J . Joosse, R.M. Buijs and F.J.H. Tilders (Eds.) Progress i n Brain Research, Vol. 92 0 1992 Elsevier Science Publishers B.V. All rights reserved.

361 CHAPTER 30

The eclosion hormone system of insects James W. Truman Department of Zoology, University of Washington, Seattle, WA 98195, U.S.A.

Introduction

Peptides and the neurons that produce them play key roles in regulating physiology, development and behavior. These peptidergic systems range through a broad spectrum in their complexity on the cellular, the molecular, and the functional levels. At one end of the spectrum are peptide genes such as the proopiomelanocortin gene in vertebrates (Nakanishi et al., 1979) or the genes for FMRFamide (Schaefer et al., 1985; Nambu et al., 1988; Schneider and Taghert, 1988) and egg-laying hormone (ELH; Scheller et al., 1983; Vreugdenhil et al., 1988) in invertebrates. The prohormones encoded by these genes are processed to yield a number of biologically active peptides, the spectrum of which may vary amongst cells (see Sossin et al., 1989 for a review). These systems are complex on a cellular level with the gene being expressed in a diverse set of neurons distributed throughout the CNS. In some cases, such as the control of egg laying in molluscs, it is possible to relate the complexity of the peptide products to the diverse cellular and neuronal responses that accompany the behavior (Mayeri and Rothman, 1985; Ter Maat, 1992). In other complex systems such as the FMRFamide family, a unitary theme has not yet emerged to account for either the variety of products or for the cellular distribution. Indeed, such a unitary theme may not exist. The ecolosion hormone (EH) system of insects, which is the topic of this review, stands at the other end of the spectrum. The functional role of the E H

system is very circumscribed - its only known function is to coordinate the physiological and behavioral processes necessary for shedding the old exoskeleton at the end of a molt, a process termed ecdysis (Reynolds, 1980). The behaviors displayed at ecdysis are highly specialized and are used at no other time. Indeed, in many insects the neurons and muscles that are associated with these behaviors degenerate after the final ecdysis to the adult stage. Associated with this functional simplicity, the E H system is also simplified on both the cellular and molecular levels. In terms of neuronal distribution, this peptide is expressed by only 2 to 4 neurons in the CNS. At the molecular level, E H is the only product of the eclosion hormone gene (Horodyski et al., 1989). Because of this extreme simplicity, the E H system readily illustrates ways by which various features of a peptidergic system are adapted to the physiology of the animal. Eclosion hormone

The first indication for the hormonal control of ecdysis behavior came from studies on the circadian control of adult ecdysis in giant silkmoths (Truman and Riddiford, 1970). Experiments involving brain removal and reimplantation showed that the brain was required for the proper form and timing of the behavior but that this control could be exerted even when the brain was transplanted to the abdomen. Thus, the ecdysis system could be dissociated into two parts - a brain-centered component containing

362

photoreceptors and a circadian clock, and a component in the ventral ganglia comprised of the effector circuits for the behavior. The effectiveness of the implanted brain suggested that the first component could act on the latter through a circulating factor. The presence of such a factor with ecdysis-stimulating activity was demonstrated in the brain of the moth (Truman and Riddiford, 1970) and also in the blood of animals in the process of ecdysis (Truman, 1973). This material was dubbed eclosion hormone (EH) because of its association with the adult ecdysis (given the specialized term of “eclosion”) but it later became clear that this hormone was involved in triggering the ecdyses of all stages (Truman et al., 1981). E H proved to be a peptide and its amino acid sequence was determined for two moths: the tobacco hornworm, Manduca sexta, (Fig. 1; Marti et al., 1987; Kataokaet al., 1987; Terziet al., 1988)and the commercial silkworm, Bombyx mori (Kono et al., 1987; 1991). The EHs from both species are unblocked peptides comprised of 62 amino acids with 3 internal disulphide bridges (Kono et al., 1990a). The positions of the cysteine bridges are conserved for the 2 species and the overall sequence identity is 80%. The EHs show no significant sequence similarity to any other known peptides. Based on the amino acid sequence of EH, a unique 72-nucleotide probe was made and used to isolate the E H gene from a Manduca genomic library (Horodyski et al., 1989). Subsequent analysis of genomic and cDNA clones showed that E H is made by a single copy gene that is comprised of 3 exons which extend over 7.8 kb of DNA (Fig. 1). The mature mRNA is about 0.8 kb in size and codes for an 88 amino acid “preEH” consisting of a 26-amino-acid signal sequence followed by a single copy of EH. Thus, in contrast to other insect neuropeptides that have been cloned to date (e.g. Nambu et al., 1988; Schulz-Aellen et al., 1989; Iwami et al., 1989; Kawakami et al., 1990), the E H gene codes for only a single secretion product and the hormone is already in its mature form once the signal peptide is removed in the endoplasmic reticulum. The size of E H and its internal disulphide bridges have made it difficult to synthesize the hormone by conventional means. However, the cloning of the

E H gene made it possible to produce synthetic material using recombinant DNA techniques. This has been accomplished in a bacculovirus system for the E H fromManduca (Eldridge et al., 1991) and in bacterial systems (Kono et al., 1990a) for Bombyx EH. Besides providing material for physiological studies, the fact that the recombinant material showed full E H activity confirmed that the reported sequence was indeed that for EH. Tests of CNS extracts from a variety of insects on the Manduca pupal ecdysis assay showed that ecdysis-stimulating activity can be found widely throughout the insects (Truman et al., 1981). The cloning of the E H gene from Manduca provided a molecular avenue for rapidly determining the structure of these EHs from other species (Horodyski, Riddiford and Truman, unpublished, as cited in Trumanetal., 1991). OnestrategywastouseaDNA fragment that contains the Manduca E H coding region to identify cross-hybridizing clones from a Drosophila genomic library. Hybridization at low stringency resulted in isolation of a clone that contained the major portion of the Drosophila E H gene. This gene codes for a 0.8-kb mRNA and conceptual translation of the coding region revealed 69% identity to Manduca EH. Comparison of the structure of Drosophila E H with those from the moths showed a conservation of sequence in the regions of residues 14 - 21 and 49 - 57. Mixed synthetic oligonucleotides were made to these two regions and used as primers for the polymerase chain reaction on the DNA isolated from various insects. Amplification products of the appropriate size, that were recognized by an internal probe, were produced from the DNA from a number of insects including Acheta domestica, Aedes aegypti, Tenebrio molitor, and Bombyx mori. The products from Aedes and Tenebrio have been sequenced and shown to be homologous to the corresponding region of Manduca E H (F.M. Horodyski, L.M. Riddiford and J.W. Truman, unpublished). Comparison of the structures of the EHs from a variety of insects will eventually provide insight into the regions of the molecule that are important for biological activity.

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Fig. 1. The eclosion hormone (EH) system of the mothhfunducasexta. Left: the location of the 4 neurons that contain EH in the nervous system of the pupa. Their cell bodies lie in the ventromedial brain and their axons extend through the chain of ventral ganglia to release sites in the proctodeal nerves. Right: organization of the EH gene. The top line shows a restriction map of the portion of a genomic clone containing the 3 exons of the EH gene. Subsequent lines show the relationship of the mature mRNA, the EH precursor (PreEH), and EH. The amino acid sequence of EH is given below. For the mRNA, the numbers refer to the number of nucleotides in each exon; for preEH, the numbers refer to the amino acids contained in the signal sequence (ss) or EH. Restriction enzymes are: b, BgnI;E, EcoRI; H, HindlII; S , Sau3A. Redrawn from Horodyski et al. (1989).

The eclosion hormone neurons

EH activity is distributed throughout the CNS of prepupal Manduca. Surprisingly, this wide distribution of the peptide is due to only 4 neurons the cell bodies of which are located in the ventromedial region of the brain (Fig. 1; Truman and Copenhaver, 1989). Staining of these ventromedial (VM) cells with antibodies raised against EH show sparse

arborization in the brain. Their axons project the length of the ventral nervous system, exiting the CNS via the terminal nerve and projecting into the proctodeal nerve where they end in an extended neurohemal site. The anatomical projection of these brain neurons to the ventral CNS is consistent with the experimental findings that transection of the CNS at any level results in loss of EH activity caudal to the cut and accumulation on the rostra1 side

364

(Truman and Copenhaver, 1989). These four cells are also the only neurons in the CNS that have detectable levels of E H mRNA as revaled by in situ hybridization using anti-sense probes (Horodyski et al., 1989; L.M. Riddiford, unpublished). Although in immature stages the VM neurons only project to the proctodeal nerves, during the pupaladult transformation they extend axon collaterals to the corpora cardiaca-corpora allata complex (J.W. Truman, unpublished). Hence, at adult ecdysis they release E H from this anterior site as well as from the posterior proctodeal nerve. In Manduca a group of Iateral neurosecretory cells become immunoreactive to the EH polyclonal antiserum during metamorphosis (Copenhaver and Truman, 1986). In situ hybridization studies at various times during adult development fail to show E H mRNA in these cells (L.M. Riddiford, personal communication). Also, a monoclonal antibody directed against Bombyx E H recognizes in Bombyx only the 4 neurons that correspond to the VM cells of Manduca (Kono et al., 1990b). These data suggest that the immunostaining in the lateral cells of Manduca does not reflect the presence of EH but perhaps a peptide that shares a similar epitope to EH. The VM cells are adapted for a single, massive bout of EH release once during each molt -intermolt cycle. Bioassays of the hormone content in the proctodeal nerves show a 95% drop in stored E H activity during the 2- to 3-hour period that proceeds pupal ecdysis (Truman and Morton, 1990). E H immunostaining of the proctodeal nerve declines markedly at this time (Hewes and Truman, 1991) and at the ultrastructural level, there is a severe depletion of secretory granules in the terminals of the E H cells (P. Brunner, J.S. Edwards and J.W. Truman, unpublished). Studies monitoring the rate of peptide appearance in partially dissected pupae suggest that the bulk of this depletion occurs over a span of about 30 min (Hewes and Truman, 1991). After such a release episode, the VM cells begin to accumulate E H in preparation for the next molt and the release sites in the proctodeal nerve gradually become re-stocked with hormone. Neurons with a morphology similar to the ven-

tromedial cells have been described from a wide variety of insects (the M3 neurons of Panov, 1983). The cautery of these neurons in dragonflies results in the permanent blockage of ecdysis behavior (Charlet and Schaller, 1976). Immunocytochemical studies using antibodies against Manduca E H show EH-immunoreactivity in the corresponding cells of both crickets and Drosophila although in the latter there is only one pair of cells rather than 2 (J.W. Truman and R.S. Hewes, unpublished). The VM cells in the fly have also been shown t o contain E H mRNA (L.M. Riddiford, F.M. Horodyski and J.W. Truman, unpublished). Thus, these cells appear to have been associated with E H and the control of ecdysis throughout most of insect evolution.

Coordination of the eclosion hormone system A successful ecdysis requires that EH release occurs at a precise phase of the molt cycle. For larval and pupal molts in Manduca ecdysis is linked to the developmental time table of the animal, occurring a fixed number of hours after the molt is initiated by the actions of the prothoracicotropic hormone and the ecdysteroids (Truman, 1972). For adult ecdysis there is an additional input from a circadian clock as well as the developmental influences. The developmental cue appears to be supplied by the steroid hormones that drive the molt - the ecdysteroids (Truman et al., 1983). Each release of E H is preceeded by a major release of ecdysteroids (Fig. 2). Interestingly, the cue for E H release is not the time of appearance of the steroid but rather the time that the ecdysteroid titer subsequently declines. Thus, if the steroid withdrawal in delayed by an injection of 20hydroxyecdysone (20-HE) during the phase of ecdysteroid decline, then there is a dose-dependent delay in the time of the subsequent ecdysis (Truman et al., 1983). In the case of larval and pupal ecdyses the magnitude of the delay is a continuous function of dosage injected whereas for adult ecdysis the delay is discontinuous with the animals shifting from one circadian gate to the next (Fig. 2). This saltatory response at adult ecdysis reflects the added influence of a circadian clock to control this par-

365

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Fig. 2. Relationship of the titer of circulating ecdysteroids to the timing of ecdysis. Top: relative titer of ecdysteroids through the last two-thirds of the life history of Munducu sextu showing the major steroid peaks that cause the molts to the 5th stage larva (5th L), the pupa and the adult. ecd, ecdysis of the respective stages; inset shows structure of 20-hydroxyecdysone (20-HE). Arrows show the times that 20-HE was administered to delay ecdysis. Bottom: an expanded time scale around the time of ecdysis of the pupa (left) and the adult (right) showing the ability of injections of 20-HE (at arrow) to delay the subsequent ecdysis in a dose-dependent fashion. The timing of pupal ecdysis is referenced to the number of hours after the start of tanning of a set of dorsal metathoracic bars in the prepupa; that of the adult is referenced to the developmental age of the animal and the ambient light-dark cycle (bar under the time-line represents darkness). Numbers refer to the dosage of 20-HE in yg/animal. Data from Truman et al., 1983.

ticular ecdysis. Thus, the hormones that cause molting also act on the EH system to insure EH release and ecdysis at the end of the molt. They d o so by acting on both the release and the response sides of the system. Coordination of EH release

The successive delays in ecdysis seen after steroid treatment reflect delays in the time of EH release (Truman et al., 1983). In the case of adult ecdysis, examination of EH blood titers in insects treated

with 20-HE during the ecdysteroid decline shows that EH fails to appear during its scheduled circadian gate but then is released about 20 h later when the next gate opens. This effect of ecdysteroids is apparently through a direct action on the brain since ecdysis in debrained animals with implanted brains is normal with respect to both circadian time and in its coordination with the events of the molt cycle. In the case of pupal ecdysis, injections of 20-HE up to about 8 h prior to the normal time of EH release were effective in blocking or delaying release but after this time steroid treatment was without effect.

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It is not known what in the brain changes in response to steroid withdrawal and how these changes then result in the massive bout of secretion about 7 - 8 h later. When Manduca at various stages are caused to ecdyse prematurely by injection of exogenous EH, the VM cells to not then release their store of peptide (e.g. Truman, 1978a). Thus, EH release appears to be inhibited by a feedback action of itself, although it is not known whether this is a direct action of EH on the VM cells. Once the release system has been shut off, it must go through a bout of steroid exposure before it is again competent to release EH. Coordination of EH responsiveness

Besides showing a highly restricted time of EH release, insects are correspondingly restricted in the times that they are responsive to EH (Truman et al., 1983; Morton and Truman, 1986,1988a). For example, Manduca are responsive to the peptide only during a very narrow temporal window that immediately preceeds the time of EH release. The timing of this response window is also a function of the steroid titer and treatment with 20-HE delays or blocks the onset of responsiveness. The relationship between ecdysteroids and EH sensitivity has been most extensively studied for pupal ecdysis in Manduca. During the 24 h preceeding pupal ecdysis, the insect experiences a declining titer of ecdysteroids. These animals become responsive to EH treatment late in the decline at about 8 h before the expected ecdysis (Truman et al., 1983; Morton and Truman, 1988b). When Manduca are given a large dose of 20-HE 24 h prior to ecdysis, then challenged with EH about 24 h later, they do not respond to the peptide. If the same dosage of 20-HE is given at progressively later times, it continues to be effective in delaying responsiveness until about 13 h before ecdysis, after which the treatment is ineffective. These data suggest that the steroid decline triggers some event at about - 13 h which results in the subsequent appearance of EH responsiveness at - 8 h. Ecdysteroids appear to make the CNS responsive to EH by inducing some of the components of the

biochemical cascade that mediates EH action. The steps thought to be involved in the action of EH are summarized in Fig. 3. EH exposure results in a rapid elevation in the levels of guanosine 3’,5’ cyclic monophosphate (cGMP; Morton and Truman, 1985). The latter, working through a cGMP-dependent protein kinase, then causes the phosphorylation of two endogenous phosphoprotein substrates, the EGPs (Morton and Truman, 1988a). The EGPs are associated with the membrane fraction of the CNS but their nature and the manner by which their phosphorylation relates to the behavioral responses are unknown. Studies of the steps involved in EH action have provided some insight into how ecdysteroids “prime” the CNS to respond t o EH. The nervous system of an intermolt last-stage larva of Manduca lacks at least 2 components of the EH response pathway (Fig. 3). No EGPs can be detected (Morton and Truman, 1986,1988a). Nor does challenge with EH result in any change in levels of cGMP (Morton and Truman, 1985). Thus there seem to be deficits at both ends of the pathway - at the level of coupling EH reception with the elevation of cGMP levels and at the level of the presumed output proteins that are modified by this system. When nervous systems are challenged with EH at progressively later times through the intermolt period and into the early stages of the molt to the pupal stage, the first time that EH induces a response in the cGMP system is late in the molt period, at about 24 h before the expected time of ecdysis, at a time that the ecdysteroid titer is starting its decline (Morton and Truman, 1985). The development of this biochemical responsiveness is blocked by preventing the prepupal peak of ecdysteroids but it can be subsequently induced by infusion of 20-HE. Interestingly, the biochemical responsiveness appears even when the infusion is prolonged and the steroid titers remain high. The changes that occur to allow EH to stimulate an increase in cGMP are not known. The simplest hypothesis is that it represents the appearance of E H receptors but this possibility has yet to be tested. Importantly, both the timing of the onset of biochemical responsiveness and its endocrine re-

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quirements are different from those seen for behavioral responsiveness. Thus, although the coupling of EH reception with the second messenger system is an essential step in rendering the CNS behaviorally responsive to EH, it is not sufficient and other factors in the cascade must be examined. The levels of the cGMP dependent protein kinase do not change during this responsive period but the levels of their substrate change dramatically. EGPs were not detected during most of the molt and intermolt period and first became apparent in the CNS of Manduca at about 8 h prior to pupal ecdysis, the L A S T L A R V A L ECDYSIS

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time when behavioral responsiveness also appears (Morton and Truman, 1988a). A similar correlation is seen for ecdysis to the fifth larval stage. The appearance of the EGPs is linked to ecdysteroids but requires their withdrawal (Morton and Truman, 1988b). This is most convincingly seen in nervous systems from - 24 h prepupae that are maintained in organ culture for 24 h in the presence or absence of physiological levels of 20-HE. The EGPs are absent at the onset of the culture period and continue to be absent if the CNS is maintained in the presence of 20-HE. By contrast, matched nervous systems PUPAL I

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Fig. 3. Relationship of the ecdysyteroid titer to the assembly of the cascade that mediates EH action. Top: relative fluctuations in ecdysteroids and EH titers during the larval-pupal transition. The two ecdysteroid peaks are the commitment and prepupal peaks, respectively. Bottom: a model showing the steps thought to be involved in EH action on its target cells (right). The notched box on the neuronal membrane represents a hypothetical EH receptor. Binding of EH to its receptor results in the activation of guanylate cyclase (GC) and the formation of cyclic GMP (cGMP) which then activates a cGMP-dependent protein kinase (gPK). The active kinase (gPKa) then phosphorylates a set of endogenous proteins (the EGPs). The manner by which the phosphorylation of the EGPs relates to the activation of target neurons is unknown. Left: during the intermolt period, target cells apparently possess only the GC and gPK,. EH receptors are then thought to be added in response to the prepupal peak of ecdysteroids but the subsequent withdrawal of steroid is necessary for the induction of the EGPs and the resulting establishment of a fully responsive system.

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sequence in order for the animal to accomplish a specific goal. A good example of this ordering is provided by molluscs such as Lymnaea stagnalis (Ter Maat, 1992) and Aplysia (Strumwasser et al., 1980) in which peptides trigger a series of behaviors which eventually result in the deposition of an egg mass. EH also elicits a sequence of behaviors which enable the insect to escape from the old cuticle and to expand its new exoskeleton. The duration of this behavioral sequence is a number of hours, a duration that outlasts the presence of the hormonal signal that was the trigger. Some of the most extensive behavioral analyses have been carried out for adult ecdysis of the Cecropia silkmoth, Hyalophora cecropia (Truman, 1971, 1978b). As illustrated in Fig. 4, EH triggers a sequence of 3 discrete behaviors: (1) the pre-eclosion behavior begins about 10- 15 min after EH treatment and involves abdominal movements that apparently loosen the connections between the old and the new cuticles. During the initial 30 min of this behavior, the animal shows frequent rotary movements of the abdomen followed by a 30 min period of relative or complete quiescence; (2) the eclosion behavior starts at about 75 - 90 min and brings about the actual shedding of the old cuticle. The principle movements are waves of peristalsis that move anteriorly up the abdomen. Each wave

maintained in vitro in the absence of 20-HE show an induction of the EGPs. In vivo experiments involving injection of 20-HE at various times prior to ecdysis show that steroid treatment blocks the appearance of the EGPs when given up to about 13 h prior to ecdysis - a similar cut-off to that for blocking behavioral responsiveness. Studies with drugs that block protein and RNA synthesis suggest that the appearance of the EGPs is due to their de novo synthesis (D.B. Morton and J.W. Truman, unpublished). There is no way at present to block the function of the EGPs, so it is not possible to directly test their involvement in inducing ecdysis behavior. However, the close correlation between the presence of these proteins and the responsive state suggests that the two are intimately related. Thus the behavioral context of the EH system is established by using the steroid titer as a common cue for both the release side and the response side. This reliance on a common cue insures that the massive release of EH occurs when the animal is in a condition that it can successfully respond to it.

EH system and the temporal aspect of the behavioral response In a behavioral context, hormones often trigger a sequence of behaviors that must occur in a stereotyped

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Fig. 4. The sequence of behaviors shown by pharate adult Hyalophora cecropia in response to an injection of eclosion hormone (EH). The drawings show the behavior of the insect during various phases of the response. The bottom shows a record of the abdominal movements of an animal injected with EH 10 min before the start of the recording and lasting through the periods of the pre-eclosion and eclosion behaviors. Complete excursions of the trace during the first phase were due to rotary movements of the abdomen; the high frequency movements at the end were eclosion movements. The record ends when the animal shed the pupal cuticle.

terminates with a vigorous flexing of the wing bases accompanied by a pronounced extension of the abdomen; (3) the wing inflation behavior begins at about 120 min. It also has a stereotyped duration and involves a tonic abdominal contraction, to aid in the movement of blood into the thorax and wings, associated with a stereotyped series of wing movements. The relationship of E H to the overall sequence is best illustrated by the first two behaviors, the preeclosion and eclosion behaviors. The system can be simplified because abdomens isolated from animals prior to adult ecdysis do not then show spontaneous ecdysis behavior but can be induced to show the preeclosion and eclosion behaviors by EH injection (Truman, 1971,1978b). The timing and form of the behaviors are like in the intact animal except that during the eclosion phase the abdomens show only the peristaltic waves and not the abdominal extensions that are driven from thoracic centers (e.g. Mesce and Truman, 1988). The ability of isolated abdomens to give a coordinated response to EH showed that the abdominal CNS contains the requisite neuronal circuits for the behavioral response. The system was further simplified by reducing it to the isolated abdominal CNS (Truman, 1978b). In response to EH, the abdominal CNS generates a program of spontaneous motor activity that mimics both spatially and temporally that seen during the pre-eclosion and eclosion behaviors (Fig. 5). Motor bursts typically begin about 20 min after addition of EH to the bath. Bouts of bursts having a rotary pattern occur at relatively high frequency for the first 30-40 min but then the interval between bouts lengthens as the nervous system enters its quiet phase. After an interval of comparable length to the active period, the quiescence is terminated and bursting spontaneously resumes but these new bursts have the peristaltic patterning characteristic of eclosion. Eclosion bursts may then continue for well over an hour. The times of ecdysis occur at unique moments in the life of the insect when it is covered by two cuticles - the new cuticle that it has just produced and the old cuticle to be shed. The isolated CNS experiments

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