Circadian systems, II. The oscillation in the individual Drosophila pupa; its independence of developmental stage.

The circadian rhythmicity of adult emergences in a population of Drosophila pupae has been the object of considerable study.1-16 The facts that the experimental system is a population and that the act of emergence occurs only once in the life of the individual have been responsible for some confusion in the literature.8-10 Harker9 appears to deny the existence of a circadian oscillation in the individual: "the eclosion rhythm is a population effect," and "the final eclosion rhythm is dependent on whatever processes control the developmental rate throughout the pupal stage" (p. 336). This paper demonstrates the reality of that oscillation without recourse to the use of population rhythmicity. It treats the circadian oscillation as a gating oscillation fully independent of rate and stage of morphogenesis and differentiation;1' 17 it gates the act of emergence to a restricted phase of the oscillation; and since the oscillation itself locks on to the light cycle in nature, it therefore gates adult emergence to a limited time of day. Populations of pupae which manifest a rhythm of emergence activity consist of individuals which are not synchronous developmentally, but are fully synchronous in their circadian oscillations. While such populations have been the principal, most useful, tool for studying the (individual's) oscillation which underlies and causes the (population's) rhythm, they are by no means necessary for that task. Populations of developmentally synchronous pupae can be created by selecting newly formed puparia within a short time interval. These populations do not, of course, display a rhythm of emergence; their eclosion activity is compressed into a single peak (or, at most, two). They do, however, lend themselves well to very explicit demonstrations of the presence of a circadian oscillation throughout pupal life and its control of the eventual time of emergence; and in representing, as they do, an individual pupa merely replicated several hundred times, they minimize the potential conceptual difficulties of utilizing a population to analyze the properties of an individual. Materials and Methods.--All of the experiments were conducted with Drosophila pseudoobscura (PU stock 301). About ten pairs of parents were placed in vials (25 X 95 mm) which contained 1 in. of MAluller's fly food, and the females were allowed to deposit eggs for a 24-hour period. The parents were then transferred to fresh food vials; for each experiment 2 or 3 transfers were made. The cultures were maintained at 20 i 10C and in constant light. About 11 days after egg laying, mature 3rd-instar larvae crawl up out of the food onto the sides of the vials. They form puparia which are white at first but SOOn tan. Just after puparium formation the prepupae can be removed very easily with a spatula, without injury, from the walls of the vials. In our experiments prepupae (before tanning of the cuticle begins) were so removed during a