Representative locomotor activity determined by mean distance travelled ± SEM of Amphiprion ocellaris in 11–13 dph old larvae (n = 15–23), ca. 100 dph old juveniles (n = 9–18) and several years old adults (n = 4). Bluish-grey background refers to lights off. The mean locomotor activity of two consecutive days (larvae and juveniles) in m/6 min or two independent days (adults) in m/1 min are shown, dependent on zeitgeber time (h). Movement of larvae and juveniles was tracked continuously and the distance covered was summed up per 6 min. Adults were tracked manually half-hourly for one minute.

Mean normalized expression of six clock genes (orange traces): bmal1, clocka, cry1b, per1b, per2, per3 and three genes of the photoreactivation DNA repair mechanism (blue traces): cpd photolyase, cry dash, 6-4-photolyase of Amphiprion ocellaris cells, larvae and juveniles. Expression was quantified using quantitative real time PCR at zeitgeber time (h) 3, 9, 15 and 21. Lights were switched off at zeitgeber time 12, indicated by bluish-grey background. Per timepoint, three samples for larvae and juveniles and two samples for cells were analysed. The cycling expression pattern of clock genes as well as genes of the photoreactivation repair mechanism was similar between larvae and juveniles. The expression pattern of the period genes and cry1b, as well as the photolyase genes in the EAO cell line resembled those in the larvae and juveniles. However, expression of clocka and bmal1 showed a non-cycling pattern in the EAO cells compared with the high amplitude rhythmic expression observed in the animal samples.

Luciferase assay showing rhythmic bioluminescence of Amphiprion ocellaris EAO (orange) and zebrafish PAC-2 cells (blue) transfected with D-box_Cry1a-Luc (above) or zfPer1b-Luc (below) constructs. A negative control of non-transfected cells is shown in grey. For each condition, the mean of eight independently transfected wells and standard deviation is shown. Cells were exposed to a 12:12 h LD cycle, indicated by bluish-grey background and black stripes. Both cell lines, transfected with zfPer1b-Luc showed a comparable robust daily rhythm, even under DD. Cells transfected with D-box_Cry1a-Luc were only rhythmic during the LD cycle.