Ecdysis Triggering Hormone Receptor Research Articles

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, 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. Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

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.

Molecular identification of the first insect ecdysis triggering hormone receptors

The Drosophila Genome Project website ( www.flybase.org) contains an annotated gene sequence (CG5911), coding for a G protein-coupled receptor. We cloned the cDNA corresponding to this sequence and found that the gene has not been correctly predicted. The corrected gene CG5911 has five introns and six exons (1–6). Alternative splicing yields two cDNAs called A (containing exons 1–5) and B (containing exons 1–4, 6). We expressed these splicing variants in Chinese hamster ovary cells and found that the corrected CG5911-A and -B cDNAs coded for two different G protein-coupled receptors that could be activated by low concentrations of Drosophila ecdysis triggering hormones-1 and -2. Ecdysis (cuticle shedding) is an important behaviour, allowing growth and metamorphosis in insects and other arthropods. Our paper is the first report on the molecular identification of ecdysis triggering hormone receptors from insects.