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The emergence of the adult Manduca sexta moth is followed by the loss of almost half of this insect's abdominal motoneurons and interneurons (Truman, 1983). This programmed cell death completes the transformation of the nervous system of the caterpillar into that of the moth. The death of these neurons has been previously shown to be a response to an endocrine signal: the decline in ecdysteroids that occurs at the end of metamorphosis (Truman and Schwartz, 1984). Our current research is focussed on the regulation of the fate of a pair of identified motoneurons, the MN-12 cells, in the third abdominal ganglion. Isolation of this ganglion from anterior parts of the nervous system can prevent the death of these cells at the time when they would normally die in response to the decline in ecdysteroids. Transection of the ventral nerve cord at various levels revealed that the source of this regulatory "death signal" is the fused pterothoracic ganglion and that it is transmitted via the interganglionic connectives. We hypothesize that the factors mediating this effect may act in concert with the ecdysteroid decline to specify the exact time of death for individual neurons.

At the end of metamorphosis, the intersegmental muscles of the moth Antheraea polyphemus undergo rapid degeneration in response to the peptide eclosion hormone (EH). Muscle death was preceded by a 22-fold increase in muscle guanosine-3',5'-cyclic monophosphate (cGMP) titers, which peaked 60 min after peptide exposure; adenosine-3'5'-cyclic monophosphate (cAMP) titers remained unchanged. EH induced a dose-dependent increase in muscle cGMP content with a threshold dose similar to that needed to induce cell death. Exogenous cGMP, but not cAMP, mimicked the action of EH. Sodium nitroprusside, a potent stimulator of guanylate cyclase, and methylated xanthines, a class of 3',5'-cyclic-nucleotide phosphodiesterase inhibitors, also induced the selective death of these muscles. It is concluded that an elevation of cGMP level is involved in EH-induced muscle degeneration. The intersegmental muscles become sensitive to EH at the end of adult development in response to the declining titers of the steroid molting hormones, the ecdysteroids. At earlier times, treatment with EH, exogenous cGMP, sodium nitroprusside, or methylated xanthines was ineffective in causing cell death. Nevertheless, treatment with EH at this time resulted in a marked increase in intersegmental-muscle cGMP. Thus, the onset of physiological responsiveness to the peptide hormone presumably results from biochemical changes distal to the EH receptors and guanylate cyclase.

Eclosion hormone (EH) is a 62-amino acid neuropeptide that initiates the ecdysis behavior of insects. The EH-encoding gene of the tobacco hornworm, Manduca sexta, was isolated by using a designed 72-mer oligonucleotide probe. Sequence analysis of this gene and its corresponding cDNA showed that the EH gene is 7.8 kilobases and consists of three exons. Exon I is totally nontranslated; exon II contains a 26-amino acid signal peptide and amino acids 1-4 of the EH peptide, and exon III encodes the remainder of the peptide. The EH gene is present in a single copy per haploid genome and transcribes an 0.8-kb mRNA that is expressed in larval, diapausing pupal, and developing adult brains but not in the ventral nerve cord or in nonneural tissues. In situ hybridization showed that the EH gene is expressed in two pairs of ventromedial neurosecretory cells in brains of both larvae and developing adults.

Neurogenesis in the ventral CNS of Drosophila was studied using staining with toluidine blue and birth dating of cells monitored by incorporation of bromodeoxyuridine into DNA. The ventral CNS of the larva contains sets of neuronal stem cells (neuroblasts) which are thought to be persistent embryonic neuroblasts. Each thoracic neuromere has at least 47 of these stem cells whereas most abdominal neuromeres possess only 6. They occur in stereotyped locations so that the same neuroblast can be followed from animal to animal. The thoracic neuroblasts begin enlarging at 18-26 hr of larval life, DNA synthesis commences by 31-36 hr, and the first mitoses occur shortly thereafter. Mitotic activity continues through the remainder of larval life with the neuroblasts showing a minimum cell cycle time of less than 55 min during the late third larval instar. By 12 hr after pupariation each neuroblast has produced approximately 100 progeny which are collected with it into a discrete packet. The progeny accumulate in an immature, arrested state and only finish their differentiation into mature neurons with the onset of metamorphosis. Most of the abdominal neuroblasts differ from their thoracic counterparts in their minimum cell cycle time (less than 2 hr) and the duration of proliferation (from about 50 to 90 hr of larval life). Neurons produced during the larval stage account for more than 90% of the cells found in the ventral CNS of the adult.

The emergence of the adult Manduca sexta moth is followed by the programmed degeneration of about 50% of the insect's abdominal interneurons and motoneurons. Neurons in implanted ganglia undergo degeneration in concert with neurons of the host, thereby indicating that a hormonal cue is important. The neuronal death follows the normal decline in the levels of the steroid hormones, ecdysteroids, that occurs at the end of metamorphosis. Manipulations that cause a precocious ecdysteroid decline result in an early death of the neurons. Also, prolongation of the ecdysteroid titer extend the life of the neurons. By properly timed steroid application it was possible to dissociate the neuronal degeneration from the behavioral events and muscle degeneration that also occur at emergence. Also the stereotyped sequence of death among identified motoneurons could be stopped at various points in the sequence by the carefully timed application of steroid. It was concluded that the signal for neuronal death was the withdrawal of ecdysteroids at the end of metamorphosis and that the death was probably a direct response of the individual neurons to the hormone withdrawal.

SYNOPSIS. The end of the molting process in the tobacco hornworm includes the rapid digestion of the old cuticle, molting fluid resorption, ecdysis of the old cuticle, and expansion and hardening of the new cuticle. The coordination of these processes is accomplished by three hormones. Each ecdysis during the life of Manduca appears to be triggered by eclosion hormone. Depending on developmental stage, the hormone comes either from the brain-corpora cardiaca complex or from the chain of ventral ganglia. The neural programs triggered by eclosion hormone include a neuroendocrine event, the release of the tanning hormone, bursicon, thereby ensuring that tanning of the new cuticle must follow ecdysis. Ecdysis, itself, appears to be controlled by the ecdysteroid levels since ecdysteroid injections delay ecdysis at physiological concentrations and in a dose dependent fashion. This delay is due to inhibition of eclosion hormone secretion and to the retardation of the terminal phases of the molt including the digestion of the old cuticle and the onset of sensitivity to eclosion hormone. Thus, eclosion hormone secretion and the ecdysis it triggers are coordinated with the end of development because both are influenced by the same endocrine signal—the decline in the ecdysteroid titer.

The segmental ganglia of adults of the moth, Manduca sexta, are constructed both from remodeled larval neurons and from adult-specific cells. The latter are produced by identified stem cells (neuroblasts) during larval life and then differentiate to form functional neurons during metamorphosis. The mitotic activity of the larval neuroblasts could be irreversibly blocked by the DNA-synthesis inhibitor hydroxyurea (HU). Treatment on day 1 of the third larval stage resulted in 80-90% of the neuroblasts being blocked before they produced any progeny while leaving the functional larval neurons unaffected. Treated larvae finished growth, underwent metamorphosis, and produced an adult CNS that contained the normal set of remodeled larval neurons but lacked most of the new adult-specific cells. When HU treatment was delayed until the start of the fourth or fifth larval stage, the neuroblasts produced the early portions of their respective lineages before they were blocked. The immature neurons that were generated prior to treatment survived to contribute adult-specific neurons to the moth CNS, but the remainder of each lineage was missing. This technique therefore enables one to produce adult nervous systems containing the basic set of remodeled larval cells plus defined sets of adult-specific neurons.

— Extraretinal photoreceptors are widespread among insects and function in the photoperiodic control of development and in the entrainment of circadian rhythms. The effects of light on the daily and seasonal regulation of brain neuroendocrine activity are mediated solely through extraretinal photoreceptors. In primitive insects, the eyes participate in the entrainment of nonendocrine circadian rhythms such as the locomotor rhythm. In more advanced forms, however, extraretinal pathways appear to be the only pathway for the entrainment of all rhythms thus far examined. But even in this latter case, the eyes sometimes effect a masking of the expression of the overt rhythm. An exact localization of the extraretinal receptors has not been accomplished, but in all studies to date they appear to be associated with the cerebral lobe region of the brain. Action spectra for photoperiodic responses have been determined for a number of insects. In general the responses are maximally sensitive in the blue with a marked decline in the red although exceptions do exist. Complete action spectra for circadian responses have been determined only for two insects. In both cases a plateau of sensitivity extends through the blue with a steep drop at longer wavelengths. From the action spectra data, the extraretinal receptors appear to have a threshold sensitivity less than 3 times 10-2 J/m2. The pigment nature of the receptor is unknown although it appears not to be a carotenoid derivative.

1. Late in the fifth instar, Manduca sexta larvae cease feeding and become ‘wandering larvae’ which are morphologically characterized by an ‘exposed heart’ and the appearance of a pink pigment along the dorsal midline. Two days later ocellar retraction signals the beginning of the prepupal period and 3 days thereafter the pupal ecdysis occurs. 2. The timing of the endocrine events which are responsible for these changes was determined by ligaturing animals of the appropriate developmental stage at various times of day. The times of prothoracicotropic hormone (PTTH) release by the brain were determined by neck ligations. Estimates of the times of prothoracic gland activity were obtained through the isolation of abdomens. 3. It was found that the fifth stage larva releases PTTH on two occasions. The first release lasts approximately 3.5 h and triggers the transformation to the wandering stage. The second release occurs two days later, lasts at least 7 h, and provokes the onset of the pupal moult. 4. The prothoracic glands are involved in triggering the same two changes. In the first instance the glands apparently require the continuing influence of the brain and consequently secrete for about 3.5 h. During the stimulation of the pupal moult the prothoracic glands become ‘turned-on’ and continue to secrete for at least 10 h after the time when the brain is no longer required. In this latter instance the total time of prothoracic gland activity may be as long as 17 h.

In the moth Hyalophora cecropia, injection of the eclosion hormone into pre-emergence (pharate) animals releases a stereotyped series of behaviours that assist the moth in escaping from the pupal cuticle and cocoon. The preeclosion behaviour begins 15 min after injection and lasts for 60 min. The first 30 min is an active period consisting of frequent abdominal rotations; a 30 min quiet period follows. This is followed by the eclosion behaviour which consists of rhythmic peristaltic waves which move up the abdomen at a frequency of 3−5 per min. The same behaviours can be elicited by injection of hormone into isolated pharate abdomens. The completely isolated abdominal CNS responded to the eclosion hormone by the generation of a programme of motor activity that mimicked that expected during the pre-eclosion and eclosion behaviours. The duration of the pre-eclosion behaviour programme in the isolated CNS was related to the general excitatory state of the preparation and varied from 57 to 325 min. In the latter instances, the behaviour lengthened as a unit with proportional increases occurring in the lengths of both the active and the quiet periods. But the structure of the individual rotational bursts appeared to be independent of these changes in the overall timing of the pre-eclosion programme. The frequency of bursts during the eclosion behaviour of the isolated CNS was always lower than that seen in intact animals. The frequency was not correlated with the length of the preceding pre-eclosion behaviour. It was concluded that the eclosion behaviour represented a behavioural programme that was distinct from the pre-eclosion behaviour. The structure of the eclosion bursts was independent of the frequency of bursting. Isolated pharate abdomens that had been aged for 2 days, often lost the ability to perform the pre-eclosion behaviour but still showed eclosion in response to hormone injection. In these cases the eclosion programme did not begin until 70−90 min after injection. Similar results were obtained with an isolated CNS preparation from an aged abdomen. Hormone was added to isolated CNS preparations and then washed-out at various times thereafter. After an initial exposure of only a few minutes, the hormone could be removed without interference with the initiation or play-out of either the pre-eclosion or eclosion behaviours. It was concluded that the eclosion hormone acts directly on the CNS to trigger two distinct behavioural programmes. The sequential arrangement of these programmes is due primarily to their respective latencies. Each programme has a two-tier hierarchical arrangement which includes a burst timer and a burst pattern generator. Sensory feedback appears to affect different components of the two programmes. In the pre-eclosion programme, sensory input alters the pattern of the rotary bursts but apparently not the number of bursts generated during the behaviour. In the case of the eclosion programme, sensory feedback influences the frequency of bursting but not the pattern of the individual bursts.

The present study describes 2 phosphoproteins, both with an apparent molecular weight of 54 kDa, in the CNS of the tobacco hornworm, Manduca sexta. Their phosphorylation is regulated by a neuropeptide, eclosion hormone (EH), and the second messenger cGMP, which thus have been named the EGPs (eclosion hormone- and cGMP-regulated phosphoproteins). Although cAMP was more effective than cGMP at stimulating the phosphorylation of the EGPs in CNS homogenates, in the intact CNS cGMP was more effective. Since cGMP mediates the action of EH, this strongly suggests that cGMP is the second messenger that stimulates the phosphorylation of the EGPs in vivo. The EGPs can only be phosphorylated in vitro during discrete time periods during the life of Manduca. During the larval and pupal molts, the EGPs can first be phosphorylated just prior to ecdysis. Their ability to be phosphorylated is correlated with the time when the insect is sensitive to EH. This close temporal correlation suggests that the ability to phosphorylate the EGPs determines when the insect can first respond to EH. During adult development, the EGPs first appeared on fluorograms 6 d before sensitivity to EH, suggesting that at this stage other factors may also be involved in the regulation of sensitivity. For the ecdyses of all 3 stages, EH appeared to stimulate the phosphorylation of the EGPs at ecdysis. The EGPs were found in all regions of the prepupal nervous system that were investigated, but only in the abdominal and pterothoracic ganglia of the developing adult. Fractionation of nervous system homogenates by ultracentrifugation revealed that one of the EGPs was present only in the pellet fraction, whereas the other was approximately equally distributed between pellet and supernatant. Furthermore, the EGPs in the pellet fraction could be partially solubilized with detergents and high salt concentrations.

Eclosion hormone activity was found in the brain and ventral ganglia of pharate pupae of Manduca sexta. No activity was detected in the corpora cardiaca-corpora allata complex. At the time of ecdysis the store of activity dropped by 50−75 % in the ventral cord whereas the hormone level in the brain remained unchanged. Also, larvae whose brains were removed at the wandering stage subsequently showed pupal ecdysis behaviour and also had essentially normal levels of hormonal activity in their blood at the start of the behaviour. It was concluded that at pupal ecdysis the hormone responsible for the initiation of the behaviour is released from the ventral nerve cord rather than from the brain. The chemical characteristics of the pharate pupal eclosion hormone were determined. The factors from the brain and ventral nerve cord were both active in a number of adult and pupal eclosion hormone bioassays. Both showed an apparent molecular weight of 8500 daltons and an isoelectric point of about 5−0, values essentially the same as that seen for the adult form of the hormone. We concluded that pupal ecdysis and adult eclosion are triggered by the same hormone but for the former it is released from the ventral nerve cord and, for the latter, from the brain. The choice of release site may depend on whether or not the release is under circadian control.

The nervous system of an adult moth is comprised of retained larval neurons that are remodeled during metamorphosis and a set of new adult specific neurons. The new neurons arise from a stereotyped array of stem cells (neuroblasts) that divide during larval life to generate nests of up to 100 arrested postmitotic immature neurons, the imaginal nest (IN) cells. At the onset of metamorphosis, some of the IN cells die while the remainder differentiate into mature functional neurons. Metamorphosis in insects is regulated by 2 classes of hormones, the ecdysteroids and the juvenile hormones. The transition from larva to pupa requires the disappearance of juvenile hormones followed by 2 releases of ecdysteroids: a small "commitment peak" and a larger "prepupal peak." Through a series of endocrine manipulations, we demonstrate that the death and differentiation observed among the abdominal IN cells at metamorphosis are both influenced by these hormonal cues. If the abdomen was isolated from the hormonal sources in the anterior half of the larva before the onset of metamorphosis, death and differentiation of the IN cells were prevented. Infusion of ecdysteroids into such abdomens, to mimic the prepupal peak, resulted in the IN cells showing the same fate as seen in control animals during the early phases of metamorphosis. The response of the IN cells to the small commitment peak of ecdysteroids was heterogeneous. Exposure to this small peak of steroids caused some cells to become committed to resume their development, making them resistant to juvenile hormone application.(ABSTRACT TRUNCATED AT 250 WORDS)

When abdomens were isolated from larvae during a critical period of the molting cycle, only parts of the abdominal epidermis had seen sufficient ecdysone to render them capable of forming a new cuticle independent of the prothoracic glands. By isolating abdomens at various times, it was found that the epidermis of a segment gains independence according to a reproducible pattern. The pattern of independence seen for a larval molt is roughly the reverse of that seen for a pupal molt. The presence or absence of juvenile hormone commits the epidermis towards the synthesis of larval or pupal cuticle, respectively. Under the influence of juvenile hormone, the pattern by which the various areas of the epidermis become committed to make larval cuticle is very similar to the larval pattern of independence from the prothoracic glands. This commitment to larval differentiation occurs at approximately the time when the epidermis becomes independent. In the absence of JH (in preparation for the pupal molt), the pattern by which the epidermis is committed to synthesis of pupal cuticle is the reverse of that seen for the larval molt. Moreover, the epidermis becomes committed to pupal differentiation 2 days prior to the time that it actually becomes able to secrete a pupal cuticle in the absence of the prothoracic glands.

Insect metamorphosis is controlled by a small ensemble of developmental hormones including a class of steroids--the ecdysteroids. In the tobacco hornworm, Manduca sexta, the progression from the larval to pupal to adult stages is controlled by the relative blood titers of ecdysteroids and juvenile hormone (JH). The cellular events in the nervous and muscular systems which accompany metamorphosis resemble those of embryonic development, but they occur in an animal which is larger and experimentally more tractable than an embryo. In this paper we review the role of ecdysteroids in directing the metamorphosis of the nervous and muscular systems in Manduca, and how JH modifies the cellular responses to the steroids. In particular, we describe how these hormones control muscle degeneration, changes in the structure and function of identified neurons, and programmed neuron death. One general finding is that interactions between cells (e.g., neurons and their target muscles) are not involved in their hormonal responses, but rather the hormones act independently and in parallel at the different sites. Another key finding is that the critical periods and hormonal requirements for the commitment to a particular differentiative pathway, and the phenotypic expression of that pathway, can differ, and are therefore experimentally separable. Finally, we find that the significance of a hormonal signal (e.g., a rise in blood ecdysteroids) is interpreted differently depending upon the previous history of hormone exposure of a neuron or muscle. This progressive change in the interpretation of hormonal signals is a major mechanism by which a limited number of hormones can orchestrate a complicated phenomenon such as metamorphosis.

Larval and pupal ecdyses of the moth Manduca sexta are triggered by eclosion hormone (EH) released from the ventral nervous system. The major store of EH activity in the latter resides in the proctodeal nerves that extend along the larval hindgut. At pupal ecdysis, the proctodeal nerves show a 90 % depletion of stored activity, suggesting that they are the major release site for the circulating EH that causes ecdysis. Surgical experiments involving the transection of the nerve cord or removal of parts of the brain showed that the proctodeal nerve activity originates from the brain. Retrograde and anterograde cobalt fills and immunocytochemistry using antibodies against EH revealed two pairs of neurons that reside in the ventromedial region of the brain and whose axons travel ipsilaterally along the length of the central nervous system (CNS) and project into the proctodeal nerve, where they show varicose release sites. These neurons constitute a novel neuroendocrine pathway in insects which appears to be dedicated solely to the release of EH.

The hawkmoth, Manduca sexta, undergoes periodic molts during its growth and metamorphosis. At the end of each molt, the old cuticle is shed by means of a hormonally-activated ecdysis behavior. The pharate adult, however, must not only shed its old cuticle but also dig itself out from its underground pupation chamber. To accomplish this, the adult performs a series of abdominal retractions and extensions; the extensions are coupled with movements of the wing bases. This ecdysis motor pattern is distinct from the slowly progressing, anteriorly-directed, abdominal peristalses expressed by ecdysing larvae and pupae. We have found that the ability to produce the larval-like ecdysis pattern is retained in the adult. Although this behavior is not normally expressed by the adult, larval-like ecdysis could be unmasked when descending neuronal inputs, originating in the pterothoracic ganglion, were removed from the unfused abdominal ganglia. Transformation of the adult-specific ecdysis pattern to the larval-like pattern was accomplished by transecting the connectives between the pterothorax and the abdomen, or by reversibly blocking neuronal activity with a cold-block. A comparative analysis of the ecdysis motor patterns expressed by larvae and by isolated adult abdomens indicates that the two motor patterns are indistinguishable, suggesting that the larval ecdysis motor pattern is retained through metamorphosis. We speculate that its underlying neural circuitry is conserved through development and later modulated to produce the novel ecdysis pattern expressed in the adult stage.

We have examined the morphology and neuronal elements of the cerebral neuroendocrine system in the larval, pupal, and adult stages of the moth Manduca sexta with a variety of neuroanatomical techniques. The larval brain contains several discrete groups of neurosecretory and non-neurosecretory cells that project to the associated neurohemal organs (the corpora cardiaca-allata complex, or CC-CA) and to a variety of more peripheral structures. A previously undescribed set of cells in the subesophageal ganglion have also been found that project out the neurosecretory nerves. During metamorphosis, the cerebral neuroendocrine system undergoes a dramatic structural reorganization, including the reduction or loss of many larval nerves and a repositioning of the cell groups and their dendritic fields. Despite these changes, most of its central elements are retained. In addition, by the completion of adult development a new cluster of cells can be found on either side of the dorsal midline of the brain. We have also determined the relative contributions of the different cell groups to the moth neuroendocrine system by intracellular iontophoresis of dye into individual cells. Within the dorsal protocerebrum, five separate morphological types of cells can be recognized, each with a distinctive pattern of dendritic arborization in the brain and terminal neurohemal processes that project to the CC, the CA, the aorta, or to a combination of these regions. The large intrinsic cells of the CC have also been filled, revealing an unusual set of morphological features in these peripheral neurosecretory cells.

Metamorphosis of the tobacco hornworm moth is followed by the death of many identified neurons in a stereotyped temporal pattern. The degeneration program is under hormonal control and can be either made to occur early or prevented completely by various endocrine treatments. This ability to manipulate the fate of identified cells may eventually lead to insights into the cellular mechanisms that bring about neuronal death.

The distribution and molecular properties of the tanning hormone bursicon were studied using an isolated wing biological assay. Wing cuticle tanning activity was measured in saline homogenates of the central nervous system and selected muscles. Activity was detected in all ganglia of the nervous system in both the prepupal and pharate adult developmental stages. In both stages, this activity was predominantly located in the abdominal portion of the nervous system. The titres in all ganglia, except the suboesophageal, increased during metamorphosis. Of the various non-neural tissues examined, only the closer muscle of the spiracle contained detectable levels of activity. All activity in the nervous tissue was sensitive to proteolytic digestion; it could not be mimicked by any of the 17 putative transmitter and/or hormonal substances tested. Partial purification of the tanning activity indicated an apparent molecular weight of 20–30 K. The partially purified material (from gel filtration) resembled the hormone bursicon in the following three ways : its titre in the tissue declined following normal bursicon release ; it could be recovered from the haemolymph during normal bursicon release; it could be released from isolated nervous tissue following high potassium stimulation in a calcium-dependent manner. It was concluded that the hormone bursicon represents most if not all the tanning activity present in the central nervous system, that it is widely distributed in that tissue and that it is present as a peptide (or class of peptides) with homogeneous size and charge.

The final step in the moulting of all insects is ecdysis, the shedding of the cuticle of the previous instar, which is triggered in Lepidop-tera by the neurosecretory peptide eclosion hormone1. This hormone acts directly on the nervous system to release the appropriate motor patterns for larval, pupal and adult ecdyses2–5, but there are only brief periods near the end of each moult when the nervous system is competent to respond to the hormone. Previous experiments have shown that the action of eclosion hormone on the nervous system at pupal ecdysis in the tobacco hornworm, Manduca sexta, is mediated by the second messenger cyclic GMP1. Here we report that the hormone-stimulated increase in cGMP results in the phosphorylation of two proteins, each with an apparent relative molecular mass (Mr) of 54,000. Moreover, the brief periods during which the central nervous system (CNS) is responsive to eclosion hormone seem to result from the transient presence of these substrate proteins within the nervous system. This provides a novel mechanism by which hormonal responsiveness can be regulated.

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.

The various physiological and behavioral roles of neuropeptides have caused a general revision in our understanding of the importance of these neurochemical messengers in the control of CNS function. Yet little is known about the functional regulation of neuropeptides at the level of the individual peptidergic cell. Here we discuss three inter-twined peptidergic systems that are involved in the control of several related behaviors in the tobacco hawkmoth, Manduca sexta. We present experiments that led to the identification of the individual peptide-containing neurons and discuss some of the mechanisms that regulate peptide release for each peptidergic system.

The abdominal ganglion neurones responsible for the secretion of the peptide hormone bursicon from the transverse nerves of the tobacco hornworm Manduca sexta have been localized. Each unfused abdominal ganglion produced bursicon during adult development. The hormone was then transported to the next posterior transverse nerve for subsequent release following adult emergence. Of the two efferent pathways that connect ganglia to posterior transverse nerves, only one had ultrastructural features consistent with a neurosecretory function. All axons in this pathway contained numerous, elementary, dense-cored granules with diameters ranging from 100 to 300 nm. In pharate adults, cobalt back-filling of this neurosecretory pathway revealed two groups of cell bodies in abdominal ganglia : three dorso-laterally and eight at the mid-line. Hormonal activity was present in lateral regions of the ganglion but not in mid-line regions. In the dorso-lateral region, four blueish cell bodies could be seen in the living tissue. The two combined clusters of four cells from both sides of the ganglion contained more than 60% of the bursicon activity present in the entire ganglion. More careful dissection of the cluster detected the presence of activity in cell pairs and individual members. Individual dye-fills of these blue cells demonstrated that three of these were the three lateral neurones that were back-filled from the transverse nerve. The fourth blue cell may be identical to a neurone whose axon travels in the descending, contralateral connective and exits the nervous system via the next posterior ganglion. On the basis of dendritic geometry, each of the neurones in the bursicon cell cluster could be identified as a unique, individual neurone.

The steroid hormone 20-hydroxyecdysone regulates many aspects of nervous system development in the moth Manduca sexta, including stage-specific neuronal morphology and stage-specific neuronal death. We have used steroid hormone autoradiography to study the distribution of cells that concentrate ecdysteroids in the ventral nervous system of this insect. The ligand was [3H]-ponasterone A, a bioactive phytoecdysone. Tissue was examined from three stages of development: the end of larval life (first day of wandering), the end of metamorphosis (pharate adult), and 4-day-old adults. In the abdominal ganglia of wandering larvae and pharate adults, a subset of neurons including both motoneurons and interneurons exhibited a nuclear concentration of radiolabeled hormone. The pattern of binding was reproducible but stage-specific, with a greater proportion of neurons showing binding in the larvae than in pharate adults. No labeled neurons were found in abdominal ganglia from mature (4-day-old) adults. In the case of the pharate adult ganglia, the ecdysteroid receptor content of specific, identified motoneurons was determined. These results are discussed in light of the responses of these neurons to physiological changes in levels of circulating ecdysteroids.

Re-investigation of the role of the corpora cardiaca in the reproductive behaviour of the giant silkmoths, Hyalophora cecropia and Antheraea polyphemus, showed that this pair of glands plays no essential role, either in “calling” behaviour by virgin females or in increased oviposition due to mating. Removal of corpora cardiaca-corpora allata complexes, either from diapausing pupae or from freshly eclosed adult females, had no effect on the calling behaviour or on its timing in either species. Moreover after mating, these operated females laid eggs in the typical mated oviposition pattern. Furthermore, females in which there was only a nervous connection between the brain and the abdomen but no haemolymph circulation called normally and oviposited after mating. Although the corpora cardiaca were not essential for calling behaviour, hormogenates of corpora cardiaca-corpora allata complexes and blood from calling or ovipositing females induced a typical “calling” response in 30–60% of the isolated virgin H. cecropia abdomens tested. This activity was not species-specific as it was also found in Manduca sexta, but the restriction of major activity to corpora cardiaca extracts and haemolymph suggested that a neurosecretory factor may modulate the normal neural control of calling behaviour.

The tobacco hornworm moth, Manduca sexta, shows extensive degeneration of abdominal neurons after the adult moth emerges (ecloses) from the old pupal skin. Death of interneurons begins about 2 hours before eclosion and continues through the next 30 hours. Motorneuron degeneration starts slightly later, commencing at about 8 hours after eclosion. Analysis of the death of identified motorneuons showed that there was a precise spatial and temporal program of neuronal death. Particular cells invariably either continued to live or died and, among the latter, the time of death varied from cell to cell but for a given cell was constant. This program of cell death was somewhat flexible in that the fates of certain cells could be adaptively modified by behaviors that might naturally occur in the life of the insect.

1. Metamorphosis in the mothManduca sexta culminates with the secretion of the peptide eclosion hormone (EH), which triggers the stereotyped behavior of adult emergence (eclosion) from the pupal cuticle. In restrained but spontaneously behaving animals, the release of EH occurred shortly before the onset of subjective night (Fig. 3) and coincided with a depletion of EH from the neurohemal organs of the brain, the corpora cardiaca-corpora allata complex (CC-CA; Fig. 4). 2. EH is produced by neurons within a bilaterally paired group of brain neurosecretory cells (Group Ia) which project to the CC-CA via the nervi corporis cardiaci-1+2 (NCC-1+2; Fig. 1). Electrical stimulation of the NCC-1+2 caused a marked increase in the levels of EH secreted from isolated CC-CA (Fig. 2), while stimulation of the other nerves innervating the neurohemal organs did not. 3. Electrical activity in the NCC-1+2 paralleled that of the cerebral neurosecretory cells (Fig. 1). Chronic extracellular recordings revealed a sudden increase in the tonic firing of several units within this nerve approximately 2 to 3 h before normal eclosion (Fig. 5), coincident with the release of EH bioactivity from the CC-CA (Fig. 6). 4. The Group Ia neurons were electrically inactive on the day before eclosion (Day −1), but on the day of eclosion (Day 0) a subgroup of these cells exhibited both enhanced synaptic input and elevated rates of tonic firing during the normal time of EH release (Fig. 7). 5. No significant differences in resting membrane potential or spike waveform characteristics were detected among the various subsets of Group Ia cells on either Day −1 or Day 0, while a significant increase in the resting input resistance was seen in the active subgroup on Day 0 (Fig. 8). This increase may be due to the regulatory effects of the steroid 20-hydroxyecdysone, which inhibits the release of EH and may act by preventing the synaptic activation of the EH neurons until the final day of adult development.

The relationship between the ecdysteroid titre and eclosion hormone was explored for the pupal and adult ecdyses of Manduca sexta . Ecdysteroid treatment late during either moult caused a dosedependant delay in the time of ecdysis. Sensitivity to exogenous steroid treatment dropped off as the respective moults neared completion and in both cases coincided with the time of the low point in the endogenous ecdysteroid titre. It was concluded that an ecdysteroid decline is a normal prerequisite for the ecdyses of both stages. The steroid drop is important for two aspects of the eclosion hormone system: it causes target tissues to become sensitive to the peptide and it is a prerequisite for the subsequent release of eclosion hormone itself. Thus, the dual action of the declining ecdysteroid titre insures that when eclosion hormone is released, the tissues will be competent to respond to it.

1. When the pupal cuticle was peeled from aroundAntheraea pernyi andHyalophora cecropia moths on the last day of adult development, the animals showed reduced levels of adult behavior. Clawing and extension movements of the legs as well as wing flapping were seen, but complex behaviors such as the righting response and walking were absent (A. pernyi) or only occasionally observed (H. cecropia). 2. Injection of the eclosion hormone into “peeled” moths was followed 2 to 3 hours later by the onset of the eclosion movements. Walking behavior and the righting response appeared abruptly at the time of eclosion. 3. The development of certain adult behaviors was examined by peelingA. pernyi moths that were in the 14th through 19th (last) day of adult development. Animals peeled on days 16 or 17 showed distinct eclosion movements which started within a few minutes after peeling. But on day 18, this behavior became suppressed and could no longer be elicited by peeling. 4. Sensitivity to the eclosion hormone did not appear until day 18 and became fully developed on day 19. 5. Injection of picrotoxin into peeled day 19A. pernyi resulted in the abrupt onset of walking behavior. 6. It was concluded that certain adult behaviors such as eclosion and walking become complete and functional during adult development. But as development progresses, these then become repressed—presumably through neural inhibition. The eclosion hormone apparently acts by turning off this inhibition.

Invertebrates use hormones in diverse ways to organize their behavior. Examples considered include a programming of the duration of a behavior by the length of a prior endocrine signal, the use of sequential endocrine signals or differential latencies to organize behavioral sequences, and the organization of complex behavioral patterns through the release of multiple bioactive materials. (Accepted for publication 11 March 1983)

A stereotyped series of behavioural changes occurs in preparation for pupation in Manduca sexta. Feeding declines over an 8-h period, after which the larva coats its body with a labial gland secretion. The animal then begins a 10-to 30-h wandering behaviour during which it constructs a pupation chamber underground. Wandering behaviour starts during a specific temporal gate which is determined by an internal circadian timer. The scotophase of the day prior to wandering has the major influence on the timing of internal processes which activate the behaviour. Wandering duration is correlated with larval size, reflecting a possible influence of juvenile hormone. The larva appears to be irrevocably committed to begin wandering by an event that occurs about 15 h previously, a time that corresponds to the second of three prothoracicotropic hormone (PTTH) pulses and the accompanying elevation of ecdysteroids as measured by Gilbert et al. (1981). We conclude that both the initiation and duration of wandering behaviour are governed primarily by processes which are internal to the larva.

Each larval moult in Manduca sexta consists of an identical series of developmental and behavioural events leading up to ecdysis. Injections of eclosion hormone into staged larvae in any instar resulted in the premature elicitation of the larval pre-ecdysis behaviour, comprising a rhythmic sequence of muscle contractions, followed by the larval ecdysis behaviour. A marked depletion of eclosion hormone stores form the ventral chain of ganglia coincided with each larval ecdysis and in the moult to the fifth instar, eclosion hormone activity appeared in the blood at the onset of the pre-ecdysis behaviour. Responsiveness to eclosion hormone for pre-ecdysis and ecdysis behaviour developed about 12 and 6 hr before normal ecdysis, respectively. Elicitation of ecdysis behaviour by exogenous hormone inhibited both subsequent behavioural responses to eclosion hormone and endogenous hormonal release. In conclusion, the behavioural programme involved in each larval ecdysis appears to be controlled by the eclosion hormone.

The blood titres of eclosion hormone in Manduca sexta indicate that the hormone is liberated into the blood during a span of about 20 min and then disappears with a half-life of about 45 min. Eclosion normally follows 2·5 h after the appearance of the hormone in the blood. Bursicon appears in the blood within 2 min after the newly emerged moth comes to rest at a wing spreading site. Hormone is apparently released during the following 10 min and then disappears with a of 40–50 min. The target tissues for the two hormones become responsive during the last day of development. The wing epidermis and nervous system appear to become sensitive to the eclosion hormone at about the same time, approximately 4 h before the hormone’s release. Wings become responsive to bursicon about 2 h earlier. During the last day of adult development, Manduca show a precise onset of responsiveness to bursicon and eclosion hormone followed a few hours later by the gated release of the two hormones. This appearance of hormone sensitivity is likely to be due to a photoperiodically gated event which occurs late in adult development but prior to eclosion hormone release. The nature of this event is unknown.

The eclosion hormone (EH) of Manduca sexta is a 62 amino acid peptide which causes the behavioral and physiological changes that occur at ecdysis. EH was purified from the corpora cardiaca-corpora allata (CC-CA) complexes from pharate adult moths using acidic methanol extraction followed by two high performance liquid chromatography separation steps. One CC-CA complex was found to store about 3.5 ng of EH. Different techniques for estimating the molecular weight of the peptide, gel filtration chromatography and SDS-urea polyacrylamide electrophoresis, gave values of about 10 and 4 kD, respectively. Both were significantly different from the calculated molecular weight of 6813 dalton. The experimentally determined pI of the peptide was 4.8. Treatment of EH with various reagents showed that intact disulfide bridges are required for biological activity.

1. At the culmination of each molt, insects shed their old cuticle by a behavior called ecdysis, which generally involves rhythmic peristaltic body movements. We have compared the abdominal peristalsis behaviors produced by the tobacco hornworm,Manduca sexta, during larval-larval molts (‘larval ecdysis’) and at the larval-pupal molt (‘pupal ecdysis’), to see whether the morphological changes during metamorphosis were accompanied by alterations in the motor patterns. 2. At larval ecdysis, caterpillars shed their old cuticle by means of rhythmic peristaltic contractions which run anteriorly along the body, accompanied by coordinated retractions and extensions of the abdominal prolegs (Fig. 2). During the larval-pupal transformation the prolegs are lost, so that at pupal ecdysis the cuticle is shed by peristalsis alone (Fig. 1). 3. Cinematographic analysis of ecdysing larvae and pupae revealed that the peristaltic movements resulted from each abdominal segment being sequentially pulled forward and partially telescoped into the next anterior segment, due to the contraction of intersegmental muscles (ISMs) in the more anterior segment. Despite the marked changes in body morphology which occurred during the larval-pupal transformation, the pattern of the peristaltic contractions produced at larval and pupal ecdysis was the same (Table 1). 4. The neural basis of these behaviors was investigated in semi-intact, deafferented preparations which produced ecdysis motor patterns in response to a peptide hormone, eclosion hormone (EH), which is the normal blood-borne trigger for ecdysis (Fig. 3). The motoneurons which generated the peristaltic motor patterns in larvae and pupae were identified by recording extracellularly from various nerve branches (Figs. 4, 5, 6, 8) and intracellularly from neuronal somata (Figs. 7, 9). 5. The pattern of the motoneuron bursts was the same during both larval and pupal ecdysis (Table 2). The only apparent difference at the two stages was that pupae exhibited a lower level of tonic background motor activity than did larvae (e.g., Figs. 4, 8). 6. Thus, both behavioral and electrophysiological data indicated that the pattern-generating circuitry underlying abdominal peristalsis remained stable during the larval-pupal transformation. In contrast, the loss of the prolegs at pupation and their consequent absence from pupal ecdysis behavior did suggest a possible stage-specific change in the neural circuitry; this possibility is investigated in an accompanying paper (Weeks and Truman 1984).

1. A preliminary analysis of the silkmoth eclosion clock demonstrated that the endogenous, free-running cycle of the clock could be divided into two parts: an initial synchronization period and a dark decay period. 2. A 1/2 hour light pulse applied during the synchronization period served to restart the free-running cycle. By proper timing of light pulses, a given cycle of the eclosion clock could be restarted ad infinitum. 3. When the length of the interrupting pulse was progressively increased up to 24 hours, the subsequent eclosion rhythms showed a constant phase relationship to the end of the interruption, irrespective of its duration. This phase relationship was the same as observed when the eclosion clock was started by transfer of moths from continuous light to continuous darkness. 4. It is concluded that the synchronization period involves a photoreversible process. During this period light rapidly stops the clock and the cycle can begin only with the onset of darkness.

1. In the giant silkmoths, adult eclosion is immediately preceded by a stereotyped series of abdominal movements - the pre-eclosion behaviour. The pattern of movements is species-specific and has a duration of about 1 ¼ h. 2. The pre-eclosion behaviour is followed sequentially by eclosion, the release of the labial gland secretion, the post-eclosion activity, and the spreading of the wings. These five elements of behaviour make up the emergence sequence. 3. The progression from one part of the behavioural sequence to the next is independent of stimuli provided by the pupal cuticle or cocoon, and therefore must be due to an internal programme. Thus, when the pupal cuticle was removed from pharate moths 12 h before their normal eclosion time, these ‘peeled’ animals continued to behave in a pupal fashion. But upon the arrival of the eclosion gate, the entire emergence sequence was displayed. 4. Through surgical manipulations the brain was shown to trigger the pre-eclosion behaviour. Moreover, this action of the brain was mediated by a neurosecretory hormone - the eclosion hormone. 5. Injections of extracts with eclosion-hormone activity triggered the precocious display of the emergence sequence by pharate moths. 6. When the eclosion hormone was injected into the isolated abdomens of pharate moths, these fragments performed the pre-eclosion behaviour and then shed the surrounding piece of pupal cuticle. The information for the pre-eclosion behaviour and for the abdominal movements associated with eclosion must therefore be programmed in the abdominal ganglia. Moreover, after its ‘activation’ the abdomen can switch from one behaviour pattern to the next without influence from the higher centres.

1. A semi-quantitative biological assay for the eclosion hormone is described. 2. In the pharate moth hormonal activity is confined to the brain and the corporacardiaca (CC). 3. During the life history of the moth, activity is practically absent from the brain and CC of larval and pupal stages and appears only in preparation for adult emergence. 4. The titer of eclosion hormone in the brain and CC of Pernyi moths was followed through adult development. In both structures activity first appeared on about day 7. Titers then increased for the next 3 to 5 days followed by a plateau. On the morning of the day of emergence, the titer in the brain fell and that in the CC correspondingly reached its highest level. At the time of eclosion in the evening, the CC titer then dropped and hormone appeared in the blood. 5. Tests on brains and CC from cockroaches and bugs failed to show activity. 6. It was concluded that the eclosion hormone is used only for the pupal-adult ecdysis and, thus, is probably restricted to the holometabolous orders of insects.

Juvenile hormone inhibits cuticular melanization in tobacco hornworm larvae. This action of juvenile hormone was used as the basis of a new bioassay which is sensitive to less than 0·01 ng (3 × 10−14 moles) of topically applied hormone and requires only 2 days.

1. Motor neurons in the fourth abdominal ganglion were identified by diffusing cobalt into the ganglion through the cut ends of the peripheral nerves. 2. The ganglion from the last instar larva had a total of 74 motor neurons. These were divided into 10 groups which were characterized by the position of the cell body in the ganglion and the root through which the axon travelled to the periphery. 3. In the transition to the pupal stage, 12 larval neurons were lost and 8 new motor neurons appeared. 4. There was no change in the number of motor neurons during adult development. Most of the larval neurons persist through metamorphosis and are used by the adult at emergence. At this stage there are 70 motor neurons. 5. After adult eclosion there was a loss of 40 motor neurons. Only “larval” neurons degenerated. Those which remained included both those which were larval in origin and those which appeared in the pupa. After emergence the interneuron number dropped from approximately 380 to 200. 6. At the time of emergence the eclosion hormone “turns-off” pupal behavior and “turns-on” adult behavior. It also triggers the breakdown of various muscle groups in the abdomen. It is concluded that the neural death was the end result of this aspect of eclosion hormone action.

1. In silkmoths the gating of adult eclosion by light-dark cycles persists after extirpation of the compound eyes, frontal ganglion, subesophageal ganglion, or corpora allata-corpora cardiaca complex. Removal of the brain abolishes gating and such moths emerge irrespective of time of day or night. 2. Implantation of a brain into the abdomen of a debrained animal restores the ability both to entrain to a light-dark cycle and to free-run under conditions of continuous darkness. 3. Implantation of pieces of brain which contain the cerebral lobes likewise restored to debrained moths the ability to entrain and to free-run. But the synchrony was not as sharp as that observed in moths which had received implants of the entire brain. 4. Transection of the brain lateral to each median neurosecretory cell cluster yields pieces which are apparently incapable of gating the eclosion of debrained moths. Both moths receiving the median piece and those receiving the lateral pieces showed a randomized distribution of eclosion. 5. Experiments involving brain transplantation and selective illumination of parts of the body showed that the brain directly receives the light information which is necessary for the entrainment of the clock. 6. In theCecropia moth, the lights-on signal serves to stimulate eclosion and thus partially masks the output of the clock. This effect is mediated solely by light which is perceived by the compound eyes.

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.

SummaryWe report the larval CNS expression patterns for 6,650 GAL4 lines based on cis-regulatory regions (CRMs) from the Drosophila genome. Adult CNS expression patterns were previously reported for this collection, thereby providing a unique resource for determining the origins of adult cells. An illustrative example reveals the origin of the astrocyte-like glia of the ventral CNS. Besides larval neurons and glia, the larval CNS contains scattered lineages of immature, adult-specific neurons. Comparison of lineage expression within this large collection of CRMs provides insight into the codes used for designating neuronal types. The CRMs encode both dense and sparse patterns of lineage expression. There is little correlation between brain and thoracic lineages in patterns of sparse expression, but expression in the two regions is highly correlated in the dense mode. The optic lobes, by comparison, appear to use a different set of genetic instructions in their development.

Adult emergence at the end of metamorphosis in the moth Manduca sexta is followed by the death of abdominal interneurons and motoneurons. Abdominal ganglia removed from insects before this period of naturally occurring cell death and maintained in vitro showed neuronal death confined to the same cells that normally die in vivo. Addition of physiological levels of the steroid 20-hydroxyecdysone to the culture system prevented the selective death of these motoneurons.

In the tobacco hornworm, many larval motoneurons become respecified and supply new muscles in the adult. Changes in the morphology of one such neuron were examined through metamorphosis. The dendritic pattern of the adult comes about both by outgrowth from the primary and secondary branches of the larval neuron and by the development of new branches that are unique to the adult.

An adult moth sheds its pupal skin only during a specific period of the day. The brain is necessary for the synchronization of this behavior with the environmental photoperiod. This function is fully preserved when all the brain's nervous connections are severed or when a "loose" brain is transplanted into the tip of the abdomen. By appropriate experiments it was possible to show that the entire mechanism is brain-centered. The components include a photoreceptor mechanism, a clock, and a neuroendocrine output. The clock-controlled release of the hormone acts on the central nervous system to trigger a species-specific behavior pattern which culminates in ecdysis.

The emergence of the adult cecropia silk moth from the pupal skin involves a stereotyped series of abdominal movements-the pre-eclosion behavior. This behavior, triggered by a neurosecretory hormone, consists of three phases that are characterized by the relative frequency and pattern of movements. Electrical recordings from a nerve cord with severed peripheral nerves demonstrate that the pre-eclosion behavior is prepatterned in the abdominal ganglia. In response to the hormone, the entire 1.25-hour behavioral program can be activated and "read off" in the absence of sensory feedback.