(A–C) Silencing most of the clock network abolishes rhythmic LD behavior. GAL4 control (A) and UAS control (C) show bimodal activity patterns with M anticipation and E anticipation. Silenced flies (B) show no sign of anticipation neither in the morning nor in the evening. Flies show short activity increases at the transitions of day/night and night/day which are considered masking. Values in upper right-hand corner indicate the number of analyzed flies. (D) Behavior of per01 flies in LD 12:12. per01 mutants show behavior similar to clk856>Kir with no M anticipation, reduced E anticipation and short reactions to the light transitions. (E–F) Silencing PDF neurons alters LD behavior. (E) PDF-GS>Kir on Vehicle food does not express Kir. Flies show the typical bimodal activity with M and E anticipation peaks. The M peak is close to lights-on (ZT0) whereas the E peak is close to lights-off (ZT12). (F) Silencing the PDF neurons by adding RU 486 to the food causes an advanced E peak, similar to pdf01 flies. Values indicate the number of analyzed flies. (G) Morning Anticipation (MA) and Evening Anticipation (EA) calculated from A-C. Both controls show values significantly above 0.5 (p<0.0001 for all) indicating prominent anticipation to both peaks. Clk856>Kir flies on the other hand show no signs of anticipation as indicated by anticipation indices indistinguishable from 0.5 (p>0.6937) (H) Morning Anticipation (MA) and Evening Anticipation (EA) calculated from D-E. per01 flies show no MA (p=0.5744), whereas there is a slight increase of activity toward lights-off (p=0.0027). Silencing the PDF neurons did not abolish MA (p=0.0004) and showed a significantly bigger EA compared to per01 flies (p=0.0391). (I-K) PER protein cycling is largely unaffected by neuronal silencing in LD. PER cycling in control brains (black data points ± SEM, pooled GAL4 and UAS, n = 6–8) is highly synchronized with peak levels around ZT0. Silencing the clock network (red data points ± SEM, n = 5–7) had little effect on LD PER rhythms in sLNvs (F(1,49)=0.93, p=0.3391) (I) and LNds (F(1,49)=0.35, p=0.5547) (J). DN1s appear dampened after silencing but 2-way ANOVA shows no difference between control and experimental line (F(1,36)=2.98, p=0.0930) (K). (L-N) PDP1 protein cycling is largely unaffected by neuronal silencing in LD. PDP1 cycling in control brains (black data points ± SEM, pooled GAL4 and UAS, n = 5) is highly synchronized with peak levels around ZT18. Silencing the clock network (red data points ± SEM, n = 5) slightly increased LD PDP1 rhythms in sLNvs (F(1,32)=15.74, p=0.0004) (L) but no effect on LNds (F(1,32)=2.71, p=0.1093) (M) and only a slight effect on DN1s (F(1,32)=6.06, p=0.0194) (N).