BIOLOGICAL OVERVIEW

At the end of each developmental stage, insects perform a stereotypic behavioral sequence leading to ecdysis (molting or shedding) of the old cuticle. While ecdysis triggering hormone (ETH) is sufficient to trigger this sequence, it has remained unclear whether it is required. This study shows that deletion of ETH, the gene encoding ETH in Drosophila, leads to lethal behavioral and physiological deficits. Null mutants (eth^-) fail to inflate the new respiratory system on schedule, do not perform the ecdysis behavioral sequence, and exhibit the phenotype buttoned-up, which is characterized by incomplete ecdysis and 98% mortality at the transition from first to second larval instar. Precisely timed injection of synthetic ETH1 (one of the peptides encoded by the ETH gene) restores all deficits and allows normal ecdysis to occur. These findings establish obligatory roles for ETH and its gene products in initiation and regulation of the ecdysis sequence. The ETH signaling system provides an opportunity for genetic analysis of a chemically coded physiological and behavioral sequence (Park, 2002a).

Insect development proceeds through a series of stages from egg to reproductive adult, each punctuated by ecdysis of the old cuticle. Initiation of the molt, which culminates in ecdysis, coincides with one or more bouts of ecdysteroid-induced gene expression appropriate to the next stage. The end of the molt is signaled by declining steroid levels, leading to ecdysis of the old exoskeleton surrounding the body and lining the respiratory system and gut (Park, 2002a).

Ecdysis is controlled by a genetic program specifying a precisely timed developmental sequence. Among the genes involved are those encoding peptide hormones that activate central pattern generators for pre-ecdysis and ecdysis behaviors. Ecdysis in Saturniid moths is triggered by a peptide factor from the brain, identified as eclosion hormone (EH: see Drosophila Eclosion hormone). EH causes secretion of ecdysis-triggering hormones (ETHs) from endocrine Inka cells (Zitnan, 1996; Kingan, 1997), which act on the CNS to trigger centrally patterned pre-ecdysis and ecdysis behaviors. It is well established that ETHs trigger ecdysis upon injection into moths and flies (Zitnan, 1996; Ewer, 1997; Baker, 1999; Park, 1999; Zitnan, 1999; Zitnan, 2000). These studies demonstrate the sufficiency of ETHs in triggering ecdysis, but do not prove their necessary involvement in the natural process (Park, 2002a).

During the hour preceding ecdysis of 1st instar Drosophila larvae, three main events occur: sclerotization of new mouthparts, tracheal dynamics and a defined behavioral sequence. Appearance of the new mouthparts, including mouthhooks and vertical plates, precedes ETH release, and no disruption of these events occurs in eth^– flies. However subsequent events are triggered by ETH release from a system of Inka cells homologous to those previously described in moths (Park, 2002a).

As direct gene products, many peptide signaling molecules provide a link between gene expression and behavior. Orchestration of the ecdysis behavioral sequence depends on coordinated induction and suppression of genes essential to its properly timed initiation. This includes induction of ETH gene expression in Inka cells and CNS sensitivity to ETH 1-2 days before ecdysis (Zitnan, 1999; Zitnan, 2000). The timing of ecdysis initiation is accomplished by ecdysteroid suppression of secretory competence in Inka cells until the animal is ready to perform the behavioral sequence and escape the old cuticle (Kingan, 2000). Once ecdysteroids drop to low levels during the hours preceding ecdysis, peptide hormones, including ETHs, eclosion hormone and Cardioacceleratory peptide, are released to initiate the behavioral sequence. Orchestration of ecdysis behavior therefore depends on coordinated expression of genes that are involved in peptide signaling (Park, 2002a and references therein).

Each of these peptides is capable of initiating ecdysis, but it has remained unclear to what extent they play obligatory roles. To test the hypothesis that ETH is required for ecdysis, genetic tools in Drosophila were used to delete its gene, ETH (Park, 1999). Genetic null Drosophila mutants carrying micro-deletions in the ETH locus are described. The consequences of this selective hormonal deficit are severe, and include failure both to inflate the new respiratory system and to perform the ecdysis behavioral sequence. Virtually all animals fail to survive the first ecdysis. These findings establish an obligatory role for eth and its gene products, and demonstrate how lack of a specific chemical signal results in a lethal behavioral deficit (Park, 2002a).

Larval ecdysis in Drosophila culminates a sequence of precisely timed morphological, physiological and behavioral events. These include appearance of new mouthparts, tracheal inflation, and pre-ecdysis and ecdysis behaviors. Park (2002) details a time line of these events, which occur during the 1 hour interval preceding ecdysis from 1st to the 2nd larval instar. Visible morphological changes become apparent ~1 hr prior to ecdysis, including the appearance of new mouth hooks and vertical plates. The first event is sclerotization of new mouth hooks adjacent to the old structures, referred to 'double mouth hooks'. About 30 minutes later, new vertical plates appear, leading to 'double vertical plates' (dVP). dVP was chosen as a reference point (time zero) to which all other events are related (Park, 2002a).

Before the appearance of dVP, the old tracheal lining or 'intima' in the main dorsal tracheal trunk separates from the new intima. About 10 minutes after the dVP stage the old intima collapse, becoming coiled in appearance. Tracheal collapse is followed immediately by inflation of the new trachea with air, which takes ~2-5 minutes. Before this time, the space between the old and new intima is filled with liquid, presumably molting fluid (Park, 2002a).

Pre-ecdysis behaviors commence upon completion of tracheal air filling. The first stage of pre-ecdysis consists of repetitive 'anteroposterior' contractions (A-P) beginning 15 minutes after dVP. This behavior is characterized by alternating telescopic contractions of 2-5 seconds duration and relaxations lasting 10-18 seconds. The second stage of pre-ecdysis beginning at 19 minutes involves rolling contractions called 'squeezing waves'. Squeezing waves (SW) are visible from the dorsal aspect and travel from posterior to anterior at 5-6 second intervals, ending with head retractions. During the A-P and SW behaviors, vigorous muscle contractions pull the mouthparts alternately in the posterior and anterior directions. It is believed that these movements may be crucial for later detachment of old vertical plates and mouthhooks from the new apparatus during subsequent ecdysis behavior (Park, 2002a).

Ecdysis behavior begins 25 minutes after dVP, with one or two forward head thrust movements, which detach old mouthparts and plant them in the substrate. The forward movement also coincides with shedding of old tracheal linings through segmental spiracular pits. Upon planting the old mouthparts onto the substrate, the forward thrust is followed by three to five vigorous backward thrusts to detach the old posterior spiracles. The behavior is interrupted by a 2-5 minute rest period, and is completed by a forward, lateral turning escape movement, freeing the animal from the old cuticle. Some variation in this pattern is observed, in which subunits of the ecdysis behavior are repeated or even entire recapitulations of the behavioral sequence are observed. The patterned behavior just described is also accompanied by some irregular behaviors prior to anterior-posterior contractions (A-P) with large variations between individuals and time of onset. These are (1) head swinging, (2) dorso-ventral contractions and (3) alternating anterior and posterior peristaltic squeezing (Park, 2002a).

Ecdysis from second to third instar follows a similar pattern. Even the time of behavioral onset is similar, with the exception that double mouth hooks appear at -104 minutes relative to dVP instead of -30 minutes as observed in the 1st to 2nd instar ecdysis (Park, 2002a).

The peptides Drosophila ETH1 and ETH2, which trigger adult ecdysis (Park, 1999), were injected into late first instar larvae to assess their ability to induce ecdysis at this stage of development. Injection of ETH1 (0.01 to 10 fmol) at the double mouth hooks stage induces the premature appearance of physiological and behavioral events accompanying ecdysis. The first response to peptide injection is premature tracheal collapse and inflation, with a latency of 3 minutes and 4 minutes, respectively, followed by pre-ecdysis and ecdysis behaviors. Weak anterior-posterior contractions were observed in 33% of injected animal. Strong squeezing waves occurred in all injected animals, with an average latency of 14±2 minutes. Ecdysis behavior consisting of forward thrust, backward thrust and turning escape movements appeared with a latency of 24±3 minutes (Park, 2002a).

The timing of Drosophila ETH1 injection is critical for successful ecdysis. All animals injected at the dVP stage underwent successful ecdysis. However injections performed earlier, for example at double mouth hooks, induced tracheal collapse and inflation, anterior-posterior contractions, squeezing waves and repeated bouts of ecdysis behavior that were unsuccessful in shedding the mouthparts, leading ultimately to death. These observations indicate that precise timing of events in the ecdysis sequence is critical for successful ecdysis (Park, 2002a).

ETH2 injections were less effective in eliciting tracheal dynamics and behaviors. At relatively high doses (>10 fmol), ETH2 induces tracheal collapse and inflation with a latency of 3 minutes and 4 minutes, respectively, and ecdysis behaviors with a latency of 34±5 minutes. Strikingly, ETH2 (10 fmol) elicits neither anterior-posterior contractions nor squeezing waves. Lower doses of ETH2 (1 fmol) induce tracheal collapse and inflation with latencies of 4 minutes and 5 minutes, but have no behavioral effects (Park, 2002a).

Inka cells of Drosophila express the gene ETH and contain the peptide ETH. Expression of ETH is evident in transformed Drosophila carrying the chimeric transgene eth3-egfp, where EGFP fluorescence and ETH-like immunoreactivity are colocalized in Inka cells. No other cells exhibit EGFP fluorescence, although some CNS neurons also show ETH-IR. The most intense EGFP fluorescence is observed as larvae approach the first ecdysis, whereas neither EGFP fluorescence nor ETH-IR is detected during embryonic development or early 1st instar. This, together with the observation that no hatching deficiency occurs in eth^- mutants, indicates that ETHs are probably not required for early development, including the patterned behavior associated with egg hatching. EGFP fluorescence and ETH-IR are observed throughout the adult stage, suggesting possible mating or reproductive functions (Park, 2002a).

ETH triggers tracheal collapse and inflation. This conclusion is indicated by two observations. (1) EGFP fluorescence in Inka cells sharply declines just minutes before these events. Since the subcellular distribution of EGFP fluorescence and ETH-IR in Inka cells is identical, it is concluded that EGFP and processed ETHs are sorted into secretory granules of Inka cells (Klein, 1999), and that these peptides are co-released. (2) Injection of either Drosophila ETH1 or DrmETH2 into wild-type larvae or eth^- mutants induces tracheal collapse and air inflation within minutes. These observations strongly implicate ETH in the control of tracheal dynamics before ecdysis (Park, 2002a).

It is notable that Inka cells and associated components of the epitracheal endocrine system are situated directly on tracheal tubes (Zitnan, 1996). While it has been recognized for some time that ETHs act directly on the CNS to elicit centrally patterned behaviors, this report documents for the first time functions for these peptides in respiratory physiology. What is the functional significance of Inka cell placement directly on tracheal tubes? It is speculated that their location in some way senses the readiness of the respiratory system to switch over from old to new trachea in preparation for pre-ecdysis and ecdysis behaviors. Whether the Inka cells indeed have such a sensory function remains to be demonstrated (Park, 2002a).

The mechanism of tracheal inflation is not known, but it is hypothesized that dissolved gas is liberated as a consequence of fluid movement out of the tracheal lumen. The hydrophobic surface of the cuticle lining the tracheal tube may then facilitate a reverse capillary force to complete air filling. This is the first indication that these processes are under hormonal control. Further work is needed to define the signaling steps involved in this process (Park, 2002a).

Upon completion of tracheal inflation, a behavioral sequence ensues consisting of pre-ecdysis and ecdysis behaviors. These behaviors and successful ecdysis are triggered upon injection of Drosophila ETH1, consistent with an earlier study showing that ETH injection induces premature eclosion behavior in the pharate adult (Park, 1999). It is striking that the same chemical signal initiates two quite different behaviors separated by two metamorphic molts (Park, 2002a).

Drosophila ETH1 is sufficient to evoke the ecdysis sequence in larval Drosophila, as was previously observed for adult Drosophila and for MasETH in M. sexta and BomETH in B. mori (Zitnan, 1996; Kingan, 1997; Park, 1999; Zitnan, 1999). The lethal phenotype observed in eth^- deletion mutants provides confirmation that, in Drosophila, ETH is necessary for ecdysis. The same physiological and behavioral deficiencies occur in both eth^25b and eth¹⁹⁶ lines, where tracheal collapse and inflation are delayed for hours, and pre-ecdysis behaviors are absent. Ecdysis behavior is abnormal and occurs prematurely, soon after the dVP stage. These physiological and behavioral deficits contribute to the buttoned-up phenotype (Park, 2002a).

It is remarkable that tracheal dynamics and the behavioral sequence absent in eth^- mutants can be completely restored by injection of Drosophila ETH1. Animals rescued by injection of Drosophila ETH1 shed the cuticle normally and develop through the second instar, although a higher mortality is observed in eth¹⁹⁶ mutants. Since this line also has a significant deletion of the adjacent orc4 gene, it may suffer additional defects which may account for this. The deletion phenotype and its facile rescue by injection of Drosophila ETH1 provide the best evidence thus far that this blood-borne peptide is a necessary signal in the orchestration of key developmental events culminating in ecdysis. Interestingly, Drosophila ETH2 injection also rescues the buttoned-up phenotype, but without triggering pre-ecdysis behaviors. It therefore appears that the pre-ecdysis behaviors in Drosophila larvae are not crucial for completion of ecdysis (Park, 2002a).

An obvious physiological consequence of ETH deletion is failure of the respiratory system to inflate on schedule. This is consistent with the finding that ETH injection regulates collapse of old trachea and inflation of new trachea, and confirms that this peptide plays a vital signaling function for tracheal dynamics associated with ecdysis. Significantly, tracheal collapse and inflation are markedly delayed, but not eliminated in eth^- larvae. Therefore ETH may act indirectly through downstream regulatory processes, which eventually succeed in mediating tracheal inflation. One candidate signal for tracheal inflation is Eclosion hormone. Adult Drosophila that lack functional eclosion hormone neurons fail to inflate the tracheal system properly upon adult eclosion and are reported to be insensitive to ETH (Park, 2002a).

The actual causes of lethality observed in eth^- larvae could be a combination of factors, including respiratory and behavioral deficits. Lack of tracheal inflation and incomplete shedding of the old tracheal lining would obviously compromise respiratory functions. Failure to shed old mouthparts at the appropriate time is likely the main factor in production of the buttoned-up phenotype. This condition appears in null mutants, as well as in wild-type flies upon premature injection of Drosophila ETH1. In either case, the buttoned-up phenotype prevents complete ecdysis and further ingestion of food. All of these factors could be jointly involved in the lethality observed (Park, 2002a).

Of special significance is the fact that ETH deletion mutants engage in a premature ecdysis-like behavior. Although quite variable and different from authentic ecdysis, these sporadic bouts gradually became more similar to authentic ecdysis more than 2 hours after dVP. Animals are able to shed the old cuticle partially, albeit with buttoned-up mouthhooks. The early appearance of ecdysis-like behavior provides further evidence that ETH not only triggers ecdysis, but delays its onset until the appropriate time. Decapitation or removal of cephalic or thoracic ganglia accelerates ecdysis, supporting the hypothesis that ETH activates both excitatory and inhibitory centers in the CNS (Baker, 1999; Zitnan, 2000), with inhibition functioning to delay ecdysis until the appropriate time. In the absence of ETH, these inhibitory centers may not be triggered, leading to premature ecdysis or ecdysis-like behavior (Park, 2002a).

The ETH gene encodes three predicted amidated peptides: ETH1, ETH2 and ETH-AP. Two of these peptides, ETH1 and ETH2 have biological activity in both larval and adult stages (Park, 1999). In wild-type larvae, injection of Drosophila ETH1 elicits the entire sequence of ecdysis-related events, although for reasons not yet clear the A-P phase of pre-ecdysis is rather weak. Similarly, Drosophila ETH1 alone rescues the entire sequence in eth- mutants, including tracheal dynamics, anterior-posterior contractions (A-P), squeezing waves (SW) and ecdysis. ETH2 also elicits tracheal dynamics and ecdysis behavior, but only at higher doses. This peptide fails to trigger pre-ecdysis (A-P and SW). Lower doses of ETH2 (~1 fmol) induce only tracheal dynamics, without pre-ecdysis or ecdysis. With regard to relative potency, these findings are consistent with earlier work showing that Drosophila ETH1 is more active than DrmETH2 in triggering adult eclosion (Park, 1999). However, no functional differences were apparent in that study (Park, 2002a).

Given that Drosophila ETH1 is sufficient to trigger the entire sequence, the functional role of DrmETH2 is unclear. It might have been expected that DrmETH2 is involved in pre-ecdysis behavior, given that Drosophila ETH1 injection elicits only weak A-P contractions. However, DrmETH2 fails to elicit pre-ecdysis at all, yet elicits tracheal collapse and air filling at lower doses than are needed to generate ecdysis behavior. The higher potency of this peptide in eliciting tracheal collapse and air filling suggests that it may regulate tracheal dynamics in a way that has thus far escaped attention. It is also possible that because ETH is expressed in the adult stage, DrmETH2 plays some role in mating or reproduction. Further work is needed to resolve these issues (Park, 2002a).

In M. sexta, the ETH gene also encodes three peptides, PETH, ETH and ETH-AP (Zitnan, 1999). PETH induces only pre-ecdysis I, whereas ETH triggers pre-ecdysis II and ecdysis. It is also known that injection of ETH into naive animals elicits all three behavioral steps, including pre-ecdysis I, pre-ecdysis II and ecdysis (Zitnan, 1996). It would therefore be predicted that, if the ETH gene were deleted in M. sexta, ETH alone might be sufficient to rescue the missing behavioral sequence, leaving the role (necessity) of PETH similarly unclear. It might be possible to approach this issue using RNAi followed by injection of each peptide (Park, 2002a).

The phenotype of eth^- null mutants results in 98%-100% lethality at the first larval ecdysis. This is striking in comparison with the phenotype of EH-cell knockout flies that show only ~50% lethality in larval stages, uncoordinated behaviors during adult eclosion and accumulated lethality of ~70%. Similarly, ablation of crustacean cardioactive peptide (CCAP: Gammie, 1997)-cells leads to no obvious defects during larval stages but major lethality at pupation. It has been proposed that ETH acts through central release of EH, because although wild-type flies show premature eclosion behavior upon injection of ETH, EH-cell knockout flies are insensitive to ETH. Furthermore, CCAP is seen as a downstream signal whose release is caused by EH (Park, 2002a).

If EH is an obligatory signal downstream of ETH why is the larval mortality of eth^- animals as shown in this study so much higher than that of EH knockouts? Analysis of behavioral phenotypes in larval ecdysis of CCAP-cell knockout and EH-cell knockout flies may provide invaluable information to aid in the understanding of the roles of each peptide and signaling cascade for the ecdysis (Park, 2002a).

The cascade of peptides involved in regulation of insect ecdysis is growing. It is clear that ETH participates in a complex cascade that includes EH, and crustacean cardioactive peptide (CCAP) (Gammie, 1997). These molecules appear to be broadly conserved among Lepidoptera, Drosophila and other insects, and may be expandable to other arthropods such as crustaceans. A conceptual framework for the peptide signaling cascade regulating ecdysis has been provided by studies of Manduca sexta (Ewer, 1997; Gammie, 1999; Zitnan, 1999; Zitnan, 2000) and Drosophila. It is thought that a positive feedback pathway between peripheral secretion of EH from the CNS and ETH from Inka cells elevates circulating levels of both peptides (Ewer, 1997). Elevated levels of ETH acting on downstream targets within the CNS recruit sequential pre-ecdysis and ecdysis behaviors. Among these targets are CCAP-containing neurons (Gammie, 1997), which activate the central pattern generator for ecdysis behavior (Park, 2002a).

Ecdysis provides an excellent model system for analysis of a chemically coded behavioral sequence. The behavioral deficits associated with ETH deletion provide a particularly clear illustration of behavioral failure in the absence of the requisite signaling molecule. It is remarkable that complete rescue of the behavior occurs on simple injection of ETH. These findings indicate that the performance of innate, stereotypic behavioral sequences may depend upon achieving proper levels of peptides and other endogenous signals in the nervous system so as to activate and bias central pattern generators appropriate to developmental and sensory context. Other specific examples of how peptides may function as behavioral orchestrators in vivo include egg-laying, feeding and maternal behaviors (Park, 2002a).

Several developmental mutants of Drosophila display phenotypes defined by molting deficiencies. Many of these display 'double mouth hooks', suggesting a defect in an aspect of the ecdysis program. Many if not most of these phenotypes result from defective events upstream of ETH signaling, and it seems likely that they may result in ETH deficiency and the buttoned-up phenotype (Park, 2002a).

Mutations that lead to the double mouthhooks phenotype fall into three general categories: ecdysone synthesis and secretion, downstream transcriptional signaling and peptide processing enzymes. Ecdysone synthesis mutants include ecd (ecdysoneless), dare (defective in avoidance of repellents) and itpr (IP-3 receptor). Transcription factors regulated by ecdysone signaling include EcR-B, USP (ultraspiracle), ßFTZ-F1 (ftz transcription factor 1) and crc (cryptocephal). Finally, a mutant displaying the buttoned-up phenotype carrying defective peptide processing enzymes is amontillado, which encodes the enzyme prohormone convertase 2; these mutants are deficient in larval molting. Some mutants also are deficient in hatching behavior. Mutations of PHM (peptidylglycine alpha-hydroxylating monooxygenase), which is required for alpha-amidation at the C-terminal end, also generate a buttoned-up phenotype. It will be interesting to determine whether many if not most of the above mutations lead to ETH deficiency. The ETH deletion mutant and the ETH reporter fly line 2eth-egfp also provide opportunities to investigate roles for ecdysone in regulation of expression, processing and secretion of ETH, and its downstream pathways up to ecdysis behavior (Park, 2002a).

GENE STRUCTURE

cDNA clone length - 735

Exons - 2

Bases in 3' UTR - 134

PROTEIN STRUCTURE

Amino Acids - 203

Structural Domains

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster, has been defined. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (Drosophila ETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of ETH in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior (Park, 1999).

date revised: 26 April 2002

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