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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). We show here that 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., (© 2013 Published by Elsevier Inc.)

Ecdysis, the shedding of cuticle at the end of each life stage, is critical to the postembryonic development of insects. The endocrine regulation of ecdysis has been highlighted by the recent description of the epitracheal endocrine system in the tobacco hornworm Manduca sexta, which produces ecdysis-triggering hormone (Mas-ETH). This peptide hormone initiates pre-ecdysis and ecdysis through a direct action on the central nervous system. Here we show that ETH-immunoreactivity and ecdysis-triggering activity in epitracheal glands of the silkworm Bombyx mori are attributable to a 23 amino acid peptide, Bom-ETH. The complete amino acid sequence of Bom-ETH is SNEAFDEDVMGYVIKSNKNIPRM-NH2. Synthetic Bom-ETH was prepared and shown to be chemically and biologically identical to the native substance. Injection of Bom-ETH leads to pre-ecdysis and ecdysis in B. mori pharate larvae and pupae as well as comparable stages of M. sexta. Exposure of the isolated nervous system to Bom-ETH triggers pre-ecdysis and ecdysis burst patterns corresponding to the natural behavior. Bom-ETH belongs to an extended family of multifunctional neurohormones and hormones found in arthropods and molluscs.

Antibodies generated against bombyxin and prothoracicotropic hormone (PTTH) of Bombyx mori and allatotropin, allatostatin, and diuretic hormone (DH) of Manduca sexta react with distinct sets of cells in the central nervous system of Drosophila larvae, pupae, and adults. Brain neurons immunoreactive with antibodies to bombyxin, PTTH, and DH are in strikingly similar positions to their lepidopteran counterparts, indicating that at least some Drosophila neuroendocrine cells are homologous to those of lepidopterans. Allatotropin and allatostatin-immunopositive neurons of Drosophila differ from those of lepidopterans, but many of them are identical with neurons that express the FMRFamide gene. Antibodies to bombyxin, PTTH, allatostatin, and DH also stain axons and axon terminals in the neurohemal part of the ring gland, and all tested antibodies except that against bombyxin show positive reaction in the neurohemal area of the ventral ganglion. Although immunoreactivity with all antibodies is variable during development, the highest levels of staining are found at developmental stages when the neuropeptides would be expected to be functioning. A genetic analysis of neuropeptide expression and function has been initiated by analyzing immunoreactivity in mutants that have prolonged larval life associated with imaginal disc overgrowth. Two of these mutants, dlg and dco, show abnormally strong immunoreactivity for allatotropin during the extended larval period and the former also show increased staining with the PTTH antibody. The reduced ecdysteroid titer and delayed or blocked metamorphosis in the mutants may be a result of altered neuropeptide production, which is probably secondary to the imaginal disc overgrowth.