Shedding light on the circadian clock of the threespine stickleback

Abstract

The circadian clock is an internal timekeeping system shared by most organisms, and knowledge about its functional importance and evolution in natural environments is still needed. Here, we investigated the circadian clock of wild-caught threespine sticklebacks (Gasterosteus aculeatus) at the behavioural and molecular levels. Although their behaviour, ecology and evolution are well studied, information on their circadian rhythms are scarce. We quantified the daily locomotor activity rhythm under a light:dark cycle (LD) and under constant darkness (DD). Under LD, all fish exhibited significant daily rhythmicity, while under DD, only 18% of individuals remained rhythmic. This interindividual variation suggests that the circadian clock controls activity only in certain individuals. Moreover, under LD, some fish were almost exclusively nocturnal, while others were active around the clock. Furthermore, the most nocturnal fish were also the least active. These results suggest that light masks activity (i.e. suppresses activity without entraining the internal clock) more strongly in some individuals than others. Finally, we quantified the expression of five clock genes in the brain of sticklebacks under DD using qPCR. We did not detect circadian rhythmicity, which could indicate either that the clock molecular oscillator is highly light-dependent, or that there was an oscillation but that we were unable to detect it. Overall, our study suggests that a strong circadian control on behavioural rhythms may not necessarily be advantageous in a natural population of sticklebacks and that the daily phase of activity varies greatly between individuals because of a differential masking effect of light.

Keywords: Circadian rhythms; Clock genes; Interindividual variation; Locomotor activity; Natural population; Threespine stickleback.

Fig. 1.

Experimental design. Group 1 was used to quantify locomotor activity under a 12 h:12 h light:dark cycle (LD, lights on at 06:00 and lights off at 18:00) and under constant darkness (DD). To that end, group 1 was exposed to LD for 8 days, then to DD for 10 days. Groups 2 and 3 were used to quantify locomotor activity under LD and brain gene expression under DD. Groups 2 and 3 were thus exposed to LD for 8 days, then to DD for 1 day (day 9). The day following the switch to DD (day 10), we sampled the brain of four randomly selected individuals every 6 h throughout a 24-h cycle (06:00, 12:00, 18:00, 00:00).

Fig. 2.

Under LD, sticklebacks display significant daily rhythmicity, but show variable activity patterns. Double-plotted actograms (A–C) of three representative individuals under a 12 h:12 h light:dark cycle (LD) and their corresponding activity profile (D–F) and χ² periodogram (G–I). The white and black bars at the top of the actograms and the activity profiles represent the light and dark phases, respectively. From left to right, respectively, individuals display 77%, 65% and 55% of their daily activity during the dark phase. Activity profiles show the average locomotor activity (number of movements) for each 10-min bin over the 8 days in LD. Q_p values on the χ² periodograms quantify the rhythmic component of the activity and the red horizontal line indicates the significance threshold (set at P=0.05).

Fig. 3.

Under DD, most individuals are arrhythmic and only a few individuals show circadian rhythmicity. Double-plotted actograms (A,B) of two representative individuals under constant darkness (DD) and their corresponding activity profile (C,D) and χ² periodogram (E,F). The grey and black bars at the top of the actograms and activity profiles represent the subjective light and dark phases, respectively. Under DD, most sticklebacks do not display circadian rhythmicity, as represented by the individual on the left of the figure. On the right, we show one of the three individuals that exhibited significant circadian rhythmicity. Activity profiles show the average locomotor activity (number of movements) for each 10-min bin over the 10 days. Q_p values on the χ² periodograms quantify the rhythmic component of the activity and the red horizontal line indicates the significance threshold (set at P=0.05).

Fig. 4.

Under LD, sticklebacks are mostly nocturnal, but show large interindividual variation in the phase of activity and in the total daily activity. The most nocturnal fish are also the least active. (A) Average night activity of each individual under a 12 h:12 h light:dark cycle (LD) for 8 days. Night activity corresponds to the percentage of the total daily activity displayed during the dark phase. Error bars represent the standard error of the mean. (B) Average total daily activity (movements per day) of each individual under LD for 8 days. (C) Correlation between the night activity (%) and the total daily activity (movements per day) under LD (average for 8 days). Note that axes do not start at zero. Pearson's correlation test, r=−0.3, P=0.04, n=48.

Fig. 5.

The expression of clock genes does not significantly vary during a 24-h period in the brain of sticklebacks in DD. Time-of-day-dependent relative expression of (A) bmal1a, (B) clock1b, (C) clock2, (D) per1b and (E) cry1b measured by qPCR in the brain of sticklebacks after 1 day in constant darkness (DD). The letter ‘a’ denotes the absence of significant difference between time points for the five genes (one-way ANOVA, P>0.05). The black line in the middle of each boxplot indicates the median and each dot represents an individual. Sample size is shown for each time point.