The role of eclosion hormone in the larval ecdyses of Manduca sexta

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.     

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

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

Hormonal control of larval coloration in the armyworm, Leucania separata

Leucania separata larvae show various degrees of darkening depending on the population density. A ligature applied behind the thorax of crowded or yellow solitary larvae caused black or reddish brown pigmentation in the anterior part after the larval ecdysis. Extirpation of the brain, the corpus cardiacumcorpus allatum complexes, or the suboesophageal ganglion reduced the degree of melanization in the crowded larvae, lack of the suboesophageal ganglion having a particularly striking effect. Transplantation of 3 complexes of brain-corpora cardiaca-corpora allata-suboesophageal ganglion induced intense black pigmentation in the isolated abdomens of crowded larvae and reddish brown pigmentation with some melanization in the isolated abdomens of yellow solitary larvae, though the melanization in the latter was weaker than in the former. Implantation of these organs or of the suboesophageal ganglia into yellow solitary larvae caused black and reddish brown pigmentation after a larval ecdysis. In the pieces of integument implanted into the body cavity of crowded larvae, melanization occurred after ecdysis, whereas it did not occur in most of the fragments implanted in yellow solitary larvae. Transplantation of corpora allata and other organs from solitary larvae or injection of juvenile hormone into crowded larvae did not inhibit melanization.     

Temporal organization of endocrine events in relation to the circadian clock during larval-pupal development in Samia cynthia ricini

The temporal organization of secretion of the prothoracicotropic hormone (PTTH) and ecdysone during larval-pupal development of Samia cynthia ricini was studied by ligations, with particular attention to the circadian control of the timing of hormone release. PTTH and ecdysone are required first for the induction of prodromes of pupation and again later for pupal-cuticle formation. PTTH release in the first step occurs during the second or third photophase after the last-larval ecdysis under a photoperiod of 12 hr light and 12 hr darkness and is thought to be under the control of a circadian clock. Ecdysone release follows 1.5 days later, i.e. during the scotophase that precedes the gut purge. In the second secretory step, PTTH is released 2 days after purging the gut, and ecdysone release follows 6 hr later. The PTTH release at this time occurs at a fixed time after the gut purge irrespective of light conditions, accounting for light insensitivity of the timing for pupal ecdysis. Possible mechanisms relating to the inconsistent association of a circadian clock with PTTH release, and those underlying the determination of timing of the gut purge are discussed.     

Neuropeptide-stimulated cyclic guanosine monophosphate immunoreactivity in the neurosecretory terminals of a neurohemal organ

The neuropeptide eclosion hormone acts on the nervous system of the tobacco hornworm, Manduca sexta, to increase cyclic guanosine monophosphate (cGMP) levels. In this study I describe the localization of some of the sites where these increases occur. Prior to pupal ecdysis, eclosion hormone stimulates an increase in cGMP in a network of fibers in the transverse nerve of each abdominal ganglion. Double-label experiments with propidium iodide suggest that the cGMP immunoreactivity is primarily localized in neurosecretory nerve endings. The time course of the increase in cGMP immunoreactivity and its requirement for lipid metabolism is similar to that of the cGMP increase measured by radioimmunoassay. The cGMP response in the transverse nerve is stage-specific, occurring prior to pupal ecdysis and not prior to larval or adult ecdysis. © 1996 John Wiley & Sons, Inc.     

Metamorphosis of the ecdysis motor pattern in the hawkmoth, Manduca sexta

Summary The hawkmoth,Manduca sexta, under-goes 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.Abbreviations A(n) nth abdominal segment - DL dorsal longitudinal - EH eclosion hormone - ISMs intersegmental muscles - MN motoneuron - SEG subesophageal ganglion - T1,T2,T3 prothoracic, mesothoracic, and metathoracic ganglion - TSMs tergosternal muscles - TX thorax     

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Interaction Between Ecdysteroid, Eclosion Hormone, and Bursicon Titers inManduca sexta

SYNOPSIS. The end of the molting process in the tobacco hornwormincludes the rapid digestion of the old cuticle, molting fluidresorption, ecdysis of the old cuticle, and expansion and hardeningof the new cuticle. The coordination of these processes is accomplishedby three hormones. Each ecdysis during the life of Manduca appearsto be triggered by eclosion hormone. Depending on developmentalstage, the hormone comes either from the brain-corpora cardiacacomplex or from the chain of ventral ganglia. The neural programstriggered by eclosion hormone include a neuroendocrine event,the release of the tanning hormone, bursicon, thereby ensuringthat tanning of the new cuticle must follow ecdysis. Ecdysis,itself, appears to be controlled by the ecdysteroid levels sinceecdysteroid injections delay ecdysis at physiological concentrationsand in a dose dependent fashion. This delay is due to inhibitionof eclosion hormone secretion and to the retardation of theterminal phases of the molt including the digestion of the oldcuticle and the onset of sensitivity to eclosion hormone. Thus,eclosion hormone secretion and the ecdysis it triggers are coordinatedwith the end of development because both are influenced by thesame endocrine signal—the decline in the ecdysteroid titer.     

New Functions of Arthropod Bursicon: Inducing Deposition and Thickening of New Cuticle and Hemocyte Granulation in the Blue Crab,Callinectes sapidus

Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.     

Bursicon release and activity in haemolymph during metamorphosis of the cockroach,Leucophaea maderae

Bursicon activity first appears in the haemolymph of the cockroach, Leucophaea maderae, early in ecdysis as the old cuticle splits and separates over the thorax. Hormonal activity reaches high levels in the haemolymph before ecdysis is complete and remains so for about 1·5 hr, with a gradual decline and disappearance by 3 hr. The sensory mechanism controlling bursicon release is located in the thorax and appears to be stimulated as the ecdysial split widens for emergence of the thorax. If the abdomen is isolated before this time no tanning of abdominal cuticle occurs, while the isolated thorax proceeds to tan. Therefore the thoracic ganglia seem to be a site of release for bursicon. Release of the hormone from abdominal and head ganglia may also occur after neural stimulation from the thoracic system. Bursicon activity was found in all ganglia of the central nervous system and the corpora cardiaca-allata complex. Removal of the old cuticle prior to the start of ecdysial behaviour does not result in tanning of the new cuticle. However, if the old cuticle is removed after the insect begins to swallow air in preparation for ecdysis, then the new cuticle tans. Mechanical prevention of ecdysis and later removal of the old cuticle also does not result in tanning of the new cuticle. Therefore, shedding of the old cuticle only activates the release of bursicon in conjunction with other normal ecdysial events.     

Effect of omeprazole-evoked hypergastrinemia on ultrastructure of enterochromaffin-like cells in the stomach of portacaval-shunted rats

Immunocytochemical staining of the nervous system of larva, pupa, and adult stage of Tenebrio molitor with anti-insulin serum demonstrated insulin-like peptides in the protocerebrum, corpora allata, and suboesophageal ganglion. During pupal development, marked changes in staining intensity of the protocerebral cells were detected. The staining pattern suggests release of insulin-like peptides early on day 0 and again on day 3 of the stadium. Injections of anti-insulin at these times caused significant delays in the timing of pupal/adult ecdysis. An immunoblot of haemolymph from day-3 pupae revealed a 6.5-kDa insulin-like molecule. These results suggest that the prothoracicotropic hormone of T. molitor is an insulin-like molecule.     

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of ～50–70 ng/ml at 3–4 hr and this level is maintained until day 5–6, the time of the head-critical period in Rhodnius. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of ～ 3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, ～ 30 ng/ml. Basal levels of ecdysteroids, ～ 15 ng/ml, were detectable in young adult males and females. A survey of haemolymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxy-ecdysone. For the large peak preceding larval-adult ecdysis, 20-hydroxy-ecdysone was the predominant hormone. These results indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rhodnius, one immediately following the blood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of moulting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rhodnius during moulting.     

Bursicon and the metabolism of tyrosine in the moulting cycle of Pieris larvae

In fourth instar larvae of Pieris brassicae the haemolymph tyrosine level begins to rise about 1 day before apolysis to reach a level about treble that in the middle of the instar. Between apolysis and ecdysis the haemolymph tyrosine level appears to decline, until just before ecdysis another steep rise occurs. About 30 min after ecdysis a steep decline starts, levelling off gradually until the level in the middle of the instar is restored.Bursicon assays show that this hormone operates in the haemolymph both during apolysis and after ecdysis; but during the actual ecdysis no bursicon activity can be demonstrated in the haemolymph.Indications have been found that the bursicon activity can restore itself spontaneously in the haemolymph of newly ecdysed larvae. This would suggest that during ecdysis a bursicon inhibitor of restricted life is operating.     

Bursicon in the mealworm, Tenebrio molitor L. and its role in the control of postecdysial tanning

Using the adult Calliphora bioassay, we found that the tanning hormone, bursicon, is present in the blood of pupal and adult Tenebrio only at the time of ecdysis, when it is released massively from the thoracic and abdominal central nervous system. The hormone's half life in the blood is short (about 1–2 h). Contrary to the findings of other workers, we could find no evidence for the presence of the hormone in the haemolymph during pharate adult development, before ecdysis begins. When newly ecdysed pupae were ligated about the neck, adult development of the thorax and abdomen proceeded normally, but postecdysial tanning of the adult cuticle was almost completely prevented. This failure to tan was not due to lack of bursicon as the hormone was released normally in the ligated animals at the time of ecdysis. This suggests that a pre-ecdysial signal may be required for the development of epidermal competence to respond to bursicon.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Developmental and regional-specific expression of FLRFamide peptides in the tobacco hornworm,Manduca sexta,suggests functions at ecdysis

The developmental profile of a family of three FLRFamide (Phe-Leu-Arg-Phe-NH₂) peptides in the tobacco hornworm, Manduca sexta, revealed regional-specific expression patterns within the segmental ganglia. Levels of the three peptides—F7G (GNSFLRFamide), F7D (DPSFLRFamide), and F10 (pEDVVHSFLRFamide)—were always higher in the thoracic than abdominal ganglia. The predominant peptide also differed regionally, with F7G being highest in the thoracic ganglia and F7G and F10 being equivalent in the abdominal ganglia. Furthermore, we found regional-specific transient declines in ganglion peptide levels temporally correlated to ecdysis. Thoracic ganglion peptide levels declined at each molt, while abdominal ganglion levels declined over a period of 2 days after ecdysis. The decline in central levels was accompanied by an increase in levels in peripheral neurohemal sites, the transverse nerves (TNs). These observations suggest peptides were released from neurosecretory cells (NSCs) at ecdysis. Distinct sets of thoracic and abdominal NSCs and their processes in peripheral neurohemal sites were immunoreactive, supporting the biochemical data. These results also suggest the regional differences may arise from cellular-specific expression patterns for this family of peptides. In addition, fine immunoreactive processes were observed traveling between TNs and skeletal muscles, suggestive of myotropic actions. We propose that the release of different M. sexta FLRFamides from regionally distinct NSCs leads to a coordinated modulation of skeletal and visceral muscles that facilitate ecdysis. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 469–485, 1998     

A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)

The levels of ecdysteroids in Sarcophaga bullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa, the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle.Assays for the cuticle tanning hormone, bursicon, in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed.     

Ecdysteroids regulate the release and action of eclosion hormone in the tobacco hornworm, Manduca sexta (L.)

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.     

The roles of central and peripheral eclosion hormone release in the control of ecdysis behavior in Manduca sexta

1. Ecdysis, a behavior by which insects shed the old cuticle at the culmination of each molt, is triggered by a unique peptide hormone, eclosion hormone (EH). In pupal Manduca sexta, EH is released into the hemolymph just prior to ecdysis, and circulating hormone is sufficient to elicit this behavior. 2. Removal of the proctodeal nerves in prepupal animals eliminated the appearance of blood-borne EH, but ecdysis behavior occurred on schedule. Therefore, circulating EH is not necessary for the triggering of ecdysis. 3. In contrast, a set of dermal glands failed to show their expected bout of secretion after proctodeal nerve removal. Injection of exogenous EH rescued this secretion. Thus, circulating EH appears necessary for action on peripheral but not central targets. 4. A major reduction in EH immunostaining is seen in the proctodeal nerves just preceding ecdysis; this coincides with a greater than 90% reduction in extractable EH from this structure and the appearance of circulating EH. A similar, concomitant reduction was seen in central EH cell processes, suggesting release of peptide within the CNS. 5. Antidromic stimulation of the proctodeal nerve stumps following proctodeal nerve removal triggered precocious ecdysis. This result further supports the conclusion that centrally released EH is sufficient to trigger the motor program.