Ecdysis triggering hormone signaling in arthropods.

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling.

Corazonin receptor signaling in ecdysis initiation.

Corazonin is a highly conserved neuropeptide hormone of wide-spread occurrence in insects yet is associated with no universally recognized function. After discovery of the corazonin receptor in Drosophila, we identified its ortholog in the moth, Manduca sexta, as a prelude to physiological studies. The corazonin receptor cDNA in M. sexta encodes a protein of 436 amino acids with seven putative transmembrane domains and shares common ancestry with its Drosophila counterpart. The receptor exhibits high sensitivity and selectivity for corazonin when expressed in Xenopus oocytes (EC(50) approximately 200 pM) or Chinese hamster ovary cells (EC(50) approximately 75 pM). Northern blot analysis locates the receptor in peripheral endocrine Inka cells, the source of preecdysis- and ecdysis-triggering hormones. Injection of corazonin into pharate larvae elicits release of these peptides from Inka cells, which induce precocious preecdysis and ecdysis behaviors. In vitro exposure of isolated Inka cells to corazonin (25-100 pM) induces preecdysis- and ecdysis-triggering hormone secretion. Using corazonin receptor as a biosensor, we show that corazonin concentrations in the hemolymph 20 min before natural preecdysis onset range from 20 to 80 pM and then decline over the next 30-40 min. These findings support the role of corazonin signaling in initiation of the ecdysis behavioral sequence. We propose a model for peptide-mediated interactions between Inka cells and the CNS underlying this process in insect development.

Molecular cloning and function of ecdysis-triggering hormones in the silkworm Bombyx mori.

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 behavioural sequence. In the present study, we report the identification of three peptides in these extracts, pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP), which 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 behavioural sequence through the direct action on abdominal ganglia; cephalic and thoracic ganglia are not required for the transition from pre-ecdysis to ecdysis behaviour. Our in vitro data provide evidence that these peptides control the entire ecdysis behavioural 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 behaviour without a detectable release of EH, suggesting that an additional central factor(s) may be involved in the activation of this process. Our findings provide the first detailed account of the natural and hormonally induced behavioural sequence preceding larval, pupal and adult ecdyses of B. mori and highlight significant differences in the neuro-endocrine activation of pre-ecdysis and ecdysis behaviours compared with the related moth, Manduca sexta.