Eggs were collected for 24 hours and allowed to develop for up to 7 days (156 ± 12 hours AEL) for anatomy analysis and up to 8 days (180 ± 12 h AEL) for Feret diameter measurement, except for control w1118 larvae that were dissected at 5 days AEL. The ratios area of NE/area of the brain lobe (ratio NE/BL) and area of CB/area of the brain lobe (ratio CB/BL) were calculated by using images acquired with an SP8 Leica confocal image microscope and by measuring the areas corresponding to the NE, the CB, and the brain lobe using ImageJ software. The Feret diameter measurement of the brain lobe pairs was performed by analyzing the images of brains taken using a Leica EC3 camera coupled to a Nikon SMZ800 stereoscope. The images were analyzed by a purpose-made macro (31) written in ImageJ software to measure the maximum brain Feret diameter of the brain lobe pair. Ventral ganglions were digitally masked before measurement. The results were represented in boxplots, and P values were calculated by nonparametric Mann-Whitney U tests using GraphPad Prism 7.00 for MacOS X (GraphPad Software, La Jolla, CA, USA) (www.graphpad.com). All genotypes and crosses were done as described above.

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