Fig 1.
Daily expression of clock genes in central tissues from turbot.
mRNA relative expression profiles of per2, cry1, clock and per1 in hypothalamus (A) and eye (B) of fish acclimatized to LD and regular feeding time at ZT2 (LD, black line), 24h in DD and regular feeding time at CT2 (DD, green line) and 24h DD and random feeding time during the light phase (R DD, red line) measured by qPCR. Data represent mean ± SEM of three (eye) or five animals per time point. The dotted black lines delimit the duration of the night in LD conditions or the duration of the subjective night in DD. Different letters of the same colour indicate statistical difference inside a group (p<0.05 One-way ANOVA followed by Tukey’s test). Asterisks indicate a statistical difference from LD conditions (*p<0.05, **p<0.01, ***p<0.001 Two-way ANOVA followed by Bonferroni’s test). Hash indicates statistical difference from regular feeding time (#p<0.05, ##p <0.01, ###p<0.001 Two-way ANOVA followed by Bonferroni’s test). These time points correspond to ZT 3, 6, 9, 15, 18, 21, 27h. ZT0 represents lights on and ZT12, lights off.
Table 1.
Rhythmic parameters of clock gene expression.
Fig 2.
Acrophase distribution of per2, cry1, clock and per1 from turbot.
Fish kept under schedule feeding time and LD (black points) or DD (green square) and under free-running conditions (red triangle). Only the acrophases of the genes that showed significant rhythms as confirmed by cosinor analysis were represented. The cry1 acrophase was shifted under DD in muscle, meanwhile the per1 acrophase shifted under free-running conditions in gut and under DD and schedule feeding and free running conditions in liver. The dashed line on the Y axis shows the boundary between the light phase and the dark phase. ZT0 is defined as the time when lights are switched on.
Fig 3.
Daily expression of clock genes in peripheral tissues from turbot.
mRNA relative expression profiles of per2, cry1, clock and per1 in gut (A), liver (B) and muscle (C) of fish adapted to LD and regular feeding time at ZT2 (LD, black line), 24h DD and regular feeding time at CT2 (DD, green line) and 24h in DD and random feeding time during the light phase (R DD, red line) measured by qPCR. Data represent mean ± SEM of five animals per time point. The dot black lines delimit the duration of the night in LD or the duration of the subjective night in DD. Plots and statistical analysis results are indicated as described for Fig 1.
Fig 4.
Diurnal locomotor activity of turbot in LD-DD-LD lighting conditions.
A, D) Representative actograms of turbot swimming activity. Recording activity during 33 days is represented by double plot of two consecutive days. The white and black bars above the actograms indicate light and dark periods. The arrows above the white bar indicate the time where food is delivered. Fish were kept in LD and fed at ZT2 for 33 days (A), or randomly fed during the light phase (D). From day 12 to 14 fish were in DD and then returned to LD. Fishes showed daily periodicity in LD (B, E), but not in DD (C, D). Turbot displayed its activity mainly during the light phase.
Fig 5.
Preprandial locomotor activity in LD (top), DD (mid) and LL (bottom). Activity levels during the photophase (0–12) and scotophase (12–24) of a 24-h cycle from turbot. Within the photophase, activity levels were analyzed 2h before lunch time (0–2) and during the entire light phase (0–12) either in the group with a schedule feeding time at ZT2 or in the group randomly fed, in LD (top), DD (mid) and LL (bottom). Bar graphs show mean ± SEM. Significant differences between groups are indicated by letters. Scheduled feeding significantly stimulates increases in locomotor activity before feeding time under LD and LL but not under DD conditions (Kruskal-Wallis one way anova: H = 73.73, df 3, p<0.05 and post hoc by the Dunn’s test p<0.001). No changes in locomotor activity were observed upon the onset of light period compared with the whole light phase in randomly fed animals.
Fig 6.
Diurnal locomotor activity from turbot in LD-LL-LD lighting conditions.
Recording activity during 18 days is represented by double plot of two consecutive days and represented by actograms (A, D). The white and black bars above the actograms indicate light and dark periods. The arrows above the white bar indicate the time where food is delivered. Fish were kept in LD and fed at ZT2 for 8 days (A), or randomly fed during the light phase (D). From day 9 to 12 fish were in constant light (LL) and then returned to LD. Fish showed daily rhythmicity in activity under LD conditions (B, E), but they were arrhythmic in LL (C, F). Turbot displayed activity during the light phase in LD.
Fig 7.
Diurnal locomotor activity from turbot under short photoperiod (SP).
Recording activity during 8 days is represented by double plot of two consecutive days and represented by actograms. The white and black bars above the actograms indicate light and dark periods. The arrows above the white bar indicate the time where food is delivered. Fish were kept in SP and fed at the end of the scotophase (ZT23) (left), or randomly fed during the light phase (right). Fish showed rhythmic activity in SP.
Fig 8.
Feeding behaviour in fish under four different lighting regimes.
One group of animals were entrained to eat at ZT2 under LD, at CT2 under DD or LL, and at ZT23 under short photoperiod (left) and a second group was fed at random times (right) during the scotophase or subjective scotophase. Bars represent activity levels: 2h before the arrival of food (0–2) under LD, DD or LL and 1h before the arrival of food under short photoperiod; when meal is provided (2–3) under LD, DD or LL and (24) during short photoperiod; during the rest of the day (3–12) under LD or subjective day under DD or LL, and during the entire light phase (0–8) and during the entire dark phase (9–24) under short photoperiod. Turbot failed to show food searching behavior when food was supplied regularly in the dark phase. Instead animals were very active when food was presented, independently of the light regime. (Kruskal-Wallis one way anova: H = 18.92 LD; H = 6.16 DD; H = 8.11 LL; H = 25.42, df 4, p<0.0001 and post hoc by the Dunn’s test p<0.05).
Fig 9.
Summary outline from behavior experiments of the inputs regulation (scheduled feeding and light) of the outputs (Locomotor activity (LA) and FAA) in turbot juveniles under laboratory conditions.
The rectangle represents the light phase (white) and the dark phase (black) within a period of 24h.