Proceedings of the National Academy of Sciences Vol. 68, No. 3, pp. 595-599, March 1971 Hour-Glass Behavior of the Circadian Clock Controlling Eclosion of the Silkmoth Antheraea pernyi JAMES W. TRUMAN The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Communicated by Carroll M. Williams, December 18, 1970 ABSTRACT The emergence of the Pernyi silkmoth thereby established (3). The phase assumed by the new from the pupal exuviae is dictated by a brain-centered, rhythm proves to be a function of the time at which the flash photosensitive clock. In continuous darkness the clock displays a persistent free-running rhythm. In photoperiod is administered during the free-running cycle. The experimen- regimens the interaction of the clock with the daily light- tally determined function permits one to plot a "phase-re- dark cycle produces a characteristic time of eclosion. sponse curve." For our present purposes, the phase-response But, in the majority of regimens (from 23L:ID to 4L:20D), relationship is of interest because it has been used success- the eclosion clock undergoes a discontinuous "hour- fully by Pittendrigh to document the operation .of the "eclo- glass" behavior. Thus, during each daily cycle, the onset of darkness initiates a free-running cycle of the clock. sion clock" throughout the days preceding eclosion (5). The next "lights-on" interrupts this cycle and the clock In the case of Drosophila, exposure to continuous light comes to a stop late in the photophase. The moment causes a dissolution of the eclosion rhythm over the course of when the Pernyi clock stops signals the release of an a few cycles. Pittendrigh demonstrated that this "damping" eclosion-stimulating hormone and is demonstrated to be a function of the time when the free-running cycle is effect of light occurs, not because the clock uncouples from the interrupted by lights-on. Moreover, the width (duration) overt rhythm, but because the clock stops (5)! Moreover, this of the eclosion peak in a photoperiod is shown to be de- effect occurs after a surprisingly short exposure to light. As a pendent upon the length of the dark phase, and, conse- result, the Drosophila eclosion clock stops during the final part quently, upon the amount of the free-running cycle that that a 12 is completed. This relationship demonstrates that the of each photophase has duration of or more hours (5). free-running cycle may be divided into two parts. The After the eclosion clock stops, it can be promply restarted attainment of maximal accuracy (and thus the narrowest by placing Drosophila in darkness. The resultant free-running eclosion peak) is dependent upon the completion of only rhythm then routinely shows a characteristic phase relation- the first 2 hr of the free-running cycle. The completion of ship to the "lights off" signal (5). It is also important to note succeeding portions of the cycle, while having an effect upon the time of eclosion, no longer affects the accuracy that, after the onset of the free-running rhythm, the first of the clock. A mechanistic model of the eclosion clock is cycle traversed by the clock is identical to that observed in presented. succeeding cycles (5). Thus, in continuous darkness the free- running cycle or free-running behavior of the eclosion clock is an invariant that becomes established immediately after dark- appears to be among The capacity to "keep-time" ubiquitous ness restarts the clock. organisms (1). Among the most striking examples of this As in other rhythmic phenomena the timing of Drosophila are circadian which temporal organization rhythms-events eclosion varies with the photoperiod (3, 4). A central problem in the absence of external cues occur at intervals of approxi- is the role of the "circadian" clock in this timing. As men- 24 The emergence ecdysis) of the fruit mately hr. (eclosion, tioned above, the Drosophila eclosion clock stops during pseudoobscura) from the puparium is one such fly (Drosophila longer than 12 hr and is subsequently restarted event that has received considerable attention during the past photophases in a free-running manner at lights-off. Near the end of the 15 years (2-5). Eclosion in this case terminates an intricate next photophase the clock again stops-and so on at daily in- series of developmental processes that transform the maggot tervals. Under this kind of discontinuous motion the timing into the fly. Although the fly may complete development at mechanism resembles not so much a clock as an "hour-glass" any time of the day, there is only a certain period during which it may emerge. Thus, eclosion is said to be "gated" (5). (5). In the case regimens having less than Studies of the role of photoperiod in the gating of eclosion of daily photoperiod 12 hr the Drosophila clock appears as a continuous have pinpointed several important properties of the control- of light, oscillation and a new cycle is started immediately upon the ling clock. Thus, when populations of flies are transformed termination of the preceding one. Thus, the situation is sim- from a photoperiod into continuous darkness, they display a photo- persistent "free-running" rhythm of eclosion. When this free- ilar to that seen in continuous darkness except that the period forces the clock into an exact 24 hr periodicity (4). The running population is exposed to a flash of light, the time of physiological mechanism for this entrainment is unknown. eclosion is either advanced or delayed and a new rhythm is. The analysis of the Drosophila eclosion rhythm by Pitten- drigh and his coworkers has provided a substantial basis for Abbreviation: #L: #D, a light-dark regimen that alternates the further studies of the mechanism of the eclosion clock. How- specified number of hours of light with the specified hours of ever, because of its small size, Drosophila is an unsatisfactory darkness. animal for investigating the physiological and biochemical as- 595 Downloaded by guest on September 28, 2021 596 Zoology: J. W. Truman Proc. Nat. Acad. Sci. USA 68 (1971) A then transferred to their respective photoperiod regimens at 260C. The methods of recording eclosion have been described (6). K////-///I * I Adult eclosion of saturniid moths is triggered by a neuro- secretory hormone released from the brain; the release in the case of the Pernyi moth takes place about 1.5 hr before eclo- sion (10). Since hormonal release, rather than eclosion per se, is the event controlled by the clock, the average time of re- lease in each photoperiod is assumed to occur 1.5 hr prior to the corresponding average eclosion time. RESULTS The Relationship of Eclosion to Photoperiod. Chilled Pernyi pupae were removed from their cocoons and placed at 260C under a 17L: 7D regimen. After the initiation of adult develop- ment, the pharate moths were placed in one of eight photo- period regimens, which ranged from 1L: 23D to 23L: ID. In all cases the developing moths were exposed to at least 10 cycles of the photoperiod prior to their eclosion. The moths usually emerged over a 4- to 5-day period. The cumulative data for each photoperiod is summarized in Fig. 1. In all of these regimens, the time of eclosion, and thus the timing of eclosion-hormone release, was characteristic of the particular photoperiod. Moreover, in most regimens, the majority of the moths emerged within a specific 3.5-hr period G of the day. Finally, we may note that the eclosion of males preceded that of females by about 1 hr. For reasons discussed *0 z z' zi I muI~ m j1.0/Z..Z I Pi below, the 1L:23D regimen disobeyed this generalization. The effects of continuous light or darkness were assessed on I I flulin * m pharate moths exposed to at least 10 cycles of a particular photoperiod. When moths were transferred from the 17L: 7D I regimen into continuous light, eclosion became arrhythmic N.- I 0 =I a E after one transient cycle (Fig. 2C). By contrast, pharate 0 6 12 Is 24 TIME (HOURS AFTER LIGHTS-OFF) moths transferred to continuous darkness developed a dis- tinct free-running rhythm that showed a period of 22 hr. As FIG. 1. The ecdysis of Pernyi moths under various photo- with Drosophila, identical rhythms were established when period conditions: A., first day of continuous darkness after moths were placed in darkness from photoperiods having prior exposure to a 12L:12D regimen; B., 1L:23D; C., 4L:20D; 8L: 12L: 22L:2D; moderate to long photophases (12L: 12D and 17L: 7D, respec- D., 16D; E., 12D; F., 17L:7D; G., 20L:4D; H., 2A first I., 23L: 1D. The white bar gives the estimated range of eclosion tively; Fig. and B). During the free-running cycle, hormone release for each regimen. The filled circles are the hormone release by the males and females occurred 21 and 22 average release times. The release time for males and for females hr, respectively, after the rhythm was started by lights-off. are presented separately in the iL: 23D regimen (see Discussion). DISCUSSION AND INTERPRETATION The hatched and white areas refer to dark and light, respectively. Termination of the clock cycle under photoperiod conditions pects of eclosion. By contrast, the giant silkmoths of the In photoperiods that have a day-length of 12 hr or more, the family Saturniidae have proven to be well suited for such behavior of the Drosophila eclosion clock is discontinuous in studies. Indeed, the photoreceptor, the clock, and a neuro- that the clock stops at some point late in the photophase. endocrine trigger necessary for eclosion have been localized to Pittendrigh demonstrated that in the 12L:12D regimen.