Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila

Abstract

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period⁰¹ mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period⁰¹ mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling (Pdf⁰¹ and han⁵³⁰⁴ PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling.

Keywords: PDF signaling; adaptive behavior; behavioral rhythms; circadian dominance; clock plasticity; eclosion; natural conditions.

Figure 1.

Eclosion profiles and autocorrelation analysis of WT_CS, per01, and PDF signaling mutants under laboratory conditions. Left half: light entrainment (LD12:12), right half: temperature entrainment (WC12:12). WT_CS flies eclose rhythmically under ZT conditions (a-a′) as well as under DD (a″), yet quickly loose rhythmicity under constant temperature (a‴). In contrast, per01 clock mutants show impaired rhythmicity under all conditions (b-b′: ZT conditions, b″-b‴: constant conditions). Under WC entrainment, however, ultradian rhythmicity appeared with a period of 12 h (b‴). Flies lacking either PDF (c-c‴) or the PDF receptor (han5304, d-d‴) eclose rhythmically during ZT conditions, but increasingly lose rhythmicity during constant conditions (c″-c‴, d’’-d‴). (***= < 0.001), #/# indicates N experiments/n flies. Abbreviations: WT_CS = wild-type Canton S; LD = light:dark; DD = constant darkness; WC = warm:cold; RI = rhythmicity index; LS: Lomb-Scargle; C = cosinor amplitude and zero-amplitude test significance level.

Figure 2.

Autocorrelation analysis of eclosion rhythmicity under quasi-natural conditions in (a) WT_CS, (b) per01, (c) Pdf01, and (d) han5304 flies. The left column (a-d) summarizes the rhythmicity of the individual experiments under quasi-natural conditions (see Suppl. Table 1) and in LD12:12 and WC12:12 entrainment in the laboratory (see Figure 1). Numbers in the columns indicate N. The middle column (a′-d′) shows the mean RI ± SD for the different conditions. A Lomb-Scargle analysis of the data is shown in Supplementary Figure 1. The right column (a″-d″) shows circular plots of the data with mean vector (large black arrow) and sum of eclosed flies per hour over all experiments under quasi-natural conditions (small blue arrows). (e) Winfree’s RI for the different genotypes under quasi-natural conditions. (f) Comparison of the outdoor results obtained by autocorrelation (RI, left columns), Lomb-Scargle (power, middle columns), and cosinor (amplitude, right columns) analyses. While differences in RI between genotypes are not statistically significant, there is a significant difference in the Lomb-Scargle power and cosinor amplitude between per01 and WT_CS flies (p < 0.05). (g) Effect size for parameters that show a significant interaction effect with the genotype. Independent variables scaled to mean; hour is scaled to 1200 h = 0. Darker colors indicate significant differences in WT_CS controls. Note that effect strengths cannot directly be compared between factors. Small letters indicate statistical significance (p < 0.05). a′ has already been published in a different context (Ruf et al., 2017). Abbreviations: WT_CS = wild-type Canton S; LD = light: dark; WC = warm: cold; RI = rhythmicity index.

Figure 3.

Combined eclosion profiles (left) and autocorrelation (right) under quasi-natural conditions. All experiments during the experimental season are combined, ignoring differences in day length and time of sunrise and sunset between the individual weeks. (a) WT_CS, (b) per01, (c) Pdf01, and (d) han⁵³⁰⁴ flies. Note the broader eclosion windows and less-defined eclosion peaks as well as arrhythmic RI and low LS power in the per01 mutants. (*** = <0.001), #/# indicates N experiments/n flies. Abbreviations: WT_CS = wild-type Canton S; RI = rhythmicity index; LS: Lomb-Scargle; C = cosinor amplitude and zero-amplitude test significance level.

Figure 4.

WT_CS flies were kept in DD at a humidity cycle of 12 h 30% and 12 h 70% RH. (a) Despite the humidity cycle, flies eclosed arrhythmically, indicating that humidity changes are unable to entrain eclosion rhythmicity. (b) Percentage of flies that either successfully eclosed (left) or extended wings among those that did eclose (right). Small letters indicate statistical significance (p < 0.05). Abbreviations: DD = constant darkness; RH = relative humidity; CS = Canton S.