A radioimmunoassay (RIA) for the recently discovered crustacean cardioactive peptide (CCAP) has been developed and used to determine contents of CCAP in different parts of the nervous system of the shore crab Carcinus maenas. Immunoreactive material is detected throughout the nervous system. In contrast to the main ganglia (brain and thoracic ganglion), which contain low levels of approximately 1.4 pmol CCAP/mg protein, a high concentration is found in a neurohemal structure, the pericardial organs (PO) (868 pmol/mg protein). A predominantly neurohormonal role of CCAP thus suggested is further supported by in vitro release studies. Incubation of POs in high (K+) saline shows that CCAP is secretable in considerable amounts by a Ca++-dependent release mechanism (Stangier, 1988).
On the basis of detailed analyses of morphological characteristics and behavioral events associated with ecdysis in a crab (Carcinus maenas) and a crayfish (Orconectes limosus), a comprehensive substaging system has been introduced for the ecdysis stage of the molt cycle of these decapod crustaceans. In a remarkably similar stereotyped ecdysis sequence in both species, a passive phase of water uptake starting with bulging and rupture of thoracoabdominal exoskeletal junctions is followed by an active phase showing distinct behavioral changes involved in the shedding of the head appendages, abdomen and pereiopods. Together with an enzyme immunoassay for crustacean cardioactive peptide (CCAP), the substaging has been used to demonstrate a large, rapid and reproducible peak in hemolymph CCAP levels (increases of approximately 30-fold in the crab and more than 100-fold in the crayfish compared with intermolt titres) during the later stages of active ecdysis. It is suggested that the release of CCAP (accumulated in late premolt) from the crab pericardial organs or the crayfish ventral nerve cord accounts for many of the changes in behavior and physiology seen during ecdysis and that this neurohormone is likely to be of critical importance in crustaceans and other arthropods (Phlippen, 2000).
The amino acid sequence of a cardioregulatory peptide from Manduca sexta has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl-terminus is amidated. The structure of this peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and it is proposed that this peptide be named Manduca CCAP (Cheung, 1992).
Crustacean cardiactive peptide of Manduca sexta has been characterized. A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was used to quantify CCAP-like immunoreactivity in the nervous system. The CCAP-like immunoreactivity from the abdominal CNS was then purified, and its sequence was ascertained by amino acid analysis, mass spectral analysis, and HPLC. These studies show that the nervous system of M. sexta contains a peptide with the sequence Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, identical to CCAP as originally isolated and sequenced from the shore crab Carcinus maenas. The actions of CCAP on the isolated heart of M. sexta and the extensor-tibia muscle of Schistocerca americana were tested. Crustacean cardioactive peptide has excitatory actions on both preparations: a dose-dependent increase in the rate of contractions was observed on the heart, and an increase in the rate of the myogenic rhythm was observed in the leg muscle. Moreover, purified and synthetic CCAP has identical effects on the isolated heart. It is concluded that CCAP occurs in M. sexta and exerts potent neurotransmitter or neurohormonal actions on a variety of muscles (Lehman, 1993).
An antiserum against crustacean cardioactive peptide was used to map immunoreactive neurons at various stages of postembryonic development of the hawkmoth Manduca sexta. About 90 immunoreactive neurons were identified. Many of these cells are immunoreactive at hatching and persist into the adult stage; others become immunoreactive late in postembryonic development. During adult development, transient immunoreactivity is expressed in several cells in the subesophageal and thoracic ganglia. Two sets of immunoreactive neurons are found in the protocerebrum of larvae, but only one of these sets persists into the adult stage. Paired lateral interneurons and neurosecretory neurons are segmentally repeated in the abdominal ganglia and are present from the first larval stage to the adult; the abdominal interneurons project contralaterally to arborizations in adjacent ganglia, and some ascend to tritocerebral arborizations. The abdominal neurosecretory cells, which correspond to a pair of cells reported to contain bursicon, project posteriorly to neurohemal release organs. Motor neurons of dorsal external oblique abdominal muscles become immunoreactive in the fourth larval stage. Paired median neurosecretory cells of abdominal ganglia become immunoreactive during the fifth larval stage. The immunoreactive median and lateral abdominal neurosecretory cells are a subset of a group of cells known to contain cardioactive peptides. Paired lateral neurosecretory cells of the subesophageal ganglion become immunoreactive during pupation and project to the corpora cardiaca and aorta of the adult. Many of the neurons identified in this study are comparable to crustacean cardioactive peptide-immunoreactive cells described previously in locusts and the mealworm beetle (Davis, 1993).
At the end of each instar, insects shed their old cuticle by performing the stereotyped ecdysis behavior. In the moth, Manduca sexta, larval ecdysis is accompanied by increases in intracellular cyclic 3', 5'-guanosine monophosphate (cGMP) in a small network of 50 peptidergic neurons within the ventral central nervous system (CNS). Studies on a variety of insects show that this cGMP response has been associated with ecdysis throughout most of insect evolution. In the mealbeetle (Tenebrio, Coleoptera) and the mosquito (Aedes, Diptera), all 50 neurons show increases in cGMP immunoreactivity (-IR) at ecdysis, and all are immunopositive for crustacean cardioactive peptide (CCAP). Other insects vary with respect to their cGMP response at ecdysis and their Ccap-IR. In more primitive insects, such as the silverfish (Ctenolepisma, Zygentoma) and the grasshopper (Locusta, Orthoptera), an abdominal subset of these neurons do not show detectable cGMP-IR at ecdysis, although the neurons are Ccap-IR. Conversely, whereas Ccap-IR is severely reduced in the thoracic and subesophageal neurons of Lepidoptera larvae and may be absent in a subset of the corresponding abdominal neurons in crickets (Gryllus, Orthoptera), the ecdysial cGMP response occurs in all 50 neurons. The most extreme case was found in cyclorrhaphous flies, in which most of the 50 neurons are Ccap-IR, although none show increases in cGMP at ecdysis (Ewer 1996).
A methanolic extract of 7000 desert locust (Schistocerca gregaria) brains contains several factors that stimulate the in vitro release of adipokinetic hormone (AKH) by glandular cells of locust (Locusta migratoria and Schistocerca gregaria) corpora cardiaca. The most potent has now been fully identified. The primary structure of the peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, is identical to that of a previously identified crustacean cardioactive peptide. This myotropin was first isolated from the shore crab, Carcinus maenas, and later from several insect species, but was never reported in the context of AKH release. The present study shows that synthetic crustacean cardioactive peptide induces the release of AKH from corpora cardiaca in a dose-dependent manner when tested in concentrations ranging from 10(-5)-10(-9) M. This is the first demonstration in invertebrates of a peptide neurohormone controlling the release of a second peptide hormone (Veelaert, 1997).
Studies on the isolated nervous system of Manduca sexta show that two peptides [ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP)] elicit the first two motor behaviors, the pre-ecdysis and ecdysis behaviors, respectively. Exposing isolated abdominal ganglia to ETH results in the generation of sustained pre-ecdysis bursts. By contrast, exposing the entire isolated CNS to ETH results in the sequential appearance of pre-ecdysis and ecdysis motor outputs. ETH activates neurons within the brain that then release eclosion hormone within the CNS. The latter elevates cGMP levels within and increases the excitability of a group of neurons containing CCAP. The ETH-induced onset of ecdysis bursts is associated with a rise in intracellular cGMP within these CCAP neurons. CCAP immunoreactivity decreases centrally during normal ecdysis. Isolated, desheathed abdominal ganglia responded to CCAP by generating rhythmical ecdysis bursts. These ecdysis motor bursts persist as long as CCAP is present and can be reinduced by successive application of the peptide. CCAP exposure also actively terminates pre-ecdysis bursts from the abdominal CNS, even in the continued presence of ETH. Thus, the sequential performance of the two behaviors arises from one modulator activating the first behavior and also initiating the release of the second modulator. The second modulator then turns off the first behavior while activating the second (Gammie, 1997b).
This study is the first demonstration that CCAP acts centrally to elicit the rhythmical ecdysis bursts. The motor bursts produced by the CNS in response to CCAP show no initial pre-ecdysis patterning, but they start with a defined ecdysis patterning. In the longest experiments (90 min), the continued presence of CCAP results in the production of ecdysis bursts throughout the entire experiment, a duration well beyond that normally seen in intact animals. The ecdysis bursts changed only slightly through the recording period. The frequency of motor bursts did not change with different CCAP concentrations (109-106 M). Additionally, because 1010 M CCAP did not elicit ecdysis motor bursts, CCAP may act as an all-or-none switch. Importantly, this effect is reversible, because the ecdysis program can be turned on and off repeatedly by the addition and withdrawal of the peptide. Thus, CCAP acts tonically, with a sustained presence required for continuous behavioral output (Gammie, 1997b).
The effects of CCAP are not stage specific. The abdominal CNS of intermolt larvae, when treated with CCAP, generates ecdysis bursts identical to those produced by animals late in the molt. The lack of stage specificity for CCAP action is in marked contrast to both EH and ETH, which show relatively narrow sensitive periods for their action on the CNS (Gammie, 1997b).
Unlike ETH, which apparently can traverse the blood-brain barrier, CCAP is not effective when injected into animals or applied to the intact CNS in vitro. Thus, a central release of CCAP is required. The immunocytochemical data support this idea. There is a marked depletion in central CCAP during the course of normal ecdysis, with the magnitude of depletion proportional to the duration of the behavior. Experiments with girdled larvae show that CCAP continues to be released if the animals become trapped and cannot shed their cuticle. These trapped larvae eventually stop their ecdysis attempts, presumably because of the exhaustion of their CCAP stores (Gammie, 1997b).
Both behavioral data and the electrophysiological data show an invariant association of the cGMP elevation in the Cell 27/704 group with ecdysis when the behavior is initiated by either ETH or EH. This cGMP increase, however, is not seen when CCAP is used to evoke ecdysis motor bursts from the abdominal CNS. This result is consistent with the hypothesis that cGMP upregulation is upstream of CCAP release and the induction of ecdysis (Gammie, 1997b).
CCAP also causes the premature termination of the pre-ecdysis motor program. ETH application to an abdominal CNS results in the long-lasting performance of pre-ecdysis bursts, with an average duration >80 min. Application of CCAP to the abdominal CNS shortly after ETH application, however, results in a brief period during which both pre-ecdysis and ecdysis bursts occur together, followed by only ecdysis bursts. In vivo, the transition from pre-ecydsis to ecdysis may be facilitated by the normal rundown of ETH action, but in these experiments the average duration of ETH-induced pre-ecdysis bursting is significantly shortened by the presence of CCAP. Also, because ETH is unable to disrupt ecdysis bursts or trigger stable pre-ecdysis patterning when applied after CCAP, it is concluded that CCAP has a dominant action on the CNS compared with ETH (Gammie, 1997b).
In an intact Manduca, the pre-ecdysis behavior (which loosens the cuticle) is followed by the ecdysis behavior (which sheds the cuticle). Recordings from the isolated CNS likewise show this sequence. ETH secretion is likely the normal stimulus to initiate the pre-ecdysis behavior. ETH also stimulates EH release and vice versa, and this positive feedback interaction results in both EH and ETH undergoing essentially complete release. Lower concentrations of ETH initiate pre-ecdysis before higher concentrations activate the ecdysis pathway. The ETH-induced release of EH then stimulates cGMP elevation in the Cell 27/704 group, which increases their excitability and leads to CCAP release centrally. CCAP then acts in a hierarchical manner on the CNS and activates the ecdysis motor output while it terminates pre-ecdysis (Gammie, 1997b).
Release of ETH from the Inka cells both initiates pre-ecdysis and excites the ventromedial (VM) neurons that contain eclosion hormone. Because they are part of a positive feedback loop, the Inka cells and VM neurons release almost all of their peptide stores. EH release within the CNS triggers cGMP upregulation in the Cell 27/704 group, causing the central and peripheral release of CCAP. Centrally released CCAP both activates the ecdysis motor program and terminates pre-ecdysis. Sensory input ( possibly from bristle hairs deformed by the pressure of the old cuticle) may maintain excitation of the Cell 27/704 group to insure CCAP release and the continuation of ecdysis until the cuticle is shed. Removal of the cuticle eliminates the sensory input, resulting in the cessation of CCAP release and of ecdysis behavior (Gammie, 1997b).
Unlike the phasic release of EH, the immunocytochemical data on CCAP depletion along with data from intracellular recordings suggest that this peptide is released in a lower-level, tonic manner. Indeed, at the end of a normal ecdysis, the central arbors of the CCAP cells still retain about half of their pre-ecdysis levels of peptide. This residual peptide may provide a safety factor, so that if the animal encounters difficulties in shedding the cuticle, such as in the girdled larvae, there will still be CCAP present that can be used to maintain an extended ecdysis motor pattern. It is suggested that mechanosensory input stimulated by the presence of the old cuticle might impinge directly onto the CCAP cells. In Manduca, cGMP levels remain elevated in some cells for up to 3 hr after ecdysis, and it is hypothesized that as long as their cGMP levels remain elevated, this sensory input would maintain the firing of the CCAP cells and hence prolong CCAP release and the consequent expression of the ecdysis motor pattern. No direct information from Manduca is available that prolonged ecdysis prolongs the upregulation of cGMP (Gammie, 1997b).
Finally, the sequential production of the two motor programs is facilitated by modifications on at least three levels. (1) The concentration of ETH required to elicit the pre-ecdysis motor pattern is much lower than that needed to cause ecdysis. Consequently, early low levels of ETH will start pre-ecdysis before the later high levels activate the pathway leading to ecdysis. (2) ETH release and action have already peaked by the time the Cell 27/704 group is first activated by EH. (3) The response of the CNS to the two peptides is hierarchical, because CCAP turns off the behavior produced by ETH as well as turns on the second behavior in the sequence (Gammie, 1997b).
Three insect peptide hormones, eclosion hormone (EH), ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP), 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).
The crustacean cardioactive peptide (CCAP) gene was isolated from the tobacco hawkmoth Manduca sexta. The gene has an open reading frame of 125 amino acid residues containing a single, complete copy of CCAP. Analysis of the gene structure revealed three introns interrupting the coding region. A comparison of the M. sexta CCAP gene with the Drosophila melanogaster genome database reveals significant similarities in sequence and gene structure. The spatial and temporal expression patterns of the CCAP gene in the M. sexta central nervous system were determined in all major post-embryonic stages using in situ hybridization techniques. The CCAP gene is expressed in a total of 116 neurons in the post-embryonic M. sexta central nervous system. Nine pairs of cells are observed in the brain, 4.5 pairs in the subesophageal ganglion, three pairs in each thoracic ganglion (T1-T3), three pairs in the first abdominal ganglion (A1), five pairs each in the second to sixth abdominal ganglia (A2-A6) and 7.5 pairs in the terminal ganglion. The CCAP gene is expressed in every ganglion in each post-embryonic stage, except in the thoracic ganglia of first- and second-instar larvae. The number of cells expressing the CCAP gene varies during post-embryonic life, starting at 52 cells in the first instar and reaching a maximum of 116 shortly after pupation. One set of thoracic neurons expressing CCAP mRNA shows unusual variability in expression levels immediately prior to larval ecdysis. Using previously published CCAP immunocytochemical data, it was determined that 91 of 95 CCAP-immunopositive neurons in the M. sexta central nervous system also express the M. sexta CCAP gene, indicating that there is likely to be only a single CCAP gene in M. sexta (Loi, 2001).
The role of calcium as a second messenger in the crustacean cardioactive peptide (CCAP)-induced contractions of the locust oviducts was investigated. Incubation of the oviducts in a calcium-free saline containing a preferential calcium cation chelator, or an extracellular calcium channel blocker, abolishes CCAP-induced contractions, indicating that the effects of CCAP on the oviducts are calcium-dependent. In contrast, sodium free saline did not affect CCAP-induced contractions. Co-application of CCAP to the oviducts with preferential L-type voltage-dependent calcium channel blockers reduces CCAP-induced contractions by 32%-54%. Two preferential T-type voltage-dependent calcium channel blockers both inhibit CCAP-induced oviduct contractions although affecting different components of the contractions. Amiloride decreases the tonic component of CCAP-induced contractions by 40%-55% and flunarizine dihydrochloride decreases the frequency of CCAP-induced phasic contractions by as much as 65%, without affecting tonus. Flunarizine dihydrochloride does not alter the proctolin-induced contractions of the oviducts. Results suggest that the actions of CCAP are partially mediated by voltage-dependent calcium channels similar to vertebrate L-type and T-type channels. High-potassium saline does not abolish CCAP-induced contractions indicating the presence of receptor-operated calcium channels that mediate the actions of CCAP on the oviducts. The involvement of calcium from intracellular stores in CCAP-induced contractions of the oviducts is likely since, an intracellular calcium antagonist decreases CCAP-induced contractions by 30%-35% (Donini, 2002a).
Hindguts of Locusta migratoria female 5th instar larvae, young adults (1-2 days) and old adults (>10 days) are equally sensitive to the crustacean cardioactive peptide (CCAP), with changes in contraction occurring at a threshold concentration of 10(-9)M and maximal responses observed at concentrations ranging between 10(-7) and 5x10(-6)M. An immunohistochemical examination of the locust gut with an antiserum raised against CCAP revealed an extensive network of CCAP-like immunoreactive processes on the hindgut and posterior midgut via the 11th sternal nerve arising from the terminal abdominal ganglion. Anterograde filling of the 11th sternal nerve with neurobiotin revealed extensive processes and terminals on the hindgut. Retrograde filling of the branch of the 11th sternal nerve, which innervates the hindgut with neurobiotin, revealed two bilaterally paired cells in the terminal abdominal ganglion which co-localize with CCAP-like immunoreactivity. Results suggest that a CCAP-like substance acts as a neurotransmitter/neuromodulator at the locust hindgut (Donini, 2002b).
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 AG17, 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 TG13 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).
Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, systemic RNA interference (RNAi) experiments were performed utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods (Arakane, 2008).
The earliest peptide signal for ecdysis behavior in Manduca so far identified is corazonin, which triggers the neuroendocrine cascade by inducing the release of ETH from the epitracheal glands. However, in Tribolium and also in other coleopteran species, corazonin is apparently absent because there has been no report of immunoreactivity with corazonin antiserum. Furthermore, no Tribolium sequences encoding this peptide or its receptor have been reported so far. Thus, the signal initiating ecdysis in this coleopteran must be something other than corazonin. In the case of the albino locust, which is believed to lack corazonin, there is no ecdysis deficiency reported, implying that corazonin may be lepidopteran-specific as a signal for ETH release (Arakane, 2008).
Severe deficiencies in preecdysis behavior were observed in Tribolium after treatment with either dseh, dseth, dsethr or dsethr-a. There were some occasional twitching-like D–V contractions in these insects, which may have been caused by incomplete suppression of the targeted mRNA. This deficiency in preecdysis behavior resulted in the failure of subsequent ecdysis behavior, which in turn resulted in failure to eclose and finally in death. The ETH signal has been found to be necessary and sufficient in Drosophila for both preecdysis and ecdysis behaviors. In Manduca, the sufficiency of ETH for inducing premature preecdysis and ecdysis behaviors also supports the conclusion that ETH is one of the earliest ecdysis-initiating molecules. This study also supports the notion of ETH being an early essential signal for ecdysis in Tribolium. In addition, dseth was found to cause deficiencies in larval and pupal ecdysis depending on the time of injection (Arakane, 2008).
Two subtypes of ETH receptors, A and B, arising from mutually exclusive alternative exon usage, are highly conserved in insects. Studies with Manduca and Drosophila showed that ethr-a is expressed mainly in numerous peptidergic cells in the CNS, while ethr-b is expressed in poorly-characterized interneuron cells. Exon-specific dsRNA in Tribolium showed that dsethr-a-treated insects had significantly fewer D–V contractions, a phenotype identical to that obtained following treatment with dseth or dseh. However, eclosion of insects injected with dsethr-b occurred normally, with no substantial reduction in preecdysial D–V contractions. Therefore, ethr-a, which activates downstream peptidergic signals, is a necessary component in Tribolium eclosion, whereas the role of ethr-b remains unclear. Switching from one behavior to the next within a certain time interval in the behavioral sequence had been thought to involve inhibitory neurons, it was not possible to determine whether premature ecdysis behavior occurred in the dsethr-b-injected Tribolium as a result of defects in inhibitory neurons (Arakane, 2008).
Positive feedback between EH and ETH has been found in Manduca. Release of ETH is triggered by corazonin as the initiator of the EH-ETH feedback loop. EH-associated positive feedback induces a massive release of ETH for the initiation of ecdysis motor patterns. However, a positive feedback loop was not found in Drosophila. Rather, Drosophila EH apparently acts downstream of ETH and is the factor triggering ecdysis behavior, a conclusion based on the timing of the cellular response of EH cells, which show Ca2+ elevation upon treatment with ETH during pupal ecdysis. Surprisingly, the EH-cell-knockout in Drosophila resulted in only a partial impairment during adult eclosion, with a significant proportion of the insects dying before pupation. In Tribolium, EH was required for early preecdysis behavior. Thus, the ETH-EH feedback loop, if it occurs, probably occurs during preecdysis in Tribolium, as it does in Manduca (Arakane, 2008).
In Drosophila, the ccap null mutant did not show any abnormality during development or ecdysis, whereas ccap-cell ablation resulted in deficiencies in both pupal and adult ecdysis. Therefore, it was concluded that other neuropeptides, which are co-expressed in the CCAP cells, are probably responsible for the phenotypes of the ccap cell-knockout. Thus, the role of CCAP in Drosophila ecdysis remains unclear. The neuropeptides co-expressed in the CCAP cells with presumed functions in ecdysis are bursicon, partner of bursicon and myoinhibitory peptide (Arakane, 2008).
In contrast to Drosophila, ccap RNAi in Tribolium resulted in a lethal arrest during ecdysis. The ecdysis deficiency was associated with significantly weaker behaviors, including reverse-bending, wing air-filling and A–P contraction, whereas these insects underwent normal preecdysis behavior. The dsccapr-2 treatment resulted in the same phenotype as that of dsccap, whereas dsccapr-1 treatment did not produce any abnormalities. Therefore, CCAP and CCAPR-2 are in the signaling pathway for ecdysis behavior, while the function of CCAPR-1 remains unknown (Arakane, 2008).
The bur and pbur genes in Tribolium form a tandem pair in the genome, separated by only ~1.3 kb. This arrangement is similar to the bur/pbur gene structure in the honeybee. Previously, it was proposed that the honeybee bur/pbur gene consisted of one long open reading frame encoding a multi-domain protein including both bur and pbur. Subsequently, however, different transcription units for bur and pbur were reported. Using RT-PCR this study determined that Tribolium bur and pbur are probably separate transcription units. Results from gene-specific RNAi for both bur and pbur support the hypothesis that two different transcription units exist. In addition, whereas mosquito bur was found to undergo trans-splicing, this study found that the Tribolium bur and pbur genes contain complete open reading frames, excluding the possibility of trans-splicing (Arakane, 2008).
A heterodimeric complex Bur/pBur consisting of the products of the bur and pbur genes is a cysteine knot family hormone that has been reported to initiate two different functional activities in Drosophila, namely cuticle tanning and wing expansion after adult eclosion. Drosophila mutants for bur and receptor mutant rickets (rk) showed deficiencies in both tanning and wing expansion (Arakane, 2008).
This study discovered that treatments with dsbur, dspbur or dsrk all produced similar postecdysis defects, namely weak postecdysis activity, wrinkled elytra and a failure to retract the hindwing, but none of these caused lethality within the observation time of 2–3 weeks after eclosion. In Drosophila, bursicon induces wing cell death and wing expansion after eclosion. The wrinkled elytra and the deficiency in proper folding of the hindwing after RNAi in Tribolium may be equivalent to the Drosophila phenotype. Interestingly, RNAi of these genes resulted in significantly diminished strengths in preecdysis behavior. The data imply that bur/pbur and their putative receptor rk in Tribolium are involved in the regulation of preecdysis behavior, and even more in postecdysis behavior. An additional unique phenotype was found only in insects injected with dsbur, which exhibited weaker A–P contractions during ecdysis and consequently an extended duration for completion of the shedding of the exuvium. These observations suggest an unknown but separate function for bursicon in addition to its role as a component of dimeric Bur/pBur acting through its receptor Rickets. Alternatively, the phenotypic variation could have been caused by different dosages of remaining transcripts in RNAi or by stability of the protein that had been produced earlier (Arakane, 2008).
Perhaps the most interesting observation in this study is that normal tanning occurs in beetles subjected to RNAi for the group of genes encoding the neuropeptides described in this study. Maturation of the cuticle is a gradual process of pigmentation and sclerotization during the first five days after eclosion. A recent study involving Drosophila has shown that bursicon acts through phosphorylation of tyrosine hydroxylase, which catalyzes an early step of catecholamine production for cuticle tanning. It has been shown previously that RNAi of Tribolium laccase 2, which is a phenoloxidase downstream of tyrosine hydroxylase in the same metabolic pathway, suppressed cuticle tanning. This result indicated that a similar cuticle tanning pathway exists in both Drosophila and Tribolium. It is concluded, therefore, that the Bur/pBur signaling pathway is required for proper wing expansion and folding in Tribolium but not for tyrosine hydroxylase/laccase-mediated tanning. Recent study in the silkworm also reported that RNAi of bur found no distinct tanning phenotype, while a deficiency in wing expansion was observed. In addition, the regulation of cuticle tanning in Tribolium appears to be different from that of Drosophila, even though the tanning pathway itself is probably conserved (Arakane, 2008).
This has been a study of key peptidergic signaling systems for insect ecdysis in T. castaneum, representing a more basal holometabolous order (Coleoptera) relative to the species of Lepidoptera and Diptera studied previously. RNAi in Tribolium followed by behavioral analysis revealed differences in the roles of EH, CCAP and bursicon compared to those found in Drosophila. Both ETH and EH are necessary for preecdysis and ecdysis behaviors in Tribolium, while an essential role of EH has not been found in Drosophila. CCAP is necessary for ecdysis behavior in Tribolium, whereas the Drosophila ccap null mutant shows normal ecdysis. In Tribolium Bur/pBur is necessary for postecdysis behavior, including wing expansion and folding, whereas, unlike the case in dipterans, it does not have a role in cuticle tanning (Arakane, 2008). Bur/pBur signaling is involved in preecdysis behavior. Only bursicon appears to have an additional role in ecdysis behavior in Tribolium (Arakane, 2008).
The differences in the precise roles of each peptidergic component among Tribolium, Drosophila and Manduca in controlling innate ecdysis behavior and cuticle tanning can be interpreted as a consequence of evolution. The loss of essential roles for EH and CCAP as well as a gain in function for bursicon in Drosophila may be associated with modifications of the requirements of those neuropeptidergic signals in these processes, whereas the more ancestral Tribolium and possibly Manduca strictly require EH and CCAP signaling systems. A comparative analysis of the functions of peptidergic signals from additional taxa will provide further insights into the evolution and regulation of ecdysis and tanning in the Ecdysozoa (Arakane, 2008).
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