Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus)

The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D: 12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10: 00 h); (2) fish fed with a 12-h shifted schedule (at 22: 00 h), (3) fish fed at 10: 00 h throughout the experiment, except the last day when fed at 22: 00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, gPer1a, gPer2a, gPer3, and gCry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock. (Author correspondence: eisornaa@bio.ucm.es)