Eclosion assays are experimental procedures used to study the process of eclosion in insects, particularly in the model organism drosophila (fruit flies). Eclosion is the process in which an adult insect emerges from its pupal case, or a larval insect hatches from its egg. In holometabolous insects, the circadian clock regulates the timing of adult emergence. The daily rhythm of adult emergence in these insects was among the first circadian rhythms to be investigated. The circadian clock in these insects enforces a daily pattern of emergence by permitting or triggering eclosion during specific time frames and preventing emergence during other periods.
The purpose of an eclosion assay is to count the number of flies that emerge over time from a developing population, which provides information on the circadian clock in the experimentally manipulated drosophila. For example, with an eclosion monitor, scientists can study how knocking out a certain gene changes the behavioral expression of a drosophila's biological clock. Additionally, the circadian rhythm of adult insect emergence was among the earliest chronobiological phenomena to be examined, significantly impacting the field of chronobiology through its contributions to understanding temperature compensation, phase response curves, and reactions to skeleton photoperiods. The eclosion assay serves as a vital tool for researchers delving into chronobiology studies.
Bang box
The bang box is the first experimental assay developed to measure eclosion in fruit flies. The first model of the bang box was developed at a Princeton University laboratory, mainly accredited to Colin Pittendrigh, to measure the time that adult drosophilids emerged from pupae populations in a controlled light and temperature environment. This original model works by securing pupae on plastic boxes that can be temperature controlled. The pupae are harvested and attached to a brass holding plate. The holding plate is then secured to face a brass mounting plate that can be temperature-controlled and then covered with an acrylic glass cover that has a tapered mouth. The tapered mouth is aligned above a vial containing detergent solution. The mounting plate is then placed atop a solenoid activated every 30 minutes. The vials of detergent are placed in a circular tray that is rotated at a rate of one vial per hour. The activation allows the mounting plate to be lifted and dropped against a rubber stopper that shakes out all of the emerging flies from the last 30 minutes into the vial of detergent. Researchers counted the number of flies in each vial to determine the times of day where eclosion activity was highest. Pittendrigh used this model to demonstrate that the circadian clock in drosophila is temperature-compensated (meaning its period is stable over a broad range of temperatures) and to design an early theoretical model for phase response curves.
The bang box was the primary means of investigation in chronobiology in the 1960s and 1970s. The application of this technique includes, but is not limited to: determining the impact of external stimuli on flies' internal biological clock, measuring the circadian rhythm of eclosion, and defining how gene alleles such as period genes contribute to the presence or shifts in the Drosophila circadian rhythm. The bang box allows for visualization of what happens to circadian rhythms when a gene gets knocked out. Using this method, the researchers were able to collect a large data sample on the number of eclosed individuals every fixed time period. Furthermore, the bang box was used by Pittendrigh earlier to conclude that the oscillation phase assay is affected by the phase response curve. Later research builds on this relationship in order to use eclosion assays to study circadian rhythms. For example, the bang box was used to measure eclosion activity in order to determine Clock mutants on the X chromosome of Drosophila that drastically change the period of the traditional 24-hour circadian rhythm.
Modern systems
Some modern eclosion monitors employ infrared counting electronics. In these systems, the pupae are glued to an elevated disk just as in the original bang box, and emerging flies fall into one tube due to gravity. The flies fall through infrared lasers in the base, which contain electronics to record the timing of eclosion. This technique has been used as recently as 2021 to evaluate how the central circadian clock regulates eclosion by coupling to an endocrine pacemaker in the prothoracic gland.