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SUMMARYAdult insects achieve their final form shortly after adult eclosion by the combined effects of specialized behaviors that generate increased blood pressure, which causes cuticular expansion, and hormones, which plasticize and then tan the cuticle. We examined the molecular mechanisms contributing to these processes in Drosophila by analyzing mutants for the rickets gene. These flies fail to initiate the behavioral and tanning processes that normally follow ecdysis. Sequencing of rickets mutants and STS mapping of deficiencies confirmed that rickets encodes the glycoprotein hormone receptor DLGR2. Although rickets mutants produce and release the insect-tanning hormone bursicon, they do not melanize when injected with extracts containing bursicon. In contrast, mutants do melanize in response to injection of an analog of cyclic AMP, the second messenger for bursicon. Hence, rickets appears to encode a component of the bursicon response pathway, probably the bursicon receptor itself. Mutants also have a behavioral deficit in that they fail to initiate the behavioral program for wing expansion. A set of decapitation experiments utilizing rickets mutants and flies that lack cells containing the neuropeptide eclosion hormone, reveals a multicomponent control to the activation of this behavioral program.

SUMMARYThe sequential behaviours shown by insects at ecdysis are due to the sequential release of various hormones, but the transition from one phase to the next can be fine-tuned by inhibitory influences. The ecdysis sequence in the moth Manduca sexta was initiated by injecting sensitive animals with the neuropeptide ecdysis-triggering hormone (ETH). Exposure to ETH stimulates the release of eclosion hormone (EH) which, in turn, activates a set of neurons containing crustacean cardioactive peptide (CCAP) by elevating their levels of intracellular cyclic GMP. We characterized a set of non-CCAP containing neurons that also appear to be EH targets because of their response to cyclic GMP at ecdysis. The neurons did not display leucokinin-,diuretic-hormone- or FMRFamide-like immunoreactivity. They are probably the bursicon-containing cells described previously. After release of EH, there is a transient inhibition of the abdominal centers responsible for ecdysis. Transection experiments suggested that this suppression is viadescending inhibitory units from the suboesophageal and thoracic ganglia. The duration of this inhibition appears to depend on the levels of cyclic GMP and can be extended by pharmacologically suppressing cyclic GMP breakdown. We further found that brief exposure to CO2 caused premature ecdysis. Since the CO2 treatment was effective only after EH release, it probably acts by suppressing descending inhibition. Studies on adult eclosion suggest that CO2, given at the appropriate time, can uncouple the basic larval motor program from modulatory influences provided by the adult pterothoracic ganglion. CO2 therefore appears to be a novel and non-invasive tool for studies of ecdysis behavior in insects.

In insects, ecdysis is thought to be controlled by the interaction between peptide hormones; in particular between ecdysis-triggering hormone (ETH) from the periphery and eclosion hormone (EH) and crustacean cardioactive peptide (CCAP) from the central nervous system. We examined the behavioral and physiological functions of the first two of these peptides in Drosophila melanogaster using wild-type flies and knockout flies that lacked EH neurons. We used ETH from Manduca sexta (MasETH) to induce premature ecdysis and compared the responses of the two types of flies. The final release of EH normally occurs approximately 40 min before ecdysis. It is correlated with cyclic guanosine monophosphate (cGMP) production in selected neurons and tracheae, by an elevation in the heart rate and by the filling of the new tracheae with air. Injection of developing flies with MasETH causes all these events to occur prematurely. In EH cell knockouts, none of these changes occurs in response to MasETH, and these flies show a permanent failure in tracheal filling. This failure can be overcome in the knockouts by injecting them with membrane-permeant analogs of cGMP, the second messenger for EH. The basis for the 40 min delay between EH release and the onset of ecdysis was examined by decapitating flies at various times relative to EH release. In flies that had already released EH, decapitation was always followed within 1 min by the start of ecdysis. Immediate ecdysis was never observed when the EH cell knockout flies were decapitated. We propose that EH activates both ventral central nervous system elements necessary for ecdysis (possibly the CCAP cells) and descending inhibitory neurons from the head. This descending inhibition establishes a delay in the onset of ecdysis that allows the completion of EH-activated physiological processes such as tracheal filling. A waning in the inhibition eventually allows ecdysis to begin 30–40 min later.

A successful ecdysis in insects requires the precise coordination of behaviour with the developmental changes that occur late in a moult. This coordination involves two sets of endocrine cells: the peripherally located Inka cells, which release ecdysis triggering hormone (ETH), and the centrally located neurosecretory neurones, the VM neurones, which release eclosion hormone (EH). These two sets of endocrine cells mutally excite one another: EH acts on the Inka cells to cause the release of ETH. ETH, in turn, acts on the VM neurones to cause the release of EH. This positive-feedback relationship allows the Inka cells and the VM neurones to be the peripheral and central halves, respectively, of a decision-making circuit. Once conditions for both halves have been satisfied, their reciprocal excitation results in a massive EH/ETH surge in the blood as well as a release of EH within the central nervous system. This phasic signal then causes the tonic activation of a distributed network of peptidergic neurones that contain crustacean cardioactive peptide. The relationship of the latter cells to the subsequent maintenance of the ecdysis motor programme is discussed.

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.

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.

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 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 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.

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.

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.

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. The larval ecdyses of the tobacco hornworm, Manduca, and of the oak silkmoth, Antheraea, occur during specific times of the day. These times are instar-specific and species-specific and depend upon the photoperiod and temperature conditions. 2. The larval ocelli are not required for the synchronization of larval ecdysis with the photoperiod. 3. The synchrony observed in larval ecdysis does not arise from a gating of the ecdysial event per se by a biological clock. Rather, it is an outcome of the gating of the endocrine events which initiate the moulting process. 4. Application of neck ligatures to fourth-instar hornworm larvae at various times of day showed that prothoracicotropic hormone (PTTH) secretion occurred only during a gate in the early to middle portion of the night. The times of the gates for second- and third-instar larvae were similar. 5. When neck ligatures were applied to fourth-instar larvae within the first 1-5 h after PTTH release, the larvae underwent precocious metamorphosis rather than Moulting to the Fifth Instar. A Consideration of the Number of Larval-Pupal Intermetiates Produced Indicated that with in Span of 15 min Sufficiant JH is Liberatd from the Corpora Allata to Ensure Full Larval Differentiation. 6. Larval abdomens which were isolated during the period from 3 to 6 h after PTTH release underwent a mosaic larval moult-i.e. they became a patchwork of new and old cuticle. Thus, the epidermal structures of the abdomen vary as to the time that each becomes independent of the prothoracic glands. The first structures which continue fifth-instar differentiation in the absence of the prothoracic glands are the crochets. These are then followed in order by the prolegs, the ventral midline, the intersegmental areas, and, by 6 h after PTTH release, the remainder of the abdomen. During this 3 h period the prothoracic glands are presumably secreting ecdysone.