The effects on male sex pheromones suggested that the oenocyte clock may play a role in regulating the reproductive behavior of Drosophila. To investigate this possibility, we utilized a group-mating assay in which six virgin males were housed with six virgin females and allowed to interact continuously over a 24 hr testing period. Under these conditions, individual wild-type females will remate multiple times over a single 24 hr reproductive episode. The temporal distribution and overall number of rematings of oeclock- males was compared to UAS-cycΔ/+ and oe-Gal4/+ heterozygous Veliparib controls when separately grouped with wild-type females. Mating assays were performed under constant conditions on DD1. The

temporal distribution showed that oeclock- males and controls remated at roughly the same frequency for the first 6–8 hr of the 24 hr testing cycle (Figure 7D). Thereafter, the remating frequency for oeclock- males flattened, remaining constant for the rest of the subjective night and continuing into the next day. In contrast, the remating frequency of the UAS-cycΔ/+ and oe-Gal4/+ control males continued to increase before peaking sharply during the

middle-to-late portion of the subjective night (CT 16–22). The mean number of rematings per male for oeclock- was significantly different then that for oe-Gal4/+, but not UAS-cycΔ/+ controls ( Figure 7E), suggesting that differences in the temporal pattern of remating behavior are not dependent on the total number of matings per individual. Thus, the loss of a functioning oenocyte clock resulted in a temporal difference in remating see more behavior, without affecting the total number of matings. The oenocyte clock, as shown above, is necessary for normal sex pheromone Mephenoxalone expression and mating behavior. This raised the question: what role does the modulation of the oenocyte clock and its physiological outputs by PDF signaling play in the regulation of mating behavior? To address this question, we again used the group-mating assay. Here, the temporal

distribution and overall number of rematings of control Canton-S males were compared to that of Pdf01 males. Mating assays were performed in a light/dark cycle (LD 12:12) to more closely simulate the light conditions that flies might typically experience in nature. The temporal distribution showed that Canton-S and Pdf01 males when grouped with Canton-S females mated at roughly the same rate for the first 6–8 hr of the 24 hr testing cycle ( Figure 8A). Thereafter, Pdf01 males sustained a higher frequency of remating than Canton-S during the late night and continued to remate for several hours past dawn (zeitgeber time [ZT] 2–4). Corresponding to this temporal difference, Pdf01 males mated more on average than Canton-S, amounting to >1 additional remating per Pdf01 male (p = 0.0085) relative to Canton-S controls when paired with Canton-S females ( Figure 8C, left).