InteractiveFly: GeneBrief

Ecdysis triggering hormone: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

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).

Rescheduling behavioral subunits of a fixed action pattern by genetic manipulation of peptidergic signaling

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. This study presents evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. These findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP (Kim, 2015).

Innate behaviors are stereotypic patterns of movement inherited from birth that require no prior experience for proper execution. Among such behaviors are fixed action patterns that, once initiated, run to completion independent of sensory inputs. Examples include courtship rituals, aggression displays, and ecdysis. Ecdysis represents a 'chemically-coded' behavioral sequence triggered by peptidergic ecdysis triggering hormones (ETH), which orchestrate a downstream peptidergic cascade leading to sequential activation of central pattern generators underlying patterned motor activity. The term FAP has fallen into disuse, since innate behaviors generally exhibit considerable plasticity. However the invariant nature of the ecdysis behavioral sequence makes it a clear example a classic FAP. In depth analysis of ecdysis behavior may provide a more thorough understanding of how hormones assemble and regulate behavioral circuitry in the brain, in particular circuits that operate sequentially (Kim, 2015).

ETHs are released by peripheral peptidergic Inka cells in response to declining levels of the steroid hormone 20-hydroxyecdysone. Presence of Inka cells in more than 40 species of arthropods, along with the sequence similarity of ETH peptides in diverse insect groups, suggests that ETH signaling is highly conserved in insects. Identification of the Ecdysis Triggering Hormone receptor (ETHR) gene in Drosophila melanogaster enabled elucidation of a complex downstream signaling cascade triggered by ETH. The ETHR gene encodes two functionally distinct subtypes of G protein coupled receptors (ETHR-A and -B) through alternative splicing. The presence of two ETH receptor subtypes has been observed in all insect species thus far examined. The two receptor subtypes show differences in ligand sensitivity and specificity and are expressed in separate populations of central neurons, suggesting that they have distinctive roles in ETH signaling (Kim, 2015).

A diversity of ETHR neurons in the moth Manduca sexta and fruitfly Drosophila melanogaster has been identified. One of the most striking properties of ETHR-A neurons is that they are virtually all peptidergic and conserved across insect orders. Groups of ETHR-A 'peptidergic ensembles' express a range of different neuropeptides, including kinins, diuretic hormone (DH), eclosion hormone (EH), FMRFamide, crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIPs), bursicon (burs and pburs), neuropeptide F (NPF), and short neuropeptide F (sNPF). It is hypothesized that released ETH acts directly on the CNS to activate these peptidergic ensembles for control of specific central pattern generator circuits that elicit stereotyped ecdysis behaviors. However evidence for direct actions of ETH on these target ensembles is yet to be reported (Kim, 2015).

Likely functions of certain ETHR-A peptidergic ensembles in Manduca have been inferred from pharmacological manipulations. For example, serially homologous L3/4 neurons of abdominal ganglia in Manduca express a cocktail of kinins and diuretic hormones; exposure of the isolated CNS to these peptides elicits a fictive pre-ecdysis I-like motor pattern. Similarly, the IN704 peptidergic ensemble that co-expresses CCAP and MIPs is implicated in initiation of ecdysis behavior, since co-application of these two peptides elicits fictive ecdysis behavior. Homologous peptidergic ensembles in Drosophila exhibit characteristic patterns and time courses of calcium mobilization indicative of electrical activity coincident with successive steps in the ecdysis FAP. Of particular interest are observations that bursicon, a hormone co-expressed in a subset of CCAP neurons, is required for ecdysis behavior (Kim, 2015).

This study tested hypotheses that two central peptidergic ensembles -- kinin neurons and a subset of CCAP neurons (CAMB) that co-express CCAP, Allatostatin CC, Myoinhibitory peptide, and Bursicon -- are direct targets of ETH and schedule pre-ecdysis and ecdysis behavior components of the ecdysis FAP, respectively in the fruit fly Drosophila. Manipulation of ETHR expression levels and signal transduction specifically in these ensembles influences scheduling of the FAP. Finally, possible mechanisms were examined underlying timing of the switch from pre-ecdysis to ecdysis behavior, and a model is proposed to explain mechanistically how these behaviors are sequentially activated (Kim, 2015).

The aims of this study were to implicate identified ETHR neuronal ensembles with specific steps in the ecdysis FAP. Through genetic manipulation of all known ETHR-A central neuron ensembles and subsets thereof, kinin and CAMB neurons were implicated in scheduling of pre-ecdysis and ecdysis, respectively. These findings confirm that these ensembles are targeted directly by ETH. A key observation is timing of calcium mobilization in kinin and CAMB ensembles following ETH application: kinin neurons mobilize calcium within minutes, while activity in CAMB neurons is delayed. CAMB neurons mobilize calcium only after kinin neuron activity ceases, some 10 minutes later. These temporal patterns of cellular activity correspond well with those of pre-ecdysis and ecdysis behaviors observed in vivo (Kim, 2015).

The findings demonstrate that the kinin peptide ensemble is necessary for proper scheduling of pre-ecdysis behavior, if not itself sufficient to elicit it. Kinin cell ablation abolishes pre-ecdysis behavior in a significant percentage (25%) of animals. The remaining 75% of individuals showed highly variable pre-ecdysis duration, ranging from 3-22 minutes, whereas duration of this behavior in control animals is tightly regulated at 9.1 ± 0.9 min. Furthermore, a 30% reduction in kinin receptor expression, caused by a piggyBac-element insertion into the promoter region of the gene, also disrupts fidelity of pre-ecdysis regulation; this phenotype is rescued by precise excision of the piggyBac insertion. Finally, RNA silencing of the kinin receptor in peripheral neurons using the Peb-Gal4 driver leads to reduced intensity of the behavior and greatly increases variability of pre-ecdysis duration. This is the first report demonstrating that kinin signaling affects scheduling of the ecdysis behavioral sequence via actions on peripheral neurons. Pan-neuronal silencing of kinin receptors also disrupted pre-ecdysis scheduling, but to a lesser extent (Kim, 2015).

Manipulation of ETHR expression levels in kinin neurons also alters scheduling of pre-ecdysis behavior significantly, confirming that these neurons are targeted directly by ETH and that they play an important role in pre-ecdysis regulation. It was reasoned that knockdown of receptor expression in these neurons would lead to a lower density of ETHR in the plasma membrane, thereby reducing sensitivity to ETH and delaying onset of pre-ecdysis. The experimental results demonstrated a reduction of pre-ecdysis duration. This reduction was attributed to a delay in pre-ecdysis onset, brought about by the need for higher ETH levels for neuronal activation. Since timing of the switch to ecdysis (controlled by CAMB neurons; see below) is unaffected, pre-ecdysis duration is shortened. In contrast, overexpression of ETHR in kinin neurons led to prolongation of pre-ecdysis duration. Following the same reasoning, this would result from premature kinin neuron activation attributable to higher sensitivity to rising ETH levels and overall longer pre-ecdysis (Kim, 2015).

Kinins were identified originally using bioassays for myotropic and diuretic functions and they have well-known actions on muscle and transport activity in epithelia. More recent studies demonstrated diverse functional roles for kinin signaling, including feeding, olfaction, and locomotory behavior. ETHR-A expression was previously demonstrated in kinin neurons of both Drosophila and Manduca, implicating them as direct targets of ETH. Imaging studies have shown that abdominal kinin neurons in fly larvae exhibit periodic calcium oscillations under normal conditions and are involved in turning behavior. These kinin neurons project to a terminal plexus in close association to kinin receptors, suggesting it functions as a site of peptide release. Interestingly, this same ensemble of kinin neurons in the pre-pupal preparation used in this study showed no such periodic activity, but instead exhibited synchronized calcium oscillations activity following exposure to ETH. This difference could be unique to the pharate stage (i.e., preceding ecdysis) animal, during which insects generally are unresponsive to external stimuli. In imaging studies, it was observed that ETH-induced calcium dynamics are initiated from the terminal plexus region and subsequently moving anteriorly to the cell bodies. These observations suggest that this plexus serves critical functions in both sending and receiving signals. Evidence presented in this study for regulation of pre-ecdysis behavior by kinin neurons demonstrates a new function for this peptide in Drosophila, which is reinforced by previous observations in Manduca that application of kinin causes a fictive pre-ecdysis motor pattern in the isolated CNS (Kim, 2015).

How do kinin neurons function in the promotion of pre-ecdysis behavior? While manipulation of kinin signaling clearly affects behavioral intensity and duration, attempts to initiating pre-ecdysis through temperature-dependent activation of kinin neurons expressing either TRPM8 and TRPA1 were unsuccessful. It is concluded that, while kinin functions as a modulatory influence necessary for proper scheduling of pre-ecdysis behavior, other as yet unidentified signals are necessary for behavioral initiation (Kim, 2015).

This study has demonstrated that CAMB neurons are both necessary and sufficient for the switch from pre-ecdysis to ecdysis behavior. This conclusion rests on results from a combination of experiments. First, CAMB cell ablation abolishes the switch to ecdysis, suggesting these neurons are necessary for the switch. Failure of ecdysis initiation is attributable to bursicon deficiency, since a previous report showed clearly that expression of the bursicon gene is required for initiation of pupal ecdysis. Calcium mobilization in CAMB neurons is delayed for ~10 min after onset of activity in kinin neurons, which fits well with the ~10 min delay before appearance of ecdysis behavior following onset of pre-ecdysis behavior observed in vivo. Altered levels of ETHR expression in CAMB neurons clearly affects timing of the switch to ecdysis behavior: receptor knockdown delays the switch, whereas overexpression accelerates it. In vitro experiments confirm that altered ETHR expression levels affect timing of calcium mobilization in CAMB neurons in register with changes in behavioral timing (Kim, 2015).

Finally, it was shown that activation of CAMB neurons through temperature-sensitive TRPM8 expression initiates ecdysis behavior in vivo. Thus, CAMB neurons are both necessary and sufficient for the switch to ecdysis behavior. However, activity in CAMB neurons alone does not result in robust ecdysis behavior. Expression of ecdysis behavior with parameters corresponding to that observed in wild-type flies requires activation of the entire CCAP ensemble (Kim, 2015).

It is interesting that, in all TRPM8 activation experiments, removal of the temperature stimulus led to re-capitulation of the entire ecdysis FAP. This might be explained by positive feedback influences on the Inka cell to release ETH, possibly via EH neurons. Alternatively, the ecdysis motor circuit may exert negative feedback on the pre-ecdysis circuit, which when removed, causes a post-inhibitory rebound leading to activation of the pre-ecdysis circuit and the entire FAP. Attempts to demonstrate such negative feedback here were inconclusive (Kim, 2015).

CAMB neurons express a combination of CCAP, Ast-CC, MIP, and bursicon. In Manduca, application of a CCAP/MIP cocktail is sufficient to elicit fictive ecdysis behavior. It would be parsimonious to extrapolate this result to Drosophila, since both of these peptides are found in CAMB neurons. Nevertheless, in Drosophila it is clear that bursicon is a key signaling molecule necessary for ecdysis initiation. It remains to be demonstrated precisely how absence of the bursicon gene blocks the switch to ecdysis. It will be interesting to elucidate possible functional roles of co-expressed peptides in CAMB neurons CCAP, Ast-CC, MIP in activation of the motor circuitry encoding the ecdysis motor pattern (Kim, 2015).

How is timing of the switch to ecdysis determined? Since both kinin and CAMB ensembles express ETHR, one would expect ETH to activate both ensembles simultaneously. Several previous studies provide evidence for the role of descending inhibition from cephalic and thoracic ganglia in setting the delay in the switch to ecdysis behavior. This study shows that expression of pertussis toxin in CAMB neurons accelerates the switch to ecdysis, consistent with disinhibition of Gαi/o input(s). It is hypothesized that a balance of excitatory and inhibitory inputs to the CAMB neurons contributes to the delay in their activity, excitatory input coming from ETH via Gαq signaling and Gαi/o from an as yet unidentified transmitter descending from cephalic and/or thoracic ganglia. The finding that RNAi-knockdown of MIP neurons lying outside the CCAP ensemble accelerates the switch to ecdysis behavior suggests one such possible inhibitory input (Kim, 2015).

It is possible, if not likely that ETH drives both inhibitory and excitatory inputs to CAMB neurons, with ETHR-B-expressing inhibitory inputs preceding excitatory input. Such a scenario follows from the fact that sensitivity of Drosophila ETHR-B to ETH was shown to be ~450-fold higher than that of ETHR-A (Park, 2003). Therefore, as ETH levels rise in the hemolymph, ETHR-B-expressing inhibitory neurons would be activated well before ETHR-A neurons. ETH would effectively inhibit CAMB neurons indirectly prior to direct excitation via ETHR-A activation (Kim, 2015).

Such a scenario pre-supposes that the EC₅₀ values governing activation of ETH receptors determined previously from heterologous expression in mammalian CHO cells are valid in Drosophila neurons. Data presented in this study suggests this is so. The EC₅₀ value for ETH1 against ETHR-A was found to be ~414 nM, while the EC₅₀ for ETH2 was determined to be ~4.3 μM. A combination of ETH1 and ETH2, each at a concentration of 300 nM, was applied to the isolated CNS, and a pattern was obtained of calcium dynamics in kinin neurons lasting for ~10 min, which matches the duration of pre-ecdysis behavior under natural conditions. Furthermore, the switch to ecdysis behavior occurs ~10 min after initiation of calcium mobilization in kinin neurons, which corresponds to timing of the switch to ecdysis behavior in vivo. Doubling concentrations of the ETH1/ETH2 cocktail reduced the duration of calcium dynamics in kinin neurons to 5.5 min and accelerated the switch to ecdysis behavior. These results make it likely that the relative sensitivities of ETHR-B and ETHR-A are as established in Park (2005) and consequently activity in ETHR-B neurons would precede that of ETHR-A neurons (Kim, 2015).

This study has shown that altered levels of ETHR expression have significant consequences for timing of pre-ecdysis duration and timing of the ecdysis switch. These findings raise the possibility that scheduling of sequential steps in the ecdysis FAP may be a consequence of different sensitivities to the peptide ligand. In other words, delay in the switch to ecdysis could result from a lower density of ETHR in CAMB neurons, making them less sensitive to ETH. Possible differential sensitivity to ETH could be tested in variety of way, including assessing timing of responses to the ligand by acutely dissociated neurons and/or single cell PCR (Kim, 2015).

A mechanistic model is proposed to explain neural mechanisms underlying the Drosophila pupal ecdysis FAP (see A model depicting functional roles of kinin and CAMB neurons in scheduling of the ecdysis FAP.). Principle players in orchestration of pre-ecdysis and ecdysis behaviors are the kinin and CAMB ETHR ensembles, respectively. As ETH levels rise in the hemolymph, ETHR-B neurons are activated due to their high sensitivity (EC₅₀ ~ 1 nM). These neurons release inhibitory signals acting through Gα_i/o to inhibit CAMB neurons. As ETH levels rise further, kinin neurons receive direct excitatory input from ETH signaling via ETHR-A and Gα_q to mobilize calcium from intracellular stores, leading to electrical activity in these neurons. ETH acts simultaneously on CAMB neurons, but inhibition from ETHR-B neurons descending from anterior ganglia prevents them from becoming active. As inhibition wanes, CAMB neurons become active, initiating the switch to ecdysis behavior (Kim, 2015).

Neuromodulatory connectivity defines the structure of a behavioral neural network

Neural networks are typically defined by their synaptic connectivity, yet synaptic wiring diagrams often provide limited insight into network function. This is due partly to the importance of non-synaptic communication by neuromodulators, which can dynamically reconfigure circuit activity to alter its output. This study systematically mapped the patterns of neuromodulatory connectivity in a network that governs a developmentally critical behavioral sequence in Drosophila. This sequence, which mediates pupal ecdysis, is governed by the serial release of several key factors, which act both somatically as hormones and within the brain as neuromodulators. By identifying and characterizing the functions of the neuronal targets of these factors, this study found that they define hierarchically organized layers of the network controlling the pupal ecdysis sequence: a modular input layer, an intermediate central pattern generating layer, and a motor output layer. Mapping neuromodulatory connections in this system thus defines the functional architecture of the network (Diao, 2017).

Using the Trojan exon method to selectively target populations of hormone receptor-expressing neurons for manipulation and monitoring of activity, this study investigated the neuromodulatory connectivity of the circuitry governing pupal ecdysis behavior in Drosophila. The sites of action of the neuromodulators ETH, Bursicon, and CCAP identify essential functional components of the network architecture, defining three hierarchically organized layers from the sites of hormonal initiation to the sites of motor neuron output. In addition, it was found that descending neuromodulatory signaling from the ETHR-expressing input layer not only governs the basic motor rhythms of the ecdysis sequence by modulating the intermediate CPG layer, but also modulates activity of the CCAP-R-expressing motor neurons of the output layerd. Neuromodulators thus act broadly within, as well as across, network layers. The finding that the functional architecture of the ecdysis network can be decoded from its patterns of neuromodulatory connectivity provides further evidence that characterizing neuromodulatory connectomes is a valuable strategy in elucidating neural networks (Diao, 2017).

Major components of the pupal ecdysis circuitry are shared by the three motor programs A schematic (ETHRB-expressing and non-CCAP/ETHRA neurons regulate Phase I) broadly augments existing models of the pupal ecdysis network, both by providing a more comprehensive description of the input layer than has previously been possible and by identifying the motor circuits on which this layer acts. A principal finding reported in this study is that the downstream targets of Bursicon and CCAP are shared components of the pupal ecdysis network and are used to generate all three motor rhythms. The results draw particular attention to the centrality of neurons that express the Bursicon receptor (Rk), which are absolutely required for all pupal ecdysis behavior. A role in central pattern generation is indicated both from the effects of their suppression, which eliminates all motor activity, and from their pattern of ETH1-induced Ca++ activity, which matches the phases of ecdysis behavior. The fact that ETH1-induced Ca++ activity is observed in the excised nervous system and thus in the absence of sensory feedback, demonstrates that it is centrally generated and further supports the identification of the VNC-Rk neurons as central pattern generators. Conclusive evidence that some or all VNC-Rk neurons participate in central pattern generation will require more precise observations and perturbations than those performed in this study, as will determining the functional roles of individual neurons. However, the preliminary observation that regions containing at most small numbers of VNC-Rk neurons exhibit activity that is phasically coupled to two or more motor patterns argues that the ecdysis circuitry includes multifunctional CPG neurons that express Rk and are subject to modulation by distinct input layer modules. Similar architectures have been described in other motor networks where two CPGs formed from overlapping pools of neurons can switch between activity states to generate distinct behaviors (Diao, 2017).

How input layer neurons modulate the pupal ecdysis CPG is exemplified by the control of Phase II by ETHRA/CCAP neurons. Direct activation of these neurons induces Phase II-like rhythmic activity in the VNC-Rk neurons, an observation that is easily explained if Bursicon secreted from ETHRA/CCAP neurons shifts the mode of activity of the VNC-Rk CPG. This mechanism is consistent with the neuromodulatory control of CPGs described in numerous other systems and accounts for the long-standing observation that CCAP- and Bursicon-expressing neurons are important for pupal ecdysis, including Phase II ('ecdysis') initiation and Phase I ('pre-ecdysis') termination. The CCAP- and Bursicon-expressing neurons are known to express additional neuropeptides, including Myoinhibitory Peptides and Allatostatin C, and it is likely that these neuromodulators also play a role in regulating these phases. The mixed activity patterns that define the transition from Phase II to Phase III ('post-ecdysis') are also readily interpreted as a period of bistability in which CPG modes transiently alternate, perhaps as Bursicon and/or other co-released neuromodulator concentrations fall (Diao, 2017).

In addition to neurons that switch CPG activity from Phase I to Phase II, the input layer must also contain neurons that initiate pupal ecdysis by inducing Phase I. The search for such neurons has focused primarily on those that express ETHRA, but no components of this group have yet been identified that are required for ecdysis initiation. To identify the ETH targets responsible for Phase I, ETHR-expressing neurons were systematically parsed into three, nearly mutually exclusive subsets that together cover the entire input layer. The results indicate that the largely uncharacterized neurons that express the B-isoform of ETHR are required to initiate Phase I, and that the non-CCAP/ETHRA neurons are important for maintaining that phase (Diao, 2017).

The essential role of ETHRB-expressing neurons in Phase I initiation is consistent with the significantly higher affinity for ETH peptides of ETHRB compared with ETHRA. ETHRB-expressing neurons may thus initiate Phase I by responding to rising titers of ETH earlier than neurons expressing ETHRA. How they regulate the VNC-Rk CPG neurons remains to be determined, but their mechanism of action appears to be different from that of the ETHRA/CCAP neurons insofar as the Phase I motor program cannot be evoked by TrpA1-mediated activation. It could be that this manipulation fails to induce the correct pattern of activity in ETHRB-expressing neurons. Preliminary imaging results show that ETHRB-expressing neurons respond to ETH1 with oscillatory activity, and it is possible that these neurons directly couple to the Rk-expressing neurons through synaptic or electrical contacts and participate in generating Phase I behavior. However, further characterization of the activity of both the ETHRB- and non-CCAP/ETHRA neurons will be required to determine how they modulate VNC-Rk CPG activity (Diao, 2017).

Two input layer neurons that are common to the ETHRB- and non-CCAP/ETHRA groups express the major ecdysis neuromodulator, EH (Diao, 2015). The EH-expressing neurons, which are among the few cells to express both ETHRA and ETHRB, respond to ETH1 application at the onset of Phase II, and evidence from other insects indicates that EH targets CCAP-expressing neurons. EH is thus thought to be responsible for the release of CCAP and Bursicon, but this has not yet been verified in Drosophila where the EH receptor has yet to be identified. It was thus not possible to target EH receptor-expressing neurons in this study, but the identity and function of such neurons is likely to be critical to understanding the progression of the ecdysis sequence (Diao, 2017).

In general, it is worth noting that the neuromodulators regulating the ecdysis sequence are of the type called 'extrinsic,' because they are released from neurons that do not function in the circuits upon which they act. Extrinsic neuromodulatory neurons, however, must be components of the broader neural networks that generate behaviors, and the mechanisms that organize their activities are only beginning to be understood. In some cases, these mechanisms are surprising. For example, the neuromodulatory connections between neurons that govern two foraging states in C. elegans are orthogonal to the sensory-to-motor synaptic connections between these neurons, which are not involved in the state decision. There are currently few studies that jointly examine patterns of neuromodulatory and synaptic connectivity, and to understand how extrinsic neuromodulatory neurons integrate into the broader networks in which they function more examples of such networks are required. Elucidating the interactions of neurons in the input layer of the ecdysis network (in addition to interactions of the input layer with neurons in other layers) should provide insight into this general problem (Diao, 2017).

The finding that the motor output of the pupal ecdysis network is mediated by neurons that express the CCAP-R provides insight into the hitherto poorly understood mechanism of action of CCAP. This neuropeptide plays critical roles in the ecdysis of other insects, but genetic data demonstrate that in Drosophila it plays a subsidiary role to Bursicon, acting synergistically with that hormone to render pupal ecdysis more robust. The current results indicate that it does so by acting on motor neurons, and because CCAP is co-released with Bursicon from the ETHRA/CCAP neurons to govern the CPG transition at Phase II, this suggests a role for feed-forward signaling in the pupal ecdysis circuit (Diao, 2017).

Neuromodulatory feedforward pathways have been previously described and appear to be a common motif in motor network architectures. Feedforward loops of the type posited here for Bursicon and CCAP may be important in adjusting the coupling between Rk-expressing CPG neurons and their downstream motor neuron targets during Phase II. Compensatory adjustments in CPG, motor neuron and muscle activity by a single neuropeptide released from two different nodes in a feedforward loop have been described in the Aplysia feeding network where they guarantee stability of network output. Coordinating CPG activity with motor neuron activity may be particularly important for multifunctional CPGs, in which individual neurons participate in multiple motor patterns, as for example, in the leech swim/crawl network in which multifunctional neurons fire in phase with the contraction of one muscle group during swimming, but not necessarily during crawling (Diao, 2017).

The architecture of the pupal ecdysis network revealed in this study is similar to that of other motor circuits, such as those governing locomotion, feeding, and breathing in which higher order neurons modulate the activity of core CPGs to generate varied motor patterns. What is striking about neuromodulator action in the ecdysis circuit is its broad scope. ETH acts throughout the input layer to control different phases of pupal ecdysis behavior; Bursicon similarly regulates a large and essential set of neurons constituting the ecdysis CPG; and CCAP acts on many motor neurons necessary for generating the rhythmic ecdysis movements. The observation that Bursicon and CCAP signal from the input layer speaks to an organizational logic in which the ecdysial neuromodulators function together to provide coherence to the operation of the pupal ecdysis network by acting both within each hierarchical layer and by acting coordinately across layers. This organization is consistent with a generalized role for neuromodulatory systems in organizing neural activity to generate behavior (Diao, 2017).

The results also support the rationale of mapping neuromodulatory pathways as a strategy for identifying essential network circuits and their functional organization. It is worth noting that this mapping of the pupal ecdysis network was done without reference to patterns of synaptic connectivity. Synaptic connectomes have proved difficult to interpret, in part due to their dense interconnectivity. If, as has been previously emphasized, this interconnectivity reflects the multifunctionality of the underlying networks, and if the functional configuration of a network at any given time is determined by where and how neuromodulators are acting on its components, then patterns of neuromodulatory connectivity may provide a necessary complement to synaptic maps to render them interpretable. A key challenge will lie in identifying which neuromodulator systems play critical roles in establishing a network's output, but as the work here demonstrates, when these are known, the neuromodulatory connections can deliver substantial insight into how a neural network is organized (Diao, 2017).

Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation

Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. This study reports that stress-induced elevation of ecdysone is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as described in this study for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability. These findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity (Meiselman, 2018).

Evidence presented in this study establishes a new paradigm for Drosophila reproduction, wherein stressful conditions arrest egg production via a hormonal cascade involving reciprocal ecdysone and ETH signaling. As steroid levels fluctuate in response to stress, so too does ETH, a consequence of steroid-regulated changes in Inka cell secretory competence. ETH activates two downstream targets: the JH-producing corpus allatum and modulatory OA neurons innervating the ovary and oviducts. This study characterized the nature of ETH dependence, and assigned function and context to a newly recognized hormonal axis governing reproductive responses to stress (Meiselman, 2018).

Previous report showed that ETH is an obligatory allatotropin, promoting oogenesis and fecundity through JH production; consequently, ETH deficiency results in low JH levels and arrested oogenesis (Meiselman, 2017). The present work demonstrates that ovulation of stage 14 oocytes depends upon ETH activation of OA neurons innervating the ovary and oviduct. A comprehensive explanation is offered for the change in distribution of vitellogenic oocytes reported in EcR mutants or under conditions of high or low ecdysone, depending on stress levels. ETH deficiency or ETHR knockdown results in accumulation of stage 14 oocytes in the ovary due to ovulation block, and a mechanistic link between altered endocrine state and ovulation is provided (Meiselman, 2018).

ETH promotes ovulation through activation OA neurons to induce contractions in the ovary and relaxation of the oviducts. It is interesting that ETH triggers calcium dynamics in vitro on distal axonal projections, suggesting ETH-stimulated OA release results from direct action of ETH on axons and/or nerve terminals. While ovary contractions in response to ETH exposure occur in both virgin and mated females, this study chose virgin females for analysis due to higher spontaneous contractile activity in mated females. This is likely due to actions of ovulin after insemination, which stimulate outgrowth of octopaminergic neurons innervating the oviduct. In virgin females, concentration-dependent ETH actions on the ovary are in the range predicted for activation of ETHR-A receptors (Meiselman, 2018).

Acting through OA neurons, ETH mobilizes calcium in the epithelium enveloping the ovary, initiating bursts of contractions in the peritoneal sheath at the base of the ovary associated with ovulation. Although bath-applied ETH and OA are both sufficient to induce calcium mobilization in the oviduct epithelium, they induce distinctive response patterns. OA causes a rapid, sustained calcium wave with a slowly waning plateau following the peak response. ETH actions occur with longer latency and induce oscillatory calcium dynamics, which could be a consequence of periodic synaptic reuptake of OA by nerve terminals. No changes in intensity were observed between treatments or at different doses, suggesting a possible threshold effect. It is also interesting to note that calcium waves spread through the epithelial layer, suggesting that the epithelium is a functional syncytium, which undoubtedly aids in coordination of relaxation (Meiselman, 2018).

Injection of mated females with either ETH or OA induces ovulation in vivo, whereas injected virgin females respond much more weakly. In order for ovulation to occur, OA causes follicle rupture inside the ovaries, a process requiring one to several hours ex vivo. It is hypothesized that mated females are in the proper endocrine state for ovulation, and thus follicle rupture may already be in progress before application of ETH or OA. As follicle rupture is the critical first step for egg-laying, this limiting factor would explain the length of time (up to 60 min) elapsed after physiological levels of ETH/OA are reached for in vivo ovulation to occur, given that ovary contraction and oviduct relaxation occur within seconds (Meiselman, 2018).

Agents previously implicated in oviduct contractions were also examined, including tyramine, glutamate, and proctolin. While the ineffectiveness of tyramine and glutamate is not surprising, the negative result with proctolin is at variance with prior literature. Examination of proctolin-induced contractions revealed that they are localized to the distal tip (germaria) of the ovaries. Moreover, proctolin does not stimulate ovulation in vitro. It appears that the role of proctolin in Drosophila ovaries is more limited than in the well-studied locust oviduct (Meiselman, 2018).

This study has shown that elevated ecdysone levels in response to heat and nutritional stress are associated with a drop in circulating ETH levels. It was previously hypothesized that the Inka cell secretory competence model governing ecdysis signaling during developmental stages may persist into adulthood (Meiselman, 2017). The results presented in this study support this hypothesis (Meiselman, 2018).

Both stress and ETH deficiency have similar consequences for reproduction, namely arrested oogenesis and reduced ovulation, resulting in increased stage 14 egg retention and lower egg production. Progression of mid-oogenetic oocytes is directly correlated with JH levels, while OA release from reproductive tract neurons is necessary for ovulation. This study shows that arrested oogenesis and ovulation contributing to the ovariole profile observed in heat-stressed flies can be explained by ETH deficiency, which has a dual role in regulating JH levels and activity of OA neurons innervating ovaries and oviducts. Indeed, arrest of both oogenesis and ovulation deficiencies can be rescued by ETH, either through TRPA1 activation of Inka cells or direct injection of ETH1 (Meiselman, 2018).

The mechanism through which elevated ecdysone leads to ETH deficiency was examined by performing rescue experiments designed to (1) suppress steroid signaling in Inka cells and (2) express the transcription factor βFTZ-F1, which confers secretory competence of Inka cells and is suppressed by high ecdysone levels. Although somewhat variable in their effectiveness, these manipulations resulted in clear rescue of oogenesis and ovulation in heat-stressed females, confirming that the thermal stress response operates through the influence of ecdysone on Inka cell secretion (Meiselman, 2018).

Methoprene treatment increases progression of oogenesis but does not increase oviposition in stressed animals. In fact, this study observed a significant increase in eggs retained after methoprene treatment, suggesting that synthesis of mature eggs resumes with JH treatment, but ovulation remains impaired under conditions of elevated ecdysone and ETH deficiency. This suggests that ovulation provides a gating mechanism under stressful conditions, limiting egg production while conditions are suboptimal. A recent report suggested that normal ecdysone levels stimulate follicle rupture and ovulation, but that elevated levels inhibit follicle rupture (Knapp, 2017). The present work provides an additional mechanism for suppression of ovulation associated with elevated ecdysone levels: repression of ETH release leading to reduced OA neuron activity (Meiselman, 2018).

It is interesting to note that wet starvation reduces ecdysone levels and increases ETH levels, whereas sugar starvation increases ecdysone levels and, as is shown in this study, increases ETH levels. Wet-starved females were precisely synchronized in mating on day 4, and began starvation (no nutrient source, wet KimWipe) 24 h later for an additional 24 h. mino acid-deprived females were group-raised until day 3, and groups were placed on agar + 10% sucrose for 24 h. Mating was not controlled in sugar-starved females, though it is known to influence ecdysone levels dramatically in the short term. Arguably the most interesting result is that ecdysone decrease led to elevated circulating ETH. This adds credence to the hypothesis that ETH and ecdysone levels are generally inversely correlated (Meiselman, 2018).

Unique stresses may garner different endocrine responses because different types of cues require differential behavioral adaptation. The ability of a hormone to coordinate a wide variety of target tissues to change in state makes it a perfect tool for stress adaptation. As an organism encounters a new type of stress, they may adapt a new endocrine state to coordinate a tissue-wide response. Many hormones in closely related insects play markedly different roles, which evolve as rapidly as behavioral niches, but an endocrine core in E-ETH-JH is highly conserved, similar to the hypothalamic-pituitary-gonadal (HPG) axis among vertebrates. A hormonal network with competence to adjust reproductive output in response to environmental changes is undoubtedly a common phenomenon among multicellular organisms. The discovery of a stress response hormonal axis and, more aptly, a peptide hormone with the potential to alleviate stress-induced deficits in reproduction could be of particular relevance to the honey bee Apis mellifera. In recent years, Apis reproductives have been producing fewer progeny due to a variety of stressors, including temperature extrema. While proctolin has already been found to be a short-term reproductive stimulant in Apis queens, ETH is attractive as it can alter JH levels, which in turn may rescue poor pheromone production, the proximal cause of supersedure (Meiselman, 2018).

REGULATION

Transcriptonal Regulation

The regulation of neuropeptide and peptide hormone gene expression is essential for the development and function of neuroendocrine cells in integrated physiological networks. In insects, a decline in circulating ecdysteroids triggers the activation of a neuroendocrine system to stimulate ecdysis, the behaviors used to shed the old cuticle at the culmination of each molt. Two evolutionarily conserved transcription factor genes, the basic helix-loop-helix (bHLH) gene dimmed (dimm) and the basic-leucine zipper (bZIP) gene cryptocephal (crc), control expression of diverse neuropeptides and peptide hormones in Drosophila. Central nervous system expression of three neuropeptide genes (Dromyosuppressin, FMRFamide-related and Leucokinin) is activated by dimm. Expression of Ecdysis triggering hormone (ETH) in the endocrine Inka cells requires crc; homozygous crc mutant larvae display markedly reduced ETH levels and corresponding defects in ecdysis. crc activates ETH expression though a 382 bp enhancer, which completely recapitulates the ETH expression pattern. The enhancer contains two evolutionarily conserved regions, and both are imperfect matches to recognition elements for activating transcription factor-4 (ATF-4), the vertebrate ortholog of the CRC protein and an important intermediate in cellular responses to endoplasmic reticulum stress. These regions also contain a putative ecdysteroid response element and a predicted binding site for the products of the E74 ecdysone response gene. These results suggest that convergence between ATF-related signaling and an important intracellular steroid response pathway may contribute to the neuroendocrine regulation of insect molting (Gauthier, 2006).

DIMM has been proposed as a direct regulator of neuroendocrine gene expression in most neuropeptidergic cells. Quantitative RTPCR results, supplemented by in situ hybridization, show that DIMM upregulates the levels of mRNAs derived from at least three neuropeptide genes, Fmrf, Lk and Dms. These findings provide strong support for DIMM as a key regulator of multiple neuroendocrine genes. The LIM-homeodomain gene apterous (ap) also controls Fmrf and Lk gene expression. ap acts cell-autonomously to stimulate dimm expression, but the AP and DIMM proteins can also physically interact, and they may function together in regulating Fmrf. Several other factors, including the transcriptional co-factors encoded by dachshund and eyes absent, the zinc-finger gene squeeze, and the retrograde bone morphogenetic protein (BMP) pathway, act in combinatorial fashion with dimm and ap to control Fmrf expression. However, other neuropeptidergic cells appear to use only portions of this code. For example, ap and dimm appear to contribute to the expression of Lk in Fmrf-negative cells (the segmental cells A1–A7 and possibly the brain lobe cells Br1). Even within the population of Lk cells, loss of dimm results in very different effects in different neurons, with a reduction in Lk transcript levels in cells A1–A7, and an increase (or no change) in Lk levels in the Br1 and the subesophageal SE neurons. How do these relatively widely expressed factors interact with other regulatory proteins to produce cell type-specific patterns of neuropeptide gene expression? It will be of interest to determine which other elements of the combinatorial pro-Fmrf code are used to control Lk and Dms expression, and to identify additional factors that interact with dimm to control expression of these neuropeptide genes (Gauthier, 2006).

Does dimm control neuropeptide levels through an additional indirect mechanism? No changes were detected in levels of three neuropeptide biosynthetic enzyme mRNAs, Phm, Fur1 and amon, in the qRTPCR analysis. This is in contrast to earlier immunocytochemical studies, in which a marked reduction was observed in the protein products of these genes in dimm mutant CNS. In some cases, these differences may reflect the spatial insensitivity of the qRTPCR methods, as was confirmed by in situ hybridization analysis of Lk expression. Phm, in particular, may belong in this category. Although levels of PHM and DIMM expression are strongly correlated, PHM is also highly expressed in many other tissues that do not express dimm. Any dimm-dependent change in Phm expression may have been obscured by the much larger pool of dimm-independent Phm mRNA in whole-animal qRTPCR analysis (Gauthier, 2006).

DIMM may regulate levels of other neuroendocrine proteins through a route that does not involve interactions between DIMM and cis-regulatory elements in the respective genes. Evidence was obtained in support of this hypothesis in an earlier analysis of an ectopically expressed neuropeptide in dimm mutant cells; levels of ectopic PDF protein were strongly reduced while dimm had no effect on levels of the cognate Pdf mRNA. This study showed that larvae homozygous for a specific loss-of-function mutation in dimm displayed reduced levels of endogenous ETH-like protein(s), but not ETH mRNA, in the endocrine Inka cells, a site of dimm gene expression. This may occur simply through a dimm-dependent change in levels of one secreted protein, such as PHM, that may disrupt the formation of multi-protein aggregates required for neuropeptide sorting into secretory granules. Alternatively, recent studies on the mouse ortholog of dimm, Mist1, suggest that dimm may play a more direct role in the management of secretory granule budding from the trans-Golgi network. In Mist1 knockout mice (Mist1^KO), pancreatic exocrine cells display reduced intracellular organization. Moreover, the Mist1^KO phenotype is partially phenocopied in animals mutant for the Rab3D gene, a small GTPase involved in secretory granule trafficking. Further studies on the regulation of ETH, PHM and Rab3-like proteins, and on the biochemical interactions among them, may shed light on the cellular mechanisms underlying the indirect actions of DIMM (Gauthier, 2006).

Mutations in the crc gene result in pleiotropic defects in ecdysone-regulated events during molting and metamorphosis. Many of the morphological defects are associated with a failure of the insect to properly complete ecdysis, a stereotyped set of behaviors required for shedding of the old cuticle at the culmination of each molt. Several neuropeptides and peptide hormones, including ETH, play critical roles in organizing and triggering ecdysis behavior (Gauthier, 2006).

This study provides four independent lines of evidence that demonstrate a crucial role for crc in the upregulation of ETH mRNA levels. First, a marked reduction by qRTPCR is observed in levels of ETH transcripts [but not in mRNAs encoding CCAP or EH, two other neuropeptides involved in the neuropeptide hierarchy controlling ecdysis in crc mutant larvae. Second, in situ hybridization revealed a strong reduction in ETH mRNA levels in the endocrine Inka cells in crc mutant larvae. Third, the intensity of anti-PETH immunoreactivity was markedly reduced in crc¹/crc¹ homozygotes. Fourth, EGFP fluorescence driven by an ETH-EGFP reporter gene was reduced in crc mutant larvae. Therefore, CRC is a strong activator of ETH gene expression, and loss of CRC results in a corresponding reduction in levels of the ETH protein (Gauthier, 2006).

Despite the molecular identification of the crc locus, almost six decades after the original description of the first crc allele, the causes of the molting and metamorphosis defects in crc mutants remained unclear. The current results suggest a simple model to explain the crc mutant phenotype. Strong hypomorphic or null mutations in crc and ETH both severely disrupt ecdysis. These defects include weak, irregular and slower ecdysis contractions and a failure to shed old cuticular structures, leading to retention of two and sometimes three sets of mouthparts into the next larval stage. These similarities in molting defects, taken together with the observation that crc is required for normal expression of ETH mRNA and ETH protein, point to the loss of ETH signaling as the likely proximate cause of the ecdysis defects observed in crc mutants (Gauthier, 2006).

Despite the specific effects of crc on ETH transcription in the Inka cells, crc is widely expressed, suggesting a cellular housekeeping function. The vertebrate ATF-4 protein is also ubiquitously expressed. In addition, the upregulation of ATF-4 constitutes a milestone of many cellular stress response pathways including oxidative stress, amino acid deprivation, and hypoxia. In the tobacco hornworm, Manduca sexta, levels of ETH fluctuate during the molts and are regulated by circulating ecdysteroids. It is hypothesized that CRC contributes to the regulation of ETH gene expression during this period, perhaps in response to signals from the tracheae (Gauthier, 2006).

Peaks in circulating levels of the ecdysteroid hormone, 20-hydroxyecdysone (20HE), initiate and coordinate each molt. A subsequent decline in 20HE levels is required for ecdysis, and the activation of these behaviors involves a hierarchical cascade of peptide hormone and neuropeptide signals that is triggered by ETH. Is CRC required in order to maintain ETH expression, or is CRC involved in regulating transcription of the ETH gene during the molts? While it is not known whether ETH mRNA levels fluctuate during Drosophila post-embryonic development, the regulation of ETH levels by ecdysteroids in molting Manduca sexta, and the analysis of the conserved region sequences CR1 and CR2 (located 91-171 bp upstream of the ETH translational start site), provides tantalizing clues to possible coordinate regulation of ETH gene expression by CRC and ecdysone response genes. There is substantial overlap between the predicted CRC binding site in CR1 and a putative ecdysteroid response element (EcRE). In addition, a potential binding site in CR2 for products of the E74 early ecdysone-inducible gene. E74 expression is induced directly by 20HE, and it encodes transcription factors that regulate other ecdysone response genes. Mutations that specifically disrupt E74B, which likely binds the same consensus as E74A, display defects associated with pupal ecdysis that closely phenocopy crc. In future, studies will focus on whether ETH expression is regulated by elements in both CR1 and CR2 in an ecdysteroid-dependent manner, and whether CRC, E74B and other factors in the ecdysone-response pathway interact competitively or cooperatively at these sites (Gauthier, 2006).

Protein Interactions

G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous 'orphan' GPCRs have become available for functional analysis. This study analyzes two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins (FXPRXamide), Cap2b-like peptides (FPRXamide), or ecdysis triggering hormones (PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (CCAP), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors (Park, 2002b).

Genes encoding Drosophila signaling peptides having PRXa C-terminal motifs were located by using BLASTP and TBLASTN searches with parameters for finding short matching sequences. Various insect PRXa peptides previously described were used for query sequences. Mature peptides were predicted by the C-terminal sequence motif PRXG(K/R): G for amidation followed by a mono- or di-basic cleavage site. N termini were predicted after the dibasic cleavage sites (K/R)(K/R) in upstream positions proximal to the PRXG(K/R) motif. A total of three genes encoding seven mature peptides were predicted. It was not possible to identify sequences similar to AVP or to the locust AVP-like insect diuretic hormone in database searches with similar search parameters as above (Park, 2002b).

The PRXa C-terminal motif is found in a number of invertebrate and vertebrate peptides. In the invertebrates, these include the PBAN-like FXPRXa motif characteristic of the pyrokinin group, FPRXa exemplified by small cardioactive peptide and CAP2b, and PRXa of ETH. Vertebrate PRXa peptides consist of pancreatic polypeptide (36 aa with C-terminal NMLTRPRYa), AVP (NXPRXa), and NMU-25 or -8 (25 or 8 aa with C-terminal FXPRXa) (Park, 2002b).

The Drosophila genome database (www.fruitfly.org/blast/) was searched for all genes encoding peptides with C-terminal amino acid PRXa motifs and for G protein-coupled receptors likely to be activated by these ligands. The search for peptides yielded three genes: hugin (CG6371, GenBank accession no. AJ133105), cap2b-like (CG15520, capability, GenBank accession no. AF203878), and eth (CG18105; GenBank accession no. AF170922). The gene hugin encodes two peptides, referred to here as Hug and Drm-PK-2, whose C-terminal motifs are related to the insect pyrokinins. The cap2b-like gene encodes three putative peptides related to cardioacceleratory peptides (CAPs), referred to here as CAP2b-1, -2, and -3. CAP2b-1 and CAP2b-2 contain a common C-terminal motif (FPRXa), whereas the C terminus of CAP2b-3 (GLWFGPRLa) is identical to that of the diapause hormone of Lepidoptera. The peptides ETH1 and ETH2 encoded by the gene eth possess a C-terminal PRXa motif (Park, 2002b).

Analysis of the three vertebrate PRXa peptides, NMU, AVP, and pancreatic polypeptide (PP) shows that the PRXa motifs are strictly conserved in NMU and AVP, whereas that of PP is likely a consequence of converging evolution from NPY/PYY/PP family, which includes Drosophila neuropeptide F [C-terminal motif (PH)R(YF)amide]. In this fashion, the search for PRXa-activated GPCRs in Drosophila was narrowed to those related to the AVP and NMURs (Park, 2002b).

Phylogenetic analysis reveals that NMURs occur in a monophyletic clade with four Drosophila GPCRs: CG8784, CG8795, CG9918, and CG14575. Three Drosophila GPCRs homologous to AVP receptors are CG6111, CG11325 (also known as gonadotropin releasing hormone receptor), and CG10698. CG6111 is orthologous to the vasopressin/oxytocin receptor gene family (Park, 2002b).

Putative Drosophila GPCRs in the database were amplified by RT-PCR using primers based on gene predictions in the FGENESH gene finder. Conceptual translations of these genes aligned with other GPCRs present complete seven transmembrane domains. Sequences confirmed by at least two independent RT-PCR experiments revealed several polymorphic sites compared with the Celera Drosophila genomic sequences (Park, 2002b).

Oocytes injected with cRNAs for the GPCRs generated inward currents up to 2.5 µA upon activation with appropriate ligands. It is presumed that ligand-activated inward currents in these experiments result from Gq activation of phospholipase C, liberation of inositol trisphosphate, and activation of chloride current by mobilization of intracellular calcium stores (Park, 2002b).

Drosophila GPCRs in the NMUR clade were activated by PRXa peptides with various levels of sensitivity and specificity. CG14575 was the most selective within this group, responding only to CAP2b-1 (EC50 150 nM) and CAP2b-2 (EC50 230 nM), which have an identical C-terminal VFPRVamide motif. All other peptides were inactive on application at 10 µM. In contrast, CG8795 responds to a relatively wide range of ligands, including Drm-PK-2, hug, CAP2b-3, and ETH1, listed in order of decreasing potency. Drm-PK-2 and Hug appear to have highest potency, but also induce the most severe receptor desensitization. The high level of desensitization complicated efforts to produce quantitative determinations of potency for these ligands. In contrast, ETH1 and CAP2b-3 treatment produces little or no desensitization.

CG9918 and CG8784 were insensitive to most ligands applied. CG9918 responded only to the highest concentration of CAP2b-3 applied (10 µM), and was otherwise insensitive to all other ligands applied at this concentration. Similarly, CG8784 was activated only by Drm-PK-2 or Hug applied at 10 microM (Park, 2002b).

Thus Drosophila GPCRs in the NMUR group respond to the PRXa peptides, Hug, Drm-PK-2, CAP2b-1 to -3, and ETH. Non-PRXa peptides such as proctolin, FMRFamide, and diuretic hormone produced no response at 10 µM, the highest concentration tested. The range of ligand concentrations sufficient to activate each GPCR ranged from low nanomolar to micromolar. CG14575 was the most ligand-selective receptor in this group, responding only to low nanomolar concentrations of CAP2b-like peptides CAP2b1 and CAP2b-2 having FPRXa motifs, whereas CAP2b-3, a mature peptide from the same gene having FXPRXa motif had no effect on CG14575 at 10 microM (Park, 2002b).

It seems likely that CG14575 is involved in ion transport functions associated with diuresis in Drosophila. It has been shown that Drosophila CAP2b-1 and -2 act on principal cells of Malphighian tubules, stimulating fluid secretion through the calcium-nitric oxide-cGMP pathway. It will be interesting to determine whether CG14575, the putative CAP2b-1/CAP2b-2 receptor from this study, is expressed in Malpighian tubules (Park, 2002b).

CG8795 responds to a different set of nonoverlapping PRXa peptides, being most sensitive to Hug and Drm-PK-2. These peptides produce activation at low nanomolar concentrations accompanied by marked receptor desensitization, making it difficult to ascertain a reliable EC50 value for these peptides. CG8795 also shows moderate sensitivity to ETH1 and CAP2b-3, responding to mid- to high nanomolar concentrations. Interestingly, ETH2 had no effect at 10 µM. The responses of CG8795 to a wide range of peptides were unexpected. Although Drm-PK-2 was most active, Hug, ETH1, and CAP2b-3 also produced robust responses. The ligands active on this receptor also include Manduca sexta MasETH and Heliothis virescens HezPBAN at 10 microM concentration. However, some obvious selectivity was apparent, with no responses registered to CAP2b-1 and -2, ETH2, and Manduca PETH applied at 10 microM (Park, 2002b).

Activation of CG8795 by both Hug and ETH1 raises the possibility of its involvement in ecdysis. Such a possibility is indicated not only by its sensitivity to ETH1 (which is known to be obligatory for ecdysis signaling). Ecdysis deficiency is induced by ectopic expression of the hugin gene. Furthermore, the hugin gene product Hug mimics ETH1 by inducing ecdysis behavior in wild-type flies and by rescuing ecdysis deficiency in buttoned-up eth null mutants. Given that Hug and ETH1 activate both CG8795 and ecdysis behavior, several interpretations are possible. CG8795 may be involved in ecdysis signal transduction, and both Hug and ETH1 are ecdysis signaling molecules. Alternatively, CG8795 is not involved in ecdysis, but can be activated by relatively high concentrations of ETH1 acting as a Hug agonist. According to this alternative scenario, CG8795 could be involved in other physiological functions such as pheromone biosynthesis as a Hug and/or Drm-PK-2 receptor. Further work is needed to clarify an authentic role for CG8795 and function of Hug in the ecdysis signaling pathway (Park, 2002b).

The remaining GPCRs in the NMU group, CG8784 and CG9918, respond only to high levels (10 microM) of Hug and Drm-PK-2, and CAP2b-3, respectively. It is possible that the endogenous signal transduction machinery in the Xenopus oocyte is inappropriate for mediation of functional receptor activation for CG8784 and CG9918. This assay system generates a presumed calcium-activated chloride current known to be activated exclusively by Gq coupled pathways. GPCRs can be coupled to a variety of G proteins, including Gi/o, Gs, and Gq, with various degrees of efficiency and specificity. Poor coupling of heterologously expressed GPCRs to Gq in the Xenopus oocyte clearly could result in artifactually low affinity estimates. In particular, CG9918 and CG8784 were found to be largely insensitive to all ligands tested (Park, 2002b).

The functions of PRXa peptides known thus far in the vertebrates include activation of ion transport and contractile activity in intestine and arterial musculature via the NMUR. In invertebrates, functions for many of the PRXamide peptides remain uncertain, biological activity having been inferred from standard assays for visceral muscle contraction. For example, early demonstrations of activity for the pyrokinins (FXPRXa) were based on stimulation of gut, oviduct, and heart, whereas more recent data implicating them in pheromone biosynthesis and cuticle melanization are more suggestive of authentic physiological functions. The FPRXa peptides, including small cardioactive peptides and cardioacceleratory peptide (CAP2b), were isolated based on their activity in heartbeat modulation but may be involved in water and ion transport. Finally, although all other PRXa peptides are produced in the central nervous system, ETH (PRXa) is produced peripherally in epitracheal Inka cells and acts on CNS to trigger central pattern generators leading to ecdysis behavior. Knowledge of the expression patterns of the receptor GPCRs will likely provide new insights into the true physiological functions for the PRXa peptides (Park, 2002b).

DEVELOPMENTAL BIOLOGY

Immunohistochemical staining using antisera raised against Drosophila ETH1 has revealed segmentally repeated cells in both larval and adult stages. These cells appear to be homologous to 'Inka cells' previously identified in M. sexta (Zitnan, 1996), and henceforth are referred to by the same name. An identical staining pattern was observed using an antiserum raised against the C-terminus of the M. sexta peptide, MasPETH (Zitnan, 1999). In larvae, cells exhibiting ETH-like immunoreactivity (ETH-IR) occur along each of the two dorsal tracheal trunks at the main branch points of transverse connectives. A total of seven Inka cell pairs occur consistently in each tracheal metamere Tr1 and Tr4 through Tr9 in the larval stage. In adults, cells showing ETH-IR also occur at homologous positions, but vary in shape and location. Depletion of ETH-IR is observed at each larval ecdysis (Park, 2002a).

Antisera to Drosophila ETH1 and MasETH also stain ~20 neurons and axons in the CNS. Staining in the CNS presumably results from cross-reactivity with neuropeptides containing the conserved C-terminal sequence motif -PRXamide, which is shared by ETHs (Drosophila ETH1, DDSSPGFFLKITKNVPRLa; Drosophila ETH2, GENFAIKNLKTIPRIa; MasPETH, SFIKPNNVPRVa; MasETH, SNEAISPFDQGMMGYVIKTNKNIPRMa), the cardioactive peptide CAP2b, pheromonotropic and diapause hormones in moths, and the Drosophila neuropeptides CG15520 and CG6371 (Park, 2002a).

To examine the cellular expression pattern of ETH, a fly line carrying the chimeric transgene 2eth3-egfp was constructed. This transgene occurs on the 2nd chromosome and contains the sequence of ETH up to the 3rd amidation site with chimeric egfp encoding the enhanced green fluorescent protein. EGFP fluorescence in 2eth3-egfp flies is observed in both larval and adult stages, but is confined to the constellation of Inka cells showing ETH-IR. No EGFP fluorescence occurs in any other cell or tissue in larvae or adults. These data are consistent with cell-specific expression of eth. Observations under laser confocal microscopy revealed an identical distribution of EGFP fluorescence and ETH-IR in Inka cells of wandering 3rd instar, suggesting that EGFP and processed ETHs are located in the same subcellular compartments (Park, 2002a).

In 1st instar larvae, peak EGFP fluorescence occurs at dVP (the time of appearence of douple vertical plates), and declines sharply to 16±3% of peak emission just before tracheal inflation. A further drop of EGFP emission to 11±3% occurs by the squeezing wave stage. Loss of EGFP fluorescence suggests that ETH is released naturally in vivo during the time interval between dVP and tracheal collapse (Park, 2002a).

In insects, ecdysis is thought to be controlled by the interaction between peptide hormones; in particular between ecdysis-triggering hormone (ETH) from the periphery and eclosion hormone (EH) and crustacean cardioactive peptide (CCAP) from the central nervous system. The behavioral and physiological functions of the first two of these peptides was studied in Drosophila melanogaster using wild-type flies and knockout flies that lacked EH neurons. ETH from Manduca sexta (MasETH) was used to induce premature ecdysis and the responses of the two types of flies were compared. The final release of EH normally occurs approximately 40 min before ecdysis. It is correlated with cyclic guanosine monophosphate (cGMP) production in selected neurons and tracheae, by an elevation in the heart rate and by the filling of the new tracheae with air. Injection of developing flies with MasETH causes all these events to occur prematurely. In EH cell knockouts, none of these changes occurs in response to MasETH, and these flies show a permanent failure in tracheal filling. This failure can be overcome in the knockouts by injecting them with membrane-permeant analogs of cGMP, the second messenger for EH. The basis for the 40 min delay between EH release and the onset of ecdysis was examined by decapitating flies at various times relative to EH release. In flies that had already released EH, decapitation is always followed within 1 min by the start of ecdysis. Immediate ecdysis is never observed when the EH cell knockout flies were decapitated. It is proposed that EH activates both ventral central nervous system elements necessary for ecdysis (possibly the CCAP cells) and descending inhibitory neurons from the head. This descending inhibition establishes a delay in the onset of ecdysis that allows the completion of EH-activated physiological processes such as tracheal filling. A waning in the inhibition eventually allows ecdysis to begin 30-40 min later (Baker, 1999).

A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles

At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. To elucidate neural substrates underlying the ecdysis sequence, neurons expressing ETH receptors (ETHRs) have been identified in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. These findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles (Kim, 2006a).

Analysis of the pupal ecdysis behavioral sequence: In Drosophila, pupal ecdysis is preceded by pupariation, whereby the prepupa contracts its body into a barrel shape to form the puparium composed of the old larval cuticle. The underlying new pupal cuticle then separates from the puparium during pupal ecdysis ~12 hr later. The stereotypic nature of pupal ecdysis and reliable developmental markers make it a favorable model for the behavioral analysis and neural imaging (Kim, 2006a).

Pupal ecdysis consists of three centrally patterned behavioral subunits performed sequentially: pre-ecdysis (~10 min), ecdysis (~5 min), and postecdysis (~60-70 min). The behavioral sequence was examined through the semitransparent puparium ('puparium-intact'), but it was found that the puparium obscures and places constraints on some movements. This made it particularly difficult to discriminate differences in abdominal swinging movements during ecdysis and postecdysis. Therefore, a 'puparium-free' preparation was used by surgically removing the puparium immediately after pre-ecdysis onset. The improved visibility and room for movement in this preparation allowed for a more complete analysis of natural and ETH-induced behavior. The following description of the natural pupal ecdysis sequence resulted from comparison of behaviors observed in both puparium-intact and puparium-free prepupae (Kim, 2006a).

Pre-ecdysis: About 5 min after in vivo ETH release, preecdysis commences with the abrupt appearance of an air bubble at the posterior end of the prepupa (time zero). Pre-ecdysis involves anteriorly directed rolling contractions along the lateral edges of the abdomen, alternating on the left and right sides of the animal. These contractions move the air bubble anteriorly to separate pupal cuticle from the puparium. This behavior is completed within ~10 min and is followed by ecdysis behavior. Preecdysis behavior is the same in puparium-intact and puparium-free animals (Kim, 2006a).

Ecdysis: In higher Diptera including Drosophila, the incipient adult head develops within the prepupal thorax. During pupal ecdysis, head eversion results from lateral swinging movements of the abdomen occurring along with anteriorly directed peristaltic contractions. In puparium-intact preparations, head eversion occurs ~1 min after the onset of ecdysis swinging and is completed within ~5s. After completion of head eversion, ecdysis contractions continue for ~15 min, facilitating expansion of wing pads and legs to their final size. The frequency of ecdysis swinging (~5 swings/min) decreases markedly after head eversion (~2 swings/min). In puparium-free animals, head eversion occurs sooner, and the duration of ecdysis behavior lasts only ~5 min. Later in ecdysis, anteriorly directed swinging contractions are often interrupted by posteriorly directed ones, indicating a transition to postecdysis (Kim, 2006a).

Postecdysis: Postecdysis behavior consists of two behavioral subroutines: postecdysis swinging and stretch-compression movements of the abdomen. Postecdysis swinging occurs along with posteriorly directed peristaltic contractions and alternates with longitudinal movements of the abdomen, referred to as 'stretch-compression.' The frequency and intensity of postecdysis contractions wane gradually until they are detected mainly in the anterior part of the abdomen; they cease w100 min after pre-ecdysis onset. Postecdysis behavior concludes with compression of the pupa at the posterior end of the puparium (Kim, 2006a).

ETH release coincides with initiation of the ecdysis sequence: To confirm the role of ETH in initiation of the pupal ecdysis sequence, its release from endocrine Inka cells was monitored in vivo by using time-lapse EGFP fluorescence imaging in pharate pupae (prepupae) carrying the chimeric transgene 2eth3-egfp. In this transgenic fly, EGFP is expressed as part of a fusion protein with the ETH propeptide precursor, and loss of EGFP fluorescence indicates ETH release. Because pharate pupae generally are immobile and Inka cells are located immediately below the semitransparent puparium, in situ imaging of Inka cell in intact pharate pupae is feasible. Two to three Inka cells were monitored simultaneously in each experiment (Kim, 2006a).

Depletion of ETH-EGFP occurs in about 50% of monitored Inka cells shortly before pre-ecdysis onset (time zero). The time course of secretory activity for each Inka cell was variable. The mean value for the timing of ETH release was 4.5 min prior to pre-ecdysis onset, and the duration of ETH secretion was 4.4. In contrast, 40% of Inka cells showed no sign of secretory activity by pre-ecdysis onset. After initiation of pre-ecdysis contractions, it was usually impossible to continue monitoring loss of EGFP fluorescence as a result of movement artifacts. All Inka cells are depleted of ETH-EGFP by the end of ecdysis sequence (Kim, 2006a).

Injection of ETH induces the ecdysis sequence: Because ETH release coincides with onset of pupal preecdysis, it was of interest to determine whether ETH injection would trigger the pupal ecdysis sequence. The Drosophila gene eth encodes a precursor producing one copy each of two peptides, ETH1 and ETH2, which share similar structure and biological activity. In vivo experiments were carried out primarily in puparium-free preparations (Kim, 2006a).

Injection of ETH1 alone into pharate pupae (~1-2 hr prior to natural ecdysis) induced within 1-3 min strong pre-ecdysis contractions followed by ecdysis and postecdysis contractions sequentially. ETH-induced pre-ecdysis showed a strong dose dependence, with higher doses inducing shorter pre-ecdysis duration and higher frequency of contractions. Similar but somewhat less pronounced dose-dependent effects were observed during ecdysis behavior, whereas the frequency of postecdysis contractions showed little or no dose dependence during the first 10 min of behavior (Kim, 2006a).

Injection of ETH2 was less efficacious for induction of the behavioral sequence compared to ETH1. ETH2 generated prolonged pre-ecdysis behavior lasting to 50 min or more, but no ecdysis behavior. In contrast, injection of the same dose of ETH1 (0.4 pmol) produced the complete behavioral sequence consisting of pre-ecdysis, ecdysis, and postecdysis. Higher doses of ETH2 (20 pmol) generated a behavioral sequence comparable to that induced by 4 pmol ETH1 in terms of pre-ecdysis duration and frequency of contractions. Behaviors after injecting a cocktail of ETH1 and ETH2 (0.4 pmol of each peptide) were also examined. Because the two peptides are processed from the same precursor, it is likely that these peptides are coreleased under natural conditions. The duration of the behavioral sequence induced by injection of the cocktail was similar to the naturally occurring sequence or one induced by 0.4 pmol ETH1 alone. It is estimated that a 0.4 pmol ETH injection into a prepupa w10 hr after puparium formation results in a concentration of ~300 nM in vivo (Kim, 2006a).

ETH receptors are expressed in diverse ensembles of peptidergic neurons: ETH acts directly on the CNS to initiate the ecdysis behavioral sequence in moths and flies via unknown signaling pathways within the CNS. A starting point for elucidation of these downstream signaling pathways is identification of primary neuronal targets of ETH. The ETH receptor gene (CG5911), first identified in Drosophila, encodes two G protein-coupled receptors, ETHR-A and ETHR-B, via alternative splicing. In situ hybridization was used for identification of central neurons expressing ETHR-A and ETHR-B by using DNA probes specific for each receptor subtype. ETHR-A and ETHR-B transcripts were located in mutually exclusive populations of neurons distributed throughout the CNS, suggesting that two subtypes of ETH receptors likely mediate different functions. Further analysis revealed that most ETHR-A neurons are peptidergic. Neurons expressing ETHR-B have not been identified thus far (Kim, 2006a).

Multiple ensembles of ETHR-A neurons are classified according to specific neuropeptides they express. Peptides expressed in these ensembles were identified by using GAL4 transgenes under control of neuropeptide promoters to drive UAS-GFP or UAS-GCaMP expression (GAL4::GFP or GAL4::GCaMP). Expression of neuropeptides in these cells was confirmed by combining immunohistochemical staining and in situ hybridization. The first ETHR-A ensemble comprises six pairs of lateral abdominal neurons producing kinin, also known as drosokinin. These cells project axons posteriorly along the lateral edge of the neuropile and then turn anteriorly along the midline of ventral nerve cord, where they arborize and form possible central release sites. Axons of these cells also exit the CNS through nerve roots, suggesting peripheral kinin release. The second ETHR-A ensemble contains three pairs of ventrolateral FMRFamide neurosecretory cells (Tv1-3 or T1-3) in the thoracic neuromeres TN1-3. These cells project axons into the dorsomedial neurohemal organs (NHOs) specialized for peptide release into the hemolymph (Kim, 2006a).

The third class of ETHR-A neurons comprises the eclosion hormone (EH)-producing VM neurons in the brain, which project one axonal branch anteriorly into the neurohemal ring gland and a second posteriorly along the dorsal midline of the entire ventral nerve cord. The fourth ETHR-A ensemble is composed of paired dorsolateral neurons producing CCAP, MIPs, and bursicon in subesophageal, thoracic, and abdominal neuromeres (SN1-3, TN1-3, AN1-7, respectively). These cells are likely homologs of moth neurons 27/704, on the basis of their anatomy, peptide coexpression profile, and functional roles during pupal ecdysis. These neurons are referred to as Drosophila neurons 27/704 and them were subdivided on the basis of peptide coexpression. In AN1-4, CCAP is colocalized with MIPs and the heterodimeric peptide hormone bursicon (composed of burs and pburs subunits). In TN2-3 and AN5- 9, CCAP is colocalized with burs, but pburs is not expressed in these neurons. Finally, CCAP is colocalized with MIPs in large paired neurons of AN8,9, but ETHR-A expression has not been confirmed in these cells. The presence of MIP mRNA in abdominal neurons 27/704 was further confirmed by in situ hybridization (Kim, 2006a).

Ca2+ imaging of primary ETH targets in transgenic flies: Having shown that ETH receptors occur in diverse ensembles of peptidergic neurons, it was asked whether these cells are activated by ETH and whether this activity coincides with specific behavioral steps of the ecdysis sequence. Calcium dynamics in each group of ETHR-A neurons was monitored by driving expression of the GFP-based Ca2+ sensor, GCaMP [23, 24], in genetically defined sets of neurons with the binary GAL4/UAS system. Ca2+ elevation induces a conformational change of GCaMP, increasing its GFP fluorescence. Using optical imaging of GFP fluorescence, [Ca2+]i dynamics of ETHR neurons were monitored, and these events were associated with each behavioral phase induced by ETH (Kim, 2006a).

An abundance of evidence indicates that the ecdysis behavioral sequence in insects is centrally patterned. In particular, the onset and duration of each behavior in the sequence (pre-ecdysis I, pre-ecdysis II, ecdysis) is the same whether observed in vivo or as fictive behavior recorded from the isolated CNS in vitro. On the basis of this evidence, [Ca2+]i dynamics of ETHR-A neuron ensembles of the isolated CNS were associated with behaviors observed in puparium-free preparations (Kim, 2006a).

FMRFamide neurons and their neurohemal endings become active early in pre-ecdysis: [Ca2+]i levels were monitored in ETHR-A/FMRFamide Tv neurons by preparing transgenic flies doubly homozygous for FMRFa-GAL4 and UAS-GCaMP. Prior to ETH1 exposure (4-6 hr prior to ecdysis), Tv cell bodies and neurohemal endings in the dorsomedial NHO exhibit low levels of basal GCaMP fluorescence (Kim, 2006a).

Exposure of the CNS to ETH1 (600 nM) elicits robust increases in calcium-associated fluorescence in cell bodies and axon terminals of all Tv neurons. At this concentration of ETH1, calcium dynamics typically are characterized by transient, spike-shaped fluctuations superimposed upon a slow upward shift of the baseline, beginning ~8 min after exposure to the peptide. This response lasts ~10-15 min, after which weaker spike-like fluctuations continue without baseline changes until the end of recordings (~40 min). It is estimated that a concentration of 600 nM ETH1 results from a dose of w0.4 pmol of the peptide in vivo. Thus the major calcium response of Tv neurons coincides with the early phase of pre-ecdysis, and weaker activity persists through ecdysis and postecdysis. In contrast, ETH2 alone (600 nM) generates Ca2+ responses after a longer delay comparable to one following exposure to 60 nM ETH1. The longer delay of Ca2+ responses after ETH2 fits with the observations of in vivo behavior, where ETH2 is a less potent agonist than ETH1. The cocktail of ETH1 and ETH2 (600 nM each) evokes Ca2+ dynamics after a delay similar to that induced by ETH1 alone. Overall, [Ca2+]i dynamics observed in Tv neurons are synchronized. In many preparations, Tv neurons from the same neuromere appear to be strongly coupled, given that they produce precisely synchronized Ca2+ dynamics. Transient Ca2+ signals are obvious in the terminal processes of Tv neurons in NHO, the release sites of FMRFamides (Kim, 2006a).

Lower concentrations of ETH1 elicit calcium dynamics after a somewhat longer delay. Interestingly, calcium dynamics are obvious first in neurohemal endings of the NHO, followed by fluctuations in cell bodies. This was particularly evident at 60 nM ETH1, where a robust calcium response in the NHO was accompanied by only a weak response in the Tv2 cell body. No calcium responses are observed in Tvs exposed to 6 nM ETH1 (Kim, 2006a).

EH neurons reach peak activity at ecdysis: VM neurons producing eclosion hormone (EH) have been implicated as primary ETH targets during fly and moth ecdysis. Expression of ETHR-A was demonstrated in VM neurons, confirming that they are primary ETH targets. To determine whether ETH elicits activity in EH neurons, transgenic flies were prepared doubly homozygous for EHup-GAL4 and UASGCaMP, that show GCaMP fluorescence only in these cells (Kim, 2006a).

EH neurons are highly sensitive to ETH1, exhibiting robust [Ca2+]i dynamics upon exposure to concentrations as low as 6 nM. No detectable fluorescence responses are observed after exposure to 0.6 nM ETH1 over a period of 50-60 min. The latency to Ca2+ responses is inversely proportional to the concentration of ETH1; higher ETH1 concentrations evoke faster responses. The cocktail of ETH1 and ETH2 (600 nM each) elicited Ca2+ responses after a ~10-15 min delay (Kim, 2006a).

Close examination of these ETH-evoked fluorescence responses reveals two components distinguished by slow and fast kinetics. The slow component is characterized by a gradual increase in baseline levels of Ca2+ followed by a decrease over 20-30 min, whereas the fast component is composed of transient, spike-like activity. Fast components have durations ranging from 5-20 s. Peak DF/F responses are quite variable, even among a group of neurons exposed to the same ETH1 concentration. No significant concentration dependence could be detected in peak response (Kim, 2006a).

Distinct subsets of neurons 27/704 are active during different phases of the ecdysis sequence: ETH-evoked Ca2+ signals of neurons 27/704 were examined in transgenic flies carrying CCAP-GAL4 and UAS-GCaMP. Use of the CCAP promoter to drive GCaMP expression resulted in a reporter pattern identical to that described previously. Upon exposure to 600 nM ETH1, distinct subsets of neurons 27/704 exhibited reproducible, stereotypic Ca2+ responses in terms of peak intensity, latency, and termination of Ca2+ dynamics. According to the magnitude of peak fluorescence intensity (peak DF/F), neurons 27/704 fall into three major groups: strong responders, weak responders, and nonresponders. The strong-responder group includes neurons 27/704 in AN1-4 (CCAP/MIPs/bursicon), AN8,9 (CCAP/ MIPs), and TN3 (CCAP). Weak responders are neurons 27/704 in SN2-3, TN1-2, and AN7 producing CCAP only. Neurons in the brain, SN1, and AN5,6 showed no reproducible Ca2+ dynamics in response to 600 nM ETH1 (Kim, 2006a).

In response to ETH1, neurons 27/704 in TN3 and AN8,9 become active within 10-15 min, whereas neurons 27/704 in AN1-4 are activated after a 15-25 min delay. Neurons in TN3 and AN8,9 are therefore activated just prior to ecdysis onset, indicating their possible roles in initiation and maintenance of ecdysis behavior. In addition, Ca2+ dynamics observed in AN8,9 neurons terminated early in postecdysis, supporting this interpretation. In contrast, Ca2+ dynamics of neurons in AN1-4 begin during ecdysis and increase in intensity during the entire postecdysis period, suggesting their roles in these events. The cocktail of ETH1 and ETH2 (600 nM each) evoked Ca2+ dynamics similar to those induced by ETH1 alone. Two groups of neurons 27/704 in abdominal neuromeres (AN1-4 versus AN8,9) exhibit differences in sensitivity to ETH and in their patterns of [Ca2+]i dynamics. It was found that 6-60 nM ETH1 activates neurons in AN1-4 (n = 4), whereas higher concentrations of ETH1 (R600 nM) are required to activate neurons in AN8,9. In addition, neurons in AN8,9 generate transient (1-2 min) Ca2+ spikes over a 15-20 min period after ETH1 activation, whereas neurons in AN1-4 generally produce slower, more persistent Ca2+ dynamics. These differences among subgroups of neurons 27/704 suggest their different functional roles during the ecdysis sequence (Kim, 2006a).

Targeted ablations of apecific ETHR neurons have behavioral consequences: To evaluate behavioral roles of specific ETHR neurons, phenotypes of the pupal ecdysis sequence were investigated in transgenic flies bearing targeted ablations of ETHR neurons, including Tv FMRFamide neurons, EH neurons, and CCAP neurons (27/704 homologs). In control flies carrying UAS-reaper and UAS-GCaMP, but lacking the GAL4 driver, pupal ecdysis was executed as in wild-type flies: pre-ecdysis (0-10 min), ecdysis (10-23 min), and postecdysis (23-100 min) (Figure 7). Given that puparium-intact animals were used, the duration of ecdysis behavior may have been overestimated. Transgenic flies bearing targeted ablations of Tv FMRFamide neurons (FMRF-KO) were generated by crossing females doubly homozygous for FMRFa-GAL4, UAS-GCaMP with homozygous UAS-reaper males. Pupal ecdysis of FMRFa-KO flies is very similar to that of control flies. Because FMRFa-GAL4 drives expression of GAL4 only in three pairs of thoracic Tv neurons and one pair of unidentified neurons in SN, FMRFa-KO flies lost only Tv neurons and not other FMRFamide neurons in the CNS. FMRFa-KO flies complete pupal ecdysis without any detectable abnormality, except that pre-ecdysis contractions appear weaker than in control flies. Pupal ecdysis of VM neuron knockout flies (EH-KO) was then examined. Behavioral analysis showed that, although they complete pupal ecdysis without any severe defects or lethality, ecdysis onset is delayed w4 min. As a result of this delay, EH-KO flies show longer pre-ecdysis than control flies. Additional parameters governing pre-ecdysis, ecdysis, and postecdysis are indistinguishable between EH-KO and control flies (Kim, 2006a).

Finally, CCAP-KO flies were generated in order to examine the functional roles of neurons 27/704 (CCAP neurons) in pupal ecdysis. As expected, CCAP-KO flies failed to initiate ecdysis contractions and could not complete head eversion. Instead, they show prolonged pre-ecdysis contractions for ~25 min, followed by weak random contractions of the abdomen (different from ecdysis and postecdysis contractions of control flies) for the next 50 min (Kim, 2006a).

Conclusions: This study has described orchestration of an innate behavior, the Drosophila pupal ecdysis sequence, by the endocrine peptide ETH. ETH release coincides with onset of behavior, and injection of ETH triggers the complete behavioral sequence, consistent with its role in ecdysis activation previously established in the moths Manduca sexta and Bombyx mori and in Drosophila larvae and adults. Absence of ETH causes lethal ecdysis deficiency, a phenotype that is rescued by ETH injection. ETH therefore functions as a 'command chemical' to orchestrate an innate behavior. Primary CNS targets of ETH was identified by using ETHR-specific in situ hybridization. ETHR-A occurs in multiple classes of peptidergic neurons producing EH, CCAP/MIPs/bursicon, FMRFamides, or kinin (Kim, 2006a).

Expression of ETHR-A was shown in VM neurons, which release EH. In response to ETH, VM neurons become active prior to ecdysis behavior and reach peak levels of activity during ecdysis. These results provide further support for a previously described positive-feedback signaling pathway between VM neurons and Inka cells. This feedback is thought to ensure depletion of ETH from Inka cells. These findings are striking because independent evidence indicates that homologous VM neurons in the moth Manduca are direct targets of ETH and that their secretory products regulate ecdysis behaviors downstream of ETH. For example, isolated EH neurons of Manduca respond to direct action of ETH with increased excitability and spike broadening. In response to ETH action, these neurons release EH, causing cGMP elevation and increased excitability in CCAP-containing neurons 27/704 of the thoracic and abdominal ganglia. CCAP and MIPs, cotransmitters produced by neurons 704, are implicated in eliciting ecdysis behavior (Kim, 2006a).

A homologous role for EH in activation of Drosophila 27/704 neurons has not been clearly demonstrated. For example, no cGMP elevation is observed in these neurons during the natural ecdysis sequence. This lack of cGMP elevation suggests that CCAP neurons are not directly targeted by EH in Drosophila. Nevertheless, EH-knockout flies exhibit a delay in ecdysis initiation, suggesting that EH may modulate excitability in 27/704 cells indirectly through release of additional factors within the CNS. It is therefore proposed that activation of EH neurons by ETH serves two purposes: (1) release of EH into the hemocoel functions as part of a positive-feedback pathway to ensure ETH depletion from Inka cells; (2) release of EH within the CNS synergizes direct ETH actions on different subsets of neurons 27/704 producing CCAP, MIPs, and bursicon, perhaps indirectly through release of downstream signals within the CNS (Kim, 2006a).

Neurons 27/704 expressing ETHR-A respond to ETH with unique patterns of Ca2+ dynamics. These neurons are subdivided by pattern of transmitter expression: CCAP/MIPs/bursicon in AN1-4; CCAP/MIPs in AN8,9; and CCAP in SN1-3, TN1-3, and AN5-7. Temporal patterns of Ca2+ dynamics were determined in each neuronal subset relevant to the behaviors observed. On the basis of these temporal patterns, it is proposed that direct action of ETH on neurons 27/704 in TN3 and in AN8,9 induces initiation and execution of ecdysis contractions and head eversion. In support of this, it is shown that ablation of CCAP neurons abolishes ecdysis contractions and head eversion. Parallel study in Manduca showed that neurons 704 expressing ETHR-A and their peptide cotransmitters, CCAP and MIPs, are implicated in control of the ecdysis motor pattern, supporting the homologous function of 27/704 neurons in Drosophila. Neurons 27/704 in AN1-4 produce CCAP, MIPs, and bursicon, and therefore a cocktail of these peptides is likely released within the CNS and into the hemolymph during postecdysis. It is suggested that centrally released peptides control postecdysis movements, whereas blood-borne CCAP/MIPs regulate heart beat and blood pressure for cuticle expansion and bursicon controls sclerotization of expanded new cuticle. Bursicon was recently identified as a heterodimeric peptide hormone regulating cuticle plasticization, sclerotization, and melanization (Kim, 2006a).

The Drosophila FMRFamide gene (FMRFa) encodes multiple FMRFamide-related neuropeptides, which are expressed in many different cell types, including neuroendocrine cells, interneurons, and perhaps motoneurons. Among these diverse FMRFamide-producing neurons, ETHR-A expression is confined to three pairs of thoracic neurosecretory neurons, Tv1-3. Results of the present study show that the Tv neurons are activated early in pre-ecdysis and that they remain active during ecdysis and postecdysis. However, FMRFa-KO flies show no differences in timing of the ecdysis behavioral sequence. Because FMRFamides enhance twitch tension of larval body-wall muscles through synaptic modulation at the neuromuscular junction, blood-borne FMRFamides released from Tv neurons likely facilitates pre-ecdysis, ecdysis, and postecdysis contractions. Thus the role of Tv neurons as primary ETH targets may be enhancement of muscle contraction during the behaviors. Further work to substantiate this is in progress (Kim, 2006a).

Expression of ETHR-A occurs in in kinin neurons of abdominal neuromeres of Drosophila. Drosophila kinin is known to be involved in water balance, but its central functions have not been described or considered. Expression of ETHR-A in kinin neurons appears to be a conserved mechanism in fly and moth; the Manduca ETHR-A is expressed in abdominal neurosecretory cells (L3,4), which produce kinins and diuretic hormones (DHs). It was further found that the isolated Manduca CNS generates the fictive pre-ecdysis motor pattern upon exposure to a cocktail of kinin and DHs. These findings suggest that ETH activates L3,4 neurons in Manduca to release kinins and DHs centrally, which initiate and execute pre-ecdysis. On the basis of the conservation between Drosophila and Manduca in spatial expression pattern of ETHR, it is proposed that ETH initiates pre-ecdysis behavior indirectly via central release of kinin in Drosophila (Kim, 2006a).

In Drosophila, pupal ecdysis is accomplished by sequential recruitment of three major behavioral units: pre-ecdysis (0-10 min), ecdysis (10-15 min), and postecdysis (15-100 min). Each behavioral unit is programmed in the CNS and sequentially activated by direct actions of ETH, which is synthesized and released from peripheral endocrine Inka cells. Around 4-5 min before pre-ecdysis onset, a sizeable portion (~50%) of Inka cells initiates secretion of ETH into the hemolymph, whereas the remaining portion completes secretion after onset of pre-ecdysis. Appearance of ETH in the hemolymph activates ETHR-A in neurons expressing neuropeptides including kinin, FMRFamides (Tv1-3), EH, or CCAP, MIPs, and bursicon, but they are not released until descending inhibition is removed at key times during the ecdysis sequence. Upon activation of ETHR, the central release of kinin initiates pre-ecdysis contractions, whereas Tv neurons secrete FMRFamides to enhance neuromuscular transmission. ETH activates neurons producing EH, CCAP, CCAP/MIPs, and CCAP/MIPs/bursicon at different times. EH cells in the brain and neurons producing CCAP in TN3 and CCAP/MIPs in AN8,9 become active ~10-13 min after pre-ecdysis initiation. EH participates in timing the activation of ecdysis neurons, whereas CCAP and MIPs from TN3 and AN8,9 control initiation and execution of the ecdysis motor program. At the end of ecdysis (25 min after pre-ecdysis onset), neurons in AN1-4 secrete a cocktail of CCAP, MIPs, and bursicon, which likely regulate postecdysis contractions and processes associated with cuticle expansion, hardening, and tanning (Kim, 2006a).

This study has mapped central ETH receptor neurons, and discovered that they comprise multiple peptidergic ensembles, which are recruited sequentially to generate each phase of the ecdysis sequence. Ensemble-specific knockout analysis supports this interpretation. Each step of the ecdysis sequence (pre-ecdysis, ecdysis, postecdysis) is driven by a central pattern generator (CPG) within the CNS in the absence of sensory input. It is known that amines and peptides can modulate and reconfigure neuronal circuits comprising CPGs so as to elicit a variety of motor patterns. It seems likely that the multiple peptidergic ensembles described in this study as targets for ETH may be involved in configuring and activating CPGs underlying each step of the ecdysis sequence (Kim, 2006a).

Processes in the brain that govern behaviors over longer time frames such as sleep, mood, sexual activities, and even learning and memory could be associated with coordinated release of neuromodulators such as peptides. Further work on activation of central peptidergic ensembles in the CNS may shed light on mechanisms underlying release of a variety of behaviors (Kim, 2006a).

Secretory competence in a gateway endocrine cell conferred by the nuclear receptor βFTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a 'gateway cell', whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline (Cho, 2014).

The splice isoforms of the Drosophila ecdysis triggering hormone receptor have developmentally distinct roles

In order to grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Triggering Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. This study used the recently introduced 'Trojan exon' technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. ETHRA and ETHRB were shown to be expressed in largely distinct subsets of neurons, and ETHRA- , but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. Several functionally important subsets of ETHR-expressing neurons were found including one that co-expresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks (Diao, 2016).

To characterize the neural circuit that governs ecdysis in Drosophila, this study exploited the Trojan exon technique to map and manipulate the ETH signaling pathway, and the effects of genetic and neuronal loss-of-function at were analysed at the level of the ETHR gene, its splice variants, and the cells that express them. Genetic disruption of ETHR expression phenocopies ETH loss-of-function, indicating that the ETHR gene encodes the sole receptor for ETH peptides in mediating ecdysis. Consistent with this, it was found that suppression of ETHR-expressing neurons blocks ecdysis at all developmental stages. Both the genetic and neuronal loss-of-function data further reveal distinct developmental requirements for the two ETHR isoforms and the neurons that express them, with ETHRB-, but not ETHRA-expressing neurons dispensable for larval ecdysis. Finally, it was demonstrated that ETHR-expressing neurons regulate both behavioral and physiological processes at pupal ecdysis (Diao, 2016).

Previous observations have shown that ETH initiates ecdysis at all stages in Drosophila development, but apart from work done on a small subset of neurons known to express ETHRA, the broader ecdysis circuit targeted by ETH and the functional roles of its receptors have remained largely uncharacterized. The data presented in this study confirm the importance of ETHRA in ecdysis, as previously demonstrated in Tribolium using RNAi knockdown. In addition, the Arakane study demonstrate, for the first time in any insect species, an essential function for ETHRB in that selective knockdown of ETHRB expression substantially blocks pupal ecdysis. The fact that larval ecdysis is largely unimpaired by this manipulation (together with the observation that restoration of ETHRA, but not ETHRB, expression compensates for loss of ETHR function at larval ecdysis) strongly argues that ETHRA and ETHRB have distinct functional roles and that their contribution to ecdysis is differentially dependent on developmental stage. These conclusions are consistent with the results of neuronal suppression using ETHRA- and ETHRB-specific Gal4 drivers: ETHRA-expressing neurons are required for ecdysis throughout development, whereas ETHRB-expressing neurons are required only after the larval stage. In addition, the differing phenotypes of pupal lethality seen with suppression of ETHRB- vs. ETHRA-expressing neurons argues that the two receptor isoforms mediate different processes (Diao, 2016).

The results also confirm and extend the conclusions drawn previously from in situ hybridization studies that ETHRA and ETHRB are expressed in distinct populations of neurons (Kim, 2006b). That this is also the case in the hawkmoth, Manduca suggests that this feature, like the generation of ETHRA and ETHRB splice isoforms itself, is highly conserved. The finding that ETHRA and ETHRB serve distinct functions that are mediated by different populations of neurons thus seems likely to represent an evolutionarily ancient characteristic of insect ecdysis circuits (Diao, 2016).

Interestingly, an exception to the mutually exclusive expression of ETHRA and ETHRB occurs in the Vm neurons, which secrete EH and occupy a unique role in the ecdysis circuit. EH acts in a well-characterized positive feedback loop between the Vm neurons and the ETH-secreting Inka cells. ETH and EH are initially released at low levels, but each reinforces the other's secretion to cause the later, massive release of both hormones at high levels, an event that is thought to drive the progression of the ecdysis sequence. It is possible that the ETHRB isoform, which exhibits higher sensitivity to ETH than ETHRA (Iversen, 2002; Y. Park, 2003), mediates the initial Vm response to ETH causing low-level EH release, while expression of the ETHRA isoform participates in the late, high-level response. Further work will, however, be required to test this hypothesis (Diao, 2016).

While the function of ETHRB-expressing neurons has been enigmatic, several groups of peptidergic neurons known to express ETHRA have been previously implicated in governing ecdysis. The results presented in this study refine and expand knowledge of two such cell groups, those that express CCAP or Lk (Diao, 2016).

CCAP-expressing neurons as a group have been shown to be required for head eversion, a defining event of pupal ecdysis. The results presented in this study provide direct evidence that the subset of CCAP-expressing neurons that coexpresses ETHRA is the one required for head eversion. A previous study of a late-differentiating CCAP-expressing neurons concluded that cells in abdominal neuromeres AN8-9 are required for head eversion. However, these neurons are not observed to express ETHRA mRNA, and it was surprising to likewise find them inconsistently represented in the expression patterns of ETHR^MI00949- and ETHRA^MI00949-Gal4 lines. The finding that both electrical suppression and ablation of these neurons leaves head eversion unimpaired forces a conclusion that CCAP-expressing neurons in AN8-9 are not responsible for this process. This conclusion is consistent with a recent report that activation of CCAP-expressing neurons that also express bursicon (and therefore do not include the AN8-9 neurons) is sufficient to induce head eversion. It is possible that the correlational nature of earlier results, which were based on the effects of stochastic ablation of subsets of all CCAP-expressing neurons, may have inadvertently suffered from sampling errors that biased the interpretation to the opposite conclusion. In general, the current demonstration that only those CCAP-expressing neurons that coexpress ETHRA block ecdysis underscores the ability of the receptor-based mapping approach described here to correctly identify critical nodes in hormonally controlled behavioral circuits (Diao, 2016).

A second critical node revealed by this analysis is the subset of ETHRA-expressing neurons that express the diuretic factor leucokinin. Based on neuronal suppression experiments using two copies of the UAS-Kir2.1 transgene, it was demonstrated that Lk-secreting neurons maintain fluid balance to support behavioral execution. It was found that ecdysis deficits induced by neuronal suppression are reversed together with fluid imbalance by feeding of tyramine suggesting that these phenotypes share a common cause in the dysregulation of fluid secretion at the level of the Malpighian tubules. A report that appeared during revision of this manuscript indicates that Lk-expressing neurons also specifically regulate the timing of pre-ecydsis behavior (Kim, 2015), which was not assayed in the current. It will be interesting to determine whether these deficits are independent of fluid imbalance and persist in tyramine-fed animals. Curiously, neither fluid imbalance nor the overt deficits in appendage extension that this study observed are described by Kim (2015) as a consequence ablating the Lk-expressing neurons. It is possible that this apparent inconsistency results from the use of different Lk-Gal4 drivers, or from the differential efficacy of neuronal suppression by UAS-Kir2.1 vs. cell killing by UAS-rpr, but in either case, the two studies support the conclusion that Lk-expressing neurons regulate physiological and/or behavioral processes important for pupal ecdysis (Diao, 2016).

Consistent with what has been previously found, this analysis suggests that peptidergic neurons, in general, are well represented within the ETHR expression pattern and are essential to the ecdysis circuit. Evidence is also provided that subsets of ETHR-expressing neurons that use the neurotransmitters acetylcholine and glutamate are functionally important. Suppression of the cholinergic subset potently blocks ecdysis at both the larval and pupal stages and may well include many neurons that also express peptides. The glutamatergic subset, however, is likely to be distinct from the cholinergic group based on the previously reported nonoverlapping expression of the cholinergic marker, ChaT, and the glutamatergic marker, VGlut (Diao, 2015). Interestingly, electrical suppression of a GABAergic subset of ETHR-expressing neurons does not result in overt ecdysis failure. It thus seems likely that inhibitory inputs previously shown to regulate the execution of different phases of the ecdysis sequence in Manduca and thought to also function in Drosophila do not derive from ETHR-expressing neurons, are not GABAergic, or are not strictly essential for ecdysis. The last possibility is favored and it is noted that this study has focused only on gross ecdysis deficits. More subtle defects that affect behavioral coordination, execution, or timing and do not result in lethality will require closer analysis. The preliminary results, however, suggest that many of the ETHR-expressing neurons identified in this study can be expected to play specific roles in ecdysis at some developmental stage (Diao, 2016).

The Trojan exon methodology used in this study to identify, manipulate, and parse the patterns of ETHR expression represents a systematic and versatile strategy for mapping functional connectivity within hormone-mediated neural circuits. In the case of the ecdysis circuit, this strategy has not only facilitated analysis of the neural substrates of behavior and physiology, but has revealed unanticipated developmental differences in the importance of the two ETHR isoforms. In the fly as in other insects, the motor patterns that mediate ecdysis vary considerably across developmental stages to accommodate differences in body plan and environmental context. However, the changes that occur in the ecdysis circuit over development remain largely unknown. The tools developed here should provide the basis for a thorough-going investigation of this, and other, important issues (Diao, 2016).

EFFECTS OF MUTATION

To test whether ETH is required for ecdysis, gene deletions were generated by imprecise excisions of EP(2)1065, a P-element located 1427 bp downstream of the ETH polyA site. These efforts led to creation of three deletion lines, eth²⁷, eth^25b and eth¹⁹⁶, all of which possess small excisions near or including ETH. The eth²⁷ line has a deletion from the P-element site in the 5' untranslated region of reg-5 (rhythmically expressed gene 5) up to the 3' untranslated region of eth, 22 bp downstream of the ETH stop codon. This deletion does not disturb the coding sequence of eth, thus serving as a useful negative control for other deletions. The eth^25b deletion removes virtually the entire ETH sequence, extending from the original P-element site up to the eth-coding region, leaving only part of the signal sequence (MRIITVLSV) (Park, 1999). The eth¹⁹⁶ deletion occurs from the P-element site through ETH to the middle of the adjacent gene orc4 (Park, 2002a).

Loss of ETH in both eth^25b and eth¹⁹⁶ lines causes recessive lethality, while eth²⁷ has no obvious phenotype. The fact that genotype eth^25b/eth¹⁹⁶ also shows the same ecdysis deficiency phenotype suggests that the ETH deletions cause this phenotype, rather than other unknown aberrations. Lethality is associated with ecdysis deficiencies, whereby double mouthhooks and dVP indicate failure to shed the old mouthparts. These animals show a shrunken body appearance, thick trachea and partial ecdysis of old cuticle both exteriorly and within the tracheal system. The phenotype resulting from eth-deletion is referred to as 'buttoned-up', which describes an inability to extricate old mouthhooks and vertical plates from the new sclerotized structures (Park, 2002a).

Further analysis has revealed disrupted respiratory dynamics and behavioral deficits in ETH null mutants. Tracheal collapse and inflation of new trachea are delayed for ~1.5 hours, and pre-ecdysis behaviors are completely absent. In the absence of these events, 'ecdysis-like' behavior occurs early, around the dVP stage with a large variation among individuals (4±23 minutes). Ecdysis-like behavior differs from wild-type ecdysis behavior in several respects. (1) Normal forward thrust movements to plant the old mouthparts in the substrate are absent. Instead, animals engage in swinging head movements, and repeated extensions and retractions of the mouth. (2) Strong backward thrust movements, which normally result in separation of the spiracles and ecdysis of trachei, are also absent. Although some backward movements are observed, animals are unsuccessful in detaching the old spiracles and tracheal linings. Some turning behavior resembling forward escape is observed, but animals are unsuccessful in this maneuver, owing to the fact that neither mouthparts nor spiracles have been detached. These ecdysis-like behaviors are repeated on an irregular basis for 1 to 3 hours. Some time after the occurrence of delayed tracheal collapse and inflation, ecdysis-like behaviors become more like normal ecdysis. Indeed, the majority of eth^- mutants are able to move through an anterior dorsal opening in the old cuticle that appears after repeated ecdysis movements. This occurs on the average at 2 hours 17 minutes±40 minutes after the dVP stage. This type of exit from the old cuticle contrasts with that of wild-type flies, which ecdyse by moving through the anterior opening created by removal of the old mouthparts. Even though many mutant larvae are able to escape the old cuticle, their mouthparts remain 'buttoned-up'. The buttoned-up phenotype remains quiescent, does not feed and dies within 1 to 2 days. A small fraction of eth^- larvae undergo successful ecdysis and development through the second instar (~2%), but all succumb following ecdysis failure at the 2nd to 3rd instar transition (Park, 2002a).

Properly timed injection of ETHs rescue ecdysis deficiencies in mutant flies and promotes successful ecdysis. Injection of Drosophila ETH1 (~ 1 fmol) into either eth^25b or eth¹⁹⁶ larvae at the dVP stage restores all missing steps in the ecdysis sequence. Specifically, Drosophila ETH1 injections induce tracheal collapse and inflation of trachea (3 and 4 minutes after the injection, respectively). Thereafter, pre-ecdysis behaviors appear, including weak anterior-posterior movements (7±2 minutes) followed by strong squeezing waves (9±2 minutes). A set of typical ecdysis behaviors, including forward and backward thrusts and forward escape, occurs at 18±2 minutes after injection. Rescued flies that succeeded in passing to the 2nd instar succumbed at the transition to the 3rd instar, owing to unsuccessful ecdysis (Park, 2002a).

Though mutants injected with Drosophila ETH1 showed a normal ecdysis behavioral sequence, some individuals were unsuccessful in completing ecdysis. Approximately 25% of eth^25b and 41% of eth¹⁹⁶ flies fail to successfully shed the old cuticle. Rescued eth^25b mutants show no further mortality during the 2nd instar, but rescued eth¹⁹⁶ mutants show significant mortality during the early 2nd instar; accumulated mortality rises from 41% to 73% (lethal phase i in the 2nd instar. The elevated mortality observed for eth¹⁹⁶ mutants during the 2nd instar may result from partial deletion of the upstream gene orc4. Maternally deposited orc4 mRNA can promote survival enough the early 1st instar, but is insufficient for development through the early 2nd instar. Further examination of this question requires genetic rescue of the eth¹⁹⁶ line (Park, 2002a).

Lethality can be reversed also by injection of DrmETH2 at relatively high doses (> 10 fmol). These treatments partially rescued behavioral deficits in eth^25b flies, including induction of tracheal collapse, inflation of trachea and ecdysis. However, DrmETH2 injections failed to induce anteroposterior contractions and squeezing waves. Lower doses of DrmETH2 (~1 fmol) induced tracheal collapse and inflation of new trachea, but were not effective in eliciting either pre-ecdysis or ecdysis behaviors (Park, 2002a).

EVOLUTIONARY HOMOLOGS

Ecdysis behavior in the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) is triggered through reciprocal peptide signaling between the central nervous system and the epitracheal endocrine system. Recent evidence indicates that eclosion hormone may initiate endocrine events leading to ecdysis through its action on epitracheal glands to cause the release of ecdysis-triggering hormone (ETH). Direct exposure of epitracheal glands to eclosion hormone in vitro leads to secretion of ETH. The threshold concentration of eclosion hormone needed to evoke release of ETH is approximately 3 pmol l-1. Eclosion hormone also induces elevation of cyclic GMP, but not cAMP, concentration in epitracheal glands at concentrations similar to those causing release of ETH. Both cGMP and 8-Br-cGMP mimic the secretory action of eclosion hormone. The sensitivity of the secretory response to eclosion hormone occurs during a narrow window of development, beginning approximately 8 h prior to pupal ecdysis. However, eclosion hormone can cause elevation of cGMP levels in epitracheal glands long before they acquire competence to release ETH, showing that the initial portion of the signal transduction cascade is in place early in development, but that the absence of a downstream step in the cascade prevents secretion. Measurements of cGMP levels in epitracheal glands during the ecdysis sequence show a sudden elevation some 30 min after the onset of pre-ecdysis, well after ETH secretion has been initiated. ETH secretion can therefore be viewed as a two-step process, beginning at pre-ecdysis when cGMP levels are relatively low, followed by a massive release resulting from a logarithmic elevation of cGMP levels (Kingan, 1997).

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, the cDNA and gene encoding the Manduca sexta preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH) that activate these behaviors, have been identified. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence (Zitnan, 1999).

Three insect peptide hormones, eclosion hormone (EH), ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (see Drosophila Cardioacceleratory peptide), have been implicated in controlling ecdysis behavior in insects. This study examines the interactions among these three peptides in the regulation of the ecdysis sequence. Using intracellular recordings, it has been found that ETH is a potent activator of the EH neurons, causing spontaneous action potential firing, broadening of the action potential and an increase in spike peak amplitude. In turn, electrical stimulation of the EH neurons or bath application of EH to desheathed ganglia results in the elevation of cyclic GMP (cGMP) levels within the Cell 27/704 group (which contains CCAP). This cGMP production increases the excitability of these neurons, thereby facilitating CCAP release and the generation of the ecdysis motor program. Extracellular recordings from isolated nervous systems show that EH has no effect on nervous systems with an intact sheath. In desheathed preparations, in contrast, EH causes only the ecdysis motor output. The latency from EH application to ecdysis is longer than that after CCAP application, but shorter than that when ETH is applied to a whole central nervous system. These data support a model in which ETH causes pre-ecdysis behavior and at higher concentrations stimulates the EH neurons. EH release then facilitates the onset of ecdysis by enhancing the excitability of the CCAP neurons (Gammie, 1999).

Ecdysis, or molting behavior, in insects requires the sequential action of high levels of ecdysteroids, which induce accumulation of ecdysis-triggering hormone (ETH) in Inka cells, followed by low levels of ecdysteroids, permissive for the onset of the behavior. High ecdysteroid levels suppress the onset of the behavioral sequence by inhibiting the development of competence to secrete ETH. In pharate pupae of Manduca sexta, Inka cells in the epitracheal glands normally develop competence to secrete ETH in response to eclosion hormone (EH) 8 h before pupation. Injection of 20-hydroxyecdysone (20E) into precompetent insects prevents this acquisition of competence, but does not affect EH-evoked accumulation of the second messenger cyclic GMP. Precompetent glands acquire competence in vitro after overnight culture, and this can be prevented by the inclusion of 20E at concentrations greater than 0.1 microg ml(-1)in the culture medium. Actinomycin D completely inhibits the acquisition of competence, demonstrating that it is dependent on transcriptional events. Cultured epitracheal glands become refractory to the inhibitory effects of 20E in the acquisition of competence at least 3 h earlier than for Actinomycin D, indicating that 20E acts on an early step in a sequence of nuclear events leading to transcription of a structural gene. These findings suggest that declining ecdysteroid levels permit a late event in transcription, the product of which is downstream of EH receptor activation and cyclic GMP accumulation in the cascade leading to ETH secretion (Kingan, 2000).

Initiation of the ecdysis behavioral sequence in insects requires activation of the central nervous system (CNS) by pre-ecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which are released from the Inka cells of the epitracheal glands. The developmental events preceding larval and pupal ecdysis of Manduca sexta involve a dual action of ecdysteroids on the epitracheal glands and CNS. The low steroid levels in freshly ecdysed and feeding larvae are associated with small-sized epitracheal glands, reduced peptide production in Inka cells and insensitivity of the CNS to ETH. The elevated ecdysteroid levels before each ecdysis led to a dramatic enlargement of Inka cells and increased production of peptide hormones and their precursors. As blood ecdysteroids reach peak levels, the CNS becomes responsive to Inka cell peptides. These effects of natural ecdysteroid pulses can be experimentally induced by injection of 20-hydroxyecdysone or the ecdysteroid agonist tebufenozide (RH-5992) into ecdysed larvae, thus stimulating peptide production in Inka cells and inducing CNS sensitivity to ETH. A direct steroid action on the CNS is demonstrated by subsequent treatment of isolated nerve cords from ecdysed larvae with 20-hydroxyecdysone and ETH, which results in pre-ecdysis or ecdysis bursts. These data show that ecdysteroid-induced transcriptional activity in both the epitracheal glands and the CNS are necessary events for the initiation of the ecdysis behavioral sequence (Zitnanova, 2001).

Inka cells of the epitracheal endocrine system produce peptide hormones involved in the regulation of insect ecdysis. In the silkworm Bombyx mori, injection of Inka cell extract into pharate larvae, pupae or adults activates the ecdysis behavioral sequence. In the present study, the identification is reported of three peptides in these extracts, pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP); these are encoded by the same cDNA precursor. Strong immunoreactivity associated with each peptide in Inka cells prior to ecdysis disappears during each ecdysis, indicating complete release of these peptides. Injection of either PETH or ETH alone is sufficient to elicit the entire ecdysis behavioral sequence through the direct action on abdominal ganglia; cephalic and thoracic ganglia are not required for the transition from pre-ecdysis to ecdysis behavior. In vitro data provide evidence that these peptides control the entire ecdysis behavioral sequence through activation of specific circuits in the nervous system. Ecdysis of intact larvae is associated with the central release of eclosion hormone (EH) and elevation of cyclic 3',5'-guanosine monophosphate (cGMP) in the ventral nerve cord. However, injection of ETH into isolated abdomens induces cGMP elevation and ecdysis behavior without a detectable release of EH, suggesting that an additional central factor(s) may be involved in the activation of this process. These findings provide the first detailed account of the natural and hormonally induced behavioral sequence preceding larval, pupal and adult ecdyses of B. mori and highlight significant differences in the neuro-endocrine activation of pre-ecdysis and ecdysis behaviors compared with the related moth, Manduca sexta (Zitnan, 2002).

The sequential behaviors shown by insects at ecdysis are due to the sequential release of various hormones, but the transition from one phase to the next can be fine-tuned by inhibitory influences. The ecdysis sequence in the moth Manduca sexta was initiated by injecting sensitive animals with the neuropeptide ecdysis-triggering hormone (ETH). Exposure to ETH stimulates the release of eclosion hormone (EH) which, in turn, activates a set of neurons containing crustacean cardioactive peptide (CCAP) by elevating their levels of intracellular cyclic GMP. This study characterizes a set of non-CCAP containing neurons that also appear to be EH targets because of their response to cyclic GMP at ecdysis. The neurons did not display leucokinin-, diuretic-hormone- or FMRFamide-like immunoreactivity. They are probably bursicon-containing cells. After release of EH, there is a transient inhibition of the abdominal centers responsible for ecdysis. Transection experiments suggest that this suppression is via descending inhibitory units from the s and thoracic ganglia. The duration of this inhibition appears to depend on the levels of cyclic GMP and can be extended by pharmacologically suppressing cyclic GMP breakdown. Brief exposure to CO₂ causes premature ecdysis. Since the CO₂ treatment is effective only after EH release, it probably acts by suppressing descending inhibition. Studies on adult eclosion suggest that CO₂, given at the appropriate time, can uncouple the basic larval motor program from modulatory influences provided by the adult pterothoracic ganglion. CO₂ therefore appears to be a novel and non-invasive tool for studies of ecdysis behavior in insects (Fuse, 2002).

Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, Inka cells were found in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviors in pharate larvae of Bombyx, indicating functional similarity of these peptides. Several PETH-immunoreactive peptides were isolated from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and the gene encoding two putative ETHs were identified in the mosquito Anopheles gambiae. Inka cells also stain with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, enzyme immunoassays show that these antisera cross-react with PETH and ETH. These results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioral sequence (Zitnan, 2003).

Central peptidergic ensembles associated with organization of an innate behavior

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 L_3,4 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 L_3,4 to induce preecdysis I through central release of DHs and kinins. At the same time, ETH acts on unidentified neurons (possibly expressing ETHR-B) to activate preecdysis II and specific ETHR-A neurons in the entire CNS to activate the ecdysis network. This ETHR-A network includes EH-producing VM neurons and IN704 in the AG1–7, which produce CCAP and MIPs. Activation of the ecdysis network is indicated by EH-mediated cGMP elevation in abdominal neurons ~30 min after onset of preecdysis I. However, descending inhibitory neurons (possibly expressing ETHR-B) in the subesophageal ganglia and TG1–3 suppress the release of CCAP and MIPs from IN704 and delay ecdysis onset. The switch from preecdysis to ecdysis is mediated by central release of CCAP and MIPs from IN704. CCAP apparently plays an excitatory role in this process and controls ecdysis execution, whereas MIPs appear to inhibit activities of other neurons not involved in ecdysis. Finally, bursicon and CCAP are released from cells 27 (NS27) to control postecdysis behaviors: cuticle expansion, hardening, and tanning. Physiological roles of other identified ETHR-A neurons expressing various neuropeptides (e.g., NPF, small NPF/MIPs, and calcitonin-like DH/MIPs) are not clear, and their functional analysis is in progress (Kim, 2006b).

It is well established that peptidergic inputs modulate central pattern generators. In the case of the crustacean stomatogastric ganglion, peptides alter the synaptic strength and intrinsic properties of neurons within the ganglion, resulting in different versions of pyloric and gastric mill rhythms. Similar regulatory mechanisms may operate for the ecdysis sequence, whereby sequential release of neuropeptides reconfigures the activity of multiple central pattern generators in succession (Kim, 2006b).

In this study, it was discovered that numerous central peptidergic ensembles express ETHRs and, consequently, are likely primary targets of ETH. Peptides produced by these ensembles are prime candidates for downstream signals in the recruitment of each centrally patterned phase of the ecdysis sequence. Possible roles of two specific peptidergic ensembles have been shown: a neuropeptide mixture consisting of DHs and kinins produced by abdominal L_3,4 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).

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).

REFERENCES

Search PubMed for articles about Drosophila Ecdysis triggering hormone

Arakane, Y., Li, B., Muthukrishnan, S., Beeman, R. W., Kramer, K. J. and Park, Y. (2008). Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum. Mech. Dev. 125(11-12): 984-95. PubMed Citation: 18835439

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Kim, Y. J., Zitnan, D., Cho, K. H., Schooley, D. A., Mizoguchi, A. and Adams, M. E. (2006b). Central peptidergic ensembles associated with organization of an innate behavior. Proc. Natl. Acad. Sci. 103(38): 14211-6. 16968777

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Knapp E, Sun J. (2017). Steroid signaling in mature follicles is important for Drosophila ovulation. Proc Natl Acad Sci 114:699-704. PubMed ID: 28439025

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Park, Y., Zitnan, D., Gill, S. S. and Adams, M. E. (1999). Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster. FEBS Lett. 463: 133-138. 10601653

Park, Y., Filippov, V., Gill, S. S. and Adams, M. E. (2002a). Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency. Development 129: 493-503. 11807040

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