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

BACKGROUND: 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. RESULTS: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) 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. CONCLUSIONS: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.     

Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. Here, we found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. Here, we establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, our data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates.     

Structure-activity relationship of ETH during ecdysis in the tobacco hornworm, Manduca sexta

In insects, ecdysis or shedding of the old cuticle, consists of a series of behaviors that are regulated by the coordinated actions of a number of neuropeptides, one of which is ecdysis triggering hormone (ETH). ETH acts directly on central pattern generators of the abdominal ganglia to trigger onset of pre-ecdysis behaviors, as well as indirectly to activate release of eclosion hormone, thereby inducing onset of ecdysis behaviors through a cGMP-mediated mechanism. We assessed the minimal C-terminal amino acids required for biological activity of ETH, by assessing: (i) onset of pre-ecdysis and ecdysis behaviors in vivo, after injection of peptide analogs, (ii) onset of fictive pre-ecdysis and ecdysis motor patterns in vitro, as recorded extracellularly, after incubation of the CNS with the peptide analogs, and (iii) accumulation of cGMP within cells of the abdominal ganglia, as assessed immunohistochemically. Amidation of ETH at the C-terminus was required to elicit a biological response in vivo and in vitro, as well as an accumulation of cGMP within the CNS. The five amino acid amidated C-terminus of ETH (NIPRMamide) was the minimal moiety able to induce a robust pre-ecdysis response in vivo and in vitro, while a seven amino acid core (NKNIPRMa) was required for induction of ecdysis, including accumulation of cGMP immunoreactivity within the CNS. Analogs smaller than 12 amino acids in length were only active at very high concentrations in vivo, suggesting that smaller fragments might be susceptible to hemolymph degradation. Some alanine substitutions or removal of internal amino acids altered the activity of ETH, as well as the time of onset of ecdysis behaviors, suggesting that internal amino acids play a role in maintaining proper folding of the peptide for successful binding or activity at the ETH receptor.     

Endocrine modulation of primary chemosensory neurons regulates Drosophila courtship behavior

The decision to engage in courtship depends on external cues from potential mates and internal cues related to maturation, health, and experience. Hormones allow for coordinated conveyance of such information to peripheral tissues. Here, we show Ecdysis-Triggering Hormone (ETH) is critical for courtship inhibition after completion of copulation in Drosophila melanogaster. ETH deficiency relieves post-copulation courtship inhibition (PCCI) and increases male-male courtship. ETH appears to modulate perception and attractiveness of potential mates by direct action on primary chemosensory neurons. Knockdown of ETH receptor (ETHR) expression in GR32A-expressing neurons leads to reduced ligand sensitivity and elevated male-male courtship. We find OR67D also is critical for normal levels of PCCI after mating. ETHR knockdown in OR67D-expressing neurons or GR32A-expressing neurons relieves PCCI. Finally, ETHR silencing in the corpus allatum (CA), the sole source of juvenile hormone, also relieves PCCI; treatment with the juvenile hormone analog methoprene partially restores normal post-mating behavior. We find that ETH, a stress-sensitive reproductive hormone, appears to coordinate multiple sensory modalities to guide Drosophila male courtship behaviors, especially after mating.     

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.     

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger 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. Here we use the recently introduced “Trojan exon” technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that 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. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here 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.     

Role of the neuropeptide CCAP in Drosophila cardiac function

The heartbeat of adult Drosophila melanogaster displays two cardiac phases, the anterograde and retrograde beat, which occur in cyclic alternation. Previous work demonstrated that the abdominal heart becomes segmentally innervated during metamorphosis by peripheral neurons that express crustacean cardioactive peptide (CCAP). CCAP has a cardioacceleratory effect when it is applied in vitro. The role of CCAP in adult cardiac function was studied in intact adult flies using targeted cell ablation and RNA interference (RNAi). Optical detection of heart activity showed that targeted ablation of CCAP neurons selectively altered the anterograde beat, without apparently altering the cyclic cardiac reversal. Normal development of the abdominal heart and of the remainder of cardiac innervation in flies lacking CCAP neurons was confirmed by immunocytochemistry. Thus, in addition to its important role in ecdysis behavior (the behavior used by insects to shed the remains of the old cuticle at the end of the molt), CCAP may control the level of activity of the anterograde cardiac pacemaker in the adult fly. Expression of double stranded CCAP RNA in the CCAP neurons (targeted CCAP RNAi) caused a significant reduction in CCAP expression. However, this reduction was not sufficient to compromise CCAP's function in ecdysis behavior and heartbeat regulation.     

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, we performed systemic RNA interference (RNAi) experiments 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.     

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.     

A single pair of interneurons controls motor neuron activity during pre-ecdysis compression behavior in larval Manduca sexta

Manduca sexta molts several times as a larva (caterpillar) before becoming a pupa and then an adult moth. Each molt culminates in ecdysis behavior, during which the old cuticle is shed. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which includes rhythmic, synchronous compressions of the abdomen. A previous study indicated that motor neuron activity during pre-ecdysis compression behavior is driven by an ascending neural pathway from the terminal abdominal ganglion. The present study describes a pair of interneurons, designated IN-402, that are located in the terminal ganglion and belong to the ascending pathway. Each IN-402 is synchronously active with pre-ecdysis compression motor bursts, and bilaterally excites compression motor neurons throughout the abdominal nerve cord via apparently monosynaptic connections. The pair of IN-402s appears to be the sole source of rhythmic synaptic drive to the motor neurons during the pre-ecdysis compression motor pattern. These interneurons play a key role in the production of larval pre-ecdysis behavior, and are candidates for contributing to the developmental weakening of pre-ecdysis behavior at pupation.Abbreviations A3, A4... abdominal ganglion 3, abdominal ganglion 4... - AT terminal abdominal ganglion - DN _A anterior branch of the dorsal nerve - EH eclosion hormone - EPSP excitatory postsynaptic potential     

Circadian regulation of the lark RNA‐binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA‐binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)‐containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA‐binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly‐emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA‐binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 14–29, 2000     

Developmental attenuation of Manduca pre-ecdysis behavior involves neural changes upstream of motoneurons and relay interneurons

Each molt in the hawkmoth, Manduca sexta, culminates in the shedding of the old cuticle at ecdysis. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which includes rhythmic, synchronous compressions in all abdominal segments. Prior to ecdysis to the pupal stage, pre-ecdysis behavior and its underlying motor pattern are markedly attenuated. A single pair of interneurons located in the terminal abdominal ganglion, the IN-402s, drives compression motoneuron activity during the pre-ecdysis motor pattern via monosynaptic excitatory connections. The present study tested the hypotheses that (1) changes in intrinsic properties (resting membrane potential, spike threshold, input resistance and excitability) of compression motoneurons, or (2) changes in the strength of synaptic connections from IN-402s to compression motoneurons, underlie the developmental attenuation of the pre-ecdysis motor pattern. Membrane potential was slightly more hyperpolarized in prepupal as compared to larval motoneurons, but no other findings supported the tested hypotheses. These results suggest that developmental weakening of the pre-ecdysis motor pattern results from changes upstream of the compression motoneurons and their synaptic connections from IN-402s. Accepted: 29 September 1999     

Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence.

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, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. 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.     

Organization of the larval pre-ecdysis motor pattern in the tobacco hornworm,Manduca sexta

The tobacco hornworm, Manduca sexta, undergoes several larval molts before transforming into a pupa and then an adult moth. Each molt culminates in ecdysis, when the old cuticle is shed. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which consists of rhythmic compressions that are synchronous along the abdomen and on both body sides, and rhythmic retractions of the abdominal prolegs. Both pre-ecdysis and ecdysis behaviors are triggered by a peptide, eclosion hormone. The aim of the present study was to investigate the neural circuitry underlying larval preecdysis behavior. The pre-ecdysis motor pattern was recorded in isolated nerve cords from eclosion hormone-treated larvae, and the effects of connective transections and ionic manipulations were tested. Our results suggest that the larval pre-ecdysis compression motor pattern is coordinated and maintained by interneurons in the terminal abdominal ganglion that ascend the nerve cord without chemical synaptic relays; these interneurons make bilateral, probably monosynaptic, excitatory connections with identified pre-ecdysis motor neurons throughout the abdominal nerve cord. This model of the organization of the larval pre-ecdysis motor pattern should facilitate identification of the relevant interneurons, allowing future investigation of the neural basis of the developmental weakening of the pre-ecdysis motor pattern that accompanies the larval-pupal transformation.Abbreviations A3, A4... abdominal ganglia 3, 4... - AT terminal abdominal ganglion - ALE anterior lateral external muscle - DN dorsal nerve - DN_A anterior branch of the dorsal nerve - DN_L lateral branch of the dorsal nerve - DN_P posterior branch of the dorsal nerve - EH eclosion hormone - TP tergopleural muscle - VN ventral nerve - VN_A anterior branch of the ventral nerve - VN_L lateral branch of the ventral nerve - VN_P posterior branch of the ventral nerve     

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.     

Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency.

At the end of each developmental stage, insects perform a stereotypic behavioral sequence leading to ecdysis of the old cuticle. While ecdysis-triggering hormone (ETH) is sufficient to trigger this sequence, it has remained unclear whether it is required. We show 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 DmETH1 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.     

Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the moth,Manduca sexta

The eclosion of the adult Manduca sexta moth is followed by a wave of cell death that eliminates up to 50% of the neurons of the central nervous system within the first few days of imaginal life. While the identity of some of the dying motoneurons has been established, that of most doomed neurons is unknown. Here, we show that the dying cells include peptidergic neurons involved in the control of ecdysis behavior. These cells belong to a small population of 50 neurons that express crustacean cardioactive peptide (CCAP), a potent regulator of the ecdysis motor program, and show increases in cyclic 3′,5′-guanosine monophosphate at each ecdysis. First, we describe new markers for these neurons and show that they are expressed in these CCAP-immunoreactive neurons in a complex temporal pattern during development. We then show that these neurons die within 36 h after adult eclosion, the last performance of ecdysis behavior in the life of the animal, via the active, genetically determined process of programmed cell death. The death of these neurons supports the hypothesis that outmoded or unused neurons are actively eliminated. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 265–280, 1998     

Developmental attenuation of the pre-ecdysis motor pattern in the tobacco hornworm,Manduca sexta

Summary At the culmination of each molt, the larval tobacco hornworm exhibits a pre-ecdysis behavior prior to shedding its old cuticle at ecdysis. Both pre-ecdysis and ecdysis behaviors are triggered by the peptide, eclosion hormone (EH). Pre-ecdysis behavior consists of rhythmic abdominal compressions that loosen the old larval cuticle. This behavior is robust at larval molts, but at the larval-pupal molt the only comparable behavior consists of rhythmic dorso-ventral flexions of the anterior body. These flexions appear to be an attenuated version of the larval pre-ecdysis behavior because (1) they show the same EH dependence, and (2) the motor patterns recorded from EH treated, deafferented larval and pupal preparations are similar except that the pupal pattern is much weaker. Both patterns are characterized by rhythmic, synaptically-driven bursts of action potentials in motoneurons MN-2 and MN-3, which occur synchronously in all segments. However, the synaptic drive to the motoneurons and their resultant levels of activity are reduced during the pupal pre-ecdysis motor pattern, especially in posterior abdominal segments. Although the dendritic arbors of both motoneurons regress somewhat during the larval-pupal transformation, this does not appear to be the primary source of diminished synaptic drive because regression is greatest in the segments in which synaptic inputs remain the strongest. The developmental weakening of the pre-ecdysis motor pattern thus may be due to changes at the interneuronal level.Abbreviations A2, A3... abdominal segments 2, 3, etc. - ALE anterior lateral external muscle - day L3 third day of the 5th larval instar - day P0 the day of pupal ecdysis - DN _a anterior branch of the dorsal nerve - EH eclosion hormone - HPLC high performance liquid chromatography - TP tergopleural muscle     

The role of eclosion hormone in the larval ecdyses of Manduca sexta

Each larval moult in Manduca sexta consists of an identical series of developmental and behavioural events leading up to ecdysis. Injections of eclosion hormone into staged larvae in any instar resulted in the premature elicitation of the larval pre-ecdysis behaviour, comprising a rhythmic sequence of muscle contractions, followed by the larval ecdysis behaviour.A marked depletion of eclosion hormone stores form the ventral chain of ganglia coincided with each larval ecdysis and in the moult to the fifth instar, eclosion hormone activity appeared in the blood at the onset of the pre-ecdysis behaviour.Responsiveness to eclosion hormone for pre-ecdysis and ecdysis behaviour developed about 12 and 6 hr before normal ecdysis, respectively. Elicitation of ecdysis behaviour by exogenous hormone inhibited both subsequent behavioural responses to eclosion hormone and endogenous hormonal release.In conclusion, the behavioural programme involved in each larval ecdysis appears to be controlled by the eclosion hormone.     

Functions of duplicated genes encoding CCAP receptors in the red flour beetle, Tribolium castaneum

Crustacean cardioactive peptide (CCAP) is a nonapeptide originally isolated from the shore crab, Carcinus maenas, based on its cardioacceleratory activity. This peptide is highly conserved in insects and other arthropods. In insects CCAP also has an essential role in ecdysis behavior. We previously identified two homologous genes, ccapr-1 and ccapr-2, encoding putative CCAP receptors in the red flour beetle, Tribolium castaneum. In contrast, some insects, including Drosophila melanogaster, carry only one gene encoding a CCAP receptor. Phylogenetic analysis of putative CCAP receptor orthologs reveals a number of independent gene duplications in several insect lineages. In this study, we confirmed that CCAP activates both putative T. castaneum receptors in a heterologous expression system. RNA interference (RNAi) of ccapr-1 and ccapr-2 revealed that ccapr-2 is essential for eclosion behavior in T. castaneum, while RNAi for ccapr-1 did not result in any abnormal phenotype. In vivo cardioacceleratory activity of exogenously applied CCAP was abolished by RNAi of ccapr-2, but not by that of ccapr-1. Thus, only ccapr-2 mediates the cardioacceleratory function, ccapr-1 having apparently lost both functions for eclosion behavior and for cardioacceleration since the recent gene duplication event.