day 6. On day 6, the ability of forskolin or 3-isobutyl-1-methylxanthine (IBMX) to stimulate ecdysteroid secretion was affected by the omission of Ca 2+ from the medium. The cAMP agonist Sp-adenosine 3′,5′-cyclic monophosphothioate (Sp-cAMPS) and the cAMP analogue dibutyryl cyclic AMP (dbcAMP) stimulated ecdysteroid secretion even in the absence of Ca 2+ from the medium. The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the cAMP antagonist Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) and the L-type Ca 2+ channel blocker verapamil. Both the Ca 2+ ionophore A23187 and the L-type Ca 2+ channel agonist S(-)•Bay K 8644 could stimulate ecdysteroid secretion. The A23187-induced ecdysteroid secretion was partially inhibited by Rp-cAMPS. The combined results indicate that Ca 2+ and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid secretion from the PGs.

Fenoxycarb application at 48 h (day 2) of the 5th instar of Bombyx mori induced permanent larvae with prothoracic glands (PGs) exhibiting weak ecdysteroidogenic activity. Although glands from control and fenoxycarb-treated larvae exhibited similar responses to dibutyl cAMP and forskolin on day 2, forskolin could not stimulate ecdysteroid secretion from PGs of fenoxycarb-treated larvae on day 3. Glands from control larvae incubated with cholera toxin (CTX) on day 3 had increased cAMP content and enhanced ecdysteroid secretion. Cholera toxin did not stimulate ecdysteroid secretion and marginally increased cAMP content in day 3 PGs of fenoxycarb-treated larvae. After application of fenoxycarb on day 2, crude brain extracts (cBRAIN) could not increase the glandular cAMP content throughout the rest of the 5th instar of the treated larvae. Fenoxycarb did not affect the basal or cBRAIN-stimulated cAMP accumulation in control PGs on day 2 and day 3 in vitro. Application of fenoxycarb on day 2 did not affect the recombinant PTTH (rPTTH)-stimulated ecdysteroid secretion on day 3, but reduced the cBRAIN-stimulated ecdysteroid secretion on day 3 to levels similar to that of rPTTH. The combined results suggest that the cAMP signalling cascade in the PGs of B. mori becomes nonfunctional after fenoxycarb application on day 2 of the 5th instar.

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2 h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30 min incubation. On the contrary, PGs incubated with cBRAIN for 30 min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca2+, rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca2+ in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca2+. Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.

The aim of this study was to clarify whether Ca 2+ plays a role in the mechanism by which recombinant prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by the prothoracic glands of the silkworm, Bombyx mori . Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect basal ecdysteroid secretion, but greatly inhibited the stimulation of ecdysteroidogenesis by PTTH. The addition of a Ca 2+ ionophore (ionomycin or A23187) stimulated ecdysteroid secretion in a dose-dependent manner. Furthermore, thapsigargin, a potent Ca 2+ -ATPase inhibitor, enhanced ecdysteroid secretion. In contrast, thimerosal, a thiol-oxidizing reagent, inhibited prothoracic gland activity. The mechanism of Ca 2+ action was examined further in relation to the cAMP effector system. Ca 2+ deprivation greatly suppressed the accumulation of cAMP in prothoracic glands treated with PTTH but had no effect on basal levels. Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect the stimulation of ecdysteroid secretion in response to dibutyryl cAMP or 1-methyl-3-isobutylxanthine. The present study suggests that specific regulatory actions of Ca 2+ in prothoracic gland cells are exerted prior to cAMP generation.

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 IP3-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.

Ovaries from house flies maintained on sucrose secrete large amounts of ecdysteroid when they are cultured with ovarian ecdysteroidogenic hormone, OEH. However, ovarian ecdysteroid secretion is reduced by incubation with both OEH and the ovarian ecdysteroidostatin (OES). A partially purified OES fraction from a semi-preparative reverse phase HPLC C18 column caused a 98% inhibition of ovarian ecdysteroid secretion in vitro at a concentration of 0.8 equivalents per μl. Ovaries can be activated to produce ecdysteroid in vivo by feeding diet containing protein to flies maintained on sucrose. Ecdysteroid secretion was inhibited when the in vivo stimulated ovaries were cultured with OES. This suggests that OES does not interfere with the OEH activation mechanism, but blocks ovarian ecdysteroid synthesis or release. Furthermore, OES inhibition is reversible and ecdysteroid secretion resumes when OES is removed. Musca OES could explain the decrease in ecdysteroid levels found in flies after mid-vitellogenesis. Both adult male and female abdomens contain OES, but OES was not transferred to females during mating. Evidence is presented that OES is not a trypsin modulating oostatic factor. Arch. Insect Biochem. Physiol. 38:166–176, 1998. © 1998 Wiley-Liss, Inc.

The prothoracic gland (moulting gland) of Periplaneta americana, the main source of ecdysteroids, was found to secrete proteins besides ecdysteroids. The course of ecdysone and protein secretion during the last larval instar was determined under in vitro conditions. Ecdysteroid production in the prothoracic gland as measured under in vitro conditions starts to increase to a first maximum between the fifteenth and twentieth days in the last larval instar. The main peak of ecdysteroid production is between the twenty-fifth and thirtieth and/or thirty-first days (i.e., at the end of the instar). Under the influence of actinomycin D, or puromycin (10−5 M) in vitro ecdysteroid release by the prothoracic gland is decreased by about 80%. On the fifth day of the last larval instar the glands show the lowest level of total protein content (5 μg per gland). Between the sixteenth and twentieth days, the protein content increases to about 9 μg, remaining at 7–9 μg per gland up to the end of the larval stadium. Prothoracic glands secrete proteins into the culture medium. Between the tenth and sixteenth days, protein secretion increases to a maximum level of about 8 μg per gland during a 4 h incubation interval. After the eighteenth day, the level decreases to 2–3 μg per incubation interval, with a transient significant increase on days 20 and 24 to about 5 μg. Some of the synthesized proteins serve a specific function in the gland cells, while some others seem to be synthesized for release into the hemolymph. Release of proteins after the tenth day of the larval stadium has no correlation to ecdysteroid release. Proteins of this period obviously have a function independent of ecdysone, perhaps as mediators. Arch. Insect Biochem. Physiol. 35:111–123, 1997. © 1997 Wiley-Liss, Inc.

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce ecdysteroids that initiate moulting and metamorphosis. During the last larval instar of holometabolous insects, a reduction in the hemolymph juvenile hormone (JH) levels is a necessary step in initiating larval-pupal transformation. Recently we have demonstrated that very low ecdysteroid levels in the early last larval instar of Bombyx mori initiate the complete inactivation of corpora allata (CA). Results presented here further indicate that PTTH signal transduction pathways undergo specific developmental changes, with a deficiency in transduction in prothoracic gland cells occurring during the early last instar. Glands from the early last instar showed no increase in either cAMP levels or steroidogenesis to the stimulation of PTTH, indicating the absence of the PTTH receptors in gland cells. We propose that this absence of PTTH receptors plays a critical role in directing larval-pupal transformation.

To investigate whether four Papilio species, Papilio xuthus, P. machaon, P. bianor and P. helenus, have two molecular forms of the prothoracicotropic hormones (PTTHs), referred to as big- and small-PTTHs, the PTTHs were extracted and fractionated from their pupal brains. The activating ability of big- and small-PTTH fractions was examined by the in vitro assay using the prothoracic glands (PGs) of 2-day-old 5th-instar larvae of their own and several other papilionid species. Big- and small-PTTH fractions activated the larval PGs of their own species to increase the ecdysteroid secretion in vitro. The doses of small-PTTH fractions for activating the larval PGs were 8- to 10-times larger than those of big-PTTH fractions. The big- and small-PTTH fractions as well as those of P. machaon activated the PGs of 2-day-old 5thinstar larvae of several heterogeneous papilionids, but the activating ability did not always decrease with the distance of the genetic (or phylogenetic) relationships. The results in...

Molt-inhibiting hormone (MIH) activity was tested in terms of the inhibitory activity of ecdysteroid secretion by cultured Y-organs of the crayfish, Procamharus clarkii. Several neuropeptides were separated from a crude extract of sinus glands of P. clarkii by means of reverse-phase high-performance liquid chromatography (HPLC) and tested for MIH activity. An extremely high level of activity was found in one fraction in reverse-phase HPLC, suggesting that the novel neuropeptide in this fraction may be a P. clarkii MIH.

The seasonal color polyphenism of the buckeye butterfly, Precis coenia, is controlled by environmental and genetic factors. Environmental stimuli, such as photoperiod and tempertaure, alter the pattern of ecdysteroid secretion. If ecdysteroids are present during a critical period early in the pupal stage the pale beige linea form develops, while in the absence of ecdysteroids the dark reddish-brown sa form develops. In this paper we report on the isolation of a genetic strain that constitutively expresses the rosa phenotype. Breeding experiments show that this effect is due to a single recessive gene. The ecdysteroid titer profile of the rosa strain is identical to that of the environmentally induced linea form, and not to that of the environmentally-induced rosa form. Ecdysteroid injections and reciprocal wing imaginai disk transplant studies between genetic rosa and wild-type strains, show that the rosa gene does not affect the endocrine system, but alters the physiological response mechanism that follows the ecdysteroid-sensitive period for linea induction and culminates in the wing-specific synthesis of ommochrome pigments.

The capacity of prothoracic glands to secrete ecdysteroids changes during the last larval stadium of the tobacco hornworm, Manduca sexta. In the present study, the protein content of prothoracic glands was observed to change significantly during this time period. Peaks in glandular protein content occurred on Day 4 (15.4 micrograms/gland) and Day 7 (14.6 micrograms/gland). These correspond to times of maximal ecdysteroid secretion in vitro. Ecdysteroid secretion in vitro was determined as a function of glandular protein content for Day 1, 3, and 7 glands. For unstimulated glands, secretion increased from 0.05 ng/microgram on Day 1, to 0.54 ng/micrograms on Day 3, to 1.37 ng/micrograms on Day 7. For glands incubated with big PTTH, secretion increased from 0.27 ng/micrograms on Day 1, to 2.05 ng/micrograms on Day 3, to 2.60 ng/micrograms on Day 7. The results suggested that developmental changes in secretory capacity are influenced by both the amount and type of glandular proteins. Glandular protein metabolism was assessed by monitoring the incorporation of [35S]methionine. A time course study revealed the rate of incorporation for Day 3 and Day 5 glands was significantly greater than the rate for Day 1 and Day 7 glands. Electrophoretic separation of radiolabeled glandular proteins revealed developmental changes in the pattern of protein synthesis. However, a band whose intensity changed in parallel with developmental changes in glandular secretory capacity was not detected. Finally, incorporation of BrdU by cells of the prothoracic glands was assessed using immunohistochemistry: Incorporation of BrdU was not observed on Days 1 or 7, occurred in only a few cells on Day 5, and was most pronounced on Day 3 (12.3% of the cells were labeled). The combined results indicate that changes in ecdysteroidogenic capacity are associated with (a) a change in glandular protein content, (b) a change in the types of proteins synthesized by prothoracic glands, and (c) a temporally restricted pulse of DNA synthesis, the latter being a possible indicant of cell proliferation.

Insect prothoracic glands are regulated by neuropeptide prothoracicotropic hormones (PTTH). In Manduca sexta PTTH exists as two size variants, big PTTH (approximately 25.5 kDa) and small PTTH (approximately 7 kDa). Previous studies indicate that both size variants employ cAMP as a second messenger and that stimulation of ecdysteroid secretion by big PTTH is Ca(2+)-dependent. In the present study, experiments were performed to assess the role of Ca2+ in small PTTH-stimulated ecdysteroid secretion by prothoracic glands from fifth instar larvae. Basal ecdysteroid secretion was not affected by Ca2+ channel blockers (verapamil or lanthanum) or by omission of Ca2+ from the incubation medium. Treatment of glands with a Ca2+ ionophore (A23187 or ionomycin) produced a concentration-dependent stimulation of ecdysteroid secretion. Stimulation of ecdysteroid secretion by small PTTH was suppressed (1) by Ca2+ channel blockers and (2) in Ca(2+)-free medium. A cAMP analog (Sp-cAMPS) stimulated ecdysteroid secretion in the presence of a Ca2+ channel blocker (verapamil) and in Ca(2+)-free incubation medium, and ionophore-induced ecdysteroid secretion appeared to be suppressed by a cAMP antagonist (Rp-cAMPS). The combined results indicate that basal ecdysteroid secretion is not dependent on external Ca2+, and suggest that small PTTH-stimulated ecdysteroid secretion is mediated by an influx of Ca2+ that precedes cAMP formation.

Gross anatomy, ultrastructure, innervation and ultrastructural alterations of the prothoracic gland (PTG) of cotton bollworm, Helicover pa armigera (Lepidoptera: Noctuidae) are illustrated for the last larval and early pupal stages as observed by light and electron microscopy. The T-shaped, paired (PTGs) consist each of 76–116 cells which are classified morphologically as large and small gland cells. In addition, another kind of small (about 6μ in diameter) gland cell was found in the PTGs of last instar larvae. The PTGs are innervated by the branches of 3 nerves! and tracheae and tracheoles are abundantly distributed to these glands. PTGs disappeared completely by the third day after ecdysis to the pupal stage (at temperature 28 C with a photoperiod L15:D9). An intercellular channel system (ICS) is formed by numerous, deep invaginations of the plasma membrane of gland cells. This ICS gradually increases in depth and width and reaches maximum development around the time of the major ecdysteroid secretion peak during the last larval instar. Numerous multivesicular sacs (MVS) with their remnants and an extensive rough endoplasmic reticulum were observed within ICS and cytoplasm, respectively, on the fourth day of the last larval instar. At that time the matrix of mitochondria became much more electron lucent. Freeze-fracture replicas of the glandular epithelium were made from last instar (4th day larvae. Dynamics of structure are related to data from others concerning secretory states of the prothoracic glands of this species.

Publisher Summary This chapter focuses on prothoracicotropic hormone (PTTH), a brain neuropeptide hormone that stimulates the secretion of the molting hormone, ecdysone, from the prothoracic glands. Hardened portions of the cuticle restrict the physical growth of insects; insects solve this problem by periodic molts in which the old cuticle is partly resorbed and partly shed and a new cuticle is laid down, allowing for further growth. In addition, many insects undergo metamorphic molts in which significant changes in structure occur. A suite of hormones controls and coordinates these episodes of rapid developmental change. Physiological, biochemical, and genetic studies on PTTH have shaped our current understanding of how insect molting and metamorphosis are controlled. This chapter reviews the discovery of PTTH followed by sections describing PTTH structure, regulation of PTTH release, and the cellular mechanisms by which PTTH stimulates ecdysteroid secretion. It ends with a brief discussion of developmental changes in the prothoracic glands and of roles played by factors aside from PTTH which have modified our current picture of the regulation of ecdysteroid secretion in insects.

The prothoracic glands of fifth instar Manduca sexta larvae respond to stimulation by the brain neuropeptide, prothoracicotropic hormone (PTTH), with a several-fold increase in the rate of ecdysteroid synthesis. Previous studies have shown that this response requires protein synthesis and that the action of PTTH can be mimicked by dibutyryl cAMP (dbcAMP) and the Ca 2+ ionophore, A23187. To further understand the role of protein synthesis in the response of prothoracic glands to PTTH, patterns of protein synthesis in stimulated glands were examined using glands incubated in vitro with [ 35 S]methionine. All three agents caused an increase in the rate of ecdysteroid synthesis as well as an increase of up to 300% in the synthesis and/or accumulation of three proteins (p100, p70, and p“50”) within 2 h of stimulation. Changes in these three proteins were specific to the prothoracic gland, were not elicited by non-brain peptides and were not simply a result of increased general protein synthesis in the gland. Exposure of the glands to A23187 alone, or concurrently with dbcAMP, resulted in increased synthesis of p100, p70, p“50” and ecdysteroids but decreased general protein synthesis. Increased synthesis of these proteins could be detected within 15 min after initiating PTTH stimulation. The behavior of these three proteins makes them candidates for modulators of ecdysteroid synthesis in the prothoracic gland. The results suggest also that PTTH may activate two biochemical pathways in the gland: one path leading to increased synthesis of the p100, p70, and p“50” proteins and increased ecdysteroid synthesis, and the second leading to increased general protein synthesis. This second trophic effect is vulnerable to intracellular Ca 2+ changes that do not inhibit the first pathway.

The general effects of temperature and nutritional quality on growth rate and body size are well known. We know little, however, about the physiological mechanisms by which an organism translates variation in diet and temperature into reaction norms of body size or development time. We outline an endocrine-based physiological mechanism that helps explain how this translation occurs in the holometabolous insect Manduca sexta (Sphingidae). Body size and development time are controlled by three factors: (i) growth rate, (ii) the timing of the cessation of juvenile hormone secretion (measured by the critical weight) and (iii) the timing of ecdysteroid secretion leading to pupation (the interval to cessation of growth [ICG] after reaching the critical weight). Thermal reaction norms of body size and development time are a function of how these three factors interact with temperature. Body size is smaller at higher temperatures, because the higher growth rate decreases the ICG, thereby reducing the amount of mass that can accumulate. Development time is shorter at higher temperatures because the higher growth rate decreases the time required to attain the critical weight and, independently, controls the duration of the ICG. Life history evolution along altitudinal, latitudinal and seasonal gradients may occur through differential selection on growth rate and the duration of the two independently controlled determinants of the growth period.

Incubation of prothoracic glands (PG) from Manduca sexta with destruxins (DTX) (principally destruxin-A) inhibited ecdysteroid secretion in vitro in a dose-dependent manner over a 6 h test period. A concentration of 0.5 μg ml −1 DTX gave ca 50% inhibition of the basal rate of secretion. The effect of a 1 h preincubation of PG with 1 μg ml −1 DTX was completely reversed by washing and incubation in normal medium. DTX (1 μg ml −1 ) effectively completely prevented (93% inhibition) the large stimulatory effects on ecdysteroid secretion of exposure to dibutyryl cAMP in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). DTX also significantly inhibited ecdysteroid secretion stimulated by the adenylyl cyclase activator forskolin, and by the calcium ionophore A23187. These results suggest that in Manduca PG, DTX may act at a level beyond these second messengers.

In Manduca sexta, basal and PTTH-stimulated secretion of ecdysteroids by prothoracic glands in vitro increases from days 1 to 4 of the fifth larval stage. Glandular content of cAMP-dependent protein kinase was analyzed to determine if the enzyme changes in concert with increased secretory response. Photoaffinity labeling with [32P]8-N3 cAMP revealed a 55-kDa cAMP-binding protein characteristic of the regulatory subunit of type-II cAMP-dependent protein kinase (RII). It appears that RII is one of a limited number of cellular proteins that is phosphorylated in the presence of [γ-35S]ATP; the thiophosphorylated protein and the photoaffinity-labeled regulatory subunit possess the same Mr and pI, and thiophosphorylation is blocked by mammalian cAMP-dependent protein kinase inhibitor. From days 1 to 4 of the fifth instar, glandular content of RII increases in conjunction with increased ecdysteroid secretory capacity. Application of JH analog on day 1 significantly inhibits the observed increase in RII. Catalytic subunit activity does not change from days 1 to 4 of the fifth instar, nor does cellular content of a 34-kDa protein previously shown to be phosphorylated in response to PTTH. While it is unlikely that increased content of RII is solely responsible for enhanced ecdysteroid secretion by the prothoracic glands, it may serve as a convenient marker for investigating the mechanism by which steroidogenic capacity is regulated.

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic hormone (CHH) family, inhibits the synthesis of ecdysteroid in Y-organ (YO) and plays a significant role in the regulation of molting and growth of crustaceans. A complete cDNA sequence encoding MIH (Ers-MIH, GenBank Accession No.: DQ341280) was cloned from eyestalk of Chinese mitten crab (Eriocheir sinensis) by 5' and 3' RACEs and PCR cloning. The full-length cDNA consists of 1457 bp with a 330 bp open reading frame, encoding 110 amino acids, containing a 75 amino acid mature peptide. The deduced amino acid sequence contains a typical CHH domain. Transcripts of Ers-MIH mRNA were detected in eyestalk by Northern blotting. The production of purified recombinant Ers-MIH (rErs-MIH) expressed in Escherichia coli was 0.3g/L. The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the deduced amino acid sequence of Ers-MIH. By in vitro assay on E. sinensis YOs, a cGMP mediated suppression of rErs-MIH on ecdysteroidogenesis could be observed. Accumulation of cGMP in YOs showed a concentration-dependent manner within 0.01-1 nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01-100 nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100 nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation.

Particulate (membrane) fractions were obtained from homogenates of prothoracic glands from staged larvae and pupae of the tobacco hornworm, Manduca sexta. The addition of cAMP to these fractions stimulated the in vitro phosphorylation of at least three phosphoproteins. This cAMP-mediated phosphorylation was blocked by the addition of the specific inhibitor of the cAMP-dependent protein kinase. When intact prothoracic glands were preincubated with [32P]O4 to label endogenous ATP, one of these proteins (34kDa) was phosphorylated in response to dibutyryl cAMP, forskolin, as well as the brain neurohormone, big prothoracicotropic hormone (PTTH). Neither cyclic GMP nor small PTTH stimulated 34 kDa protein phosphorylation, although the latter elicited increased ecdysteroid synthesis by larval glands. Further, injection of big PTTH into intact larvae previously loaded with [32P]O4 also resulted in enhanced phosphorylation of the same prothoracic gland protein. In prothoracic glands from animals at all developmental stages examined except diapausing pupae the protein was phosphorylated in vitro, although glands activated at the time of dissection showed a reduced phosphorylation response in vitro. The intensity of phosphorylation in intact glands was big PTTH dose-dependent, and the dose-response curve of phosphorylation closely followed the curve for PTTH-stimulated ecdysteroid secretion. The time course of phosphorylation is consistent with a role for this phosphoprotein as an intermediate between PTTH stimulation of adenylate cyclase and ecdysteroid biosynthesis.

1. 1. Testes of Heliothis virescens synthesized ecdysteroid in media containing low titers of calcium; the optimum calcium titer for testis sheaths stimulated to synthesize ecdysteroid in vivo was ca 1 mM, while the optimum of testes stimulated in vitro with the peptide testis ecdysiotropin was ca 0.3 mM calcium. 2. 2. Verapamil at concentrations lower than 10−3 M induced increases in ecdysteroid synthesis, indicating more efficient synthesis when calcium influx was inhibited. 3. 3. Hemolymph of H. virescens was 7 mM in calcium, while whole testes were maintained at 1–2 μM calcium.

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. Arch. Insect Biochem. Physiol. 68:197–205, 2008. © 2008 Wiley-Liss, Inc.

The involvement of protein and RNA synthesis in insect steroidogenesis was investigated using the prothoracic glands of the tobacco hornworm Manduca sexta. Ecdysone secretion stimulated by prothoracicotropic hormone (PTTH) and by cAMP analogs such as dibutyryl cAMP (dbcAMP), was suppressed by the translation inhibitors cycloheximide and puromycin, and by the transcription inhibitor actinomycin D. Inhibition of protein synthesis did not prevent the activation of glandular kinases, as indicated by continued protein phosphorylation in the presence of cycloheximide. Incorporation of radiolabeled amino acids and uridine increased within 60 min of glandular activation, suggesting that ecdysteroid secretion was accompanied by enhanced protein and RNA synthesis. One-dimensional gel electrophoresis revealed an increase in the translation of glandular proteins within 20 min of activation. The results suggest that the translation of protein from short-lived mRNA is necessary for optimal synthesis of ecdysteroids, and that the requisite proteins act beyond the activation of cAMP-dependent protein kinase.

Summary Ecdysteroid synthesis is regulated in insects by prothoracicotropic hormone (PTTH) and in crustaceans by molt-inhibiting hormone (MIH). These neurohormones exert opposite effects on their respective target tissues, PTTH stimulating the prothoracic glands and MIH inhibiting the Y-organs. The present work reviews recent progress in the neurohormonal regulation of prothoracic gland and Y-organ function. The steroid products of these glands are briefly discussed, as is current information on the structures of PTTH and MIH. Focus is placed on the mechanism of action of these hormones at the cellular level, as well as developmental changes in cellular sensitivity to PTTH. Though exerting different effects on ecdysteroid secretion, both PTTH and MIH increase cyclic nucleotide second messengers, are influenced by alterations in cellular calcium, and are likely to activate protein kinases. The contrasting steroidogenic effects of PTTH and MIH probably arise from differences in the cellular kinase substrate...

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic neuropeptide hormone family, inhibits ecdysteroidogenesis in the molting gland or Y-organ (YO). A cDNA encoding MIH of the land crab (Gel-MIH) was cloned from eyestalk ganglia (EG) by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 3'- and 5'-rapid amplification of cDNA ends (RACE). The cDNA (1.4 kb) encoded MIH prohormone containing a 35 amino acid signal peptide and a 78 amino acid mature peptide. The mature peptide had the six cysteines, one glycine, two arginines, one aspartate, one phenylalanine, and one asparagine in identical positions in the highly conserved sequence characteristic of other crustacean MIHs. Gel-MIH was expressed only in the EG, as determined by RT-PCR; it was not detected in Y-organ, heart, integument, gill, testis, ovary, hepatopancreas, thoracic ganglion, or skeletal muscle. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using a yeast (Pichia pastoris) expression system. Two constructs were designed to yield either a mature MIH fusion protein with a c-myc epitope and histidine (His) tag at the carboxyl terminus or an untagged mature protein without the c-myc and His sequences. Immunoreactive peptides were detected in Western blots of the cell culture media with both MIH constructs, indicating secretion of the processed rMIH into the medium. Culture media containing the untagged mature peptide significantly inhibited ecdysteroid secretion by YOs from land crab and green crab (Carcinus maenas) cultured in vitro, indicating that the Gel-rMIH was biologically active.