Do all animals sleep? Sleep has been observed in many vertebrates, and there is a growing body of evidence for sleep-like states in arthropods and nematodes []. Here we show that sleep is also present in Cnidaria [], an earlier-branching metazoan lineage. Cnidaria and Ctenophora are the first metazoan phyla to evolve tissue-level organization and differentiated cell types, such as neurons and muscle []. In Cnidaria, neurons are organized into a non-centralized radially symmetric nerve net [] that nevertheless shares fundamental properties with the vertebrate nervous system: action potentials, synaptic transmission, neuropeptides, and neurotransmitters []. It was reported that cnidarian soft corals [] and box jellyfish [] exhibit periods of quiescence, a pre-requisite for sleep-like states, prompting us to ask whether sleep is present in Cnidaria. Within Cnidaria, the upside-down jellyfish Cassiopea spp. displays a quantifiable pulsing behavior, allowing us to perform long-term behavioral tracking. Monitoring of Cassiopea pulsing activity for consecutive days and nights revealed behavioral quiescence at night that is rapidly reversible, as well as a delayed response to stimulation in the quiescent state. When deprived of nighttime quiescence, Cassiopea exhibited decreased activity and reduced responsiveness to a sensory stimulus during the subsequent day, consistent with homeostatic regulation of the quiescent state. Together, these results indicate that Cassiopea has a sleep-like state, supporting the hypothesis that sleep arose early in the metazoan lineage, prior to the emergence of a centralized nervous system.

Results and Discussion

6 Allada R.

Siegel J.M. Unearthing the phylogenetic roots of sleep. 7 Joiner W.J. Unraveling the evolutionary determinants of sleep. 24 Campbell S.S.

Tobler I. Animal sleep: a review of sleep duration across phylogeny. Three behavioral characteristics define a sleep state []: (1) behavioral quiescence, a period of decreased activity; (2) reduced responsiveness to stimuli during the quiescent state; and (3) homeostatic regulation of the quiescent state. Both behavioral quiescence and reduced responsiveness must be rapidly reversible to differentiate sleep-like states from other immobile states (e.g., paralysis or coma), and reduced responsiveness distinguishes sleep from quiet wakefulness. Homeostatic regulation results in a rebound response, i.e., a compensatory period of increased sleep after sleep deprivation. Here we asked whether the cnidarian jellyfish Cassiopea exhibits these behavioral characteristics.

To track behavior in Cassiopea, we designed an imaging system ( Figures S1 C–S1F) for counting the pulses of individual jellyfish over successive cycles of day and night, defined as a 12 hr period when the light is on or off, respectively. As Cassiopea pulse, the relaxation and contraction of the bell causes a corresponding change in average pixel intensity, which was measured for each frame of the recording, producing a pulse trace ( Figure 1 D). Pulse events were counted using the peak of the pulse trace, and the inter-pulse interval (IPI) was calculated as the time between the peaks ( Figure 1 D; Figure S2 ).

Figure 2 Continuous Tracking of Cassiopea Reveals Pulsing Quiescence at Night Show full caption (A) Pulsing traces for individual jellyfish during day and night over 120 s. (B) The distribution of IPI length for a 12 hr day and a 12 hr night for the same jellyfish shown in (A). Tick marks below the distribution show each IPI length during the day and night. This highlights the long-pause events, which are more common at night ( Figure S3 A; Data S1 ). (C–G) Each blue line corresponds to a single jellyfish. The black line indicates the mean activity of all jellyfish. Dark-gray shading indicates night periods. Dark tick marks on the x axis indicate the time of feeding. (C) Baseline activity (pulses/20 min) of 23 jellyfish tracked for 6 days from four laboratory replicates. (D) Normalized baseline activity for jellyfish shown in (C), where each jellyfish is normalized by its mean day activity. (E) Mean day activity versus mean night activity for each jellyfish over the 6 day experiment shown in (C). Two-sided paired t test, day versus night, p = 6 × 10−9. (F) Normalized baseline activity without feeding of 16 jellyfish tracked over 3 days from two laboratory replicates, where each jellyfish is normalized by its mean day activity. (G) Mean day activity versus mean night activity for each jellyfish over the 3 day experiment shown in (F). Two-sided paired t test, day versus night, p = 10−5. ∗∗∗p < 10−3. See also p < 10. See also Figure S3 We observed that Cassiopea pulse less at night than during the day ( Figure 2 Data S1 ). To quantify this difference in pulsing frequency, we tracked the pulsing behavior of 23 jellyfish over six consecutive days and nights ( Figure 2 C). We define “activity” as the total number of pulses in the first 20 min of each hour. Although individual jellyfish showed different basal activity levels ( Figure 2 C), all showed a large decrease in mean activity (∼32%) at night (781 ± 199 pulses/20 min, mean ± SD) compared to the day (1,155 ± 315 pulses/20 min, mean ± SD; Figures 2 C and 2E). To determine whether fast- and slow-pulsing jellyfish change their activity to a similar degree, we normalized activity of individual jellyfish by their mean day activity. Despite variations in basal activity, the relative change from day to night was similar between jellyfish ( Figure 2 D). Jellyfish activity decreased throughout the first 3–6 hr of the night, with the lowest activity occurring 6–12 hr after the day-to-night transition. Pulsing activity peaked upon feeding, occurring on the fourth hour of each day ( Figures 2 C and 2D). To ensure that day feeding does not cause the day-night behavioral difference, we tracked the activity of 16 jellyfish over three consecutive days and nights without feeding and observed results consistent with those including feeding ( Figures 2 F and 2G; Figure S3 D). These results demonstrate that Cassiopea have a quiescent state during the night. To test the reversibility of this nighttime quiescent state, we introduced a food stimulus at night, which transiently increased activity to daytime levels ( Figure S3 E). The nighttime quiescent state in Cassiopea is thus rapidly reversible, consistent with a sleep-like behavior.

th percentile of night IPI frequency distribution [gray] is 13.9 s). Such long pauses are rarely seen during the day (th percentile of day IPI frequency distribution [yellow] is 2.5 s). This pause behavior may be analogous to long rest bouts observed in Drosophila and zebrafish, which are suggested to be periods of deep quiescence with reduced responsiveness to stimuli [ 1 Hendricks J.C.

Finn S.M.

Panckeri K.A.

Chavkin J.

Williams J.A.

Sehgal A.

Pack A.I. Rest in Drosophila is a sleep-like state. 28 Zhdanova I.V. Sleep and its regulation in zebrafish. To better understand the nighttime quiescence, we compared day and night pulse traces of individual jellyfish. The day and night pulse traces of one representative jellyfish are shown in Figure 2 A. During the night, the IPI is typically longer than during the day ( Figures 2 A and 2B; Data S1 Figure S3 A). Two features contribute to this lengthening of the IPI: (1) the mode of the IPI distribution is longer at night than during the day, and (2) night pulsing is more often interrupted by pauses of variable length. These pauses are seen as a tail in the IPI frequency distribution ( Figure 2 B; 95percentile of night IPI frequency distribution [gray] is 13.9 s). Such long pauses are rarely seen during the day ( Figure 2 B; 95percentile of day IPI frequency distribution [yellow] is 2.5 s). This pause behavior may be analogous to long rest bouts observed in Drosophila and zebrafish, which are suggested to be periods of deep quiescence with reduced responsiveness to stimuli [].

Figure 3 Cassiopea Show Reduced Responsiveness to a Sensory Stimulus at Night Show full caption (A) Schematic of experiment to test sensory responsiveness. Jellyfish were lifted and held at a fixed height (h L ) and then dropped to a fixed height (h D ). h L and h D were kept constant throughout experiments. (B and C) Boxplots of time to first pulse after drop (B) for 23 jellyfish and time to reach bottom after drop (C) for 23 jellyfish during the day and night. Dots represent individual jellyfish collected from two laboratory replicates. Two-sided unpaired t test, day versus night, (B) p < 10−4 and (C) p = 5 × 10−4. (D) Time to first pulse after initial drop and after perturbation for both day and night for 23 jellyfish. (E) Time to reach bottom after initial drop and after perturbation for both day and night for 23 jellyfish. ∗p < 5 × 10−2, ∗∗∗p < 10−3). For the time to first pulse, a two-sided unpaired t test (B) and two-way ANOVA (D) were performed after log transformation ( A two-way ANOVA was performed for data shown in (D) and (E), followed by post hoc comparisons between experimental groups using Bonferroni post test (p < 5 × 10p < 10). For the time to first pulse, a two-sided unpaired t test (B) and two-way ANOVA (D) were performed after log transformation ( STAR Methods ). To test whether Cassiopea exhibit reduced responsiveness to stimuli during their nighttime-quiescent state, we designed an experiment to deliver a consistent arousing stimulus to the jellyfish. We observed in our nursery that Cassiopea prefer staying on solid surfaces as is found in nature. If Cassiopea are released into the water column, they quickly reorient and move to the bottom of the tank. We used placement into the water column as a stimulus to compare responsiveness during the night versus the day. Cassiopea were put inside a short PVC pipe with a screen bottom ( Figure 3 A). This was lifted to a fixed height, held for 5 min to allow the jellyfish to acclimate, and then rapidly lowered, placing the jellyfish free-floating into the water column. We then scored the time it took for the jellyfish to first pulse and the time to reach the screen bottom ( Figure 3 A; STAR Methods ). At night, the jellyfish showed an increase in the time to first pulse and the time to reach bottom compared to during the day (time to first pulse: day 2.1 ± 0.9 s versus night 5.9 ± 4.0 s; time to reach bottom: day 8.6 ± 2.9 s versus night 12.0 ± 3.2 s; mean ± SD; n = 23 animals) ( Figures 3 B and 3C). This increased latency in response to stimulus indicates that Cassiopea have reduced responsiveness to stimulus during the night.

To determine whether the increased latency at night is rapidly reversible, we initiated a second drop within 30 s of the first drop, that is, after the jellyfish have been aroused. Reversibility was tested during both the day and night for 23 jellyfish. During the night, there is a large decrease in the time to first pulse and time to reach the bottom after the second drop when compared to the first drop ( Figures 3 D and 3E). During the day and night, the time to first pulse and time to bottom after the second drop were indistinguishable, demonstrating that after perturbation, animals have similar arousal levels during the day and night. These results indicate that Cassiopea have rapidly reversible reduced responsiveness to a stimulus during the night.

th percentile) ( Figure 4 Homeostatic Rebound in Cassiopea Show full caption Each blue line corresponds to a single jellyfish. The black line indicates the mean activity of all jellyfish. Dark-gray shading indicates night periods. Maroon shading indicates perturbation periods with 10 s water pulses every 20 min. Jellyfish were exposed to different perturbation lengths (6 or 12 hr) at different times (day or night). The normalized activity of all jellyfish tracked over multiple days is plotted. Maroon horizontal lines show the mean activity of pre-perturbation day (solid) and pre-perturbation night (dashed). (A) Perturbation of 30 jellyfish for the last 6 hr of the night. (B) Perturbation of 26 jellyfish for the first 6 hr of the day. (C) Mean day and night activity pre- and post-perturbation for experiments shown in (A) and (B). (D) Perturbation of 16 jellyfish for an entire 12 hr night. (E) Perturbation of 16 jellyfish for an entire 12 hr day. (F) Mean day and night activity pre- and post-perturbation for experiments shown in (D) and (E). ∗p < 5 × 10−2. Both day and night 6 hr perturbation experiments include data from four laboratory replicates. Both day and night 12 hr perturbation experiments include data from two laboratory replicates. See also Black-horizontal lines in (A), (B), (D), and (E) indicate the windows of time used for calculating pre- and post-perturbation means shown in (C) and (F) for both the night (bottom lines) and day (top lines). For the 6 hr experiments, we compared the first 4 hr of the post-perturbation day to the equivalent time pre-perturbation and also compared the first 6 hr of post-perturbation night to the equivalent time pre-perturbation. For the 12 hr experiments, we compared the full 12 hr days and nights pre- and post-perturbation. Two-way ANOVA followed by post hoc comparisons between experimental groups using Bonferroni post test,p < 5 × 10. Both day and night 6 hr perturbation experiments include data from four laboratory replicates. Both day and night 12 hr perturbation experiments include data from two laboratory replicates. See also Figure S4 and Movie S2 To test whether Cassiopea nighttime quiescence is homeostatically regulated, we deprived jellyfish of behavioral quiescence for either 6 or 12 hr using a mechanical stimulus ( Figure 4 ). The stimulus consisted of a brief (10 s) pulse of water every 20 min, which caused a transient increase in pulsing activity ( Movie S2 ). This increase in pulsing activity lasts for approximately 5 min after the 10 s pulse of water. Thus, the perturbation disrupts quiescence for approximately 25% of the perturbation period (either 6 hr or 12 hr). When the perturbation was performed during the last 6 hr of the night ( Figure 4 A), we observed a significant decrease in activity (∼12%) during the first 4 hr of the following day relative to the pre-perturbation day (mean of first 4 hr of pre-perturbation day: 1,146 ± 232 pulses/20 min; compared to post-perturbation day: 1,008 ± 210 pulses/20 min; mean ± SD; n = 30 animals; Figure 4 C). This period of decreased activity is due to both decreased pulsing frequency (increased mode of IPI length) and increased pause length (increase in the IPI length 95percentile) ( Figures S4 B and S4C). This result is consistent with an increased sleep drive after sleep deprivation. After a single day of decreased activity, the jellyfish return to baseline levels of day and night activity. Similar results were observed after an entire night of perturbation (12 hr; Figure 4 D), with a large decrease in activity (∼17%) throughout the following day (mean of 12 hr of pre-perturbation day: 1,361 ± 254 pulses/20 min; compared to post-perturbation day: 1,132 ± 263 pulses/20 min; mean ± SD; n = 16 animals; Figure 4 F). The decrease in activity caused by the 12 hr perturbation was larger than that of the 6 hr perturbation, indicating that the amount of sleep rebound is dependent on the level of sleep deprivation. During periods of decreased activity after either the 6 hr or 12 hr perturbation, we also observed increased response latency to a sensory stimulus ( Figure S4 A), indicating a sleep-like state.

If the reduced activity after nighttime perturbation is due to sleep deprivation rather than muscle fatigue, then applying the perturbation during the day, when Cassiopea are much less quiescent, should not result in reduced activity. To distinguish between sleep deprivation and muscle fatigue, we performed the 6 or 12 hr mechanical stimulus experiments during the day ( Figures 4 B and 4E). We observed no significant difference between pre- and post- perturbation activity levels ( Figures 4 C and 4F), indicating that the rebound response is specific to deprivation of nighttime quiescence. Taken together, these results demonstrate that Cassiopea have a nighttime-quiescent state that is homeostatically controlled.

29 Borbély A.A.

Achermann P. Sleep homeostasis and models of sleep regulation. 4 Hill A.J.

Mansfield R.

Lopez J.M.N.G.

Raizen D.M.

Van Buskirk C. Cellular stress induces a protective sleep-like state in C. elegans. 5 Trojanowski N.F.

Raizen D.M. Call it worm sleep. 6 Allada R.

Siegel J.M. Unearthing the phylogenetic roots of sleep. 7 Joiner W.J. Unraveling the evolutionary determinants of sleep. 30 Zimmerman J.E.

Naidoo N.

Raizen D.M.

Pack A.I. Conservation of sleep: insights from non-mammalian model systems. 4 Hill A.J.

Mansfield R.

Lopez J.M.N.G.

Raizen D.M.

Van Buskirk C. Cellular stress induces a protective sleep-like state in C. elegans. 5 Trojanowski N.F.

Raizen D.M. Call it worm sleep. 31 Nath R.D.

Chow E.S.

Wang H.

Schwarz E.M.

Sternberg P.W. C. elegans stress-induced sleep emerges from the collective action of multiple neuropeptides. 30 Zimmerman J.E.

Naidoo N.

Raizen D.M.

Pack A.I. Conservation of sleep: insights from non-mammalian model systems. In many animals, sleep is regulated by both homeostatic and circadian systems [], but this is not always the case []. For instance, the nematode C. elegans exhibits a developmentally regulated sleep state, and adult C. elegans show a non-circadian stress-induced-sleep state []. A fully functioning circadian system is also not essential for sleep to occur; animals with null mutations of circadian rhythm genes still sleep, though sleep timing is altered []. To test whether nighttime quiescence in Cassiopea is regulated by a circadian rhythm, we first entrained the jellyfish for 1 week in a normal 12 hr:12 hr light/dark cycle and then shifted them to constant-lighting conditions for 36 hr. We tested low-intensity (∼0.5 photosynthetic photon flux [PPF]), mid-intensity (∼100 PPF), and full-intensity (∼200 PPF) light, as well as dark ( Figures S4 D and S4E). If jellyfish activity is regulated by a circadian rhythm, cycling activity should persist in the absence of entraining stimuli, such as light. We observed no circadian oscillation of jellyfish activity under any of the constant-light conditions ( Figure S4 D). However, we did observe circadian oscillation of activity in constant-dark conditions ( Figure S4 E). This result suggests that the quiescent state may be under circadian regulation.

7 Joiner W.J. Unraveling the evolutionary determinants of sleep. 8 Kirszenblat L.

van Swinderen B. The yin and yang of sleep and attention. 9 Dunn C.W.

Giribet G.

Edgecombe G.D.

Hejnol A. Animal phylogeny and its evolutionary implications. 10 Arendt D.

Tosches M.A.

Marlow H. From nerve net to nerve ring, nerve cord and brain--evolution of the nervous system. 11 Katsuki T.

Greenspan R.J. Jellyfish nervous systems. 12 Erwin D.H.

Davidson E.H. The last common bilaterian ancestor. 13 Hejnol A.

Rentzsch F. Neural nets. 14 Kelava I.

Rentzsch F.

Technau U. Evolution of eumetazoan nervous systems: insights from cnidarians. 15 Bosch T.C.G.

Klimovich A.

Domazet-Lošo T.

Gründer S.

Holstein T.W.

Jékely G.

Miller D.J.

Murillo-Rincon A.P.

Rentzsch F.

Richards G.S.

et al. Back to the basics: cnidarians start to fire. 16 Grimmelikhuijzen C.J.

Westfall J.A. The nervous systems of cnidarians. 15 Bosch T.C.G.

Klimovich A.

Domazet-Lošo T.

Gründer S.

Holstein T.W.

Jékely G.

Miller D.J.

Murillo-Rincon A.P.

Rentzsch F.

Richards G.S.

et al. Back to the basics: cnidarians start to fire. 16 Grimmelikhuijzen C.J.

Westfall J.A. The nervous systems of cnidarians. 17 Satterlie R.A. Do jellyfish have central nervous systems?. 18 Watanabe H.

Fujisawa T.

Holstein T.W. Cnidarians and the evolutionary origin of the nervous system. 19 Dupre C.

Yuste R. Non-overlapping neural networks in Hydra vulgaris. 20 Grimmelikhuijzen C.J.

Williamson M.

Hansen G.N. Neuropeptides in cnidarians. 32 Peres R.

Reitzel A.M.

Passamaneck Y.

Afeche S.C.

Cipolla-Neto J.

Marques A.C.

Martindale M.Q. Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone Nematostella vectensis. 33 Zhdanova I.V.

Wang S.Y.

Leclair O.U.

Danilova N.P. Melatonin promotes sleep-like state in zebrafish. 34 Brzezinski A.

Vangel M.G.

Wurtman R.J.

Norrie G.

Zhdanova I.

Ben-Shushan A.

Ford I. Effects of exogenous melatonin on sleep: a meta-analysis. 35 Tosches M.A.

Bucher D.

Vopalensky P.

Arendt D. Melatonin signaling controls circadian swimming behavior in marine zooplankton. 36 Gandhi A.V.

Mosser E.A.

Oikonomou G.

Prober D.A. Melatonin is required for the circadian regulation of sleep. Cassiopea display the key behavioral characteristics of a sleep-like state: a reversible quiescent state with reduced responsiveness to stimuli and both homeostatic and possibly circadian regulation. To our knowledge, our finding is the first example of a sleep-like state in an organism with a diffuse nerve net [], suggesting that this behavioral state arose prior to the evolution of a centralized nervous system. Though at least 600 million years of evolution separate cnidarians from bilaterians [], many aspects of the nervous system are conserved, including neuropeptides and neurotransmitters []. One such conserved molecule, melatonin [], promotes sleep in diurnal vertebrates, including zebrafish [] and humans [], and induces quiescence in invertebrates []. We observed that melatonin induces a reversible decrease in activity in Cassiopea during the day in a concentration-dependent manner ( Figures S4 F–S4H), suggesting that melatonin has a conserved quiescence-inducing effect in Cassiopea. Pyrilamine, a histamine H1 receptor antagonist that induces sleep in vertebrates [], also induces concentration-dependent quiescence in Cassiopea ( Figure S4 F). These results suggest that at least some mechanisms involved in vertebrate sleep may be conserved in Cassiopea.