Significance of activity peaks in fruit flies, Drosophila melanogaster, under seminatural conditions

Abstract

Studies on circadian entrainment have traditionally been performed under controlled laboratory conditions. Although these studies have served the purpose of providing a broad framework for our understanding of regulation of rhythmic behaviors under cyclic conditions, they do not reveal how organisms keep time in nature. Although a few recent studies have attempted to address this, it is not yet clear which environmental factors regulate rhythmic behaviors in nature and how. Here, we report the results of our studies aimed at examining (i) whether and how changes in natural light affect activity/rest rhythm and (ii) what the functional significance of this rhythmic behavior might be. We found that wild-type strains of fruit flies, Drosophila melanogaster, display morning (M), afternoon (A), and evening (E) peaks of activity under seminatural conditions (SN), whereas under constant darkness in otherwise SN, they exhibited M and E peaks, and under constant light in SN, only the E peak occurred. Unlike the A peak, which requires exposure to bright light in the afternoon, light information is dispensable for the M and E peaks. Visual examination of behaviors suggests that the M peak is associated with courtship-related locomotor activity and the A peak is due to an artifact of the experimental protocol and largely circadian clock independent.

Keywords: afternoon peak; chronoethogram; circadian rhythms; courtship; period mutants.

Fig. 1.

Activity profiles of CS under SN and various light-modified protocols under SN. (A, Top Left) Activity profile of CS under SN averaged across days and flies. Error bars are SEM. Three separate axes on extreme right represent three environmental factors measured: light intensity (L) (in lux), temperature (T) (in degrees Celsius), and relative humidity (H) (in percentage). The black arrowhead on the x axis indicates dawn (>0 lux). (A, Top and Middle) Average activity profiles when naturally varying light intensity was reduced by 50% (SN₅₀), 75% (SN₇₅), and 90% (SN₉₀). Only the amplitude of light waveform was reduced without alteration in qualitative profile of light. (A, Middle) Average activity profile under DD in otherwise SN (DD+SN). (A, Middle and Bottom) Average activity profiles under light-blocking protocols: morning cover (MC), afternoon cover (AC), evening cover (EC), and morning-plus-evening cover (MEC). The shaded horizontal bars below x axis in middle and bottom rows depict durations of light blocking under SN. (B) Phases of the M [F_(4,147) = 5.88, P < 0.0002] and E peaks [F_(4,143) = 23.03, P < 0.0002] were modulated by light-filtering protocols. The M peak was phase advanced compared with SN (P < 0.05) for the three partial light-filtering protocols, whereas the E-peak phases did not differ from each other or from SN. (C) Light-blocking protocols also modulated phase of the M [F_(4,133) = 2.75, P = 0.03], A [F_(4,129) = 5.95, P < 0.001], and E peaks [F_(5,153) = 147.89, P < 0.0001]. In B and C, error bars are 95% confidence interval (95% CI) to enable visual hypothesis testing.

Fig. 2.

A peak is an artifact of experimental paradigm. (A, Left) Schematic of experimental setup. (A, Middle) Average activity profiles of flies recorded in flatter version of DAM (DAM5) monitor, with (filled circles) or without shade (unfilled circles) in the middle. Error bars are SEM. Other details are same as Fig. 1A. (A, Right) Activity in the afternoon interval was greater in the shaded compared with unshaded tubes [F_(1,29) = 6.02, P < 0.02]. Error bars are 95% CI. (B) Visual observation of flies during daytime in the DAM2 monitor. (B, Left) Flies preferred the middle zone of the tube in the afternoon more than other times of the day [F_(11,120) = 19.5, P < 0.001, proportion of flies in the middle zone at 12 and 13 h are significantly greater than at 7–11, 17, and 18 h]. (B, Right) Visual observation of locomotion in the tubes placed in DAM2 monitor showed two peaks of locomotion [F_(11,60) = 16.17, P < 0.001]. Error bars are SEM. (C) Average activity recorded in the same DAM2 monitor showed the A peak. Other details are same as Fig. 1A. (D) Proportion of solitary flies in petri dishes exhibiting locomotion as estimated by visual observation. No detectable A peak was observed, but the M and E peaks persisted [F_(11,24) = 2.73, P < 0.03].

Fig. 3.

Examining the role of clock in regulating activity peaks under SN. (A, Left to Right) Activity profiles of CS flies under different constant light intensities, 10 (LL₁₀+SN), 100 (LL₁₀₀+SN), and 1000-lux (LL₁₀₀₀+SN) in otherwise SN conditions assayed in June 2012. (B) Average activity profiles of period mutants (per⁰, per^S, and per^L) and their genetic background controls (w¹¹¹⁸ for per⁰, and CS for per^S and per^L) under SN assayed in February 2013. Other details for A and B are the same as in Fig. 1A. (C) The A-peak phase was not different across genotypes [F_(4,87) = 1.32, P = 0.2], but the E peak was delayed in per^L compared with its control (P < 0.001). (D) Average activity profiles of per mutants and controls under DD+SN. (E) Afternoon activity level was greater in per⁰ compared with w¹¹¹⁸ (P < 0.001), although per^S and per^L did not differ from CS. Error bars in C and E represent 95% CI.

Fig. 4.

Chronoethograms of flies under SN. (Top) Profile of proportion of flies performing locomotion [F_(11,48) = 6.96, P < 0.001; Left] or resting [F_(11,48) = 9.68, P < 0.001; Right] in petri dishes under group condition of three males and three females. (Bottom) Profile of proportion of flies performing courtship-related activities such as wing expansion [F_(11,48) = 4.37, P < 0.001], chasing [F_(11,48) = 4.00, P < 0.001], and copulation [F_(11,48) = 1.21, P = 0.3]. Courtship-related behaviors peak during the morning hours. Significant effect of time was seen for all behaviors except copulation.