SUMMARY 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, we found Inka cells 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 behaviours in pharate larvae of Bombyx , indicating functional similarity of these peptides. We isolated several PETH-immunoreactive peptides from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and identified the gene encoding two putative ETHs in the mosquito Anopheles gambiae . Inka cells also are stained with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, our enzyme immunoassays show that these antisera cross-react with PETH and ETH. Our 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 behavioural sequence.
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). Here, we report that 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.