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Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up , the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a “gateway cell”, whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline.

Eclosion hormone (EH) was isolated from 1 500 pharate adult heads of oak silkmoth, An-theraea pernyi. The crude EH which weight 420 mg was obtained by 8 steps. The A. pernyi bioassay was adopted to test biological activity of crude EH, and total EH activity was about 750 EH units. The specific activity was approximately 560 μg/unit. The crude EH was inactivated by proteolytic enzymes. It was stable in heat and had non species-specific.

Wang, Y. J., L. Yurttas, B. E. Dale, D. H. Russell, G. Kinsel, L. M. Preston-Schaffter, V. Johnson and T. K. Hayes. MALDI-MS as a monitor of the purification and folding of synthetic eclosion hormone. Peptides 18(3) 337–346, 1997.—Analogues of the small protein Manduca sexta eclosion hormone (62 amino acids) were synthesized by Fmoc solid-phase methodology. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to analyze the products of the syntheses and this information was used to design an efficient purification scheme. MALDI-MS was used to monitor the target products through purification and it was also used to monitor folding of the purified materials. The folded EH analogues were shown to be biologically active proteins with an in vivo bioassay using pharate adult moths, Heliothis virescens.

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identifieid, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown. © 2011 Elsevier Ltd., Additional funds for transcriptome studies that supported the annotation were from a University of Utah SEED grant and equipment funds from the US National Science Foundation (0820985; to Richard M. Clark).

Six neuropeptides of the silkworm, Bombyx mori, have been isolated and chemically characterized during the past 10 years. They are bombyxin, prothoracicotropic hormone, pheromone-biosynthesis-activating neuropeptide/melanization-and-reddish-coloration hormone, diapause hormone, eclosion hormone, and adipokinetic hormone. Recent progress in research on these neuropeptides is described.

SUMMARYLight signals can entrain circadian clocks, but they can also mask aspects of the circadian output. We have analyzed the masking effects of a lights-on(LOn) signal on Drosophila eclosion. The LOn response results in 12–21% of the flies that emerge on a given day eclosing within 10 min of the LOn signal. Flies that lack the neuropeptide eclosion hormone (EH), or in which its release is inhibited by the tetanus toxin light chain, lack the response. Optic photoreceptors in both the ocelli and the compound eyes appear to be required for the response. The LOn signal has two effects: (1) it drastically reduces the interval between EH release and eclosion, presumably by suppressing a transient descending inhibition that immediately follows EH release, and (2) it stimulates premature EH release. The LOn signal does not influence the latency of wing spreading, an EH-regulated post-ecdysis behavior.

エリサン潜成虫および成虫頭部には,カイコガ潜成虫の羽化を誘導する羽化ホルモン活性が存在し,それぞれの羽化ホルモン力価は潜成虫で5.8 unit/head,成虫頭部で2.1 unit/headであった。また,エリサン潜成虫および成虫頭部の羽化ホルモンについて部分精製を行い化学的性状を調べた結果,それぞれの羽化ホルモンは熱に安定な分子量8,500±1,000のペプタイドであることが判明した。これらの結果から,羽化ホルモンはその生理作用において,両種間で種特異的でないものと判断された。

The extremely large number of insects and members of allied groups alive today suggests that molting--shedding of an old cuticle--may be one of the most commonly performed behaviors on our planet. Removal of an old cuticle in insects is associated with stereotyped, species-specific patterns of behavior referred to as ecdysis. It has been recognized for decades that the initiation of ecdysis is under hormonal control, but until recently many of the key peptides that regulate ecdysis were unknown. The report in 1996 of a new ecdysis-triggering hormone (ETH) sparked an era of significant advances in our understanding of the regulation of molting. This article summarizes the current model of peptide regulation of ecdysis, a model that is based on a positive feedback loop between ETH and a brain peptide, eclosion hormone. Then the relationship of these regulatory peptides to the neural circuitry that is the ultimate driver of the behavior are described. Because insects can undergo both status quo (larval-larval) and metamorphic (larval-pupal and pupal-adult) molts, differences in ecdysis behavior at different life stages are described and potential sources of these differences are identified. Most of the work described is based on studies of ecdysis in the hawkmoth, Manduca sexta, but results from studies of ecdysis in the fruit fly Drosophila melanogaster are also discussed.

A gene encoding eclosion hormone (EH) from the silkworm, Bombyx mori was chemically synthesized, inserted into a secretion vector and expressed in Escherichia coli, leading to the production of biologically active EH. Sequence analysis of cystine-containing peptides in a thermolysin digest of this EH established the locations of 3 disulfide bonds in the molecule. Evidence was also obtained that the 6 residues at the NH2-terminal are dispensable but 4 residues at the COOH-terminal play an important role in EH activity.

The eclosion hormone (EH) activities which can induce the precocious adult eclosion of the pharate-adults of Bombyx mori or Samia cynthia ricini were present in the adult heads of Pseudaletia separata, Bombyx mori, Samia cynthia ricini, Antheraea pernyi, Antheraea yamamai and Manduca sexta. These results indicated that EH is not a species-specific hormone among the lepidopteran insects.

Eclosion hormone (EH) is a neuropeptide hormone which controls the ecdysis behavior in insect. The three dimensional structure of the N-terminal fragment (1–34) of the eclosion hormone which was predicted to contain a compact region crucial for the EH activity was studied in 50% d 3 -trifluoroethanol(TFE)/50% H 2 O at pH 3 and 298 K by 1 H NMR spectroscopy with the combined use of distance geometry and molecular dynamics calculations. NMR results indicated that the fragment actually assumes an α-helix between Ala10 and Gln20, but no rigid structure is present from Cys21 through the C-terminus and for the N-terminal region (Ser1–Asp9). The elucidated structure was compared with the predicted structure of the native EH for the further development of the design of the insecticide.

The process by which a butterfly emerges from a chrysalis, called ecdysis, has been thought to be controlled by eclosion hormone. J. Truman discusses the discovery of a new insect hormone, reported in this issue of Science by hacek Zithacek nan et al. (p. 88), that can also trigger ecdysis—MasETH. The two hormones seem to act sequentially.

Publisher Summary Peptides and the neurons that produce them play key roles in regulating physiology, development, and behavior of insects. This chapter describes the eclosion hormone (EH) system of insects, which coordinates the physiological and behavioral processes necessary for shedding the old exoskeleton at the end of a molt. Circulating EH has only been found just preceding the times of ecdysis, and these are the only times, when measurable EH depletions occur in the neurohemal storage sites. The nature and context of the behaviors triggered by EH are doubtlessly responsible for some of the extreme modifications of the EH system. The performance of ecdysis behavior only makes sense when an insect is covered by an old cuticle that must be shed. The appearance of EH is rapidly followed by the inactivation of the system on both the release and response sides. Both sides of the system are only then reconstituted in response to the steroid cues that also provide the physiological context for the next bout of ecdysis.

Adult eclosion of the silkworm, Bombyx mori, occurs during a specific period of the day under the control of eclosion hormone (EH). Bombyx EH was extracted from the pupal and adult brains (Fugo, et al., 1982), and also the embryos of B. mori (Chen, et al., 1986). In 1985, Bombyx EH was first isolated from the pharate adult heads of B. mori (Nagasawa, et al., 1985). Recently the entire amino acid sequence of Bombyx EH was determined as shown in Fig. 1 through the combination of sequencing the isolated Bombyx EH (Kono, et al., 1987) and cloning the Bombyx EH gene. A monoclonal antibody against the synthetic peptide corresponding to the carboxyl-terminal 13 amino acid residues of Bombyx EH was prepared (Kono, et al., in press).

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 wings of pharate adult tobacco hornworm moths, Manduca sexta, are relatively inextensible until 3 or 4 h before emergence from the pupal case. At this time the wing cuticle becomes plasticized, so that by the time of eclosion, the wings are readily extensible. This change in the mechanical properties of the wing cuticle is shown to be under the control of a factor from the head. This factor is present in the corpora cardiaca/corpora allata complex, and in the protocerebrum of the brain, being released into the blood prior to eclosion. It is able to act directly on isolated wings. The active principle was found to be indistinguishable in a number of ways from the hormone which triggers emergence from the pupal case, the eclosion hormone. Partial purification of the eclosion hormone failed to separate activity causing eclosion from activity causing wing cuticle plasticization. It is concluded that the same hormone is probably responsible for both effects. The cuticle plasticizing activity of the eclosion hormone forms the basis for a new, highly sensitive bioassay. Another factor, distinct from the eclosion hormone, is able to cause wing cuticle plasticization. This factor is found in the abdominal nerve cord, and is only released into the blood after eclosion has occurred. It is probably identical with the tanning hormone, bursicon, which is released at this time. The factor in the nerve cord which causes cuticle plasticization is indistinguishable from bursicon in a number of ways, including partial purification by gel filtration. Bursicon evidently causes a further increase in wing cuticle extensibility after eclosion, at the time of wing inflation.

Eclosion hormone was purified 5,000-fold from the extracts of male adult heads of the silkworm, Bombyx mori. The fourteen-step purification procedure consisting of solvent extractions, fractional precipitations and chromatographies afforded a partially purified preparation of eclosion hormone, 1.8 μg of which showed activity in a Bombyx pharate adult. The hormone was inactivated by proteolytic enzymes. The molecular weight of eclosion hormone was estimated to be 8,400 ± 1,000 daltons by gel-filtration on Sephadex G-50.

カイコガ潜成虫の羽化ホルモンに対する反応性と,20ヒドロキシエクダイソン(Ecd)投与の羽化におよぼす影響について検討し,以下の結果を得た。種々の発育段階の潜成虫に羽化ホルモンを投与し,早期羽化誘導率から羽化ホルモンに対する反応性を調べた結果,予想羽化時刻の約14∼12時間前ごろから羽化時刻に近づくにつれて,反応性は高くなることが判明した。予想羽化時刻の22時間前の潜成虫に,0.01∼10μgのEcdを注射しその後の羽化状況を調べた結果,0.01∼0.1μgの範囲では羽化日は対照区と変わらなかったが,0.5∼10μgの範囲では羽化の遅れがみられ,とくに5μg以上の注射区では対照区より2日遅れて羽化する個体が多くみられた。一方,予想羽化時刻8時間前に同様のEcd処理した個体では羽化の遅延はみられなかった。予想羽化時刻の20時間前にEcdを5μg投与された個体は,羽化ホルモンに対する反応性が低下していた。

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.

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.

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.

(1987). Molecular Heterogeneity of Eclosion Hormone in Adult Heads of the Silkworm, Bombyx mori. Agricultural and Biological Chemistry: Vol. 51, No. 6, pp. 1741-1743.

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 eclosion hormone triggers a stereotyped preprogrammed pattern of behavior in silk moths. The effects of the hormone were duplicated by the injection of dibutyryl adenosine 3', 5'-monophosphate, adenosine 3', 5'-monophosphate (cyclic AMP), or guanosine 3', 5'-monophosphate (cyclic GMP) into theophylline-treated pharate moths. Treatment with theophylline reduced the latency of the response to a low dose of hormone, presumably by blocking phosphodiesterase. Endogenous levels of cyclic AMP, but not cyclic GMP, increased significantly in the central nervous system within 10 minutes after hormone injection. We conclude that an early step leading to the release of the eclosion motor program is an increase in cyclic AMP in target neurons of the central nervous system.

The neurosecretory system (NSS) of the insect brain controls numerous processes of life activity. At present it is already known to produce at least six different hormonal substances: hyperglycemic, diuretic, prothoracotropic, as well as neurohormone D, bursicon, and eclosion hormone. This has produced a great interest in the complexity of the cell composition of the brain NSS and in the elucidation of its origin during onto- and phylogenesis. However, in spite of an intense study of brain NSS during the last 25 years, these problems still remain inadequately investigated. The qualitative composition of the brain neurosecretory cells (NSC) has not yet been studied in about a half the orders of insects.

The isolated chain of four abdominal ganglia of Hyalophora cecropia has previously been shown to respond to the eclosion hormone by generating a pre-eclosion motor programme followed by the eclosion programme. Abdominal nervous systems reduced at any two ganglia could still generate both programmes. Selective addition of hormone to individual ganglia of the intact abdominal CNS resulted in the coordinated performance of both programmes by the entire CNS. In the eclosion programme, burst frequency was little influenced by which ganglion was exposed to hormone but in many cases the eclosion waves were initiated by the neurones in the treated ganglion rather than at the normal site in the terminal ganglion. When ganglia were removed from hormone-treated preparations during eclosion, the remainder of the chain showed a transient disruption in the bursting frequency but the frequency often eventually returned to pre-cut levels. Removal of anterior ganglia had no effect on the patterning of posterior bursts but removal of posterior ganglia resulted in a permanent reduction in the length of eclosion bursts in anterior ganglia. It was concluded that the information for both the pre-eclosion and eclosion programmes is patterned into each ganglion of the abdominal chain. For the pre-eclosion behaviour, the endogenous programme becomes pro-gressively longer in more posterior ganglia and each ganglion appears to have mutually exciting interactions with the other ganglia in the chain. In the intact animal the most anterior ganglion appears to drive the behaviour. By contrast, the eclosion behaviour also has segmental burst generators but they appear very similar in their properties. Each burst generator has an excitatory input on to the next anterior ganglion and inhibitory influences on posterior ganglia. Normally, the most posterior ganglion serves as driver for the eclo-sion behaviour.

1. In the tobacco hornworm, wing spreading involves a stereotyped series of movements which take 74 min to complete and which include two components, wing folding and wing inflation. 2. Moths decapitated at the moment of eclosion show neither wing inflation nor wing folding. If decapitation is delayed until 5 sec after emergence, then the full wing-spreading behaviour is displayed. 3. Newly emerged moths which are confined show intense digging-behaviour and delay the onset of wing spreading until after their release. Decapitated moths attempt to spread their wings immediately, regardless of whether or not they are confined. 4. Surgical experiments showed that the brain was not required in a neural capacity in order for wing spreading to occur; it was needed only as a source of the eclosion hormone. The neural influence of the suboesophageal ganglion was required until immediately after eclosion. 5. Newly emerged moths whose abdomens had been removed showed wing-folding behaviour. No inflation occurred and the duration of wing folding was much longer than normal. It was concluded that wing-folding behaviour was centrally programmed but that the abdomen could modify the length of the programme. 6. Injections of bursicon into abdomenless moths reduced the duration of wing-folding behaviour to almost normal levels. Therefore, in some manner, bursicon has an important role in determining when wing-folding behaviour will come to an end.

Publisher Summary This chapter discusses the role of the eclosion hormone in coordinating ecdysial events in insects. It explains two ecdysial events: adult eclosion and pupal ecdysis in Lepidoptera. It focuses on the hormone that triggers ecdysis and the central role of this hormone in coordinating the physiological and behavioral events that occur in ecdysis. The eclosion hormone activity is found in insect orders besides the Lepidoptera, but the chemical similarity of the hormones from other orders with the moth hormone is yet to be explored. The eclosion hormone is produced in two endocrine centers. Only a series of comparative studies reveal whether the choice of release site depends solely on the circadian involvement in the release or whether other factors are also important. In its action of coordinating the events of ecdysis, the hormone acts on various target tissues including the central nervous system, muscles, and epidermis in the adult. The dermal glands may also be targets in earlier stages.

Publisher Summary This chapter presents a review that aims to bring together what is known of the physiology of ecdysis with what is known of the behavior of ecdysing insects and shows how one is integrated with the other. Ecdysial behavior is probably largely programmed within the CNS, but once initiated the details of its performance may be more or less influenced by sensory feedback according to the species, and which part of the behavior is being considered. The exceptional stereotypy of the abdominal movements involved in the preeclosion and eclosion behavior of H. cecropia and the confinement of eclosion to a narrow, temporal “gate” by a circadian clock in the brain are key factors that led to the discovery of the role of the eclosion hormone in initiating eclosion behavior. The chapter provides evidence that silkmoth eclosion behavior is initiated directly by eclosion hormone. H. cecropia and A. pernyi emerge from the pupal cuticle only at a particular time of day, during relatively narrow gates determined by a circadian clock. If the pharate adults are removed from their pupal cases during the few hours before the normal eclosion gate, then they continue to show predominantly pupal behavior. In the case of A .pernyi , this is particularly striking, the peeled moth showing very little spontaneous motor activity at all. However, at the time of the normal eclosion gate, the moths go through a pantomime emergence, despite having been free of the pupal case for several hours.