To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon [Fraenkel, 1962; Cottrell, 1962), but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon (Honegger, 2002), the Drosophila gene CG13419 was identified as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted Bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-ß (TGF-ß) and glycoprotein hormones. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased Bursicon bioactivity. In situ hybridization and immunocytochemistry reveal that Bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons also lack Bursicon bioactivity. These results indicate that CG13419 encodes Bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to be assigned a defined function in invertebrates. Mutants show that the spectrum of Bursicon actions is broader than formerly demonstrated (Dewey, 2004).

bursicon transcripts are expressed in a set of the neurons that contain CCAP. Transgenic Drosophila bearing targeted ablations of CCAP neurons (CCAP KO flies) are defective in wing expansion and tanning and have low survival to adulthood (Park, 2003). In situ hybridization of the CNS of CCAP KO larvae using a bursicon antisense probe shows a significant reduction in the number of labeled neurons, as would be expected if CCAP neurons express bursicon. In some preparations, a few surviving neurons weakly express the CG13419 message in the posterior VNS, consistent with previous findings (Park, 2003). Similarly, it was predicted that homogenates of CNSs from the CCAP KO flies would not contain bursicon bioactivity if tested in the neck-ligated Sarcophaga bioassay. Indeed, homogenates from CCAP KO flies contain little or no bursicon activity in the Sarcophaga bioassay, whereas homogenates of the relevant control flies (LacZ) produced a score similar to that of wild-type flies (Dewey, 2004).

The phenotype of burs mutants is similar to that of rickets (rk) mutants, which also fail to sclerotize, pigment, and expand their wings (Baker, 2002). The rk gene encodes a G protein-coupled receptor with a large, leucine-rich extracellular domain and was proposed to be a Bursicon receptor. It belongs to a subfamily of receptors whose ligands include the vertebrate glycoprotein hormones (Baker, 2002), which are also cystine-knot proteins. The similarity in phenotype between burs and rk mutants suggests that they may represent the ligand and receptor for this hormonal signaling pathway. Nevertheless, flies that were transheterozygous for mutant alleles of both genes (rk⁴/+; burs^Z1091/+ and rk⁴/+; burs^Z5569/+) showed no defects in wing expansion or tanning. Additional detailed experiments will need to be carried out to determine whether these two molecules interact (Dewey, 2004).

The homology to peptide sequences from cockroach bursicon and other insect species, the phenotypes and reduction in Bursicon activity in mutants of this gene, and the localization of its transcripts to neurons known to produce bursicon in other insect species, make a convincing case that CG13419 encodes the tanning hormone Bursicon. Hence, 42 years after its discovery, the structure of the last of the classic insect developmental hormones has finally been elucidated. Bursicon is the first member of the cystine knot family of signaling molecules to be assigned a defined hormonal function in invertebrates. Knowledge of the molecular identity of Bursicon now provides tools for the analysis of its function, including the mechanism of its tightly timed action in triggering the biochemical events (Andersen, 1996) that lead to cuticle hardening. The colocalization of Bursicon with CCAP, a peptide that activates the ecdysis motor program, poses the fascinating problem of how these two hormones are differentially regulated to produce two temporally distinct behaviors. The understanding of the neuroendocrine control of cuticle sclerotization may also have potential for the development of novel insect-specific pest control agents and measures (Dewey, 2004).

Bursicon signaling mutations separate the epithelial-mesenchymal transition from programmed cell death during Drosophila melanogaster wing maturation

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial-mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation (Natzle, 2008. Full text of article).

Completion of wing morphogenesis in Drosophila is an active process that is part of the suite of events initiated following eclosion of the fly from the pupal case. These events are known to be controlled by release of Bursicon from specific neurons in the abdominal ganglion. This study investigated the possibility that the Bursicon/Rickets signaling pathway also plays a role in the events that remove the cellular epidermis from within the expanding wing. The approach differed from that of other labs that have studied programmed cell death in the wing in that a marker was simultaneously followed for nuclear breakdown associated with programmed cell death (DAPI chromatin staining) and another marker was followed for the behavior and integrity of adherens junctions between cells (Arm-GFP). Mutations in this signaling pathway separate the dissociation of the epithelial cells from initiation of programmed cell death. In wild-type wings, within about an hour after eclosion, redistribution of Arm-GFP from its location in the subapical adherens junctions to the cytoplasm is accompanied by changes in cell shape. Within the same time frame, chromatin condensation was also observed, indicating nuclear changes accompanying cell death, beginning in some nuclei and spreading to others. Loss-of-function mutations in several different components in the signaling pathway (rickets, bursicon, pupal) and a potential upstream component affecting the secretion of Bursicon (batone) all produce a similar phenotype marked by aberrant persistence of the epithelia with intact adherens junctions, extending at least to 24 hr post-eclosion. In contrast, in all of these mutant backgrounds, normal initiation of programmed cell death in the epithelial cells is observed with a time course similar to wild type. These results provide evidence for rk-dependent regulation of the EMT and existence of a cell-death-signaling pathway that is independent of signaling through the rk receptor. These data might also be consistent with a model in which rk activation is capable of initiating both the EMT and cell death, but cell death induction can also occur via an independent and redundant pathway in the absence of wild-type rk function (Natzle, 2008).

Elimination of the epithelial cells from the wing involves coordinated changes in the epithelia that involve delamination from the cuticle and loss of contacts between cells (Kiger, 2007), accompanied by rapid and widespread initiation of programmed cell death. Completion of the EMT is followed by removal of the cells from the wing. When the caspase inhibitor p35 is expressed in the epithelial cells, the cells complete the EMT and assume a rounded shape. A significant proportion of the disorganized cell population does not leave the wing. A recent study (Link, 2007) also demonstrated that mutations that block programmed cell death in the wing epithelium produce persistence of nuclear staining long past the time when cells are normally cleared from the wing. While Link did not employ methods that allowed visualization of the shape of whole cells or of adherens junctions, it is likely that persistence of the nuclei marked persistence of intact epithelial cells. These observations imply that completion of one or more steps in the cell death program are required in addition to the EMT for exit of epithelial cells from the wing. In this view, both activation of programmed cell death and the EMT are necessary for removal of the wing epithelium, but neither is sufficient. Link has proposed that the cells disintegrate within the wing and their debris is flushed from the wing by flowing hemolymph. The results presented in this study clearly show that EMT must accompany cell death for dispersal of the wing epithelium to occur. Programmed cell death without EMT does not cause loss of the intact wing epithelium (Natzle, 2008).

On the basis of the spindle shape assumed by individual epithelial cells and the appearance of cell streaming in wings dissected from the body, it has been suggested that active migration could facilitate removal of the cells from the wing, with the caveat that frequent grooming of the wings by the hind legs of the fly may be an important part of the process (Kiger, 2007). It is suggested that loss of adhesion and acquisition of motility, grooming, and hemolymph flow may all contribute in vivo to rapid removal of cells. It seems likely that cells in which p35 is expressed, or in which other functions necessary for programmed cell death have been impaired, may make or retain attachments to the cuticle or extracellular matrix that prevent their removal from the wing. The ability to modulate such attachments, which may be necessary for clearing cells from the wing, could depend on appropriate regulation of caspase activity. The Drosophila caspase Dronc regulates border cell migration in the ovary independently of its role in apoptosis. In the mouse, Caspase-8 is required for activation of calpain proteases, Rac, and lamellipodial assembly. A similar requirement for caspase activity could link progress of a cell death program to the release of wing epithelial cells from their substrate attachments following EMT (Natzle, 2008).

The evidence for independent activation of epithelial cell death and the EMT raises the question of how these events are coordinated at eclosion. A complete answer to this question will have to await identification of other signal(s) that initiate programmed cell death or modulate the EMT. It is likely that overall synchronization is controlled via activation of the peptidergic networks within the central nervous system and coordinated release of signals that regulate specific aspects of ecdysis behavior. For example, the same neurons that express Bursicon at eclosion also express other active signaling neuropeptides such as the crustacean cardioactive peptide (CCAP) and the myoinhibitory peptide (MIP) with potentially independent direct or indirect molecular targets. Sequential release of different regulators can also modulate a process, e.g., control of cuticle tanning via pre-eclosion CCAP stimulation of tyrosine hydroxylase (TH) protein accumulation followed by Bursicon-dependent activation of TH via phosphorylation after eclosion (Natzle, 2008).

Coordination of the post-eclosion behavior of cells within the wing epithelium also potentially involves signals to the wing epithelial cells, prior to their death and dispersal, directing additional functions such as construction of the matrix that bonds the two cuticular surfaces of the mature wing. For example, the Timp gene (tissue inhibitor of metalloproteinases) is expressed in wings of newly eclosed flies and is required for normal wing bonding. Homozygous Timp^-/Timp^- flies expand their wings and eliminate the epithelial cells normally; however, the cuticular surfaces of the wing do not bond. Small Timp^-/Timp^- clones in the epithelia, produced by mitotic recombination, exhibit no bonding defects, consistent with secretion and diffusion of Timp from neighboring Timp⁺/Timp^-cells (KIGER, 2007). In contrast, large Timp^-/Timp^- clones, produced by mitotic recombination using the Minute technique, generate improperly bonded wings similar to those of homozygous Timp^-/Timp^- flies. Thus, the genotype of the epithelial cells themselves affects bonding of the cuticular surfaces, establishing that the wing cells play a critical role in metabolizing and remodeling the wing extracellular matrix. This is in agreement with observations that premature death of the epithelial cells prevents bonding of the cuticular surfaces. The timing and initiation of these cellular behaviors that affect wing bonding may also be coordinated via signals generated by the ecdysis cascade (Natzle, 2008).

For survival of the fly following eclosion in the natural environment, it is important that the entire wing epidermis be removed quickly to ensure timely completion of wing blade bonding and formation of functional flight organs. It would be reasonable to postulate that imposition of an EMT along with initiation of programmed cell death would ensure a more rapid and uniform dispersal of the epidermis from the appendage. Indeed, it would appear that the genes for producing EMT form a cassette that can be deployed as needed during the evolution of developmental pathways: first during gastrulation, then during peripheral nervous system formation, and later in winged insects during wing maturation (Natzle, 2008).

GENE STRUCTURE

Exons - 3

PROTEIN STRUCTURE

Amino Acids - 173

Structural Domains

CG13419 was identified using a modified protein BLAST search of the Drosophila genome using the P. americana partial bursicon peptide sequences obtained by microsequencing (Honegger, 2002). Sequence analysis of the genomic clone and its corresponding cDNA has revealed that the coding sequence is 522 nucleotides long. The gene contains three short exons (130, 125, and 267 nucleotides, respectively) and two introns (64 and 58 nucleotides, respectively). The CG13419 gene product is predicted to be a 173 amino acid preprotein (19 kDa). Removal of the predicted N-terminal signal sequence of 33 amino acids would result in a mature protein of 140 amino acids, approximately 15 kDa. CG13419 is a member of the 10-membered cystine knot protein family (Vitt, 2001), which typically forms dimers. Members of this family contain six cysteine residues that form the knot and an optional additional cysteine that may be important for dimerization. They include the glycoprotein hormones, TGF-β, platelet-derived growth factor, and the mucins. The sequence of CG13419 is most similar to that of the mucin subfamily, which also includes signaling molecules such as the Bone Morphogenic Protein antagonists. Previous data indicate that bursicon functions as a 30 kDa dimer in the cockroach (Kostron, 1999). The available sequence information for the homologous partial peptide sequences from the Anopheles gambiae and Apis mellifera genomes show 83% identity to CG13419. The cysteines within the cystine knot domain are conserved in these species (Dewey, 2004).

EVOLUTIONARY HOMOLOGS

In an effort to characterize the insect molting hormone bursicon from the cockroach, Periplaneta americana, amino acid sequences with high identity of Cu,Zn-superoxide dismutase (SOD) of Drosophila virilis were identified. Antisera against a conserved region of SOD, and a sequence unique to Periplaneta SOD were produced and used to test whether bursicon might be a form of SOD. Western blots of one- and two-dimensional gels revealed that the dimeric form of SOD and bursicon have a similar molecular mass (30 kDa). The two proteins can be separated, however, according to their different isoelectric points. Bursicon is identified in two-dimensional gels by elution from four unique spots not labeled by the anti-SOD antisera. In sections of Periplaneta nerve cords, the antisera labeled glial material surrounding neuronal somata close to the neural sheath. Bursicon, however, is contained in unique cell pairs in the ganglia of the ventral nerve cord. These neurons were labeled with new antisera produced against novel sequences of one of the four above-mentioned bursicon active spots. The results show unequivocally that SOD and bursicon are distinctly different proteins. Furthermore, the anti-SOD antisera provided a tool to isolate and sequence bursicon (Kostron, 1999).

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two-dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon-containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon-containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two-dimensional gels. The 12-amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related (Honegger, 2002).

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum

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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