Shedding light on the threespine stickleback circadian clock

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. While 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 either indicate 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.

Methods
GENERAL INFORMATION

Project title: Circadian activity and gene expression in the threespine stickleback

Authors: Marie-Pier Brochu and Nadia Aubin-Horth

Date of data collection: 26/09/2019 - 09/06/2020

Location of data collection: Université Laval, Québec, Canada

PROJECT DESCRIPTION AND METHODOLOGICAL INFORMATION

Using threespine sticklebacks (Gasterosteus aculeatus) caught in the wild population of the lac Témiscouata (Québec, Canada) in June 2019, we first quantified daily locomotor activity in individual fish under a 12 h light:12 h dark cycle (LD, lights on at 6:00 and lights off at 18:00) during 8 days and under constant darkness (DD) during 10 days. To do this, we individually placed 18 fish in experimental 2 L tanks. Each tank was equipped with an infrared photoelectric sensor (E3Z-D67, Omron). Every time a fish interrupted the infrared light beam, it was counted as one movement. We quantified the locomotor activity of 54 individuals under LD (groups 1, 2 and 3) and of 18 individuals under DD (group 1). Second, we quantified the expression rhythm of five clock genes (bmal1a, clock1b, clock2, per1b and cry1b) in the brain of sticklebacks under DD using qPCR (groups 2 and 3). To do this, we sampled the brain of four randomly selected individuals every 6 h throughout a 24-h cycle (6:00, 12:00, 18:00, 0:00), n=8/ time point). Please refer to the following publication for more information on the experimental design, methods and data analysis:

Brochu, M.-P. and Aubin-Horth N. (2021). Shedding Light on the Circadian Clock of the Threespine Stickleback. J. Exp. Biol.

MISSING DATA

For all files, 6 individuals are missing because they died during experiments or were parasitized.

Group 1: ind. 10.

Group 2: ind. 02 and 11.

Group 3: ind. 05, 14 and 15.

FILES DESCRIPTION

Filename: group_1_2_3_sex.csv

Description: sex identification

Variable list:

1. Group ID

2. Individual ID

3. Sex

Filenames: group_1_LD_activity.csv; group_2_LD_activity.csv; group_3_LD_activity.csv; group_1_DD_activity.csv

Description: number of movements per 10-min interval during 8 days (LD) or 10 days (DD)

Variable list:

1. Interval (1 to 144)

2. Day

3. Hour

4. Minute (from the start of the interval)

5. Ind01

6. ...

Filename: group_2_3_gene_expression.csv

Description: mean CQ values for the five clock genes studied and the reference gene (ubc)

Variable list:

1. Group ID

2. Individual ID

3. Time of brain collection (0:00, 6:00, 12:00 or 18:00)

4. Mean CQ values for ubc

5. Mean CQ values for period1b

6. Mean CQ values for cry1b

7. Mean CQ values for bmal1a

8. Mean CQ values for clock1b

9. Mean CQ values for clock2