(A) Fluorescence confocal image of a pupal CNS wholemount. Neurons that express ETHRA, CCAP, and Bursicon are revealed by intersectional expression of UAS-6XEGFP (green, left) under the control of the CCAP-Gal4DBD∩ETHRA-p65AD hemidriver pair and anti-pBurs immunolabeling (magenta, middle). Merged image (right). Brackets, double-labeled neurons; SEZ, subesophageal zone; VNC, ventral nerve cord. Scale bar: 50 μm. (B) Suppression of ETHRA/CCAP neurons by two copies of UAS-Kir2.1 (bottom) eliminates both Phases II and III. Upper panel, pupal ecdysis behavior of control animals lacking the driver. Bars show the average durations of Phases I and II (±standard deviations, N in parentheses). The end of Phase III was not calculated. (C) Brief activation of ETHRA/CCAP neurons (1 min) using UAS-dTrpA1 terminates Phase I and initiates Phase II (bottom). Upper panel, behavior of control animals lacking the driver and subjected to the same temperature shift.