Activity monitors

All locomotor activity experiments were conducted with 2–5 days-old male flies at 25°C using Trikinetics Activity Monitors as previously described [9 (link)]. We crossed Gal4 lines to w¹¹¹⁸ to create control progeny. Locomotor activities were monitored for 6 days under different photoperiodic conditions, and then for 9 days under constant dark (DD). To analyze rhythmicity under constant conditions, we normalized the activity of flies from DD day 3 to day 9 and used χ2-periodogram analysis with a 95% confidence cut-off, as well as SNR analysis [68 (link)]. Arrhythmic flies were defined by a power value ≤ 10 and width value ≤ 2, and period outside the range, 18 to 30 hours. To analyze periods, we used Graphpad Instat (v. 8) software to run one-way ANOVA measures followed by the Tukey-Kramer Multiple Comparisons Test. We used Clocklab (Actimetrics) software to produce actograms and the Brandeis Rhythms Package [69 (link)] to produce average activity plots (group eductions). To analyze onset and offset phases of Morning and Evening activity bouts on the final two days of light:dark entrainment (LD 5&6), we followed the method of Kula-Eversole et al. [57 (link)]: [(A_n+2 + A_n+1)–(A_n-1 + A_n-2)  =  ΔActivity]. Experimental genotypes were tested in the han mutant background, pdfr⁵³⁰⁴[11 (link)], or in the pdf⁰¹ (null) mutant background [1 (link)], or in the w¹¹¹⁸ background, as noted.

10 day old males with D42-GAL4 activated RNAi against CG9172 and CG5389 or crossed with white¹¹¹⁸ laboratory strain were used for behavioral analysis. Flies were monitored with a 2 μW/cm² intensity green LED light for 7 days at 12:12 light:dark cycle, followed by 7 days of 24 hrs dark cycle in Trikinetics activity monitors (Waltham, MA). Flies were maintained at 22°C, 60% relative humidity. 32 male flies were used per condition, and activity monitor tubes contained a plug of fly food at one end. Experiments using the activity monitor were conducted in duplicate. Data were analyzed using the ShinyR-DAM program [23 (link)] and Microsoft Excel.

For behavioral experiments, adult male flies (2–4 days old) were used for testing locomotor activity rhythms. Flies were entrained for 4 full days LD cycle at 25°C, using about 500 lux light intensities, and then released into DD at 25°C for at least 6 days. Locomotor activity was measured with TriKinetics Activity Monitors in I36-LL Percival Incubators. Locomotor activity was averaged over the 4 days entrainment for LD and 6 days for DD. Analyses of period and power were carried out using FaasX software as previously described [48 (link)]. Actograms were generated using a signal-processing toolbox implemented in MATLAB (MathWorks) [49 (link)]. For GAL80^ts experiments, flies were raised at 18°C and tested at 30°C. They were entrained for 5 days and then released in DD for at least 6 days. Morning anticipation was calculated by the ratio of activity counts between 2 hrs before light on and 6 hrs before light on. We first measured the single fly activity counts obtained in twelve 30-min bins between Zeitgeber Time (ZT) 17.5 and ZT24 (6 hr before lights on) and six 30-min bins between ZT22.5 and ZT24 (2 hrs before lights on). The first value is divided by the second to obtain the morning activity index. Morning anticipations of individual flies were then averaged and plotted on the graphs.