Circadian control of prothoracicotropic hormone release in an adult insect and the induction of its rhythmicity by light cues.

The insect neuropeptide prothoracicotropic hormone (PTTH) is a critical regulator of larval development. We recently demonstrated that PTTH is also present in adult Rhodnius prolixus and is released by adult brains in vitro with a clear daily rhythm during egg development. Here, we employ a well-established in vitro bioassay, to show that the daily rhythm of PTTH release by brains in vitro is under circadian control since it persists in aperiodic conditions with a free running period of around 24h that is temperature compensated. Prolonged exposure (3weeks) of insects to continuous constant light (LL) completely eliminated PTTH release. Subsequent transfer of such insects from LL to constant darkness (DD) rapidly induced rhythmic PTTH release, indicating that the circadian rhythm of PTTH release is induced by photic cues. Western analysis identified PTTH in the adult hemolymph, suggesting that PTTH acts as a functional neurohormone in the adult insect. Dot blot analysis revealed that PTTH levels in the hemolymph also cycled with a daily rhythm that persisted in DD and was synchronous with the rhythm of PTTH release by brains in vitro. We conclude that the previously documented photosensitive clock in the brain regulates rhythmic PTTH release and thus generates the rhythm seen in the hemolymph. These results emphasize the importance of rhythmic PTTH release in the adult insect and support a role for PTTH in adult physiology and possibly within the adult circadian system.

Circadian regulation of hemolymph and ovarian ecdysteroids during egg development in the insect Rhodnius prolixus (Hemiptera).

Ecdysteroids, classically studied as the molting hormones of insects, occur at lower levels in the adult stage, but their source and significance in adult physiology is debated. In Rhodnius prolixus, a blood meal initiates a cycle of egg development and ecdysteroids appeared in the hemolymph within 1-2h of feeding. Systematic changes in hemolymph ecdysteroid titer then occurred during egg development. There was a clear circadian rhythm in the hemolymph ecdysteroid titer throughout egg development. This is the first report of an ecdysteroid rhythm in any adult female insect. Of various tissues examined in vitro, only ovaries released ecdysteroids. The amounts released were adequate to account for observed hemolymph ecdysteroid titers. Therefore, ovaries are the primary, perhaps sole, source of hemolymph ecdysteroids. Ovaries in vitro showed a circadian rhythm of changes in ecdysteroid content and release that was in synchrony with the hemolymph ecdysteroid rhythm, indicating that ovarian ecdysteroid release drives the rhythm seen in the hemolymph. Potential regulation of ovarian ecdysteroids is discussed in relation to the known rhythms in prothoracicotropic hormone and insulin-like peptides that occur during egg development. The prospect of circadian regulation of egg development itself by hormones and/or an ovarian clock is also discussed.

Rhythmic release of prothoracicotropic hormone from the brain of an adult insect during egg development.

Prothoracicotropic hormone (PTTH) is a brain neurohormone that has been studied for over 80 years. The only known target of PTTH is the prothoracic glands (PGs) of larvae, which synthesize the insect molting hormones (ecdysteroids) and a massive literature exists on this axis. The PGs degenerate around the time of adult emergence, yet presence of PTTH has been reported in the brains of several adult insects. Using an in vitro bioassay system, we confirm that PTTH is present in the adult female brain of Rhodnius prolixus. The material is electrophoretically, immunologically and biologically indistinguishable from larval PTTH. The amount of PTTH in the brain shows a daily rhythm during egg development. We show that brains in vitro release PTTH with a daily rhythm over this period of time. PTTH is released at each scotophase. This is the first report that PTTH is released from the adult brain and functions as a hormone, inviting explanation of its function. Larval PTTH is also known to be released with a daily rhythm, and the clock in the brain controls both larval and adult rhythms. The potential significance of rhythmic PTTH release in female adults is discussed in relation to the regulation of ecdysteroids, egg development and the concept of internal temporal order.