All animals appear to sleep, defined behaviorally as a state of quiescence that is rapidly reversible, associated with a postural change, and during which arousal threshold is increased; sleep loss is followed by homeostatic sleep rebound (Allada and Siegel, 2008; Campbell and Tobler, 1984). We tested whether behavioral quiescence in larvae represents a sleep state by examining these behavioral criteria. First, we asked if larval quiescence is associated with a postural change. Using frame-by-frame postural analysis of individual sleep bouts, we observed that during periods of rest, larvae frequently retract their head towards the body, resulting in a quantitative change in body length and width (Figure 2A–E; Video 4). Head retraction occurred in 76.6% of prolonged quiescence bouts (>36 s; Figure 2D) and less commonly (47.1%) in shorter bouts (18 s; Figure 2C), suggesting that postural change is related to duration of the sleep bout. The postural change most often occurred as part of a stereotyped sequence of behaviors: we found in 72.0% of sleep-associated postural changes that the animal transitioned from activity to behavioral quiescence prior to postural change; quiescence then persisted following postural change in 64.6% of episodes, prior to resumption of activity (Video 4). Less often (28.0%) a larva transitioned directly from activity to coincident postural change with quiescence, or directly from head retraction to an active phase (35.4%), consistent with the observation that posture is more likely to change in association with prolonged rest. Our data thus show that Drosophila larval quiescence is a patterned behavior with a previously unrecognized temporal sequence, and that like animals ranging from nematodes (Iwanir et al., 2013) to humans, larvae demonstrate postural changes during sleep.

Figure 2 Download asset Open asset Drosophila larval quiescence meets behavioral criteria for sleep. Image of a larva before (A) and after (B) postural change associated with sleep. Yellow arrow indicates head retraction. Quantification of postural change frequency associated with 18 s sleep bout length (C) (n = 104 sleep episodes) or ≥36 s sleep bout length (D) (n = 107 sleep episodes) (white=% sleep episodes with postural change (+PC); light gray = no postural change (-PC); dark gray = not determined). (E) Quantification of larval length/width ratio before and after postural change. (F) Percentage of larvae aroused from quiescence using a high intensity (black bar, n = 187 sleep episodes) or low intensity stimulus (gray, n = 119 sleep episodes).~20% of larvae wake spontaneously in absence of a stimulus (white, n = 312). (G) Probability of spontaneous activity (black) or continued quiescence (gray) following a defined period of inactivity (n = 274, 88, 53, 59, 52 quiescent episodes from left to right). https://doi.org/10.7554/eLife.33220.008

Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse (36x) of a second instar larva exhibiting a postural change (head retraction) associated with quiescence. Quiescence bout begins at 00:09, followed by postural change at 00:11, and then continued quiescence. https://doi.org/10.7554/eLife.33220.009

To determine whether larval rest is a rapidly reversible behavioral state, we randomly presented a high intensity blue light stimulus (irradiance 39.8 µW/mm2) for 4 s to second instar larvae and retrospectively analyzed behavioral data for larvae that were inactive at the time of stimulus presentation. We found that nearly 100% of inactive larvae were aroused (as determined by altered position in next captured frame) by this intense stimulus (Figure 2F; Video 5), indicating that behavioral quiescence is rapidly reversible. Using a low intensity blue light stimulus (irradiance 3.98 µW/mm2) to perturb larvae, we found that only ~60% of inactive larvae became active (Figure 2F), demonstrating an altered responsiveness to sensory stimuli during larval rest. Importantly, only ~20% of quiescent larvae spontaneously became active without a stimulus (Figure 2F), confirming that the low intensity stimulus arouses resting larvae more than chance. We next increased the temporal resolution of our system to monitor behavior every 2 s, with the goal of examining whether the behavioral quiescence described above is qualitatively distinct from brief (2 s) periods of inactivity. Focusing first on probability of spontaneous arousal, we found that following 2 s of inactivity, 54% of larvae became active in the subsequent frame (Figure 2G). In contrast, following 6 s of inactivity, only 19% of larvae became active spontaneously; longer bouts of quiescence were not associated with a further reduction in probability of subsequent spontaneous activity (Figure 2G). These results suggest that ≥6 s of quiescence represents an altered behavioral state in comparison to brief (2 s) periods of inactivity. To test directly whether arousal threshold is altered with ≥6 s of behavioral quiescence, we presented a 1 s high intensity blue light stimulus and assessed arousal in larvae that were inactive at the time of stimulus presentation. While 82.4% of larvae were aroused after 2 s of inactivity, only 46.5% were aroused by the same stimulus if quiescent for ≥6 s prior to light presentation (n = 108 quiescent episodes for 2 s and 357 quiescent episodes for ≥6 s; p<0.0001, Fisher’s exact test). Together, these findings indicate that larval quiescence is a reversible state during which arousal threshold is increased from baseline, and that behavioral quiescence lasting at least 6 s can be defined as sleep.

Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse (36x) of a second instar larva demonstrating rapid reversibility of quiescence. Quiescence begins at 00:02, and the light stimulus occurs at 00:05. https://doi.org/10.7554/eLife.33220.010

Does larval quiescence exhibit homeostatic properties of sleep, with rebound following enforced sleep loss? We perturbed sleep using blue light pulses over a 3 hr window, which resulted in 40% reduction in sleep during that time (Figure 3A–C). Over the subsequent 3 hr, sleep deprived larvae exhibited increased quiescence compared to non-deprived controls (Figure 3C), resulting primarily from increased quiescence bout length (Figure 3D,E). Use of a mechanical stimulus to disturb quiescence likewise resulted in subsequent quiescence rebound (Figure 3—figure supplement 1), showing that this rebound is not specific to the modality of quiescence loss. During rebound, sleep-deprived larvae also exhibit deeper sleep, as evidenced by an increased arousal threshold compared to non-deprived larvae (Figure 3—figure supplement 2). Importantly, disrupting quiescence did not cause long-lasting deficits to larval rest/activity or disruption of normal developmental progression (Figure 3—figure supplement 1), demonstrating that increased quiescence immediately following deprivation does not reflect a nonspecific impairment or injury related to deprivation.

Figure 3 with 2 supplements with 2 supplements see all Download asset Open asset Homeostatic sleep rebound following enforced sleep loss in Drosophila larvae. (A, B) Activity heat map of 8 control larvae (A) and eight sleep-deprived larvae (B) during light-based sleep deprivation assay demonstrates reduced quiescence (blue) and increased activity (yellow) throughout the 3 hr deprivation period. (C) Quantification of sleep deprivation (SD) over 3 hr with a repetitive light stimulus (white = control, n = 24 larvae; light red = deprived, n = 24) and subsequent sleep rebound (dark red = previously deprived, n = 18; gray = non deprived assayed during the same period, n = 24). (D,E) Quantification of sleep bout number and length during the 3 hr deprivation period and subsequent rebound (0–3 hr post SD) demonstrates that sleep loss derives from reduced sleep bout length (E). Increased total sleep during the rebound period is more consolidated, with reduced bout number (D) and increased bout length (E). https://doi.org/10.7554/eLife.33220.011

In most animals, including adult fruit flies, sleep timing is controlled by a circadian clock (Borbély, 1982). We examined the role of circadian factors in Drosophila larval sleep. Anatomical analysis has shown that clock cells are present in Drosophila larvae (Liu et al., 2015), and light exposure in first instars results in synchronized adult behavioral rhythms, indicating that photic cues for circadian entrainment can be perceived at this stage (Sehgal et al., 1992). However, we found that rearing animals in constant light, which disrupts the circadian clock, did not change features of larval sleep (Figure 4A; Figure 4—figure supplement 1A,B), including the propensity for increased sleep in the early second instar stage. Behavioral analysis of the circadian clock mutants clockjrk and cyc01 likewise revealed no alteration of larval sleep (Figure 4B; Figure 4—figure supplement 1C,D), suggesting that circadian factors do not play a prominent role in developmentally timed sleep in Drosophila larvae.

Figure 4 with 2 supplements with 2 supplements see all Download asset Open asset Sleep regulatory mechanisms are distinct between larval and adult stages in Drosophila. (A) Quantification of sleep in hourly bins demonstrates that sleep amount and distribution are unchanged with rearing of embryos/larvae in constant light (LL; n = 27) compared to normal 12:12 light:dark cycles (LD; n = 29). (B) Sleep is unaffected in molecular clock mutants clkJrk and cyc01 (n = 16,18,21). All sleep assays were conducted in constant dark under infrared light condition. (C) Quantification of larval total sleep over 6 hr in mutants known to be short-sleepers as adults (iso31 controls, sleepless [sss], and fumin [fmn]; n = 27,18,24 larvae from left to right). (D) No correlation is found between larval and adult sleep time (slope of linear regression line is not significantly different from zero). Sleep is sexually dimorphic in adults (Female (F) < Male (M); the red points (F) are distributed on the y axis lower than the blue points (M)), but not in larvae (red and blue points are evenly distributed on the x axis) (M; n = 34; F; n = 25). https://doi.org/10.7554/eLife.33220.014

We sought to use Drosophila larvae to understand mechanisms of sleep control during development, and first examined whether previously characterized adult short-sleeping mutants exhibit sleep deficits as larvae. Surprisingly, mutants for the gene sleepless (Koh et al., 2008), which regulates potassium channel activity, and for the gene fumin (Kume et al., 2005), which encodes a dopamine transporter, both showed normal larval sleep levels during the second instar period (Figure 4C; Figure 4—figure supplement 2), despite exhibiting severe short-sleeping phenotypes in adulthood. We next asked whether sleep as larvae is predictive of sleep in adulthood. Sleep amount was assessed in second instar larvae, which were subsequently collected and housed individually into adulthood, at which time sleep was again measured, revealing that sleep of individual larvae was not correlated with their sleep as adults (Figure 4D). Finally, in contrast to adulthood when female flies sleep less than males (Andretic and Shaw, 2005), we did not observe sexual dimorphisms to larval sleep (Figure 4D). Taken together, these data indicate that genetic controls of developmental and adult sleep are at least partially distinct.