Ecdysis behavior in the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) is triggered through reciprocal peptide signaling between the central nervous system and the epitracheal endocrine system. Recent evidence indicates that eclosion hormone may initiate endocrine events leading to ecdysis through its action on epitracheal glands to cause the release of ecdysis-triggering hormone (ETH). Direct exposure of epitracheal glands to eclosion hormone in vitro leads to secretion of ETH. The threshold concentration of eclosion hormone needed to evoke release of ETH is approximately 3 pmol l-1. Eclosion hormone also induces elevation of cyclic GMP, but not cAMP, concentration in epitracheal glands at concentrations similar to those causing release of ETH. Both cGMP and 8-Br-cGMP mimic the secretory action of eclosion hormone. The sensitivity of the secretory response to eclosion hormone occurs during a narrow window of development, beginning approximately 8 h prior to pupal ecdysis. However, eclosion hormone can cause elevation of cGMP levels in epitracheal glands long before they acquire competence to release ETH, showing that the initial portion of the signal transduction cascade is in place early in development, but that the absence of a downstream step in the cascade prevents secretion. Measurements of cGMP levels in epitracheal glands during the ecdysis sequence show a sudden elevation some 30 min after the onset of pre-ecdysis, well after ETH secretion has been initiated. ETH secretion can therefore be viewed as a two-step process, beginning at pre-ecdysis when cGMP levels are relatively low, followed by a massive release resulting from a logarithmic elevation of cGMP levels (Kingan, 1997).
At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, the cDNA and gene encoding the Manduca sexta preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH) that activate these behaviors, have been identified. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence (Zitnan, 1999).
Three insect peptide hormones, eclosion hormone (EH), ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (see Drosophila Cardioacceleratory peptide), have been implicated in controlling ecdysis behavior in insects. This study examines the interactions among these three peptides in the regulation of the ecdysis sequence. Using intracellular recordings, it has been found that ETH is a potent activator of the EH neurons, causing spontaneous action potential firing, broadening of the action potential and an increase in spike peak amplitude. In turn, electrical stimulation of the EH neurons or bath application of EH to desheathed ganglia results in the elevation of cyclic GMP (cGMP) levels within the Cell 27/704 group (which contains CCAP). This cGMP production increases the excitability of these neurons, thereby facilitating CCAP release and the generation of the ecdysis motor program. Extracellular recordings from isolated nervous systems show that EH has no effect on nervous systems with an intact sheath. In desheathed preparations, in contrast, EH causes only the ecdysis motor output. The latency from EH application to ecdysis is longer than that after CCAP application, but shorter than that when ETH is applied to a whole central nervous system. These data support a model in which ETH causes pre-ecdysis behavior and at higher concentrations stimulates the EH neurons. EH release then facilitates the onset of ecdysis by enhancing the excitability of the CCAP neurons (Gammie, 1999).
Ecdysis, or molting behavior, in insects requires the sequential action of high levels of ecdysteroids, which induce accumulation of ecdysis-triggering hormone (ETH) in Inka cells, followed by low levels of ecdysteroids, permissive for the onset of the behavior. High ecdysteroid levels suppress the onset of the behavioral sequence by inhibiting the development of competence to secrete ETH. In pharate pupae of Manduca sexta, Inka cells in the epitracheal glands normally develop competence to secrete ETH in response to eclosion hormone (EH) 8 h before pupation. Injection of 20-hydroxyecdysone (20E) into precompetent insects prevents this acquisition of competence, but does not affect EH-evoked accumulation of the second messenger cyclic GMP. Precompetent glands acquire competence in vitro after overnight culture, and this can be prevented by the inclusion of 20E at concentrations greater than 0.1 microg ml(-1)in the culture medium. Actinomycin D completely inhibits the acquisition of competence, demonstrating that it is dependent on transcriptional events. Cultured epitracheal glands become refractory to the inhibitory effects of 20E in the acquisition of competence at least 3 h earlier than for Actinomycin D, indicating that 20E acts on an early step in a sequence of nuclear events leading to transcription of a structural gene. These findings suggest that declining ecdysteroid levels permit a late event in transcription, the product of which is downstream of EH receptor activation and cyclic GMP accumulation in the cascade leading to ETH secretion (Kingan, 2000).
Initiation of the ecdysis behavioral sequence in insects requires activation of the central nervous system (CNS) by pre-ecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which are released from the Inka cells of the epitracheal glands. The developmental events preceding larval and pupal ecdysis of Manduca sexta involve a dual action of ecdysteroids on the epitracheal glands and CNS. The low steroid levels in freshly ecdysed and feeding larvae are associated with small-sized epitracheal glands, reduced peptide production in Inka cells and insensitivity of the CNS to ETH. The elevated ecdysteroid levels before each ecdysis led to a dramatic enlargement of Inka cells and increased production of peptide hormones and their precursors. As blood ecdysteroids reach peak levels, the CNS becomes responsive to Inka cell peptides. These effects of natural ecdysteroid pulses can be experimentally induced by injection of 20-hydroxyecdysone or the ecdysteroid agonist tebufenozide (RH-5992) into ecdysed larvae, thus stimulating peptide production in Inka cells and inducing CNS sensitivity to ETH. A direct steroid action on the CNS is demonstrated by subsequent treatment of isolated nerve cords from ecdysed larvae with 20-hydroxyecdysone and ETH, which results in pre-ecdysis or ecdysis bursts. These data show that ecdysteroid-induced transcriptional activity in both the epitracheal glands and the CNS are necessary events for the initiation of the ecdysis behavioral sequence (Zitnanova, 2001).
Inka cells of the epitracheal endocrine system produce peptide hormones involved in the regulation of insect ecdysis. In the silkworm Bombyx mori, injection of Inka cell extract into pharate larvae, pupae or adults activates the ecdysis behavioral sequence. In the present study, the identification is reported of three peptides in these extracts, pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP); these are encoded by the same cDNA precursor. Strong immunoreactivity associated with each peptide in Inka cells prior to ecdysis disappears during each ecdysis, indicating complete release of these peptides. Injection of either PETH or ETH alone is sufficient to elicit the entire ecdysis behavioral sequence through the direct action on abdominal ganglia; cephalic and thoracic ganglia are not required for the transition from pre-ecdysis to ecdysis behavior. In vitro data provide evidence that these peptides control the entire ecdysis behavioral sequence through activation of specific circuits in the nervous system. Ecdysis of intact larvae is associated with the central release of eclosion hormone (EH) and elevation of cyclic 3',5'-guanosine monophosphate (cGMP) in the ventral nerve cord. However, injection of ETH into isolated abdomens induces cGMP elevation and ecdysis behavior without a detectable release of EH, suggesting that an additional central factor(s) may be involved in the activation of this process. These findings provide the first detailed account of the natural and hormonally induced behavioral sequence preceding larval, pupal and adult ecdyses of B. mori and highlight significant differences in the neuro-endocrine activation of pre-ecdysis and ecdysis behaviors compared with the related moth, Manduca sexta (Zitnan, 2002).
The sequential behaviors 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. This study characterizes 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 bursicon-containing cells. After release of EH, there is a transient inhibition of the abdominal centers responsible for ecdysis. Transection experiments suggest that this suppression is via descending 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. Brief exposure to CO2 causes premature ecdysis. Since the CO2 treatment is 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 (Fuse, 2002).
Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, Inka cells were found in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviors in pharate larvae of Bombyx, indicating functional similarity of these peptides. Several PETH-immunoreactive peptides were isolated from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and the gene encoding two putative ETHs were identified in the mosquito Anopheles gambiae. Inka cells also stain with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, enzyme immunoassays show that these antisera cross-react with PETH and ETH. These results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioral sequence (Zitnan, 2003).
At the end of each developmental stage, insects perform the ecdysis sequence, an innate behavior necessary for shedding the old cuticle. Ecdysis triggering hormones (ETHs) initiate these behaviors through direct actions on the CNS. This study identifies the ETH receptor (ETHR) gene in the moth Manduca sexta; the gene encodes two subtypes of GPCR (ETHR-A and ETHR-B). Expression of ETHRs in the CNS coincides precisely with acquisition of CNS sensitivity to ETHs and behavioral competence. ETHR-A occurs in diverse networks of neurons, producing both excitatory and inhibitory neuropeptides, which appear to be downstream signals for behavior regulation. These peptides include allatostatins, crustacean cardioactive peptide (CCAP), calcitonin-like diuretic hormone, CRF-like diuretic hormones (DHs) 41 and 30, eclosion hormone, kinins, myoinhibitory peptides (MIPs), neuropeptide F, and short neuropeptide F. In particular, cells L3,4 in abdominal ganglia coexpress kinins, DH41, and DH30, which together elicit the fictive preecdysis rhythm. Neurons IN704 in abdominal ganglia coexpress CCAP and MIPs, whose joint actions initiate the ecdysis motor program. ETHR-A also is expressed in brain ventromedial cells, whose release of EH increases excitability in CCAP/MIP neurons. These findings provide insights into how innate, centrally patterned behaviors can be orchestrated via recruitment of peptide cotransmitter neurons (Kim, 2006b).
In M. sexta, the shedding of old cuticle is accomplished by the ecdysis sequence consisting of the sequential behaviors preecdysis I, preecdysis II, and ecdysis. Each individual motor program is triggered by direct actions of PETH and ETH on the CNS. The ecdysis sequence is initiated by corazonin release from the brain, causing low-level secretion of pre-ecdysis triggering hormone (PETH) and ETH from Inka cells. These initial low levels of circulating peptides activate the ETHR-A in abdominal neurons L3,4 to induce preecdysis I through central release of DHs and kinins. At the same time, ETH acts on unidentified neurons (possibly expressing ETHR-B) to activate preecdysis II and specific ETHR-A neurons in the entire CNS to activate the ecdysis network. This ETHR-A network includes EH-producing VM neurons and IN704 in the AG1–7, which produce CCAP and MIPs. Activation of the ecdysis network is indicated by EH-mediated cGMP elevation in abdominal neurons ~30 min after onset of preecdysis I. However, descending inhibitory neurons (possibly expressing ETHR-B) in the subesophageal ganglia and TG1–3 suppress the release of CCAP and MIPs from IN704 and delay ecdysis onset. The switch from preecdysis to ecdysis is mediated by central release of CCAP and MIPs from IN704. CCAP apparently plays an excitatory role in this process and controls ecdysis execution, whereas MIPs appear to inhibit activities of other neurons not involved in ecdysis. Finally, bursicon and CCAP are released from cells 27 (NS27) to control postecdysis behaviors: cuticle expansion, hardening, and tanning. Physiological roles of other identified ETHR-A neurons expressing various neuropeptides (e.g., NPF, small NPF/MIPs, and calcitonin-like DH/MIPs) are not clear, and their functional analysis is in progress (Kim, 2006b).
It is well established that peptidergic inputs modulate central pattern generators. In the case of the crustacean stomatogastric ganglion, peptides alter the synaptic strength and intrinsic properties of neurons within the ganglion, resulting in different versions of pyloric and gastric mill rhythms. Similar regulatory mechanisms may operate for the ecdysis sequence, whereby sequential release of neuropeptides reconfigures the activity of multiple central pattern generators in succession (Kim, 2006b).
In this study, it was discovered that numerous central peptidergic ensembles express ETHRs and, consequently, are likely primary targets of ETH. Peptides produced by these ensembles are prime candidates for downstream signals in the recruitment of each centrally patterned phase of the ecdysis sequence. Possible roles of two specific peptidergic ensembles have been shown: a neuropeptide mixture consisting of DHs and kinins produced by abdominal L3,4 neurons generates the preecdysis I rhythm, whereas a combination of CCAP and MIPs produced by IN704 neurons elicits the ecdysis rhythm (Kim, 2006b).
In related work, it has been shown that ensembles of neurons in D. melanogaster homologous to those in M. sexta (EH-containing VM neurons, CCAP/MIPs-containing IN704 neurons, and CCAP/bursicon-containing NS27 neurons) become active at the onset of successive behavioral subunits (Kim, 2006a). Taken together, these findings indicate that sequential recruitment of peptidergic ensembles elicits components of an innate behavior in stepwise fashion. It seems reasonable to suggest that central neuropeptide release as described here may represent a general mechanism for orchestration of behaviors (Kim, 2006b).
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