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.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Factors affecting change in sensitivity of prophoracic glands to juvenile hormone in Mamestra brassicae

The prothoracic glands of the early last-instar larva of Mamestra brassicae (day 0–3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-1, day-2 and day-3) were given juvenile hormone 5–10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-1, day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain, presumably PTTH, and ecdysone released from the prothoracic glands themselves.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Composition,synthetic and cytolytic activities of Galleria mellonella silk glands during the last-larval instar under the action of juvenile hormone

Within the first 48 hr of the last-larval instar of Galleria mellonella the silk glands grow but silk production is restrained. This ‘preparatory phase’ of the glands is probably maintained by juvenile hormone. Silk production and accumulation are stimulated in the ‘accumulation phase’ between 60 and 132 hr by unknown factors in the absence of juvenile hormone. The rate of RNA synthesis culminates at 84 hr but the RNA content increases until the end of cocoon spinning at 144 hr. In the following ‘regression phase’ (144–160 hr), when the glands exhibit high activities of acid and alkaline DN-ases and of acid phosphatase, the RNA and protein contents rapidly decrease, but that of DNA remains high. This phase is typical of moulting insects, is independent of juvenile hormone, and seems to be caused either by an increase in ecdysteroids or by lack of nutrients. The following ‘degeneration phase’ occurs when the surge of ecdysteroids terminates the larval-pupal transformation. Disintegration of silk glands by autolysis and phagocytosis is completed after pupal ecdysis (180 hr). Treatment of larvae with a juvenoid (ZR 512) at 48 or 132 hr in the last instar dramatically alter the composition, synthetic and cytolytic activities of silk glands. At the next ecdysis the glands attain a state very similar to that of the preparatory phase. They are capable of intensive silk production and completion of developmental cycle when the supernumerary larvae prepare for pupation. The results indicate that juvenile hormone can reverse the development of the silk glands.     

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.     

Induction of dauer pupae by fenoxycarb in the silkworm,Bombyx mori

Topical application of fenoxycarb (1 μg per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.     

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.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.     

Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea

In Heliothis zea, pupal diapause is not due to a deficiency of the prothoracicotropic hormone (PTTH), as it is in many other insects. However, PTTH is essential for diapause termination and adult development. Removal of the pupal brain 4 hr after larval-pupal ecdysis blocks the insect's ability to initiate adult development. Transplantation of brain neurosecretory cells restores this ability, whereas other tissues such as corpora allata have no effect. In the diapausing pupa, PTTH is released from the brain within 24 hr after larval-pupal ecdysis. Subsequent removal of the brain fails to block the ability for diapause termination, because PTTH potentiates the ability for adult development. Since diapause termination is suppressed in a temperature of 21°C, the bollworm retains the ability to initiate development in 27°C whereas it remains in diapause in 21°C. Diapause continues even though pupae are supplied with additional PTTH via neurosecretory cell transplantation.Ecdysone injection and prothoracic gland-ablation experiments indicate that the prothoracic glands are the source of the prohormone α-ecdysone, and that diapause is maintained by an α-ecdysone deficiency. This evidence, in conjunction with the above results, suggests that PTTH release potentiates prothoracic gland function in the diapausing pupa which is then regulated by a temperature dependent process.     

Temporal organization of endocrine events underlying larval-larval ecdysis in the silkworm, Bombyx mori

The timing of ecdysis in the penultimate instar of Bombyx mori was demonstrated to be under the control of a circadian clock. The temporal organization of secretion of prothoracicotropic hormone (PTTH), ecdysone and juvenile hormone was studied with particular attention to the circadian control of the timing of hormone release. PTTH release occurs, at least, in the second and third night. The latter is responsible for evoking the larval ecdysis. Prothoracic gland initiates ecdysone secretion abruptly with a very short span after the second PTTH release and secrete enough amount of ecdysone for larval moulting, which takes place 11 h later. Juvenile hormone titer is relatively high before the second PTTH release and corpus allatum becomes dispensable for ensuring the larval moulting in 1.5 h. Based on these findings, interpretations for the endocrine system underlying precocious pupation and formation of intermediates, which are produced by neck ligation, are presented.     

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.     

Temporal organization of endocrine events underlying larval-pupal metamorphosis in the silkworm, Bombyx mori

The temporal organization of endocrine events underlying larval-pupal moult was examined. After the phagoperiod of 6–7 days, the last instar larvae of Bombyx mori purged the gut contents and then pupated 4–5 days thereafter. Juvenile hormone titer was considerably high on the first day of the fifth instar, but declined to an undetectable level on day 3. Release of prothoracicotropic hormone (PTTH) began after the decline in the junvile hormone titer and completed during the night of day 4. Prothoracic glands began to secrete ecdysone on day 5. Larvae could pupate on schedule if their brains were removed after the PTTH release. One time of PTTH release during the feeding stage could satisfy the requirement for pupation occuring on schedule. The phagoperiod could be shortened by allatectomy early in the fifth instar and prolonged by the injection of a certain dose of juvenile hormone analogue before the gated PTTH release, accounting for the role of juvenile hormone in the timing of PTTH release.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Endocrine Mechanisms Regulating Post-Diapause Development in the Cabbage Armyworm,Mamestra brassicae

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.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

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.