Protein phosphorylation in the prothoracic glands as a cellular model for juvenile hormone-prothoracicotropic hormone interactions

Prothoracicotropic hormone (PTTH) is a brain peptide that initiates the molting process by acting directly at the cell membrane of the prothoracic glands to increase the intracellular levels of free Ca²⁺ and cyclic AMP (cAMP). This, in turn, leads to enhanced cAMP-dependent protein kinase activity resulting in the phosphorylation of a specific protein (M_r 34,000), and ultimately to a stimulation of ecdysone synthesis. When prothoracic glands are incubated in the presence of juvenile hormone (JH I) or (7S) hydroprene and then challenged with PTTH, the phosphorylation of the 34 kDa protein is decreased in a dose-dependent manner. The morphogenetically inactive methyl farnesoate is ineffective in preventing this downstream effect of PTTH. The JH effect does not appear to be stage specific, as early last larval, late last larval and pupal Manduca sexta prothoracic glands are similarly affected. The mechanism by which JH may prevent this PTTH-stimulated phosphorylation is discussed in terms of inhibition of phosphorylation via stimulation of an ATPase and stimulation of dephosphorylation by activation of a phosphoprotein phosphatase.     

Cellular regulation of ecdysone synthesis by the prothoracic glands of Manduca sexta

The cellular mechanism of action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH), was investigated in vitro using prothoracic glands from the tobacco hornworm, Manduca sexta. An involvement of cyclic AMP (cAMP) in PTTH-stimulated ecdysone synthesis was demonstrated as follows: (a) the steroidogenic effect of PTTH on prothoracic glands of day 3 fifth instar larvae and day 0 pupae was mimicked by agents (1-methyl-3-isobutylxanthine, dibutyryl cAMP and forskolin) which act by increasing intracellular levels of cAMP; and (b) PTTH stimulated the formation of cAMP in glands from both stages in a rapid, dose-dependent manner. However, a significant accumulation of cAMP in response to PTTH occurred only in larval prothoracic glands. In pupal glands, effects of the neuropeptide on cAMP synthesis were seen only in the presence of a phosphodiesterase inhibitor. Although cAMP is involved in PTTH action at both stages, it thus appears that the developmental state of the prothoracic glands influences the degree to which cAMP accumulates in response to the neurohormone. In addition to cAMP, it appears from the following that Ca²⁺ plays an essential role in mediating the steroidogenic effects of PTTH: (a) PTTH-stimulated ecdysone synthesis was blocked by omission of Ca²⁺ from the incubation medium; and (b) ecdysone synthesis was stimulated by the calcium ionophore A23187. Agents which act by increasing intracellular levels of cAMP enhanced ecdysone synthesis equally well in both the presence and absence of extracellular calcium. By contrast, cAMP formation stimulated by both PTTH and A23187 was completely dependent upon extracellular Ca²⁺. The results suggest a primary role for Ca²⁺ in mediating PTTH-stimulated synthesis of cAMP, with the cyclic nucleotide in turn stimulating ecdysone synthesis.     

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.     

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.     

Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts

Summary Using the techniques of intraspecific in vitro activation of prothoracic glands and ring glands by serial dilutions of prothoracicotropic hormone (PTTH) extracts from pupalManduca sexta (Lepidoptera) and larvalSarcophaga bullata (Diptera), a dose-response of activation was observed for both species. In both species maximum activation was at 0.5 brain equivalents while the number of brain equivalents necessary for half maximal stimulation (ED₅₀) was 0.20 forManduca and 0.15 forSarcophaga. When prothoracic glands or ring glands were challenged with interspecific PTTH extracts from a stage different from that of the gland donor, no dose-response of gland activation was observed. However, whenM. sexta larval prothoracic glands were challenged byS. bullata larval PTTH extract, activation was observed. The dose-response profile fell midway between the dose-response curves obtained for the intraspecific assays. Thus, PTTH extract from one insect has the ability to activate the prothoracic glands of an insect representing another order.     

Erratum

Allatotropin (AT) is a 13 amino acid neuropeptide isolated from the tobacco hornworm, Manduca sexta, which stimulates the biosynthesis of juvenile hormone (JH) in adult females of this insect. In this paper we report that AT stimulates inositol phosphate formation in both female and male corpora allata (CA) of adult Manduca sexta. Furthermore, thapsigargin, a potent Ca²⁺-ATPase inhibitor, staurosporine, a potent inhibitor of protein kinases A and C, and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca²⁺ sequestration, all stimulate JH biosynthesis without changing the basal level of inositol phosphates. These data suggest that AT activates an inositol 1,4,5-trisphosphate (IP₃) pathway. Furthermore, increases in the free intracellular Ca²⁺ concentration in the CA stimulate JH biosynthesis. © 1992 Wiley-Liss, Inc.     

Protein phosphatase activity is required for prothoracicotropic hormone-stimulated ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm,Manduca sexta

The multiple phosphorylation of ribosomal protein S6 appears to be required for prothoracicotropic hormone (PTTH)-stimulated protein synthesis and ecdysteroidogenesis by the prothoracic glands of Manduca sexta. The present study investigated the role of protein phosphatase in these phenomena by analyzing the effects of pretreatment of prothoracic glands with the phosphatase inhibitors okadaic acid and calyculin A in both basal and PTTH-stimulated glands. Okadaic acid or calyculin A treatment enhanced ribosomal S6 phosphorylation in control glands to a level similar to that observed with PTTH-stimulated glands. This treatment also prevented S6 dephosphorylation but had no apparent synergistic effect on S6 phosphorylation in PTTH-stimulated glands. Most importantly, okadaic acid or calyculin A treatment inhibited, rather than augmented, ecdysteroidogenesis in both PTTH-stimulated and non-stimulated glands. The composite data suggest that protein phosphatase activity sensitive to okadaic acid or calyculin A is required for PTTH-stimulated ecdysteroidogenesis. © 1996 Wiley-Liss, Inc.     

The steroidogenic action of haemolymph stimulatory factor in Manduca sexta: Comparisons with prothoracicotropic hormone

A recently described protein, found in the haemolymph of Manduca sexta larvae, stimulates ecdysone synthesis by both larval and pupal prothoracic glands in vitro. The mode of action of this haemolymph stimulatory factor has been investigated, particularly as it compares to the action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH). Unlike PTTH, the haemolymph factor does not stimulate ecdysone synthesis via an increase in the level of cAMP in the prothoracic glands. The haemolymph factor requires extracellular calcium for maximal stimulation of the prothoracic glands, but in contrast to PTTH, significant activity is retained in calcium-free medium. Exposure of the prothoracic glands to the haemolymph factor results in enhanced steroidogenesis within 1 min. This rapid stimulation contrasts with the 10–20 min lag period observed following PTTH exposure. However, the prolonged activation elicited by brief exposure to PTTH is not observed following exposure of the glands to the haemolymph stimulatory factor. Rather, the factor appears to be required as a sustained stimulus in order to exert its steroidogenic effects. The data indicate that the mode of action of the haemolymph factor is distinctly different from that reported previously for PTTH, and are consistent with the hypothesized role of the factor as a carrier of a sterol precursor utilized in ecdysone synthesis.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm,Manduca sexta

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.     

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

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

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta, involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.     

Production of cyclic AMP and adenosine by the brain and prothoracic glands of Manduca sexta

Relatively large amounts of cyclic AMP are produced by the prothoracic glands (source of the insect moulting hormone or moulting hormone percursor) of the tobacco hornworm, Manduca sexta. Pharate pupal glands produce more cyclic AMP than early fifth instar larval glands, and the addition of aminophylline enhances cyclic AMP accumulation. The much lower cyclic AMP level in the absence of aminophylline indicates the presence of potent cyclic AMP phosphodiesterase activity. Brains (sources of the prothoracicotropic hormone) also produce cyclic AMP but at a lower rate. Brains efficiently produce adenosine from ATP while β-ecdysone inhibits adenosine formation in early fifth instar larval brains. β-Ecdysone stimulates adenyl cyclase in brains of both stages when aminophylline and fluoride are present but has no effect on cyclic AMP accumulation in prothoracic glands. The absence of fluoride greatly reduces the amount of cyclic AMP produced by prothoracic glands when aminophylline is present. No cyclic AMP is accumulated in prothoracic glands when both fluoride and aminophylline are absent or in brains when fluoride is absent, notwithstanding the presence of aminophylline. Other insect tissues were also analysed for cyclic AMP production and none showed levels nearly as high as the prothoracic glands, suggesting a close relationship between cyclic AMP production and the function of the gland.     

Control of ecdysteroidogenesis: Activation and inhibition of prothoracic gland activity

The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insectManduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca²⁺ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a β tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the gland's potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.     

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

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

Characterization of cAMP-dependent protein kinase from the pupal wing epidermis of Manduca sexta

Cyclic AMP-dependent protein kinase (cAMP-PK) activity in the wing epidermis of day zero pupae of Manduca sexta was characterized. The preferred exogenous substrates were histones, subfractions H1 and H2b, casein and protamine sulfate; histone H2a was only phosphorylated moderately, while free base protamine and bovine serum albumin were poor substrates for cAMP-PK. cAMP-PK activity required Mg²⁺ and was optimal in the presence of 1 mM Mg²⁺. Co²⁺ and Mn²⁺ did not substitute for Mg²⁺, and Ca²⁺ inhibited cAMP-PK activity. The effective concentration of cAMP for activation of the cAMP-PK was substantially lower than that of cGMP (EC₅₀ 1.3 × 10⁻⁸ and 1.2 × 10⁻⁶ M, respectively). The type II isozyme of cAMP-PK comprised approx. 75% of the total cytosolic wing cAMP-PK as determined by DEAE anion exchange chromatography. Photoaffinity labeling of the whole cell homogenate with 8-azido cAMP revealed the presence of only type II isozyme. The distribution of the cAMP-PK isozymes was also determined for whole cell homogenates of brain, prothoracic glands, hemolymph, trachea, nerve cord, fat body, muscle and midgut.     

Manduca sexta prothoracicotropic hormone: evidence for a role beyond steroidogenesis

Prothoracicotropic hormone (PTTH) is a homodimeric brain peptide hormone that positively regulates the production of ecdysteroids by the prothoracic gland of Lepidoptera and probably other insects. PTTH was first purified from heads of adult domestic silkworms, Bombyx mori. Prothoracic glands of Bombyx and Manduca sexta undergo apoptosis well before the adult stage is reached, raising the recurring question of PTTH function at these later stages. Because Bombyx has been domesticated for thousands of years, the possibility exists that the presence of PTTH in adult animals is an accidental result of domestication for silk production. In contrast, Manduca has been raised in the laboratory for only five or six decades. The present study found that Manduca brains contain PTTH at all stages examined post‐prothoracic gland apoptosis, i.e., pharate adult and adult life, and that PTTH‐dependent changes in protein phosphorylation and protein synthesis were observed in several reproductive and reproduction‐associated organs. The data indicate that PTTH indeed plays a role in non‐steroidogenic tissues and suggest possible future avenues for determining which cellular processes are being so regulated. © 2009 Wiley Periodicals, Inc.     

An endogenous elevation of cGMP increases the excitability of identified insect neurosecretory cells

In the moth, Manduca sexta, the neuropeptide, eclosion hormone, triggers a dramatic rise in the levels of intracellular cGMP within a group of 50 neurons. The cells within this group include the segmentally repeated neurosecretory cell, Cell 27. In this study the effect of cGMP on the excitability of Cell 27 was investigated using intracellular recordings. Prior to its normal elevation in cGMP, Cell 27 exhibited a high spike threshold, but this was lowered dramatically when intracellular cGMP levels increased. The latter was also associated with spontaneous action potentials. This change in excitability did not correspond with changes in either resting potential, input resistance, or action potential amplitude. A similar lowering of threshold was induced by perfusion of 8-bromo-cGMP, whereas 8-bromo-cAMP caused the threshold to increase. Intracellular recordings using various ion substitution paradigms and channel blockers provided evidence which suggests indirectly that Ca²⁺ is mostly responsible for the depolarizing phase of the action potential while a Ca²⁺-activated K⁺ current contributes to the hyperpolarization. The results of these manipulations are consistent with the hypothesis that cGMP may partially increase excitability in Cell 27 by enhancing an inward Ca²⁺ current. Accepted: 31 October 1996     

Prothoracicotropic hormone activity in the embryonic brain of the tobacco hornworm,Manduca sexta

Summary Head segments and brains were extirpated from embryos of the tobacco hornworm,Manduca sexta, extracted and the resulting extracts assayed for prothoracicotropic hormone (PTTH) activity on prothoracic glands from day 3 fifth instar larvae and day 0 pupae. Dose-response curves were generated and indicated the presence of PTTH activity in embryonic brains and head segments, suggesting a role(s) for this neurohormone during embryogenesis. Maximal PTTH activity was found in brains from embryos 117 h post-oviposition, just prior to hatching, but activity was also noted in head segments as early as 24 h postoviposition. These data on PTTH and those on ecdysteroids and juvenile hormones in embryos suggest that these 3 classes of hormones which control insect post-embryonic development, may also be involved in the regulation of developmental processes in the embryo.