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The temporal organization of secretion of the prothoracicotropic hormone (PTTH) and ecdysone during larval-pupal development of Samia cynthia ricini was studied by ligations, with particular attention to the circadian control of the timing of hormone release. PTTH and ecdysone are required first for the induction of prodromes of pupation and again later for pupal-cuticle formation. PTTH release in the first step occurs during the second or third photophase after the last-larval ecdysis under a photoperiod of 12 hr light and 12 hr darkness and is thought to be under the control of a circadian clock. Ecdysone release follows 1.5 days later, i.e. during the scotophase that precedes the gut purge. In the second secretory step, PTTH is released 2 days after purging the gut, and ecdysone release follows 6 hr later. The PTTH release at this time occurs at a fixed time after the gut purge irrespective of light conditions, accounting for light insensitivity of the timing for pupal ecdysis. Possible mechanisms relating to the inconsistent association of a circadian clock with PTTH release, and those underlying the determination of timing of the gut purge are discussed.

Under a 12-hr light and 12-hr dark photoperiod, haemolymph ecdysteroid titre of the last(5th)-instar larva of Samia cynthia ricini begins to rise in the early scotophase preceding gut purge, which marks the larval-prepupal transition, to reach a peak titre of 7.6 ng/ml ca. 4.5 hr before gut purge. This profile of ecdysteroid increment is phase-shifted in response to phase shifts of the scotophase immediately before gut purge, in parallel with gut-purge phase shifts, suggesting that ecdysone release is under the control of a circadian clock dictating gut-purge timing. Ecdysone and 20-hydroxyecdysone (10–20 μg), injected no later than 18 hr before the normal gate of gut purge induced well-defined peaks of precocious gut purge ca. 8 hr after injection. Earlier injections caused acceleration of gut purge but the degree of acceleration was unpredictable. These results suggest that the timed surge of ecdysteroids is responsible for the gated occurrence of gut purge and that 18 hr before gut purge larvae acquire the competence to undergo gut purge in a gated fashion provided that they are exposed to a sufficient surge of ecdysteroids.

Observations on the timing of ecdysis and neck ligation experiments on larvae of Samia cynthia ricini under various light-dark conditions show that an endogenous circadian clock controls the timing of larval ecdysis and prothoracicotropic hormone (PTTH) secretion preceding it. The clock, upon reaching a specific phase point, causes the brain to secrete PTTH provided that the brain has acquired the secretory competence. This time may vary, in relation to a previous ecdysis, according to the light-dark conditions by which the clock phase is specifically determined, but is fixed relative to a subsequent ecdysis. Thus, in the case of the ecdysis to the 5th instar, PTTH is secreted [15+nτ] hr (τ: free-running period, slightly less than 24 hr) after the clock has started when the rhythm is free-running, and in the second and third nights of the 4th instar under a photoperiod of 12 hr light and 12 hr dark. Full secretion of ecdysone occurs 6 hr after PTTH secretion and ecdysis ensues 34 hr thereafter to complete the ultimate sequence of ecdysis.

The prothoracicotropic hormone (PTTH) was isolated from adult heads of Bombyx mori . Fifty micrograms of pure PTTH was obtained from 648,000 heads through a 15-step purification procedure with a 2 × 10 6 -fold purification and an 8% recovery. Chemical analyses of this PTTH have shown that it is a single-chain peptide consisting of 40–43 amino acid residues (MW, 4330–4740), the N-terminus of which is glycine. As little as 0.1 ng of PTTH elicited adult development in a debrained pupa of Samia cynthia ricini . Five picograms of PTTH directly stimulated the prothoracic glands in vitro so as to enhance ecdysone release. The hemolymph ecdysteroids of brainless Samia pupae that were developed by PTTH injection increased with essentially the same pattern as in developing normal pupae.