Figure 1.
A) Schematic diagram of the tracking system. From bottom to top: 1. IR emitter; 2. Microhole filter; 3. well plate where nematodes are held with 20 µl CeMM + FuDR 40 µM + AB 1x, 20 µl silicon oil (optional to avoid medium evaporation), tape; 4. Phototransistor. The light intensity output is converted to digital format and acquired by a microcontroller system. B) Whole-life recording of locomotor activity in wild-type (N2) and mutant clk-1(qm30) (slow moving strain) nematodes. The graph shows average activity of 48 nematodes. FuDR prevented the birth of larvae throughout the experiment (see inset picture).
Figure 2.
A) Phase histogram of nematodes entrained to a light-dark cycle (12 h∶12 h 400 lux, 18°C), before and after a 6 h shift of the photoperiod. The middle panel shows the circular statistical analysis (Rayleigh test) for both situations (LD1: lights on at 0900 h; lights off at 2100 h; p<0.05, Rayleigh’s test, n = 80; LD2: lights on at 1500 h, lights off at 0300 h, p<0.05, Rayleigh’s test, n = 81) (the dotted circle inside the plot represents the significance threshold (p = 0.05), to compare with the resultant vector amplitude). The actogram on the right corresponds to average activity of the population. Gray shaded areas represent dark phases of the photoperiod. B) Average daily activity plot corresponding to 3 days of data from 20 nematodes synchronized to a 12 h∶12 h light-dark cycle. The analysis of 3 days of average activity indicates a significant difference between diurnal and nocturnal values (Maximum value = 1.35±0.10 (10 h); Minimum value = 0.68±0.09 (20 h); ANOVA test: p<0.0001). c) Comparison of locomotor activity acrophases between LD and the initial days under constant dark (DD) conditions, each triangle corresponds to the mean acrophase (cosinor analysis) of individual nematodes, calculated with the last 3 days of the pre-entrained condition (X axis) vs. the mean acrophase of the 3 first days of the constant dark condition (Y axis). Inset: Rayleigh test showing the phase angle of the population in entrainment (dark circles; r = 0.3 phi = 1.9 (7.3 h) n = 24) and the beginning of the constant condition post entrainment (white circles) (Rayleigh test: r = 0.3 phi = 2.0 (7.6 h) n = 24).
Figure 3.
A) Acrophase histogram of nematodes entrained to a high (17°C) - low (16°C) temperature cycle, before and after a 6 h shift of the thermal cycle. The dark-white bar corresponds to cycles of low and high temperature, respectively. The middle panel shows the Rayleigh analysis for both situations: Tt1(17°C from 0900 h to 2100 h, 16°C from 2100 h to 0900 h; p<0.05 n = 32) and Tt2(17°C from 1500 h to 0300 h, 16°C from 0300 h to 1500 h; p<0.05 n = 38). The right actogram corresponds to average activity of the population. Gray shaded areas represent the phase of high temperature of the daily cycle. B) Average daily activity plot corresponding to 3 days of data from 20 nematodes synchronized to a 17∶16°C. There was a significant variation between activity values during the high and low temperature phases (Maximum value = 1.28±0.13 (1100 h) ; Minimum value = 0.78±0.07 (2200 h), p<0.03, ANOVA). C) Average daily activity plot corresponding to 3 days of data from 20 nematodes synchronized to a 20∶16°C cycle. There was a significant variation between activity values during the high and low temperature phases (Maximum value = 1.55±0.12 (1400 h), Minimum value = 0.37±0.06 (2200 h); p<0.0001, ANOVA). D) Comparison of locomotor activity acrophases between Tt (20°C∶16°C) and the initial days after release into constant darkness (DD) and constant temperature conditions (16°C). Inset: Rayleigh analysis of locomotor activity acrophase under Tt and tt cycles (Tt (black circles): r = 0.7 phi = −2.4 (14.8 h) n = 35; tt (white circles): r = 0.5 phi = −2.9 (12.9 h) n = 31).
Figure 4.
Locomotor activity rhythms in C. elegans under constant conditions.
A) Examples of individual nematodes exhibiting different circadian periods. Actograms are plotted in 24 h-days (top panel) and replotted in “tau” days (i.e., the length of each cycle corresponds to the circadian period of each animal, see middle panel). The lower panel shows average circadian activity for two cycles. B) Distribution of circadian periods in wild-type C. elegans under different temperature conditions.
Figure 5.
Representative actograms and frequency distribution of circadian periods in C. elegans.
a) lin-42(mg152) mutants (n = 26). b) lin-42(n1089) mutants (n = 51). c) Vels26 mutants (which rescue the lin-42(mg152) mutation) (n = 36). d) Average circadian periods for control and mutant strains (*p<0.05, **p<0.02 versus N2 controls).