DNA damage modulates sleep drive in basal cnidarians with divergent chronotypes

Domain: neuroscience
Published: 2026-01-06
Editorial outcome: major_revision

Abstract
Sleep is a conserved behavior across all animals with a nervous system, ranging from cnidarians to humans. Considering the survival risks, why sleep evolved in basal lineages and what essential benefits it provides to the simple nerve net of nocturnal and diurnal invertebrates remain elusive. We used behavioral criteria to empirically define sleep in the upside-down jellyfish Cassiopea andromeda and the starlet sea anemone Nematostella vectensis. Light and homeostasis were the primary drivers of sleep in C. andromeda, which slept at night and napped at midday in both the laboratory and the natural habitat. In contrast, both the circadian clock and homeostatic processes regulated sleep in N. vectensis, which increased sleep at dawn. Similar to humans, C. andromeda, wild-type (WT) and Clock mutant (NvClkΔ/Δ) N. vectensis slept about one-third of the day, irrespective of the daily timing and architecture of sleep, and melatonin promoted sleep in accordance with the species-specific chronotype. Notably, sleep deprivation, ultraviolet radiation, and mutagens increased neuronal DNA damage and sleep pressure, while spontaneous and induced sleep facilitated genome stability in both the diurnal and crepuscular cnidarians. These results suggest that DNA damage and cellular stress in simple nerve nets may have driven the evolution of sleep.

Introduction
Sleep is a fundamental and evolutionarily conserved behavioral state prevalent across the animal kingdom, ranging from cnidarians to insects and mammals, suggesting that sleep is a Cambrian metazoan adaptation1–6. Sleep deprivation (SD) and disturbances are associated with increased risks for health decline in bilaterian animals7,8. The roles of sleep range from cellular repair and energy restoration to synaptic plasticity9–12. Nevertheless, the evolution of sleep came with major fitness compromises, such as reduced awareness of the environment and vulnerability to predation, and the fundamental conserved cellular functions of sleep remain elusive1,2,13.

Sleep is defined by behavioral criteria and neuronal activity patterns. In most non-mammalian animals, sleep is characterized solely by distinct behavioral criteria, including consolidated periods of behavioral immobility accompanied by an increased arousal threshold in response to external stimuli and species-specific postures1,2,14,15. Sleep is regulated by the circadian process, which determines sleep timing, and by the homeostatic process, which compensates for SD. The presence of sleep and these regulatory processes in basal and derived nocturnal and diurnal species suggests a common evolutionary origin of sleep regulation16,17.

Sleep plays a critical role in the maintenance of genomic integrity: it reduces DNA damage, which accumulates during wakefulness in neurons of flies, fish, and mice18–21. The causes of DNA damage are diverse and include the solar ultraviolet (UV) radiation, the activity of reactive oxygen species (ROS), the inadvertent action of nuclear enzymes, and neuronal activity20,22,23. The non-dividing and irreplaceable mature excitable neurons may be particularly vulnerable to sleep loss. This cell type evolutionarily emerged in basal metazoans24 and may require sleep to facilitate cellular maintenance. Indeed, sleep-like behavior has recently been observed in two diurnal cnidarians, the upside-down jellyfish Cassiopea and Hydra vulgaris (H. vulgaris)3,4, suggesting that the development of simple nerve nets in cnidarians25,26 coincided with the emergence of sleep.

Cnidarians represent one of the most ancient animal lineages possessing neurons, which extend neurites throughout the endodermal and ectodermal layers, and contain conserved neurotransmitters and neuropeptides27,28. The jellyfish Cassiopea, which lives in obligate symbiosis with photosynthetic dinoflagellates, exhibits rhythmic daily activity that peaks during the day4. In contrast, the sea anemone Nematostella vectensis (N. vectensis), which lacks symbiotic eukaryotes, is considered nocturnal29,30. However, sleep architecture has not been empirically defined in Cassiopea, and sleep has not been characterized in N. vectensis. Thus, the mechanisms that drove the evolution of sleep in these model animals are unknown.

To dissect the origin of animal sleep and its core cellular drivers, we defined and analyzed sleep architecture in Cassiopea andromeda (C. andromeda) and N. vectensis. We found that the light/dark cycle and homeostatic pressure are the primary regulators of sleep in the diurnal C. andromeda, whereas the circadian clock and homeostatic drive are the predominant regulators of sleep in the crepuscular (active at dusk) N. vectensis. Notably, the results demonstrated that the capacity of sleep to relieve neuronal DNA damage is an ancestral trait shared by both symbiotic diurnal and non-symbiotic crepuscular cnidarians. We suggest that sleep may have evolved to enable consolidated periods of neural maintenance.

Results
Characterization of sleep in C. andromeda under controlled conditions and in their natural habitat
In Cassiopea, a sleep-like state has been characterized by monitoring the rate of pulsation activity of the jellyfish umbrella, which reduces during the night4. To quantify sleep, it is essential to determine the threshold of the pulsation frequency, which can differentiate sleep from wake behavior. An infrared (IR) camera was used to track the simultaneous behavior of multiple C. andromeda during the day and night (Fig. 1a, Supplementary Movie 1). Pulsation events were determined by monitoring the changes in pixel intensity, which reflected expansion and contraction of the C. andromeda bell diameter (Fig. 1b). Consistent with its diurnal nature, locomotor activity consistently reduced during the night in C. andromeda under 12h light:12h dark (LD) conditions with a mean pulsation rate of 36.4 ± 1.6 and 31.9 ± 1.4 pulses/min during the day and night, respectively (mean ± SEM; Fig. 1c, d). The constant pulsing behavior31 precluded the use of quiescence to define sleep in C. andromeda. To identify the arousal threshold distinguishing sleep from wake in C. andromeda, we applied a brief light stimulus at night and measured the response latency (Fig. 1e). A threshold-based optimization algorithm was used to determine the optimal behavioral parameters to distinguish between slow and fast responses (Fig. 1g, j). We found that C. andromeda, which pulsed fewer than 37 times per minute (Fig. 1f–h) for more than 3 min (Fig. 1i–k) were slower to respond to the stimulus. Thus, this threshold was used to characterize sleep in C. andromeda. Applying this criterion revealed that the average sleep time increased during the night (63.7%) compared to the day (45.3%) under LD in C. andromeda (Fig. 1l, m). Notably, similar to findings in primates and flies32,33, a midday nap was also observed in C. andromeda (Fig. 1l). The daily average sleep time rhythm was 24.2h (Fig. 1n). However, rhythmic locomotor activity was abolished under constant darkness (DD) and robustly dampened under constant light (LL) conditions (Supplementary Fig. 1a–d). Moreover, under forced desynchrony conditions (6hL:6hD:6hL:6hD, LDLD), the animals showed a 12.1h rhythm of locomotor activity (Supplementary Fig. 1e–g). These findings showed that sleep is primarily driven by the light/dark cycle in C. andromeda. To determine whether sleep in C. andromeda is regulated by a homeostatic process, we sleep-deprived the animals for 6h (ZT17-ZT23) using computer-controlled alternating repeated water turbulence at night (Supplementary Movie 2). Following SD, C. andromeda displayed sleep rebound, i.e., sleep-deprived animals slept 1.5-fold more than control animals during the day (Fig. 1o, p). In contrast, SD during the active period (ZT5-11) did not induce sleep rebound (Supplementary Fig. 1k, l). These findings show that sleep is regulated by light and homeostatic drive in the diurnal symbiotic C. andromeda.

To generalize the sleep criteria found in the laboratory to Cassiopea’s natural habitat, we monitored the pulsation behavior of wild animals (Supplementary Fig. 1m) during day and night (~15hL:~9hD) in shallow water adjacent to the seashore of Key Largo, FL. The findings revealed a strong negative association between the size of Cassiopea and their pulsation rate (Fig. 1q). We therefore normalized the average activity according to the size of each group and showed elevated activity during the day at the sea (Fig. 1r and Supplementary Fig. 1n). Notably, the pulsation threshold used to detect sleep under controlled laboratory conditions (37 pulses/min, Fig. 1f–h) closely matched the average daytime pulse rate observed in the lab (36.4 pulses/min, Fig. 1d). Given this similarity, we approximated the sleep threshold in the field by the average daytime pulse rate, adjusted according to the bell diameter, as a proxy to differentiate sleeping and awake animals. Utilizing this model, we found that the sleep architecture of Cassiopea is similar in the laboratory and the sea. In their natural habitat, the animals primarily sleep at night and nap during midday (Fig. 1s). These results enable the quantification of sleep in various sizes of Cassiopea living in the laboratory and in their natural habitat at sea.

Circadian and homeostatic control of sleep in the crepuscular N. vectensis
Although rhythmic locomotor activity has been described in N. vectensis29,30,34, sleep was not characterized in sea anemones, including N. vectensis. To detect the state of sleep, we used an IR camera and monitored the behavior of 18 N. vectensis placed individually in three 6-well plate arenas (Fig. 2a, Supplementary Movie 3). We quantified the levels of movement by calculating pixel change above a quiescence threshold, which was set based on MgCl2-paralyzed animals (Fig. 2b and Supplementary Fig. 2a–d). We analyzed 6h bins (Supplementary Fig. 4) but chose 12h windows (ZT18-6 and ZT6-18) because they encompass the full crepuscular (i.e., active at dusk) behavioral pattern and align with the 12h light/dark periods used for C. andromeda (Fig. 1m), facilitating cross-species statistics. In contrast to C. andromeda, under LD, N. vectensis showed rhythmic locomotor activity (Fig. 2c), which increased between ZT6-ZT18 and decreased between ZT18-ZT6 (Fig. 2d). These results indicate that N. vectensis is a crepuscular animal. Notably, the exposure to a sudden artificial dark-to-light transition transiently induced locomotor activity (ZT0-2, Fig. 2c). This robust response to light was used as a stimulus to identify increased arousal threshold in sleeping animals. During daytime (ZT2), N. vectensis was exposed to various light intensities and showed a negative correlation between the light intensity and response latency (Fig. 2e and Supplementary Fig. 2f–k). Using the intermediate-intensity light stimulus and optimization algorithm (Fig. 2g), we found a slower response in N. vectensis individuals that were quiescent for more than 8min prior to the light stimulus (Fig. 2f–h). To validate this arousal threshold, we tested the response to an additional sensory stimulus, i.e., a food cue. Similar to the response to light (Fig. 2f-h), quiescence lasting at least 8min resulted in a slower response and could differentiate sleep from quiet wakefulness (Fig. 2i–k). Thus, we defined sleep in N. vectensis as a quiescence state of at least 8min, which is associated with reversible behavior and increased arousal threshold.

This characterization of sleep enabled us to convert mobility data to sleep time (Supplementary Fig. 2a-e). The crepuscular N. vectensis showed rhythmic locomotor activity and an average sleep duration of 25 ± 2.3 min during ZT18-ZT6 and 14 ± 1.8 min during ZT6-ZT18, with a sleep period of 23.4h under LD conditions (mean ± SEM). Similarly, the rhythmic sleep period was 22.5h under DD (Fig. 2l-n, Supplementary Fig. 3a, b), suggesting that sleep is regulated by the circadian clock in N. vectensis. To further differentiate between light- and circadian-regulation of sleep, we monitored sleep under forced desynchrony (LDLD). While the rhythmic period of locomotor activity was 22.2h and controlled by the clock, sleep period was 11.7h (Supplementary Fig. 3e, f, i, j). These results suggest that although sleep is primarily regulated by the circadian clock it can also be modulated by light in N. vectensis.

To understand the hierarchy of light- and intrinsic clock-regulation of sleep, we monitored sleep in NvClkΔ mutant34 (NvClkΔ/Δ) N. vectensis. NvClkΔ/Δ N. vectensis showed bimodal sleep/wake rhythms (periods of 12.25h and 25.24h) under LD and this rhythm was abolished under DD (Fig. 2o–q, Supplementary Fig. 3c, d). Under LDLD, the sleep/wake cycle followed the 6h alternating light/dark rhythm (Supplementary Fig. 3k, l). These findings demonstrate that the transcription factor NvClk is essential for maintaining clock-controlled sleep in N. vectensis.

To further characterize sleep, we examined species-specific association with posture. Sleep time was monitored in animals displaying closed or open tentacle posture (Fig. 2r). Although close or open tentacle posture did not strictly differentiate sleep from wake state; i.e. animals with closed tentacles can be awake and vice versa, the closed tentacle N. vectensis slept 2.1-fold more than open tentacles N. vectensis at the peak of the sleep period (ZT23, Fig. 2s). To test whether homeostatic process regulates sleep, we sleep-deprived the N. vectensis for 8h during sleep period (ZT20-ZT4) using gradual and gentle water exchange once per hour (Supplementary Movie 4). Following SD, sleep-deprived animals slept 1.24-fold more than control animals (Fig. 2t, u), indicating a homeostatic sleep drive. In contrast, control SD during the activity period (ZT8-ZT16) did not induce a sleep rebound (Fig. 2v, w). Altogether, while sleep can be modulated by light, a combination of circadian and homeostatic processes are the primary regulators of sleep in the crepuscular N. vectensis.

Inter-species comparison of sleep architecture revealed that melatonin consolidates sleep independently of the animal chronotype
The establishment of sleep criteria in two cnidarians, which sleep at different times of the day, enabled us to study species-specific changes in sleep architecture and regulatory mechanisms. The analysis of sleep time revealed nocturnal sleep in C. andromeda, and a crepuscular rhythmic sleep/wake cycle in WT N. vectensis, which was abolished in NvClkΔ/Δ N. vectensis under constant conditions (Fig. 2). However, total sleep time over 24h was similar in C. andromeda, WT and NvClkΔ/Δ N. vectensis (Fig. 3a). To determine sleep patterns, we quantified the length and number of sleep bouts during the sleep and wake periods of each species. We found longer sleep bout durations and a higher number of sleep bouts at ZT18-6 compared to ZT6-18, under LD, in both WT (Fig. 3b, c) and NvClkΔ/Δ (Fig. 3d, e) N. vectensis. Similarly, under LD conditions, sleep bout length and the number of sleep bouts increased during the night (ZT12-0) in the diurnal C. andromeda (Fig. 3f, g). Inter-species comparison of sleep architecture revealed that although total sleep time was similar (Fig. 3a), the average sleep bout length was shorter by 2.8 min (Fig. 3h), and the mean number of sleep bouts was approximately two-fold higher (Fig. 3i) in C. andromeda compared to N. vectensis. These results show that, like humans, both cnidarian species spend approximately one-third of their lives sleeping (Fig. 3a). However, sleep is more consolidated in N. vectensis, i.e., longer and less fragmented sleep bouts, compared to C. andromeda.

The presence of circadian and homeostatic regulation of sleep in cnidarians suggests a common neuroregulatory mechanism. The melatonin hormone is secreted from the pineal gland during the night and mediates rhythmic daily physiological and behavioral processes in vertebrates35,36. However, its role in invertebrates remains poorly understood37,38. We tested the effect of melatonin on sleep in the diurnal C. andromeda and crepuscular N. vectensis. While melatonin treatment did not affect sleep during the rest periods of N. vectensis (ZT3) and C. andromeda (ZT13) (Fig. 3j–l, s–u), sleep time, sleep bout duration, and the number of sleep bouts increased during the active phase in both N. vectensis (ZT13) and C. andromeda (ZT3) (Fig. 3m–o, p–r). These results suggest an evolutionarily conserved role of melatonin in sleep regulation across animals with different chronotypes.

Sleep relieves the accumulation of DNA damage in both diurnal and crepuscular cnidarians
The presence of sleep in cnidarians indicates that sleep evolved independently of the brain. Studies in fish have shown that sleep decreases DNA damage and is essential for the health of single, non-dividing neurons18,19. To investigate cellular factors that might have driven the evolutionary emergence of sleep in cnidarians, we monitored DNA damage levels in single cells of N. vectensis and C. andromeda during the sleep/wake cycle and following SD (Fig. 4a–d). We hypothesized that DNA damage would peak and decline following extended wake and sleep, respectively. To identify the timepoints of maximal and minimal sleep pressure, we calculated the average sleep time over the past 12h that preceded each timepoint. Both C. andromeda and N. vectensis were sampled at the end of their wake (ZT11 and ZT17) and sleep (ZT23 and ZT8) phases (Fig. 4a, b), where wake and sleep accumulation peaked (Fig. 4c, d). DNA damage was quantified using immunohistochemical assays with an antibody against the phosphorylated histone H2AX (γH2AX), a well-established biomarker of cellular response to DNA damage39. The number of nuclear γH2AX foci was quantified in the neuron-enriched mesogleal surface of the peri-rhopalial tissue40, located adjacent to individual rhopalium of C. andromeda (Fig. 4e, g), and in mOrangeCAAX-positive neurons of transgenic tg(-2.4NvElav1b:mOrangeCAAX)27 N. vectensis (Fig. 4f, h). Under LD, the number of γH2AX foci increased at the end of the wakefulness period in C. andromeda (ZT11) and N. vectensis (ZT17) and reduced at the end of the sleep period in C. andromeda (ZT23) and N. vectensis (ZT8) (Fig. 4i, j). The opposite timing of DNA damage reduction between C. andromeda and N. vectensis was aligned with their species-specific chronotype and daily timing of sleep. To determine the effect of homeostatic sleep pressure, C. andromeda and N. vectensis were sleep-deprived for 6h (ZT17-ZT23) and 8h (ZT20-ZT4), respectively. Following SD, the number of γH2AX foci increased in C. andromeda (ZT23) and N. vectensis (ZT8) and subsequently decreased to baseline levels following sleep recovery in both species (Fig. 4k, l). These results demonstrate that DNA damage accumulates during wakefulness and decreases during sleep in both diurnal and crepuscular cnidarians. Furthermore, these findings suggest that decreasing DNA damage during sleep is an evolutionarily conserved cellular function that may have contributed to the emergence of sleep in early metazoans.

DNA damage increases sleep pressure in C. andromeda and N. vectensis
The correlation between sleep drive and cellular stress (Fig. 4) raised the possibility that environmental and intrinsic factors, disrupting DNA integrity, promote sleep in cnidarians. UV radiation is a potent DNA damage agent and an ancient pervasive atmospheric stressor impacting marine invertebrates41–43. To determine the effect of UV radiation on DNA damage and sleep, we exposed C. andromeda and N. vectensis to 15 min and 60 min, respectively, of UVB radiation44 (312 nm) during the day (ZT8). In C. andromeda and N. vectensis, the number of γH2AX foci increased one hour after UV exposure (Fig. 5a, b, f, g). Subsequently, nighttime and daytime sleep increased in C. andromeda (Fig. 5k, l) and N. vectensis (Fig. 5m, n), respectively. Notably, following sleep recovery, the animals remained viable, and two days later, the levels of DNA damage reduced in C. andromeda (Fig. 5a, b, f, g). These results suggest that UV-mediated induction of DNA damage increases sleep pressure. Since UVB may also affect the behavior of cnidarians in alternative pathways45, we treated N. vectensis with the mutagenic compound Etoposide46. While UVB induces oxidative stress and damage to DNA bases, Etoposide inhibits the topoisomerase II enzyme and stabilizes DNA double-strand break formation. Similar to the effect of UV, Etoposide treatment abruptly increased the number of γH2AX foci (Fig. 5c, h). The animals then increased sleep during the following day and night (Fig. 5o, p), and DNA damage was reduced (Fig. 5c, h). Consistent with these findings, melatonin treatment, which promoted sleep during the species-specific active phase in both N. vectensis (ZT13) and C. andromeda (ZT3) (Fig. 3m, p), reduced γH2AX foci levels in both species (Fig. 5d, e, i, j). Together, these results show bidirectional interactions between sleep and DNA damage in early divergent lineage. Moreover, these inter-species comparative findings suggest that environmental factors, such as UV radiation and soluble mutagenic compounds, which can induce DNA damage and cellular stress, may have contributed to the evolutionary emergence of sleep in cnidarians.

Methods
C. andromeda and N. vectensis husbandry
C. andromeda jellyfish were initially collected from the Gulf of Eilat and later raised and reproduced in the laboratory. The animals were reared in artificial seawater (ASW, OmegaSea, 39-40 ppt) at a 24°C–25°C with a pH of 8.1–8.3. A water cooler and heater were used to maintain a constant temperature. A deuterium-depleted water (DDW) compensation system was used to maintain salinity levels. Animals were raised under a 12h day/12h night cycle (LD) using 2400W Metal Halogen 14,000K light sources providing approximately 100 PPFD at the animal level, located 20 cm above the water surface. The animals were housed and monitored in a 113-liter closed water system equipped with a waste filtration system and a protein skimmer. Waste products were kept close to 0 ppm ammonia, 0 ppb phosphorus, 0 ppm nitrite, and 0 ppm nitrate. C. Andromeda were fed daily with brine shrimp (Artemia nauplii) at noon. Animals with bell diameters of 5-7.5 cm, in a relaxed state, were randomly assigned to each experimental group. The C. Andromeda genotype was validated by sequencing the cytochrome oxidase subunit 1 (CO1) gene81. Whole genomic DNA was extracted using the Minikit NucleoSpin tissue (NucleoSpin, REF 740952.50), and DNA fragment amplification was performed using the following degenerated primers: Forward: 5’-ACNAAYCAYAAAGATATHGG-3’, Reverse: 5’-TGGTGNGCYCANACNATRAANCC-3’.

N. vectensis were raised in ASW (OmegaSea, 12 ppt) in a closed system at 17°C under constant darkness and fed daily with Artemia nauplii at noon. Spawning of gametes and fertilization was performed according to established protocol82. In brief, the temperature was raised to 25°C for 9h, and males and females were exposed to strong white light. 3h later, oocytes were mixed with sperm to enable fertilization. To genotype NvClk mutant and WT N. vectensis, genomic DNA was extracted using the Minikit NucleoSpin tissue (NucleoSpin, REF 740952.50). A fragment of the NvClk gene was amplified using the following primers: forward 5’-ACCCCACTGAGTGACCTCTT-3’ and reverse 5’-ATACGCCTGCGCTATACACC-3’. The PCR products were then loaded on a 3% Tris-borate-EDTA (TBE) agarose gel supplemented with GelStar Nucleic Acid Gel Stain (Lonza, Basel, Switzerland). The genotype of each animal was determined based on the size of the PCR products: NvClkΔ/Δ - 120 bp; WT - 100 bp. The gel-based genotyping was confirmed by sequencing in selected animals.

Behavioral tracking and analysis of C. andromeda
Two modular water systems (Aquazone Ltd, Israel) were designed to monitor the behavior of 9 or 16 jellyfish simultaneously while maintaining controlled light, temperature, and salinity conditions. The systems consisted of 9 or 16 clear perspex chambers of 10x10x10 cm or 7x7x10 cm for individual jellyfish, respectively (Fig. 1a). These apparatuses were kept inside a 55x50x30 cm tank, connected to a waste filtration system, a protein skimmer, a water heater, and a DDW water compensation system. C. andromeda were kept under 12h of light (five 50W LED lights providing 100–200 PPFD at the animal level) and 12h of darkness (LD) and constantly illuminated with low-intensity IR LED lights. Prior to each experiment, C. andromeda were acclimated in the recording system, one individual per chamber, for 2-3 days. An IR camera (Dahua Technology, Hangzhou, China) with an IR filter was used to record mp4 videos at resolution of 1920×1200 and 20 frames per second (fps). The mp4 videos were converted to avi files using the FFmpeg software (FFmpeg, Irvington, NJ, USA). MATLAB Version: 9.8.0 (R2020a)83 and a lab-made script were used to determine pulsation behavior. The change in the average pixel intensity was measured for each frame, producing a pulse trace. A rectangular region of interest (ROI) was selected for each jellyfish. The smoothed average pixel intensity was calculated using a Gaussian moving window and was subtracted from the data to control for low-frequency oscillations of the data. Pulsation events were counted using the peak of the pulse trace (Fig. 1b), and the mean activity was analyzed during the first 20 min of each hour along day and night. Sleep architecture was analyzed using the full duration of the first 24h of the video recording to ensure the continuity of the data. Jellyfish that changed their horizontal to a vertical position or anomalous animals exhibiting pulsing behavior three standard deviations below or above the mean were excluded. The oscillation frequencies were evaluated based on the average values of each experiment using the Lomb-Scargle Periodogram (LSP) from the ‘Lomb’ library84 on R85 with a p<0.01 threshold. To assess the change in arousal threshold, the jellyfish were stimulated repeatedly every hour at night (ZT13-ZT19) using a 1s 100–200 PPFD light pulse. A response was defined as a reduction of 2 standard deviations in the inter-pulse intervals (IPI) compared to the distribution of IPIs before the stimulation, within a time window of 10s after the stimulus. A threshold-based optimization algorithm was used to determine the optimal behavioral parameters to distinguish between slow and fast responses (i.e., elevated arousal threshold; Fig. 1g, j). For each candidate threshold (e.g., pulsation rate), responses were split into two groups: below and above the threshold. We then summed all Euclidean distances of these responses from the mean value of their respective groups (i.e., error value). Thus, the behavioral value that optimally divided a group of fast responses and slow responses results in the lowest error value. In SD experiments, the animals were stimulated repeatedly with a gentle water pulse using thin tubes connected to a water pump and a computer clock. These 10 min pulses of water were applied every 10 min for 6h at ZT17-ZT23. Field experiments were conducted in May in the native habitat of Cassiopea at Key Largo, FL. Light intensities ranged between 400-1500 PPFD at a depth of 0.3–1.5 m at the peak of the day, salinity was 31.5 ppt and Temperature was 26.8°C. The pulsation rate was monitored manually by counting the number of pulses per minute every hour in 30–40 animals during the day and night (total of 662 animals). Subsequently, the diameter of the relaxed bell of each individual was measured.

Behavioral tracking and analysis of N. vectensis
Locomotor activity of individual N. vectensis was monitored using a custom-made temperature-controlled (20°C) water system equipped with an IR camera (Dahua, Binjiang District, Hangzhou, China). Animals of similar size (1-2 cm), genetic background, and age were distributed individually into wells of four six-well plates (Fig. 2a) and were subject to alternating white light Illumination with intensity of 17 PPFD (Sylvania Lightning Solution, Wilmington, MA, USA). MATLAB script was generated based on H. vulgaris sleep tracking methodology3 to analyze the locomotor activity and sleep. We measured changes in the pixel value between two consecutive frames at 5s intervals (Supplementary Fig. 2a-e). Noise was reduced by subtracting the minimum pixel change of the individual. To set the minimum threshold of locomotor activity, pixel changes of MgCl2-treated paralyzed animals were quantified. These changes were considered immobility. The ratio of frames with detectable movements was calculated in a 1 min bin (i.e., fraction movement) and defined as quiescence if movement was not detected in more than 50% of the frames per 1 min bin (i.e., fraction movement < 0.5). To assess the change in arousal threshold, the polyps were stimulated with either light or food. Polyps were stimulated with three different light intensities (+5, +50, +500 PPFD, above the 17 PPFD baseline) during the day (ZT10) for 30 min (Supplementary Fig. 2f–k). Food stimulation was performed by application of “Reef Energy Plus” solution (Red Sea, Israel). A response-to-stimulus was defined as 30s of continuous movement above the threshold. A threshold-based optimization algorithm was used to determine the optimal behavioral parameters to distinguish between slow and fast responses (i.e., elevated arousal threshold; Fig. 2g, j) as is described for C. andromeda. Sleep durations were summed in hourly bins, and the average and standard errors were calculated for all tested animals. Since N. vectensis showed crepuscular behavior (i.e., active at dusk), we used a data-driven approach to define sleep (ZT18-6) and wake (ZT6-18) periods in a 12h time window. Sleep architecture data were analyzed using continuous 60h movies starting at ZT18. In SD experiments, the animals were stimulated by gentle water exchange once per hour during their peak of sleep period (ZT20-ZT4) and peak of activity period (ZT8-ZT16; Supplementary Movie 4) and sleep was monitored before and after the SD. The oscillation frequencies were evaluated based on the average values of each experiment using Fourier analysis-based software LSP with a p<0.01 threshold. Open or closed posture was manually annotated from a single video frame. Animals were categorized based on the number of tentacles deployed outside of their body. Animals that showed at least two visible tentacles were classified as ‘open’.

Melatonin treatments in C. andromeda and N. vectensis
For both sleep and DNA damage assays, animals were first habituated. Specifically, C. andromeda and N. vectensis were entrained and maintained in the behavioral systems described above for 72h under LD conditions. In the C. andromeda system, water circulation was terminated one hour prior to melatonin treatment due to the high volume of constant water exchange in the system. Both species were treated with either 100 μM melatonin (diluted in ethanol) or ethanol for control, at ZT3 and ZT13. These time points were selected to correspond to their respective periods of sleep (ZT3 for N. vectensis, ZT13 for C. andromeda) and wakefulness (ZT13 for N. vectensis, ZT3 for C. andromeda) for both species. In C. andromeda, behavior was recorded 30 min before and during melatonin administration. In N. vectensis, behavior was recorded 60 min before and during melatonin treatment. The delta effect (Fig. 3j–u) was calculated by subtracting the mean post-treatment values from the mean pre-treatment values of either melatonin or ethanol treatment. In DNA damage quantification assays, animals were treated with melatonin for 3h, starting at ZT13 for N. vectensis and ZT3 for C. andromeda at their respective wake period, and were then fixed in 4% PFA. For N. vectensis, the upper body column was mounted on a slide, and cells in the epithelium layer (ectodermis) were imaged. For C. andromeda, three independent rhopalia were dissected from each animal (total of eight animals per Ctl and Mel groups) and DNA damage was quantified in cells located in the mesogleal surface of the peri-rhopalial tissue adjacent to each rhopalium.

Irradiation of UV in C. andromeda and N. vectensis
For both sleep and DNA damage assays, animals were first habituated. Specifically, C. andromeda and N. vectensis were entrained and maintained in the behavioral systems described above for 72h under LD conditions. A dark plastic barrier divided and isolated UV-treated animals from controls in the behavioral monitoring systems. For C. andromeda, sleep was recorded for 30h; for N. vectensis, 42h. At ZT8, half of the animals were exposed to 312 nm UVB (Spectroline® E-Series UV lamp, 20 cm above the animals): 15 min in C. andromeda and 1h in N. vectensis. To quantify DNA damage, at each time point, UV-treated and control C. andromeda and N. vectensis were fixed in 4% PFA before (ZT7), 1h (ZT9), and two days (ZT7 Day3) following the UV irradiation. All UV assays were performed in two independent experiments for both species. For N. vectensis, the upper body column was mounted on a slide, and the cells in the epithelium layer (ectodermis) were imaged. For C. andromeda, 2–3 rhopalia were dissected from each animal (11 animals per timepoint; ZT7, ZT9, ZT7 Day3) and DNA damage was quantified in cells located in the mesogleal surface of the peri-rhopalial tissue adjacent to each rhopalium.

Etoposide treatment in N. vectensis
N. vectensis were habituated for 72h and entrained to LD cycle in six-well plates. Sleep was recorded during 54h. At ZT8, half of the animals were treated with 10 μm Etoposide (E1383, Sigma-Aldrich, St. Louis, MO, USA) and half with control 0.01% DMSO. To quantify DNA damage, at each time point, Etoposide-treated and control animals were fixed in 4% PFA before (ZT7), 1h (ZT9), and two days (ZT7 Day3) following the treatment. Etoposide assays were performed in two independent experiments. The upper body column was mounted on a slide, and cells located in the epithelium layer (ectodermis) were imaged.

DNA damage dynamics under LD and SD conditions in C. andromeda and N. vectensis
In both species, animals were habituated in their video monitoring system for 72h and entrained to an LD cycle. SD was applied as described above. To quantify DNA damage, animals were fixed in 4% PFA at each time point. All LD and SD assays were performed in three independent experiments for both species. For N. vectensis, the upper body column of transgenic tg(-2.4NvElav1b:mOrangeCAAX)+/- animal was mounted on a slide, and neurons in the epithelium layer (ectodermis) were imaged. DNA damage quantification analyses were performed in mOrangeCAAX-positive neurons at ZT17, ZT8, and ZT17 Day 2. For C. andromeda, 2-4 rhopalia were dissected from each animal (8-10 animals per time point; ZT11, ZT23, and ZT11 Day 2) and DNA damage was quantified in cells located on the mesogleal surface of the peri-rhopalial tissue adjacent to each rhopalium.

Immunohistochemistry assays in C. andromeda and N. vectensis
tg(-2.4NvElav1b:mOrangeCAAX)+/- and wild type N. vectensis were fixed overnight in 4% PFA in 2X PBS at 4°C and washed with PBS. The umbrella tissues of C. andromeda surrounding the rhopalium were sampled and fixed for 5 min in 2% PFA in ASW at room temperature, followed by a 6h fixation in 4% PFA in PBS at 4°C and washed with PBS. For both species, the tissues were incubated with 2 μg/mL protein kinase (PK) for 10 min, then washed in PBS+0.1% Triton 3x15 min, blocked with 10% fetal bovine serum with 1% DMSO, and diluted in PBS+0.1% Triton for 1h at room temperature. After blocking, in N. vectensis, tissues were incubated with a primary antibody: rabbit anti-γH2AX (GTX127342, GeneTex, Irvine, CA, USA) 1:200 dilution and anti DsRED (Living Colors® mCherry Monoclonal mouse Antibody, Clontech, Cat# 632543) 1:200 dilution in blocking buffer for 72h at 4°C, while in C. andromeda, tissues were incubated with the rabbit anti-γH2AX 1:200 dilution. Then, for both species, samples were washed in PBS+0.1% Triton 3x15 min. N. vectensis were incubated for 1h with a goat polyclonal secondary anti-rabbit IgG H&L (Alexa Fluor® 646 nm, 2 mg/ml, Invitrogen, Waltham, MA, USA) and anti-mouse IgG H&L (Alexa Fluor® 488 nm, 2 mg/ml, Invitrogen) and with DAPI at 1:1000 dilution in blocking buffer at room temperature. C. andromeda were incubated for 1h with anti-mouse IgG H&L (Alexa Fluor® 488 nm, 2 mg/ml, Invitrogen) and with DAPI at 1:1000 dilution in blocking buffer at room temperature. Next, both species were washed in PBS+0.1% Triton 3x15 min and the slides were mounted and prepared for imaging and cellular quantification.

Confocal microscope imaging
All imaging experiments were conducted on fixed dissected tissue mounted on slides in glycerol for both species. Imaging was performed using a Zeiss LSM880 inverted and a LSM980 upright confocal/two-photon microscope (Zeiss, Oberkochen, Germany) with ×63, 1.4 NA objective. γH2AX puncta were automatically counted using an ImageJ macro code utilizing the Analyze Particles built-in ImageJ plugin (NIH, Bethesda, Maryland, USA), within DAPI-based ROIs to focus on nuclear signal. Between 10-100 cells were analyzed in each C. andromeda rhopalia and N. vectensis. In addition to DAPI and γH2AX signal, a third channel was used to detect the fluorophore mOrange expressed in the nervous system of tg(-2.4NvElav1b:mOrangeCAAX) N. vectensis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "What a treat! This was an enjoyable paper to read. The English would benefit from a more thorough proof-read as it was\nchunky at times, still the data are important and original. Here, the authors characterize sleep in upside-down jellyfish (done\nbefore by Nath et al 2017), but now also an anemone for which sleep data was indirect. They then look for the adaptive\nvalue of sleep, with focus on DNA repair. To do so they measured a DNA damage-proxy under baseline and sleep-\ndeprivation conditions, but then flip-the-table to look at exogenous stimulation (UV light) as a trigger for sleep. Very nice.\nHere are some comments I hope will strengthen their solid paper.\nLine 13: “of sleep to the simple nerve net”\nLine 14: Please give common names before the scientific name; e.g., upside-down jellyfish and starlet sea anemone.\nLine 16-17. The clock is not the dominant regulator of sleep in the anemone as you say on line 135.\nLine 26. The earliest known jellyfish comes from the Cambrian, not Precambrian Period; see Moon et al., 2023 Proceedings\nof the Royal Society B. But in any case, Precambrian is no longer an accepted term for geological epochs. This term was\nreplaced, in 2004, by the Ediacaran Period.\nLine 28. Whether sleep loss is lethal is open to debate. The rats used in the three-weeks chronic sleep deoprivation study\nwere confounded by periodic thermoregulatory challenged from repeatedly getting wet whenever they fell asleep.\nLine 37: the presence of sleep homeostasis in nocturnal and diurnal animals does not mean there is a common evolutionary\norigin. Rather the presence of sleep and circadian regulation of sleep in basal and derived animals will suggest that…\nLine 49: “symbiotic” implies that the jellyfish is the symbiont, whereas it is the photosynthetic dinoflagellates that are the\nsymbionts of the jellyfish. Similarly, it should just be stated that the anemone studied does not contain symbiotic eukaryotes.\nLine 54: It is said that the authors meticulously define sleep in the jellyfish, and yet later we find that sleep is defined simply\nas fewer than 37 pulses per minute over 3-minutes. I would argue that this is not specific at all: is sleep occurring between\nthe pulses or also during a slow pulse? Given that sleep is a prominent behaviour characterized by restfulness (first and\nforemost) it is odd to say that jellyfish are sleeping when they still move, just less. I accept the results of the sleep\nhomeostasis study in which stimulated jellyfish pulse less when allowed to behave freely, but it would be important to know\nwhat aspect of this recovery is the actual sleep: all of it, including slow pulses? Or a subset and if so, how much?\nLine 71 and throughout the Results: Please give variance when giving means in the text.\nLine 78-79: The meaning of “average sleep” is unclear: “Applying this criterion revealed that the average sleep increased\nduring the night…”\nLine 89-100: Looking at animals in the wild is a very nice component to see whether pulses in the lab are meaningful for the\nspecies in nature. I do wish you also applied the treatment in the wild too. Why not? Just bring the lights to the animals :)\nLine 107-108: This sentence is clunky and could even be removed without loss of content.\n(Line 111. In the future, you should simulate dawn and dusk in the lab with transitions between light and dark.)\nLine 121: You have characterized sleep, not defined it. Use of the word ‘define’ is circular.\nLine 124: To look at circadian regulation, normally, one would do many photoperiodic regimes, including 12:12 LD, 24:0 LL,\n0:24 DD but also 6:6:6:6 (LDLD) and others. The latter is called forced desynchrony.\nLine 144. Please remove the term ‘sleep quality’ throughout. This term originates in humans with the assumption that sleep\ncontinuity is the most important feature of sleep. In non-human animals, we have no idea what constitutes “high-quality”\nsleep or not. For instance, the recent report of 10,000 sleep bouts per day in penguins… is that high quality sleep or not?\nUltimately, we have no idea, since the term is vague and best avoided. You could just say you measured the duration of\nsleep episodes.\nLine 154. Melatonin is only sleep promoting in diturnal species, thus it is not surprising it had little effect in crepuscular\naneneomes. It is important to note also (line 158) that melatonin has already been shown to increase sleep in jellyfish (Nath\neta l 2017). That said, you could say nocturnal flatworms given melatonin show reduced activity (Omond et al 2017).\nLine 233. I think the work Symbiodiniaceae can go since you refer to them first as dinoflagelattes.\nLine 281-283. I think these last lies would benefit from expansion, even while speculative. All life is made of DNA and much\nof that life is transparent, microscopic, sea-living and exposed to sunlight rich in UV and X-rays. I’d like to hear more on the\nauthors thoughts on where they think sleep began.\nThat's it! Thanks,\nJohn", "reviewer_2": "This paper represents the third reported instance of sleep phenomena in cnidarians, might be contributing significantly to the\nclarification of the evolutionary discourse on sleep. I assume the identification of sleep phenomena in both C. andromeda\nand N. vectensis likely demanded considerable effort. Moreover, beyond the mere reporting of sleep phenomena, the paper\ndelves into the significance of sleep, noting the observed relationship between DNA damage and sleep in cnidarians,\nechoing findings in other organisms and addressing the early significance of sleep in animals.\nHowever, given the significance of the research and its findings, meticulous attention to the paper's structure, methodology,\nanalysis, statistics, and figure presentation is warranted. Particularly, the presence of notation in the statistics section that\nmay not be readily understandable is regrettable. It is imperative that the presentation be crafted to allow readers to grasp\nthe content instantaneously and clearly. Additionally, some roughness is evident in the experimental aspect concerning the\nrelationship between DNA damage and sleep, prompting cautious consideration of the paper's claims. Below, I have\nenumerated specific concerns that stand out, suggesting that addressing these issues is essential at the very least.\nMajor:\n1. The vertical axis of Fig.1c does not start from zero, giving the impression of a clear LD cycle, but in reality, the difference in\npulses per minute is very small. The increase in activity immediately after the light period and before the dark period\nsuggests that much of the difference between day and night in Fig1.d is due to this rise in activity. This bimodal activity, also\nobserved in organisms like Drosophila, often signifies time recognition by the circadian clock. However, as the authors\nthemselves note, based on the results in Extended Data Fig. 1a-d, it seems unlikely that a circadian rhythm is controlling\nbehavior in C. andromeda. In that case, how should we interpret the bimodal pattern seen in Fig1.c? Given its likely\nsignificant impact on the difference between day and night in Fig1.d, it should be adequately explained. For instance, it\nwould be advisable to investigate what activity and sleep cycles are observed when the LD cycle is changed to 6h:6h or\n3h:3h instead of 12h:12h.\n2. In Figures 1o and p, it is not easy to find explanations regarding sample size and statistics, making it difficult to properly\nevaluate the results. To assert the validity of the results, descriptions regarding sample size and statistics should be made\nexplicit.\n3. The mentioned statement, particularly \"36.4 pulses/min, Fig. 1q\", refers to a specific data point in Figure 1q where the\naverage pulse rate during the day at sea is represented. This data point is crucial for understanding the comparison being\nmade in the sentence. Additionally, it is indeed conceivable that locomotor activity (measured in pulses/min) might be\ninfluenced not only by bell size but also by temperature variations. Therefore, it would be prudent to discuss the potential\nimpact of seawater temperature (changes) on locomotor activity.\n4. In the experiments presented in Fig.2f-k, the latency to response when stimuli were applied to individuals in an active\nstate cannot be considered as a response time. Therefore, the latency to response when the Length of quiescent bout is 0\nshould be excluded. Its inclusion would naturally result in shorter response times when the quiescent bout is less than 8\nminutes, significantly affecting the definition of sleep. Especially considering that Fig.3 also measures bout length, there is\nconcern that this inclusion may influence other results as well.\n5. In Fig2.l-q, if discussing the sleep patterns of DD and NvClk mutant, it would be beneficial to include their activity rhythms\nas well. This would enhance clarity and facilitate better understanding of the data.\n6. In the experiments presented in Fig.2r, and s, how were the close tentacle and the open tentacle distinguished from the\nimaging data? Is it possible that the difference in sleep quantity between them is due to the detection of tentacle movement\nas activity, leading to an underestimation of sleep?\n7. The statistics regarding the results in Fig.2t, and u are not readily apparent, making it difficult to evaluate the results\nproperly. While it is possible that they are mentioned somewhere, it is not immediately clear, so please ensure that they are\npresented clearly for easy understanding.\n8. In Fig.3c, e, and g, why are mean transitions presented as count per hour? In this case, it seems that representing the\nnumber of bouts per 12 hours would be the simplest and most basic way of notation.\n9. In Fig3j, kl, and m, as melatonin is known to influence circadian rhythms, it is important to show the sleep patterns when\nmelatonin is added, not only to determine its effect on sleep but also to clearly distinguish its influence from that of circadian\nrhythms. Additionally, I feel a slight discomfort with the fact that detailed analyses such as day versus night or ZT6-ZT18\nversus ZT18-ZT6 have not been conducted, which would provide a comparison with previous analyses.\n10. The figures in Fig. 4a and b are difficult to understand immediately, so please make efforts to create figures that are\neasier for readers to comprehend. It is unclear why, despite both graphs having the same vertical axis labeled as 'average\nsleep time during 12h preceding each time point,' the range of transitions in (a) is approximately 40% to 60%, while in (b) it\nremains between 15% and 30%. Additionally, the statistics regarding the results in Fig.4g and h are not readily apparent, making it difficult to evaluate the results properly. While they may be mentioned somewhere, it is not immediately clear, so please ensure that they are presented clearly for easy understanding.\n11. The figures in Fig. 4g and h exhibit a considerable number of outliers, giving the impression that the discussion is based\non very marginal differences. Therefore, I believe there might be room for improvement in the analysis method or\nexperimental conditions. If the authors' claims regarding the experimental results in Fig. 4 are accurate, providing continuous\nsleep deprivation (SD) could potentially lead to a higher accumulation of DNA damage. Conducting long-term SD\nexperiments might enhance the credibility by demonstrating clearer differences.\n12. It is difficult to determine whether DNA damage is involved in sleep based solely on the experimental method in Fig. 5.\nChanges in locomotor activity due to UV irradiation should also be included. Additionally, observing the relationship\nbetween the number of γH2AX foci on the day after UV irradiation and two days after UV irradiation and sleep would provide\na more accurate discussion of the relationship between DNA damage and sleep.\nMinor:\n1. In LL and DD conditions, periodic activity is not observed in C. andromeda (Extended Data Fig. 1a-d). Does this suggest\nthat clock genes are not present in the genome? Additionally, in LL conditions, the pulse/min is extremely high at the initial\nLD stage. Moreover, the sample size is n=6, smaller compared to others, raising some doubts about the reliability of these\nresultsPlease explain what the black triangles in Fig. 1e represent, as it is unclear.\n2. Since the term 'optimization algorithm' may not be easily understood by general readers, could you provide a more\nspecific description for clarity?\n3. In LL and DD conditions, periodic activity is not observed in C. andromeda (Extended Data Fig. 1a-d). Does this suggest\nthat clock genes are not present in the genome? Additionally, in LL conditions, the pulse/min is extremely high at the initial\nLD stage. Moreover, the sample size is n=6, smaller compared to others, raising some doubts about the reliability of these\nresults.\n4. In Extended Data Fig. 1c, it should be described as 'Circadian time (h)' under constant conditions. Additionally, Extended\nData Fig. 1f should be described as 'time of day (h)' instead of 'Zeitgeber time (h).'\n5. I do not understand the meaning of the red dashed lines in Extended Data Fig. 2f-k. It is necessary to describe them more\nclearly for better understanding.\n6. L131: The text states 'ZT0-ZT8,' indicating an 8-hour sleep deprivation period during the day, while the legend mentions\n'ZT0-ZT7.' To avoid confusion, it should be clarified which one is correct.", "reviewer_3": "This manuscript examines the link between DNA damage in neurons and sleep, extending previous observations that were\nmade in zebrafish and rodent models to more “ancient” sleepers, the jellyfish and anemone. Overall, the work is intriguing\nand is suggestive of a link between sleep and DNA damage even in these animals that lack brains. In particular, the work\ndemonstrating a sleep like state in the anemone is well done (with a few minor gaps, see minor comments) and the jellyfish\nbehavioral work is a nice confirmation and extension of Nath et al., 2017. However, although problems with the data\npresentation and analysis of the DNA damage data makes it a little difficult to evaluate fully, the results don’t seem to line up\nwith the major hypothesis that DNA damage is resolved during sleep in these species, and in fact some of the data seems to\ncontradict that notion entirely.\nThe first half of the manuscript, which describes the sleep state in jellyfish and the anemone is done to a high standard, and\nall the major expected behavioral criteria are examined (e.g. circadian period, sleep deprivation and recovery, arousal\nthreshold changes). This makes for a convincing case that the two species have sleep states, although one is diurnal and\nthe other is more crepuscular. I have only minor concerns about some of the sleep deprivation methodology (see Additional\ncomments below) and a bit more concern about the interpretation of the role of melatonin in the two species. They conclude\nthat melatonin is effective as a hypnotic in the jellyfish (which was also shown by Nath et al., 2017) but not in the anemone;\nhowever, the timing of the melatonin delivery may have missed the effective window—adding melatonin at ZT2-5 puts this at\nthe sleep trough for jellyfish but at a relatively high sleep window for the anemone. In other words, sleep could be ceilinged\nout so melatonin can’t boost sleep further, giving an artificial null result (adding melatonin to diurnal species like zebrafish\nalso only really boosts sleep during the day and not at night when sleep and natural melatonin are already high). Testing\nmelatonin later in the day, when sleep is lower, should be a straightforward way to bolster this claim.\nThe second half of the manuscript then examines the link between DNA damage and sleep, and here I find the analysis and\ninterpretation of the results to be more muddled. Firstly, the independent experimental Ns for calculating DNA damage\nappears to be each cell (Figure 4g,h), but in how many independent animals? It isn’t really appropriate to treat cells from the\nsame animal as independent experimental replicates for statistical purposes; a more appropriate analysis would be average\nfoci/cell calculated for each animal, and the N would be each animal. Thus, it is difficult from the data as presented to\nconclude if there are true differences in some of the conditions following sleep deprivation. However, even if we take these\nresults as described, some of the data appears to contradict the claim that sleep is important for resolving the DNA damage.\nFor example, Figure 2t shows that a sleep deprivation from ZT0-6 leads to a substantial sleep rebound from ZT12-24. And\nyet, In figure 4h, the number of DNA damaged foci remains elevated at ZT17 following SD, even though these animals slept a\nlot more than the controls. This seems to contradict the conclusion of lines 180-183: “These experiments show that DNA\ndamage increased during wakefulness and decreased during sleep in either diurnal or crepuscular cnidarians. Furthermore,\nthese findings suggest that alleviating the levels of DNA damage, which accumulated during wakefulness, is an\nevolutionarily conserved cellular role of sleep”\nFinally, the authors use UV light to induce DNA damage and claim that sleep is increased, thus showing a bi-directional\nrelationship between DNA damage and sleep. However, the data are shown only as box-whisker summary plots, making\nthem difficult to evaluate in terms of quality of the results. Better would be to display these as timecourse data, before and\nafter UV exposure, and for a longer window than the summary—the post UV sleep is reaching 100% for jellyfish and 60/60\nminutes for the anemone, so more convincing evidence that these animals are not permanently harmed by the UV exposure\nwould be critical here. Even better would be to show that the DNA damage eventually resolves (by the next day or\nsomething) and that the sleep recovers as well.\nTo be more convincing, it would also be good to show that DNA damage induced in another manner also increases sleep—\nfor example these creatures have a host of natural behavioral responses to UV light (see for example Lilly et al., 2024, where\nthe larval anemones shift their behavior to a planktonic swimming in direct response to UV), so showing the sleep changes\nare due to the damage and not UV-activated neural pathways would go far to convince.\nTo conclude, I am convinced that anemones (and jellyfish) have a sleep state, and this alone is a nice contribution.\nHowever, I am less convinced that the DNA-damage sleep hypothesis has been so clearly demonstrated in these animals\nbased on the current data presented.\nAdditional and minor comments:\nFigure 2—The arousal threshold data, circadian and clock mutant data are all very good. The Sleep deprivation data is good\nbut a few additional controls would be useful. For example, the rebound is shown after depriving the animals of the daytime\nsiesta, but the sleep traces show a lot of sleep in the latter half of the night as well—even more in fact. Showing SD for this\nwindow would also be good, as in other species like Drosophila is not totally clear that the siesta and night sleep are\nidentically regulated. Perhaps more important is to use the SD method at a time of relative wakefulness and control for\nrebound due to physical exhaustion of being kept awake, a standard test in the field.\nFigure 2—the SD method uses light, which may be altering circadian clock function and timing as well as sleep deprivation.\nEither a non-light method for SD should be used, or show that clocks aren’t shifted by the light pulses.\nFigure 2- methods; the details for the light stimulation SD are not provided in the methods as far as I can see, including light\nlevels, etc.\nFigure 2t—if light is being used to sleep deprive, it isn’t clear to me why there is a gap in the data—it would be better to track\nthe animals during the SD to see exactly how much sleep is lost. I can understand why there is a gap in Figure 1, where the\nSD is done physically that will interfere with the tracking, but light should be straightforward to track.\nFigure 2m—defining the windows of analysis across the dark-light transition (ZT18-6 and 6-18) is a bit odd, I would just\nbreak it down into night 2nd half and day first half to be a little clearer.\nFigure 3J—100 mM melatonin must be a typo, and it is listed as 100uM in the methods (and that could still be considered\nhigh). To convince that melatonin plays no role in the sleep of the anemone, adding melatonin at different timepoints will be\ncritical (see main comments).\nThe term “sleep quality” is too loaded a term—it implies something about a “good” night or a “bad” night of sleep but it is\nimpossible to say that fragmented sleep or consolidated sleep is of more or lesser quality without showing effects on\nperformance the next day. I would recommend “sleep structure” or “sleep architecture” or similar when looking at sleep bout\ncounts and lengths.\nFigure 4: The SD experiment for jellyfish is from ZT17-23, but for N vectensis is ZT0-8. ZT0-8 does not quite line up with\nmaximal sleep in this species ZT18-24 or ZT18-6 makes better alignment. Figure 4A, showing the rolling 12 hour pervious\nsleep time is misleading on this – the figure 4b data is high already at ZT0 and peaking at ZT6, but that means the sleep is\nmaximal in the PREVIOUS 12 hours, which is when the SD should be done for testing the effect of SD on sleep. Plotting it\nthis way is visually confusing compared to Figure 2L, where the sleep timing is easier to appreciate.\nFigure 4: If sleep is really important for repairing the DNA damage, then why does it not recover in the anaemone after SD\nfrom ZT0-8? It is confusing because they sleep more from 12-17 then they even do during the “SD” ZT0-8 window, so if this\nwere all about sleep driving the recovery, then wouldn’t we expect it to be recovered? Figure 3 shows after SD, the\nanemones sleep more, so this should have resolved the foci if the interpretation were correct. This seems counter to the\nconclusion of line 180-183: “These experiments show that DNA damage increased during wakefulness and decreased\nduring sleep in either diurnal or crepuscular cnidarians. Furthermore, these findings suggest that alleviating the levels of\nDNA damage, which accumulated during wakefulness, is an evolutionarily conserved cellular role of sleep.\nFigure 4: The data is not showing independent Ns…. The legend says n=291 cells, but how many animals? One cannot use\na cell in the same animal as an independent measure for the stats unless you control for them as dependent on each other.\nWhat stats are being done in Figure 4? The legend doesn’t say. It says Tukey, but that is a post-hoc test. It also lists a lot of\ncross comparisons, how is this being controlled for multiple comparisons? There are really two-factors, SD/no SD and time,\nso a 2-factor analysis design would be clearer.\nFigure 5: the post-UV sleep data is too summarized. IT would be better to see the time course of the behavior afterwards.\nAlso, the recovery on the following day—sleep is being driven so high after UV are you sure they aren’t permanently\nharmed, i.e. sleeping a lot but actually damaged?\n5e and 5f are not presented in the same manner, one show ZT8-12 and ZT12-0, the other says Pre: Post, but over what\nwindows constitute “pre” and what consistutes “post”? The legend says 1hr pre and 1 hr post, but that seems like too short a\ntime? What would be better would be to induce UV damage, see how long the damage takes to be resolved and correlate that\ntimecourse with the sleep timecourses— i.e. once the damage is resolved, does sleep end?\nAnother method to induce DNA damage? These species are quite sensitive behaviorally to UV light directly, for example the\nanemone in the larval state are induced by UV into plaktonic swimming (Lilly et al 2024). Can you distinguish between these\ntwo models UV DNA damage sleep vs. UV sleep (independent of the DNA damage?\nThe evolutionary claim seems too strong: “Altogether, these inter-species comparative findings suggest that environmental\nstressors, such as UV radiation, which can induce DNA damage and cellular stress, drove the evolutionary origin of sleep in\nthe day or crepuscular active cnidarians.” What exactly is the evidence that this was the “driver” of evolution? Showing that\nDNA damage induces sleep and sleep resolves DNA damage in these species isn’t quite the same as saying that this\nrelationship is what “drives” evolution of sleep."}, "author_response": "We thank the reviewer for their kind words and strong support. As requested, we have performed\nadditional English language editing.\nComment:\nLine 13: “of sleep to the simple nerve net”\nResponse:\nThe typo has been corrected in Page 1, Line 12.\n” Considering the survival risks, why sleep evolved in basal lineages and what essential benefits\nit provides to the simple nerve net of nocturnal and diurnal invertebrates remain elusive”.\nComment:\nLine 14: Please give common names before the scientific name; e.g., upside-down jellyfish and\nstarlet sea anemone.\nResponse:\nThe common names have been added to the abstract, Page 1, Line 14.\n“We used behavioral criteria to empirically define sleep in the upside-down jellyfish Cassiopea\nandromeda and the starlet sea anemone Nematostella vectensis.”\nComment:\nLine 16-17. The clock is not the dominant regulator of sleep in the anemone as you say on line\n135.\nResponse:\nThis sentence has been revised in Abstract, Page 1, Line 17.\n“In contrast, both the circadian clock and homeostatic processes regulated sleep in N. vectensis,\nwhich increased sleep at dawn.”\nComment:\nLine 26. The earliest known jellyfish comes from the Cambrian, not Precambrian Period; see Moon\net al., 2023 Proceedings of the Royal Society B. But in any case, Precambrian is no longer an\naccepted term for geological epochs. This term was replaced, in 2004, by the Ediacaran Period.\nResponse:\nAs noted by the reviewer, the term referring to geological epochs has been replaced, and the\nrelevant citation has been added in Page 1, Line 28.\n“...suggesting that sleep is a Cambrian metazoan adaptation1–6.”\nComment:\nLine 28. Whether sleep loss is lethal is open to debate. The rats used in the three-weeks chronic\nsleep deprivation study were confounded by periodic thermoregulatory challenged from\nrepeatedly getting wet whenever they fell asleep.\nResponse:\nThe description of the effect of sleep loss on health has been revised in Page 1, Line 29.\n“Sleep deprivation (SD) and disturbances are associated with increased risk for health decline in\nbilaterian animals”.\nComment:\nLine 37: the presence of sleep homeostasis in nocturnal and diurnal animals does not mean there\nis a common evolutionary origin. Rather the presence of sleep and circadian regulation of sleep\nin basal and derived animals will suggest that…\nResponse:\nThis sentence has been revised in Page 2, Line 38.\n“The presence of sleep and these regulatory processes in both basal and derived nocturnal and\ndiurnal species suggests a common evolutionary origin of sleep regulation16,17”\nComment:\nLine 49: “symbiotic” implies that the jellyfish is the symbiont, whereas it is the photosynthetic\ndinoflagellates that are the symbionts of the jellyfish. Similarly, it should just be stated that the\nanemone studied does not contain symbiotic eukaryotes.\nResponse:\nThis content has been added to the Introduction in Page 2, Line 53.\n“The jellyfish Cassiopea, which lives in obligates symbiosis with photosynthetic dinoflagellates,\nexhibits rhythmic daily activity that peaks during the day. In contrast, the sea anemone\nNematostella vectensis (N. vectensis), which lacks symbiotic eukaryotes, is considered nocturnal”.\nComment:\nLine 54: It is said that the authors meticulously define sleep in the jellyfish, and yet later we find\nthat sleep is defined simply as fewer than 37 pulses per minute over 3-minutes. I would argue that\nthis is not specific at all: is sleep occurring between the pulses or also during a slow pulse? Given\nthat sleep is a prominent behaviour characterized by restfulness (first and foremost) it is odd to\nsay that jellyfish are sleeping when they still move, just less. I accept the results of the sleep\nhomeostasis study in which stimulated jellyfish pulse less when allowed to behave freely, but it\nwould be important to know what aspect of this recovery is the actual sleep: all of it, including slow\npulses? Or a subset and if so, how much?\nResponse:\nSleep in mobile animals is well-documented in migrating birds and marine mammals, which exhibit\nunihemispheric sleep1. Even humans move during sleep. Our raw data show that Cassiopea rarely\npauses for more than one minute; nevertheless, it consistently reduces activity during sleep. By\nevaluating all behavioral sleep criteria, including an increased arousal threshold, we defined sleep\nin Cassiopea as fewer than 37 pulses per minute over a three-minute period. This threshold\ncaptures a state of reduced activity and aligns with the recovery behavior observed following sleep\ndeprivation. We consider sleep to encompass the entire recovery period below this threshold,\nincluding both behavioral pulses and pauses. This is consistent with sleep definitions in basal\nanimals, where molecular and physiological markers are often limited. We agree with the reviewer\nthat characterizing potential sleep stages (pulses vs. pauses?) in Cassiopea is a promising\nresearch direction. We believe that this study lays essential groundwork for the development of\nmolecular and physiological tools to define sleep architecture at both the cellular and behavioral\nlevels in cnidarians.\nTo avoid the expectation that we would define sleep stages, we have removed the word\n“meticulously”, Page 2, Line 59. The implications for future research on sleep architecture are now\ndiscussed in the Discussion section in Page 10, Line 337.\nComment:\nLine 71 and throughout the Results: please give variance when giving means in the text.\nResponse:\nWe agree that reporting a measure of dispersion with every mean improves clarity. Accordingly,\nwe have added “mean ± SEM (n = …)” after each numerical mean in the Results section and,\nwhere appropriate, in the figure legends. For example, in the results section page 03, line 77:\n“locomotor activity consistently decreased at night, with a mean pulsation rate of 36.4 ± 1.6 pulses\nmin ¹ (mean ± SEM, n = 30) during the day and 31.9 ± 1.4 pulses min ¹ (n = 30) at night (Fig.\n1c,d).”\n⁻ ⁻\nComment:\nLine 78-79: The meaning of “average sleep” is unclear: “Applying this criterion revealed that the\naverage sleep increased during the night…”\nResponse:\nWe intended to refer to sleep time. The sentence has been revised to: “Applying this criterion\nrevealed that the average sleep time increased during the night”. Page 03, Line 86.\nComment:\nLine 89-100: Looking at animals in the wild is a very nice component to see whether pulses in the\nlab are meaningful for the species in nature. I do wish you also applied the treatment in the wild\ntoo. Why not? Just bring the lights to the animals :)\nResponse:\nCassiopea lives in open seawater, and conducting arousal threshold experiments requires video\nrecordings under controlled conditions to ensure consistent measurements. Performing such\nexperiments in the wild is technically challenging due to environmental variables such as the\npresence of other animals, light fluctuations, sediment, and water turbidity. Additionally, locating\nand maintaining the animal within the experimental area is non-trivial. While technically feasible,\nimplementing these experiments in the field would require substantial additional infrastructure and\nresources. Importantly, we found that sleep patterns observed in the laboratory closely mirror\nthose in the wild. This similarity supports the physiological relevance of the arousal threshold\nexperiments conducted in the lab and suggests that lab-based findings are applicable to natural\nsettings.\nComment:\nLine 107-108: This sentence is clunky and could even be removed without loss of content.\nResponse:\nThese sentences have been combined and revised; the updated version now appears on Page 4,\nLine 124.\n“In contrast to C. andromeda, under LD, N. vectensis showed rhythmic locomotor activity (Fig.\n2c), increased between ZT6-ZT18 and decreased between ZT18-ZT6 (Fig. 2d).”\nComment:\nLine 111. In the future, you should simulate dawn and dusk in the lab with transitions between\nlight and dark.\nResponse:\nWe agree with the reviewer’s comment and plan to implement this in future studies. Indeed, the\nanimals are highly sensitive to sleep–wake transitions. To better mimic their natural habitat, we\nwill employ a lighting system that gradually transitions between light and dark conditions. This\napproach will allow us to monitor sleep while simulating the natural light environment. Based on\nthe consistent results observed in our lab and field experiments with jellyfish, we anticipate that\nsea anemones will exhibit similar behavior under both light conditions—excluding the one-hour\nperiod immediately following light–dark transitions.\nComment:\nLine 121: You have characterized sleep, not defined it. Use of the word ‘define’ is circular.\nResponse:\nAs suggested by the reviewer, this sentence has been revised in Page 4, Line 116 to:\n“…sleep was not characterized in sea anemones, including N. vectensis.\nComment:\nLine 124: To look at circadian regulation, normally, one would do many photoperiodic regimes,\nincluding 12:12 LD, 24:0 LL, 0:24 DD but also 6:6:6:6 (LDLD) and others. The latter is called forced\ndesynchrony.\nResponse:\nIn the original version of the manuscript, we had already conducted experiments under LD, LL,\nand DD conditions. We agree with the reviewer that introducing a desynchrony experiment (LDLD;\n6L:6D:6L:6D) could provide deeper insight into how light and the circadian clock interact to\nregulate locomotor activity and sleep. As suggested, we therefore included new data in the revised\nmanuscript, examining the behavior of C. andromeda, as well as wild-type (WT) and NvClk∆/∆\nmutant N. vectensis, under LDLD conditions:\n- In C. andromeda, locomotor activity displayed a ~12-hour rhythm in LDLD (Extended Data\nFig. 1e-g), indicating that behavior is primarily driven by the light/dark cycle rather than the\ncircadian clock. This interpretation is consistent with the arrhythmic activity observed under\nboth DD and LL conditions (Extended Data Fig. 1a-d).\n- In contrast, WT N. vectensis maintained a robust ~24-hour locomotor rhythm in LDLD\n(Extended Data Fig. 3e,f), suggesting circadian control. This conclusion is further\nsupported by two observations: (1) NvClk∆/∆ mutants lost the ~24-hour rhythm under LDLD\n(Extended Data Fig. 3g-h), and (2) WT animals retained ~24-hour rhythmicity under both\nDD and LL (Extended Data Fig. 3a-d; Aguillon et al. 2024).\n- Interestingly, LDLD also induced distinct sleep/wake patterns: ~12-hour cycles in WT N.\nvectensis and ~6-hour cycles in the NvClk∆/∆ mutants. These findings indicate that while\nthe circadian clock is the primary regulator of sleep, as evidenced by the ~24-hour sleep\nrhythm observed under DD (Fig. 2l-n), light/dark transitions also play a modulatory role in\nsleep regulation in N. vectensis.\nIn the revised version of the manuscript, the LDLD results are presented in the Results section\nand illustrated in Extended Data Fig. 1 and Extended Data Fig. 2.\nPage 3, Line 89: “However, rhythmic locomotor activity was abolished under constant darkness\n(DD) and robustly dampened under constant light (LL) conditions (Extended Data Fig. 1a-d).\nMoreover, under forced desynchrony conditions (6h L:6h D:6h L:6h D, LDLD), the animals showed\na 12.1h rhythm of locomotor activity (Extended Data Fig. 1e-g). These findings showed that sleep\nis primarily driven by the light/dark cycle in C. andromeda.”\nPage 5, Line 144: “To further differentiate between light- and circadian-regulation of sleep, we\nmonitored sleep under forced desynchrony (LDLD, as in C. andromeda). While the rhythmic period\nof locomotor activity was 22.2h and controlled by the clock, sleep period was 11.7h (Extended\nData Fig. 3e, f, i, j). These results suggest that although sleep is primarily regulated by the\ncircadian clock it can also be modulated by light in N. vectensis. To understand the hierarchy of\nlight- and intrinsic clock-regulation of sleep, we monitored sleep in NvClk∆ mutant34 (NvClk∆/∆) N.\nvectensis. NvClk∆/∆ showed bimodal sleep/wake rhythms (periods of 12.25h and 25.24h) under\nLD and this rhythm was abolished under DD (Fig. 2o-q, Extended Data Fig. 3c, d). Under LDLD,\nthe sleep/wake cycle followed the 6h alternating light/dark rhythm (Extended Data Fig.3k, l). These\nfindings demonstrate that the transcription factor NvClk is essential for maintaining clock-\ncontrolled sleep in N. vectensis”\nExtended Data Fig.1a-g\nExtended Data Fig.2e-l\nComment:\nLine 144. Please remove the term ‘sleep quality’ throughout. This term originates in humans with\nthe assumption that sleep continuity is the most important feature of sleep. In non-human animals,\nwe have no idea what constitutes “high-quality” sleep or not. For instance, the recent report of\n10,000 sleep bouts per day in penguins… is that high quality sleep or not? Ultimately, we have no\nidea, since the term is vague and best avoided. You could just say you measured the duration of\nsleep episodes.\nResponse:\nAs suggested by the reviewer, we have removed the term “sleep quality” and replaced it with\n“sleep architecture” throughout the manuscript.\nComment:\nLine 154. Melatonin is only sleep promoting in diurnal species, thus it is not surprising it had little\neffect in crepuscular anemones. It is important to note also (line 158) that melatonin has already\nbeen shown to increase sleep in jellyfish. That said, you could say nocturnal flatworms given\nmelatonin show reduced activity (Omond et al 2017).\nResponse:\nThe differential effect of melatonin on sleep in nocturnal and diurnal animals remains controversial\nand inconclusive. Melatonin is a circadian marker of night and is presumed to promote sleep in\ndiurnal animals and wakefulness in nocturnal animals. However, studies have shown that\nmelatonin can promote sleep even in nocturnal mice during the night2. As noted by the reviewer,\nand in support of this observation, melatonin treatment was found to reduce nighttime activity in\nnocturnal flatworms3.\nIn the first version of the manuscript, we assessed the effect of melatonin at a single time point. In\nthe revised version, we expanded these assays to evaluate the effect of exogenous melatonin\nduring both day and night in both species. In the diurnal C. andromeda, melatonin increased sleep\nonly during the day (Fig.3p-r), while in the crepuscular N. vectensis, it promoted sleep only during\nthe night (Fig.3m-o). In both species, melatonin had no observable effect during the natural sleep\nperiod, possibly because endogenous sleep masked the pharmacological impact (Fig.3j-l and s-\nu). These findings demonstrate a conserved sleep-promoting function of exogenous melatonin,\nirrespective of chronotype, and are consistent with previous observations in rats and flatworms.\nThe new melatonin-related data are now described in the Results, Methods, and Discussion\nsections of the revised manuscript. Page 06, Line 187: “We tested the effect of melatonin on sleep\nin the diurnal C. andromeda and crepuscular N. vectensis. While melatonin treatment did not affect\nsleep during the rest periods of N. vectensis (ZT3) and C. andromeda (ZT13) (Fig. 3j-l, s-u), sleep\ntime, sleep bout duration, and the number of sleep bouts increased during the active phase in both\nN. vectensis (ZT13) and C. andromeda (ZT3) (Fig. 3m-o, p-r). These results suggest an\nevolutionarily conserved role of melatonin in sleep regulation across animals with different\nchronotypes” Page 09, Line 298: “…despite their opposing chronotypes, melatonin treatment\npromoted sleep in both species during their respective active phases, suggesting a conserved role\nfor this hormone in sleep modulation.” Page 22, Line 646: “Both species received either 100 μM\nmelatonin (diluted in ethanol) or an ethanol control at ZT3 and ZT13, corresponding to their\nrespective periods of wakefulness and activity.”\nThese new results are now shown in Fig.3j-u:\nComment:\nLine 233. I think the work Symbiodiniaceae can go since you refer to them first as dinoflagelattes.\nResponse:\nAs recommended by the reviewer, the word “Symbiodiniaceae” has been deleted in page 09, line\n287: “The light-dependence regulation may be attributed to the C. andromeda symbiosis with\nendosymbiotic dinoflagellates .”\nComment:\nLine 281-283. I think these last lines would benefit from expansion, even while speculative. All life\nis made of DNA and much of that life is transparent, microscopic, sea-living and exposed to\nsunlight rich in UV and X-rays. I’d like to hear more on the authors thoughts on where they think\nsleep began.\nResponse:\nFollowing the reviewer’s suggestion, we have expanded the Discussion, which now reads as\nfollows (page 11, line 350):\n“Moreover, considering that the interplay between the circadian clock and genomic stability has\nbeen shown in non-neuronal mammalian cultured cells, fungi and cyanobacteria78–81, sleep-like\nstates may have preceded the evolution of neurons, originally evolving to support synchronized\nand consolidate periods of cellular maintenance.”\nReviewer #2 (Remarks to the Author):\nThis paper represents the third reported instance of sleep phenomena in cnidarians, might be\ncontributing significantly to the clarification of the evolutionary discourse on sleep. I assume the\nidentification of sleep phenomena in both C. andromeda and N. vectensis likely demanded\nconsiderable effort. Moreover, beyond the mere reporting of sleep phenomena, the paper\ndelves into the significance of sleep, noting the observed relationship between DNA damage and sleep\nin cnidarians, echoing findings in other organisms and addressing the early significance of sleep\nin animals.\nHowever, given the significance of the research and its findings, meticulous attention to the paper's\nstructure, methodology, analysis, statistics, and figure presentation is warranted. Particularly, the\npresence of notation in the statistics section that may not be readily understandable is regrettable.\nIt is imperative that the presentation be crafted to allow readers to grasp the content\ninstantaneously and clearly. Additionally, some roughness is evident in the experimental aspect\nconcerning the relationship between DNA damage and sleep, prompting cautious consideration\nof the paper's claims. Below, I have enumerated specific concerns that stand out, suggesting that\naddressing these issues is essential at the very least.\nMajor:\nComment:\n1. The vertical axis of Fig.1c does not start from zero, giving the impression of a clear LD cycle,\nbut in reality, the difference in pulses per minute is very small. The increase in activity immediately\nafter the light period and before the dark period suggests that much of the difference between day\nand night in Fig1.d is due to this rise in activity. This bimodal activity, also observed in organisms\nlike Drosophila, often signifies time recognition by the circadian clock. However, as the authors\nthemselves note, based on the results in Extended Data Fig. 1a-d, it seems unlikely that a\ncircadian rhythm is controlling behavior in C. andromeda. In that case, how should we interpret\nthe bimodal pattern seen in Fig1.c? Given its likely significant impact on the difference between\nday and night in Fig1.d, it should be adequately explained. For instance, it would be advisable to\ninvestigate what activity and sleep cycles are observed when the LD cycle is changed to 6h:6h or\n3h:3h instead of 12h:12h.\nResponse:\nWe thank the reviewer for this insightful mechanistic comment. In Fig.1c, the average sleep time\nof the population never reached zero; therefore, we displayed the relevant range of data on the Y-\naxis. We agree that the difference in pulsation rate between light and dark periods is relatively\nsmall. However, this difference is consistently significant in both laboratory and field settings—\neven under gradual light/dark transitions—and is highly relevant to the animal’s physiology, as\ndemonstrated in other figures. As noted by the reviewer, monitoring behavior under 12 h light:12\nh dark (LD), constant darkness (DD), and constant light (LL) suggests that the bimodal activity\nobserved in C. andromeda is primarily light-driven. Additionally, our sleep deprivation experiments\nindicate that sleep regulation involves a homeostatic component. To further support this\nmechanism, as suggested by the reviewer, we conducted a 6L:6D:6L:6D forced desynchrony\nexperiment in the revised manuscript. The results clearly show that behavior is primarily regulated\nby the light/dark cycle. This new data is presented in Extended Data Fig. 1e–g.\nThis data has been integrated into the Results section (page 06, line 144): “To further differentiate\nbetween light- and circadian-regulation of sleep, we monitored sleep under forced desynchrony\n(LDLD, as in C. andromeda). While the rhythmic period of locomotor activity was 22.2h and\ncontrolled by the clock, sleep period was 11.7h (Extended Data Fig. 3e, f, i, j). These results\nsuggest that although sleep is primarily regulated by the circadian clock it can also be modulated\nby light in N. vectensis. To understand the hierarchy of light- and intrinsic clock-regulation of sleep,\nwe monitored sleep in NvClk∆ mutant34 (NvClk∆/∆) N. vectensis. NvClk∆/∆ showed bimodal\nsleep/wake rhythms (periods of 12.25h and 25.24h) under LD and this rhythm was abolished\nunder DD (Fig. 2o-q, Extended Data Fig. 3c, d). Under LDLD, the sleep/wake cycle followed the\n6h alternating light/dark rhythm (Extended Data Fig.3k, l). These findings demonstrate that the\ntranscription factor NvClk is essential for maintaining clock-controlled sleep in N. vectensis”. In\naddition, we add this sentence to the discussion (page 09, line 285): “Light and homeostatic drive\nwere the predominant sleep regulatory processes in C. andromeda, while homeostatic drive and\nthe circadian clock control sleep in N. vectensis.”.\nComment:\n2. In Figures 1o and p, it is not easy to find explanations regarding sample size and statistics,\nmaking it difficult to properly evaluate the results. To assert the validity of the results, descriptions\nregarding sample size and statistics should be made explicit.\nResponse:\nWe thank the reviewer for the insightful comment. In the revised manuscript, we have explicitly\nadded the sample size and statistical information as suggested. Specifically, we have updated the\nfigure legends to make these details clear and easily accessible. For example: Fig.1o and p, page\n25, line 716: “(o) Sleep percentages in control (Ctl; n=14) and sleep-deprived (SD; n=18) animals\nunder LD. (p) Average percentage of sleep in Ctl and SD animals during the day and night pre-\nSD, and during the day post-SD.” And page 25 line 725: “For panel (p): Tukey HSD (post-hoc\nanalysis following ANOVA), letters indicate significant differences (p < 0.05).”\nComment:\n3. The mentioned statement, particularly \"36.4 pulses/min, Fig. 1q\", refers to a specific data point\nin Figure 1q where the average pulse rate during the day at sea is represented. This data point is\ncrucial for understanding the comparison being made in the sentence. Additionally, it is indeed\nconceivable that locomotor activity (measured in pulses/min) might be influenced not only by bell\nsize but also by temperature variations. Therefore, it would be prudent to discuss the potential\nimpact of seawater temperature (changes) on locomotor activity.\nResponse:\nThe value of 36.4 pulses per minute cited in the manuscript represents the average daytime pulse\nrate observed in the laboratory (Fig. 1d), while the threshold of 37 pulses per minute was set to\ndistinguish between sleep and wake states (Fig. 1f–h). Given the close similarity between these\nvalues, we approximated the sleep threshold based on the laboratory-derived average to estimate\nsleep time in the field across animals of varying sizes (Fig. 1q–s). This clarification has been added\nto the Results section (Page 04, Line 106): “Notably, the pulsation threshold used to detect sleep\nunder controlled laboratory conditions (37 pulses/min, Fig. 1f-h) closely matched the average\ndaytime pulse rate observed in the lab (36.4 pulses/min, Fig. 1d). Given this similarity, we\napproximated the sleep threshold in the field by the average daytime pulse rate, adjusted\naccording to the bell diameter, as a proxy to differentiate sleeping and awake animals.”\nDespite the environmental differences between the lab (Bar-Ilan University, Israel; 24 °C, 35 ppt\nsalinity) and the field (Key Largo, FL, USA; 26.8 °C, 31.5 ppt salinity), sleep patterns in C.\nandromeda were consistent between day and night. The animals predominantly slept during the\nnight. Intriguingly, under both lab and field conditions, we observed a siesta-like sleep phase\naround midday (Fig. 1i, s). This consistency suggests that while seawater temperature and salinity\nmay influence the absolute level of locomotor activity, the overall sleep pattern is robust and\nprimarily driven by the light/dark cycle8.\nTo emphasize this point, we added the following sentence to the revised manuscript (Page 09,\nLine 281): “These animals exhibited similar sleep duration and architecture as in controlled\nlaboratory conditions, including short midday naps despite differences in salinity and temperature.”\nComment:\n4. In the experiments presented in Fig.2f-k, the latency to response when stimuli were applied to\nindividuals in an active state cannot be considered as a response time. Therefore, the latency to\nresponse when the Length of quiescent bout is 0 should be excluded. Its inclusion would naturally\nresult in shorter response times when the quiescent bout is less than 8 minutes, significantly\naffecting the definition of sleep. Especially considering that Fig.3 also measures bout length, there\nis concern that this inclusion may influence other results as well.\nResponse:\nWe thank the reviewer for pointing this out. Upon re-examining our analysis, we identified an error\nintroduced by the use of the jitter function in R. In the revised version of the manuscript, we have\ncorrected this error, which led to slight changes in Fig.2f–k.\nOriginal Fig.2f–k (initial submission)\nFig.2f-k in the revised version:\nFurthermore, as recommended by the reviewer, we excluded animals that were active at the time\nof stimulus onset. This exclusion did not alter the threshold value of 8 minutes used to identify\nsleeping animals.\nComment:\n5. In Fig2.l-q, if discussing the sleep patterns of DD and NvClk mutant, it would be beneficial to\ninclude their activity rhythms as well. This would enhance clarity and facilitate better understanding\nof the data.\nResponse:\nWe agree with the reviewer that including activity rhythms alongside the sleep data would enhance\nthe clarity and interpretation of the results. In the revised version of the manuscript, we have added\nthe locomotor activity rhythms of both WT and NvClk∆/∆ N. vectensis (Extended Data Fig. 3a-d),\nwhich now complement the sleep data presented in Fig. 2i-q.\nTo describe these data, we added the following text to the Results section (Page 05, Line 139):\n“Similarly, under DD the rhythmic sleep period was 22.5h (Fig. 2l-n, Extended Data Fig. 3a, b),\nsuggesting that sleep is regulated by the circadian clock in N. vectensis. To further differentiate\nbetween light- and circadian regulation of sleep, we monitored sleep under forced desynchrony\n(LDLD). While the rhythmic period of locomotor activity was 22.2h and controlled by the clock,\nsleep period was 11.2h (Extended Data Fig. 3e, f, i, j).”\n“NvClk∆/∆ showed bimodal sleep/wake rhythms (periods of 12.25h and 25.24h) under LD and this\nrhythm was abolished under DD (Fig. 2o-q, Extended Data Fig. 3c, d).”\nExtended Data Fig. 3A-D:\nComment:\n6. In the experiments presented in Fig.2r, and s, how were the close tentacle and the open tentacle\ndistinguished from the imaging data? Is it possible that the difference in sleep quantity between\nthem is due to the detection of tentacle movement as activity, leading to an underestimation of\nsleep?\nResponse:\nThe open or closed tentacle state was determined manually. Animals were categorized as ‘open’\nif they displayed more than two visible tentacles. Notably, while open and closed tentacle postures\nare associated with wake and sleep states, respectively, they are not definitive indicators of\nbehavioral state; for example, animals with closed tentacles can be awake, and those with open\ntentacles can be asleep. To quantify activity, we assessed the movement of the entire body rather\nthan relying solely on tentacle or body column motion, similar to approaches used in Hydra. Our\ndata indicate that tentacle posture alone is not a reliable determinant of sleep (Fig. 2r, s).\nNonetheless, in future studies, a more detailed analysis of body part-specific movements may\nhelp refine the characterization of sleep and potentially uncover sleep stages.\nTo clarify the methodology for defining the open versus closed tentacle state, we added the\nfollowing text to the Methods section (Page 22, Line 638): “Open or closed posture was manually\nannotated from a single video frame. Animals were categorized based on the number of tentacles\ndeployed outside of their body. Animals that showed at least two visible tentacles were classified\nas 'open’.\"\nComment:\n7. The statistics regarding the results in Fig.2t, and u are not readily apparent, making it difficult to\nevaluate the results properly. While it is possible that they are mentioned somewhere, it is not\nimmediately clear, so please ensure that they are presented clearly for easy understanding.\nResponse:\nAs requested, we clarified the statistical details. In the revised version of the manuscript, Fig. 2t\nand Fig. 2v now include visual indicators highlighting the time windows selected for the\nsubsequent statistical analyses shown in Fig. 2u and Fig. 2w. The statistical details are explicitly\nprovided in the legend of Fig. 2, page 26 line 755: (t) Average sleep time in control (Ctl: n=22) and\nsleep-deprived (SD: n=24) animals during the day and night before and after SD (ZT20-ZT3). The\ntime window (ZT10-16) used to compare post-SD sleep time in panel (u) is indicated above the\ncurves. (u) Average sleep time in Ctl and SD animals at ZT10-16 (Ctl: n=22, SD: n=24). (v)\nAverage sleep time in control (Ctl: n=24) and sleep-deprived (SD: n=28) animals before and after\nSD (ZT8-ZT15), serving as a mirror control for SD experiment in panel (t). The time window (ZT8-\nZT15) used for comparison in panel (w) is indicated above the curve. (w) Average sleep time in\nCtl and SD animals at ZT8-ZT15 (Ctl: n=24, SD: n=28).…For panels (h), (k), (t), (u), (w): Mann\nWhitney Wilcoxon... ****p<0.0001” In addition, we have revised the Results section accordingly\n(Page 05, Line 161): “To test whether homeostatic process regulates sleep, we sleep-deprived\nthe N. vectensis for 8h during sleep period (ZT20-ZT3) using gradual and gentle water exchange\nonce per hour (Extended Data Movie 4). Following SD, sleep-deprived animals slept 1.24-fold\nmore than control animals (Fig. 2t, u), indicating a homeostatic sleep drive. In contrast, control SD\nduring the activity period (ZT8-ZT15) did not induce a sleep rebound (Fig. 2v, w).”\nComment:\n8. In Fig.3c, e, and g, why are mean transitions presented as count per hour? In this case, it seems\nthat representing the number of bouts per 12 hours would be the simplest and most basic way of\nnotation.\nResponse:\nWe chose to present transitions as counts per hour to maintain consistency with the behavioral\nanalyses shown in Figures 1 and 2. However, we agree with the reviewer that presenting the\nnumber of bouts per 12-hour period offers better clarity for the reader. Accordingly, we reanalyzed\nthe data and quantified the number of sleep bouts and state transitions as follows:\n- During the analysis window, any sleep bout already ongoing at the end of the window was\ncounted as half a bout.\n- In contrast, a complete bout—one that both began and ended within the time window—\nwas counted in full.\n- This adjustment was necessary because a complete bout consists of two transitions,\nwhereas an incomplete bout includes only one.\n- This weighting method allows us to preserve information on sleep fragmentation while\nconverting the data into sleep bout counts.\nThe revised data are now presented in Figure 3 and are described in the Results section (Page\n06, Line 173): “To determine sleep patterns, we quantified the length and number of sleep bouts\nduring the sleep and wake periods of each species. We found longer sleep bout durations and a\nhigher number of sleep bouts at ZT18-6 compared to ZT6-18, under LD, in both WT (Fig. 3b, c)\nand NvClkD/D (Fig. 3d, e) N. vectensis. Similarly, under LD conditions, sleep bout length and the\nnumber of sleep bouts increased during the night (ZT12-0) in the diurnal C. andromeda (Fig. 3f,\ng). Inter-species comparison of sleep architecture revealed that although total sleep time was\nsimilar (Fig. 3a), the average sleep bout length was shorter by 2.8min (Fig. 3h), and the mean\nnumber of sleep bouts was approximately two-fold higher (Fig. 3i) in C. andromeda compared to\nN. vectensis. These results show that, like humans, both cnidarian species spend approximately\none-third of their lives sleeping (Fig. 3a). However, sleep is more consolidated in N. vectensis, i.e.,\nlonger and less fragmented sleep bouts, compared to C. andromeda.”\nFig. 3a-i\nComment:\n9. In Fig3j, kl, and m, as melatonin is known to influence circadian rhythms, it is important to show\nthe sleep patterns when melatonin is added, not only to determine its effect on sleep but also to\nclearly distinguish its influence from that of circadian rhythms. Additionally, I feel a slight discomfort\nwith the fact that detailed analyses such as day versus night or ZT6-ZT18 versus ZT18-ZT6 have\nnot been conducted, which would provide a comparison with previous analyses.\nResponse:\nIn response to the comments of this and other reviewers, we extended the melatonin treatments\nto include an additional time window. A limitation of long-term pharmacological experiments is that\nthe removal of melatonin—typically achieved through water exchange—can itself stimulate animal\nbehavior, as demonstrated in our sleep deprivation protocol. This stimulation may mask the effects\nof the hormone, complicating interpretation. To detect the long-term circadian effect, a different\nbehavioral set up is required including a continuous water flow alternative method of sleep\ndetection. Nevertheless, as suggested by the reviewer and to assess the differential effects of\nmelatonin during the day and night, we quantified sleep time and architecture during the active\n(ZT3 for C. andromeda; ZT13 for N. vectensis) and sleep (ZT13 for C. andromeda; ZT3 for N.\nvectensis) periods. The results showed that melatonin promotes sleep during the active period in\nboth crepuscular and diurnal cnidarians, supporting the notion that melatonin promotes sleep\nregardless of the animal’s chronotype. Similar effects have also been reported in other diurnal and\nnocturnal species2,3,9.\nThe new melatonin-related data are now described in the Results, Methods, and Discussion\nsections of the revised manuscript. Page 06, Line 187: “We tested the effect of melatonin on sleep\nin the diurnal C. andromeda and crepuscular N. vectensis. While melatonin treatment did not affect\nsleep during the rest periods of N. vectensis (ZT3) and C. andromeda (ZT13) (Fig. 3j-l, s-u), sleep\ntime, sleep bout duration, and the number of sleep bouts increased during the active phase in both\nN. vectensis (ZT13) and C. andromeda (ZT3) (Fig. 3m-o, p-r). These results suggest an\nevolutionarily conserved role of melatonin in sleep regulation across animals with different\nchronotypes” Page 09, Line 298: “…despite their opposing chronotypes, melatonin treatment\npromoted sleep in both species during their respective active phases, suggesting a conserved role\nfor this hormone in sleep modulation.” Page 22, Line 646: “Both species received either 100 μM\nmelatonin (diluted in ethanol) or an ethanol control at ZT3 and ZT13, corresponding to their\nrespective periods of wakefulness and activity.”\nComment:\n10. The figures in Fig. 4a and b are difficult to understand immediately, so please make efforts to\ncreate figures that are easier for readers to comprehend. It is unclear why, despite both graphs\nhaving the same vertical axis labeled as 'average sleep time during 12h preceding each time\npoint,' the range of transitions in (a) is approximately 40% to 60%, while in (b) it remains between\n15% and 30%.\nResponse:\nThank you for the helpful feedback. To improve clarity and make the charts in Fig. 4c and 4d\neasier to interpret, we have added average sleep time charts (Fig. 4a and 4b) above the\ncorresponding sleep accumulation charts. The data presented in Fig. 4c and 4d are derived\ndirectly from the sleep time data shown in Fig. 4a and 4b, respectively.\nThe differences in the Y-axis between Fig. 4c and Fig. 4d reflect the distinct units used to quantify\nsleep: percentage for C. andromeda and hours for N. vectensis. This dataset-specific scaling\nfacilitates the visual identification of transitions between sleep and wake states.\nThese changes are now described in the Results section, page 07, Line 201: “We hypothesized\nthat DNA damage would peak and decline following extended wake and sleep, respectively. To\nidentify the time points of maximal and minimal sleep pressure, we calculated the average sleep\ntime over the past 12h that preceded each time point. Both C. andromeda and N. vectensis were\nsampled at the end of their wake (ZT11 and ZT17) and sleep (ZT23 and ZT8) phases (Fig. 4a, b),\nwhere wake and sleep accumulation peaked (Fig. 4c, d).”\nIn addition, we modified the units in the Y axis and presented it in Fig. 4:\nComment:\nAdditionally, the statistics regarding the results in Fig.4g and h are not readily apparent, making it\ndifficult to evaluate the results properly. While they may be mentioned somewhere, it is not\nimmediately clear, so please ensure that they are presented clearly for easy understanding.\nResponse:\nThank you for the comment. As explained above, and in accordance with Nature Communications\nguidelines, we have indicated the statistical information for Fig. 4i and 4j (previously Fig. 4g and\n4h) in the corresponding figure legend. In the revised manuscript we modified the statistical details\npresentation and provided a clear statistical summary at the end of each figure legend. Letters are\nused to denote statistically significant differences. This is now indicated in the relevant legend,\nsuch as in Fig. 4 legend, page 27, line 813: “In (I, j) different lowercase letters above the bars\nindicate statistically significant at p<0.05”.\nComment:\n11. The figures in Fig. 4g and h exhibit a considerable number of outliers, giving the impression\nthat the discussion is based on very marginal differences. Therefore, I believe there might be room\nfor improvement in the analysis method or experimental conditions. If the authors' claims regarding\nthe experimental results in Fig. 4 are accurate, providing continuous sleep deprivation (SD) could\npotentially lead to a higher accumulation of DNA damage. Conducting long-term SD experiments\nmight enhance the credibility by demonstrating clearer differences.\nResponse:\nThank you for this insightful comment, which inspired us to develop a more efficient sleep\ndeprivation (SD) protocol. The observed variability in DNA damage is expected, given the distinct\nidentities, activity levels, and functional roles of individual neurons. Similar heterogeneity has been\nreported in other model organisms10–12, and such assays typically require relatively large sample\nsizes for robust interpretation.\nTo overcome these limitations and enhance the impact of SD on DNA damage, we implemented\nan improved SD protocol in the revised manuscript. N. vectensis were sleep-deprived using hourly\nwater flow stimulation, a method that proved more effective and minimized circadian interference\ncompared to light-based stimulation. SD administered during the natural sleep phase successfully\ninduced wakefulness, followed by a clear sleep rebound the subsequent night (Fig. 2t–w).\nCorrespondingly, DNA damage levels increased following SD (ZT8) and decreased after the sleep\nrebound period (ZT17). These results support the conclusion that sleep plays a protective role in\nalleviating DNA damage.\nThese findings are detailed in the Results section (Page 07, Line 210): “Under LD, the number of\nγH2AX foci increased at the end of the wakefulness period in C. andromeda (ZT11) and N.\nvectensis (ZT17) and reduced at the end of the sleep period in C. andromeda (ZT23) and N.\nvectensis (ZT8) (Fig. 4i, j). The opposite timing of DNA damage reduction between C. andromeda\nand N. vectensis was aligned with their species-specific chronotype and daily timing of sleep. To\ndetermine the effect of homeostatic sleep pressure, the C. andromeda and N. vectensis were\nsleep-deprived for 6h (ZT17-ZT23) and 8h (ZT20-ZT4), respectively. Following SD, the number of\nγH2AX foci increased significantly in C. andromeda (ZT23) and N. vectensis (ZT8) and\nsubsequently decreased following sleep recovery (Fig. 1o, p; Fig. 2t, u) in both species (Fig. 4i,\nj).” In addition, we explained the SD protocol in the methods section page 22, line 634: “In SD\nexperiments, the animals were stimulated by gentle water exchange once per hour during their\npeak of sleep period (ZT20-ZT4) and peak of activity period (ZT8-ZT16; Extended Data Movie 4)\nand sleep was monitored before and after the SD.”\nWe also modified Fig.4i, j:\nComment:\n12. It is difficult to determine whether DNA damage is involved in sleep based solely on the\nexperimental method in Fig. 5. Changes in locomotor activity due to UV irradiation should also be\nincluded. Additionally, observing the relationship between the number of γH2AX foci on the day\nafter UV irradiation and two days after UV irradiation and sleep would provide a more accurate\ndiscussion of the relationship between DNA damage and sleep.\nResponse:\nWe agree with the reviewer that continuous behavioral monitoring over multiple days is essential\nto determine the effects of UV-induced DNA damage on sleep. As requested, we monitored sleep\nin UV-treated and control animals for 30 hours in C. andromeda and 38 hours in N. vectensis.\nIn parallel, we imaged the animals and quantified DNA damage in individual neurons at multiple time\npoints: before UV exposure (ZT7), immediately after UV irradiation (ZT9), and during the recovery\nperiod (ZT3, two days later). Notably, UV irradiation led to increased DNA damage (Fig. X). During\nthe subsequent sleep phase—nighttime in C. andromeda and daytime in N. vectensis—we\nobserved a significant increase in sleep (Fig. 5g-j). Remarkably, in both species, this sleep\nrecovery period was followed by a reduction in DNA damage levels. These findings support the\nhypothesis that DNA damage induces sleep pressure and that sleep facilitates DNA repair.\nThese results are described in the Results section (Page 08, Line 229): “In C. andromeda and N.\nvectensis, the number of γH2AX foci increased one hour after UV exposure (Fig. 5a, b, d, e).\nSubsequently, nighttime and daytime sleep increased in C. andromeda (Fig. 5g, h) and N.\nvectensis (Fig. 5i, j), respectively. Notably, following sleep recovery, the animals remained viable,\nand two days later the levels of DNA damage reduced in both species (Fig. 5a, b, d, e). These\nresults suggest that UV-mediated induction of DNA damage increases sleep pressure.”\nThe experimental protocol is detailed in the methods section (page 23, line 652): “C. andromeda\nwere habituated for 72h and entrained to an LD cycle. A plastic barrier divided and isolated UV-\ntreated animals from control in the behavioral monitoring system. Sleep was recorded during 30h\nand following 1h (ZT8) half of the C. andromeda were exposed to 15min 312nm UVB light\n(Spectroline® E-Series UV lamp) located 20cm above the animals. Similarly, N. vectensis were\nhabituated for 3 days and entrained to LD cycle in six-well plates. Sleep was recorded for 42h,\nand following 2h, half of the N. vectensis were exposed to 1h of 312nm UV light (Spectroline® E-\nSeries UV lamp), located 20cm above the animals. To quantify DNA damage, at each time point,\n5-7 UV-treated and control C. andromeda and N. vectensis were fixed in 4% Paraformaldehyde\n(PFA) before (ZT7), 1h (ZT9), and 48h (ZT7 Day2) following the UV irradiation.”\nThe new data is presented in Fig. 5:\nMinor:\nComment:\n1. In LL and DD conditions, periodic activity is not observed in C. andromeda (Extended Data Fig.\n1a-d). Does this suggest that clock genes are not present in the genome? Additionally, in LL\nconditions, the pulse/min is extremely high at the initial LD stage. Moreover, the sample size is\nn=6, smaller compared to others, raising some doubts about the reliability of these results. Please\nexplain the black triangles in Fig. 1e represents, as it is unclear.\nResponse:\nAs recommended by the reviewer, and to validate our results, we repeated the constant light\n(LL) experiment with an independent cohort of animals (N = 16). We found that C. andromeda\nmaintains a weak 24-hour rhythm (Lomb-Scargle Periodogram, LSP) under LL conditions. The\npulse rate (pulses/min) during the initial LD phase of the experiment (Extended Data Fig. 1c)\nclosely resembled that observed in the primary LD experiment (Fig. 1c). However, under LL, the\nrhythmicity was only marginally significant and was largely driven by the first cycle following the\ntransition from LD to LL (Extended Data Fig. 1c, d).\nConsidering the arrhythmic behavior observed under both free-running conditions (DD and LL;\nExtended Data Fig. 1a–d), in contrast to the robust rhythms recorded under LD (Fig. 1c) and LDLD\n(Extended Data Fig. 1e), we conclude that rhythmic pulsation activity in C. andromeda is primarily\nregulated by external light/dark cycles rather than by an endogenous circadian clock.\nImportantly, the absence of behavioral rhythms under constant conditions does not preclude the\npresence of circadian clock genes in the genome. Other rhythmic outputs—molecular, cellular, or\nphysiological—may still be under circadian regulation. Motivated by the reviewer’s intriguing\nsuggestion, we searched for circadian clock gene orthologs in the genome of C. xamachana (as\nthe C. andromeda genome is currently unavailable) using the protein sequences of known N.\nvectensis clock genes. We identified several candidate orthologs:\nNvCLK: Two bHLH-PAS domain-containing proteins (Casxa1/23723 and 23306) with 28.2% and\n27.3% identity;\nNvCYCLE: Five bHLH-PAS proteins (Casxa1/20122, 14058, 859, 1772, 23306) with identities\nranging from 33% to 47%;\nNvCRYa: Two cryptochrome proteins (Casxa1/27641 and 6769) with 51.3% and 36.1% identity.\nThese putative orthologs suggest that core circadian clock components are conserved in the\nCassiopea genus. Further research will be necessary to determine their functional roles in\nregulating rhythmic processes.\nIn the revised manuscript, we described the conclusions of the LL experiment in the Results\nsection (Page 03, Line 89): “However, rhythmic locomotor activity was abolished under constant\ndarkness (DD) and robustly dampened under constant light (LL) conditions (Extended Data Fig.\n1a-d). Moreover, under forced desynchrony conditions (6h L:6h D:6h L:6h D, LDLD), the animals\nshowed a 12.1h rhythm of locomotor activity (Extended Data Fig. 1e-g). These findings showed that sleep\nis primarily driven by the light/dark cycle in C. andromeda.” We also discuss these\nresults in the discussion section (page 09, line 285): Light and homeostatic drive were the\npredominant sleep regulatory processes in C. andromeda, while homeostatic drive and the\ncircadian clock control sleep in N. vectensis.” In addition, we clarified the meaning of the black\ntriangles in Fig. 1e by adding an explanation to the Methods section (Page 21, Line 594): “A\nresponse was defined as a reduction of 2 standard deviations in the inter-pulse intervals (IPI)\ncompared to the distribution of IPIs before the stimulation, within a time window of 10s after the\nstimulus.”\nExtended Data Fig. 1a-g:\nComment:\n2. Since the term 'optimization algorithm' may not be easily understood by general readers, could\nyou provide a more specific description for clarity?\nResponse:\nAs requested we clarified this term. In the result section, we added in page 03 line 82 “A threshold-\nbased optimization algorithm was used to determine the optimal behavioral parameters to\ndistinguish between slow and fast responses (Fig. 1g, j).” and in the method page 21 line 596 “A\nthreshold-based optimization algorithm was used to determine the optimal behavioral parameters\nto distinguish between slow and fast responses (i.e., elevated arousal threshold; Fig. 1g, j). For\neach candidate threshold (e.g., pulsation rate), responses were split into two groups: below and\nabove the threshold”\nComment:\n3. In LL and DD conditions, periodic activity is not observed in C. andromeda (Extended Data Fig.\n1a-d). Does this suggest that clock genes are not present in the genome? Additionally, in LL\nconditions, the pulse/min is extremely high at the initial LD stage. Moreover, the sample size is\nn=6, smaller compared to others, raising some doubts about the reliability of these results.\nResponse:\nThis comment duplicates Minor Comment 1. Please refer to our response above for a detailed\nexplanation.\nComment:\n4. In Extended Data Fig. 1c, it should be described as 'Circadian time (h)' under constant\nconditions. Additionally, Extended Data Fig. 1f should be described as 'time of day (h)' instead of\n'Zeitgeber time (h).'\nResponse:\nThank you for the feedback. The 36-hour experiment consists of a 12 h:12 h light–dark (LD) cycle\nfollowed by constant light (LL). We acknowledge that the X-axis terminology may not have been\nclear. Following the reviewer’s suggestion, we replaced \"Zeitgeber time (h)\" with \"Circadian time\n(h)\" in Extended Data Fig. 1c, and added \"Time of day (h)\" to Fig. 1s and Extended Data Fig. 1k.\nThese annotations have also been added to Extended Data Fig. 1a-d.\nComment:\n5. I do not understand the meaning of the red dashed lines in Extended Data Fig. 2f-k. It is\nnecessary to describe them more clearly for better understanding.\nResponse:\nThe red dashed line represents the threshold used to distinguish a behavioral response from\nquiescence. As noted by the reviewer, this was not explicitly indicated in the original figure legend.\nIn the revised version, we have added the following explanation to the legend of Extended Data\nFig. 2 (Page 30, Line 887): “The red horizontal dashed line indicates the movement threshold.”\nComment:\n6. L131: The text states 'ZT0-ZT8,' indicating an 8-hour sleep deprivation period during the day,\nwhile the legend mentions 'ZT0-ZT7.' To avoid confusion, it should be clarified which one is\ncorrect.\nResponse:\nThe correct time frame is “ZT0–ZT8,” which corresponds to an 8-hour sleep deprivation (SD)\nperiod. However, following other reviewer recommendations, we revised the SD protocol to\nimprove its effectiveness. Specifically, we applied gentle hourly water exchanges for 8 hours\nduring the following time windows: ZT20–ZT4 and ZT8–ZT16.\nReviewer #3 (Remarks to the Author):\nThis manuscript examines the link between DNA damage in neurons and sleep, extending\nprevious observations that were made in zebrafish and rodent models to more “ancient” sleepers,\nthe jellyfish and anemone. Overall, the work is intriguing\nand is suggestive of a link between sleep and DNA damage even in these animals that lack brains.\nIn particular, the work demonstrating a sleep-like state in the anemone is well done (with a few\nminor gaps, see minor comments) and the jellyfish behavioral work is a nice confirmation and\nextension of Nath et al., 2017. However, although problems with the data presentation and\nanalysis of the DNA damage data makes it a little difficult to evaluate fully, the results don’t seem\nto line up with the major hypothesis that DNA damage is resolved during sleep in these species,\nand in fact some of the data seems to contradict that notion entirely.\nThe first half of the manuscript, which describes the sleep state in jellyfish and the anemone is\ndone to a high standard, and all the major expected behavioral criteria are examined (e.g.\ncircadian period, sleep deprivation and recovery, arousal threshold changes). This makes for a\nconvincing case that the two species have sleep states, although one is diurnal and the other is\nmore crepuscular.\nComment:\nI have only minor concerns about some of the sleep deprivation methodology (see Additional\ncomments below) and a bit more concern about the interpretation of the role of melatonin in the\ntwo species. They conclude that melatonin is effective as a hypnotic in the jellyfish (which was\nalso shown by Nath et al., 2017) but not in the anemone; however, the timing of the melatonin\ndelivery may have missed the effective window—adding melatonin at ZT2-5 puts this at the sleep\ntrough for jellyfish but at a relatively high sleep window for the anemone. In other words, sleep\ncould be ceilinged out so melatonin can’t boost sleep further, giving an artificial null result (adding\nmelatonin to diurnal species like zebrafish also only really boosts sleep during the day and not at\nnight when sleep and natural melatonin are already high). Testing melatonin later in the day, when\nsleep is lower, should be a straightforward way to bolster this claim.\nResponse:\nWe agree with the reviewer’s comment and, as suggested, treated both species with melatonin\nduring their respective active (C. andromeda: ZT3; N. vectensis: ZT13) and sleep periods (C.\nandromeda: ZT13; N. vectensis: ZT3). In both species, melatonin significantly promoted sleep\nduring the active phase but had no effect during their natural sleep phase (Fig. 3j–u). We conclude\nthat exogenous melatonin can promote sleep in both species, independent of their chronotype.\nSimilar effects have been reported in other diurnal species (e.g., zebrafish9) and nocturnal animals\n(e.g., mice and worms2,3).\nThe new melatonin-related data are now described in the Results, Methods, and Discussion\nsections of the revised manuscript. Page 06, Line 187: “We tested the effect of melatonin on sleep\nin the diurnal C. andromeda and crepuscular N. vectensis. While melatonin treatment did not affect\nsleep during the rest periods of N. vectensis (ZT3) and C. andromeda (ZT13) (Fig. 3j-l, s-u), sleep\ntime, sleep bout duration, and the number of sleep bouts increased during the active phase in both\nN. vectensis (ZT13) and C. andromeda (ZT3) (Fig. 3m-o, p-r). These results suggest an\nevolutionarily conserved role of melatonin in sleep regulation across animals with different\nchronotypes” Page 09, Line 298: “…despite their opposing chronotypes, melatonin treatment\npromoted sleep in both species during their respective active phases, suggesting a conserved role\nfor this hormone in sleep modulation.” Page 22, Line 646: “Both species received either 100 μM\nmelatonin (diluted in ethanol) or an ethanol control at ZT3 and ZT13, corresponding to their\nrespective periods of wakefulness and activity.”\nComment:\nThe second half of the manuscript then examines the link between DNA damage and sleep, and\nhere I find the analysis and interpretation of the results to be more muddled. Firstly, the\nindependent experimental Ns for calculating DNA damage appears to be each cell (Figure 4g,h),\nbut in how many independent animals? It isn’t really appropriate to treat cells from the same animal\nas independent experimental replicates for statistical purposes; a more appropriate analysis would\nbe average foci/cell calculated for each animal, and the N would be each animal. Thus, it is difficult\nfrom the data as presented to conclude if there are true differences in some of the conditions\nfollowing sleep deprivation.\nResponse:\nThe analysis presented in Figure 4i–j focuses on a cellular phenotype rather than a whole-\norganism behavioral phenotype. Each neuron independently accumulates and repairs DNA\ndamage and is therefore considered an individual experimental unit. Within a single animal,\nneuronal identity is highly variable and can be classified based on distinct genetic and activity\nprofiles. In both N. vectensis and C. andromeda, the nervous system comprises diverse neuronal\nsubtypes13, which may respond differently to sleep and wake states. Indeed, we observed\nrelatively high variability in neuronal DNA damage levels (Fig.4i, j). The analytical approach used\nin this study—immunostaining of nuclear γH2AX and treating individual neurons as independent\nexperimental replicates—is well established in other model systems, including zebrafish10,11,\nflies14, and mice14. In response to this and other comments raised by the reviewer, we repeated\nthe experiment, revised the sleep deprivation (SD) protocol, and are now presenting the new data\nin Figure 4i, j. Detailed statistical information and sample sizes are provided in the corresponding\nfigure legend and in the methods section.\nComment:\nHowever, even if we take these results as described, some of the data appears to contradict the\nclaim that sleep is important for resolving the DNA damage. For example, Figure 2t shows that a\nsleep deprivation from ZT0-6 leads to a substantial sleep rebound from ZT12-24. And yet, In figure\n4h, the number of DNA damaged foci remains elevated at ZT17 following SD, even though these\nanimals slept a lot more than the controls. This seems to contradict the conclusion of lines 180-\n183: “These experiments show that DNA damage increased during wakefulness and decreased\nduring sleep in either diurnal or crepuscular cnidarians. Furthermore, these findings suggest that\nalleviating the levels of DNA damage, which accumulated during wakefulness, is an evolutionarily\nconserved cellular role of sleep”.\nResponse:\nThe link between DNA damage and sleep is supported by our findings in C. andromeda, which\nshowed a reduction in DNA damage levels during the sleep rebound period (Fig. 4g, previous\nversion). However, as noted by the reviewer, this trend was not observed in N. vectensis, where\nDNA damage levels did not decrease during the recovery period (Night 2, ZT17). Upon closer\nexamination, we found that DNA damage did not significantly increase immediately after SD (ZT8)\ncompared to baseline levels from the first day (ZT17), and no clear behavioral effect (i.e., reduced\nsleep immediately post-SD) was observed (Fig. 2t in the previous version). To address these\nlimitations, we revised the sleep deprivation (SD) protocol. In the new version of the manuscript,\nanimals were sleep-deprived by applying gentle, gradual water exchange once per hour during\ntheir sleep phase (ZT20-ZT4, marked by the red horizontal dashed line in Fig. 2t). This improved\nprotocol effectively reduced sleep immediately post-SD (ZT 4), leading to a clear sleep rebound\n(Fig. 2t, u). In contrast, applying the same SD protocol during the peak of activity (ZT8–ZT15)\ndid not result in a rebound (Fig. 2v, w), confirming that the effect is sleep-phase dependent.\nImportantly, under the revised protocol, DNA damage increased immediately following SD (ZT8),\nand significantly decreased during the rebound sleep phase at ZT17 of the second night (Fig. 4j).\nThese results strengthen the conclusion that sleep facilitates the reduction of DNA damage.\nIn the revised version of the manuscript, the results are described in page 05, line 159: “To test\nwhether homeostatic process regulates sleep, we sleep-deprived the N. vectensis for 8h during\nsleep period (ZT20-ZT3) using gradual and gentle water exchange once per hour (Extended\nData Movie 4). Following SD, sleep-deprived animals slept 1.24-fold more than control animals\n(Fig. 2t, u), indicating a homeostatic sleep drive. In contrast, control SD during the activity period\n(ZT8-ZT15) did not induce a sleep rebound (Fig. 2v, w).” The behavioral sleep results are\npresented in Fig. 2t-w:\nIn the revised version of the manuscript, the results are described in page 07, line 210: “Under\nLD, the number of γH2AX foci increased at the end of the wakefulness period in C. andromeda\n(ZT11) and N. vectensis (ZT17) and reduced at the end of the sleep period in C. andromeda\n(ZT23) and N. vectensis (ZT8) (Fig. 4i, j)… Following SD, the number of γH2AX foci increased\nsignificantly in C. andromeda (ZT23) and N. vectensis (ZT8) and subsequently decreased\nfollowing sleep recovery (Fig. 1o, p; Fig. 2t, u) in both species (Fig. 4i, j)” The effect of SD on\nDNA damage is presented in Fig. 4j:\nThe SD protocol is explained in the methods section page 22, line 634: “In SD experiments, the\nanimals were stimulated by gentle water exchange once per hour during their peak of sleep period\n(ZT20-ZT3) and peak of activity period (ZT8-ZT15; Extended Data Movie 4) and sleep was\nmonitored before and after the SD.”\nComment:\nFinally, the authors use UV light to induce DNA damage and claim that sleep is increased, thus\nshowing a bi-directional relationship between DNA damage and sleep. However, the data are\nshown only as box-whisker summary plots, making them difficult to evaluate in terms of quality of\nthe results. Better would be to display these as timecourse data, before and after UV exposure,\nand for a longer window than the summary—the post UV sleep is reaching 100% for jellyfish and\n60/60 minutes for the anemone, so more convincing evidence that these animals are not\npermanently harmed by the UV exposure would be critical here. Even better would be to show\nthat the DNA damage eventually resolves (by the next day or something) and that the sleep\nrecovers as well.\nResponse:\nWe thank the reviewer for this important conceptual comment. As requested, we conducted a ~2-\nday time-course monitoring of sleep behavior and DNA damage before and after UV exposure in\nboth C. andromeda and N. vectensis. UV irradiation was applied at ZT8 (indicated by a red triangle\nin Fig. 5X–X or a red line in Fig. 5X–X), and it promoted increased sleep, which occurred during\nthe subsequent night in C. andromeda and during the day in N. vectensis, consistent with their\nrespective chronotypes. In parallel, UV exposure led to a significant increase in DNA damage at\nZT9. Following the sleep recovery period, DNA damage levels returned to baseline (Fig. 5X–X).\nThe normalization of DNA damage suggests that the animals were not permanently affected by\nthe relatively short UV exposure. These findings support the conclusion that UV-induced DNA\ndamage promotes sleep, and that sleep, in turn, facilitates the repair and reduction of DNA\ndamage.\nWe describe these findings in the Results section (Page 08, Line 229): “In C. andromeda and N.\nvectensis, the number of γH2AX foci increased one hour after UV exposure (Fig. 5a, b, d, e).\nSubsequently, nighttime and daytime sleep increased in C. andromeda (Fig. 5g, h) and N.\nvectensis (Fig. 5i, j), respectively. Notably, following sleep recovery, the animals remained viable,\nand two days later the levels of DNA damage reduced in both species (Fig. 5a, b, d, e). These\nresults suggest that UV-mediated induction of DNA damage increases sleep pressure… These\nresults show that elevated DNA damage can promote sleep in early divergent lineage. Moreover,\nthese inter-species comparative findings suggest that environmental factors, such as UV radiation\nand soluble mutagenic compounds, which can induce DNA damage and cellular stress, may have\ncontributed to the evolutionary emergence of sleep in cnidarians.”\nThese new results are presented in Fig. 5a–j.\nComment:\nTo be more convincing, it would also be good to show that DNA damage induced in another\nmanner also increases sleep—for example these creatures have a host of natural behavioral\nresponses to UV light (see for example Lilly et al., 2024, where the larval anemones shift their\nbehavior to a planktonic swimming in direct response to UV), so showing the sleep changes are\ndue to the damage and not UV-activated neural pathways would go far to convince.\nResponse:\nThe results presented in Fig. 5a–j suggest that UV-induced DNA damage promotes sleep. As\nsuggested by the reviewer, and to further support this finding, we induced DNA damage using an\nalternative method. Animals were treated with etoposide, a well-established mutagenic\ncompound. While UV exposure induces oxidative stress and DNA base damage, etoposide\ncauses DNA damage by inhibiting topoisomerase II, thereby preventing effective DNA repair.\nEtoposide treatment led to increased DNA damage, followed by an increase in sleep. Notably,\nDNA damage levels decreased after the recovery sleep period the following day (Fig. 5c, f, k, l).\nThis experiment provides an independent, light-free method to induce DNA damage and further\nsupports the conclusion that DNA damage promotes sleep (Fig. 5k, l).\nThese results are described in the Results section (Page 08, Line 234): Since UVB may also affect\nthe behavior of cnidarians in alternative pathways46, we treated N. vectensis with the mutagenic\ncompound Etoposide47. While UVB induces oxidative stress and damage to DNA bases,\nEtoposide inhibits the topoisomerase II enzyme and stabilizes DNA double strand break formation.\nSimilar to the effect of UV, Etoposide treatment abruptly increased the number of γH2AX foci (Fig.\n5c, f). The animals then increased sleep during the following day and night (Fig. 5k, l), and DNA\ndamaged was reduced (Fig. 5c, f). These results show that elevated DNA damage can promote\nsleep in early divergent lineage.”. The results of the etoposide treatment are presented in Fig. 5c,\nf, k, l. And we cite Lilly et al., 2024 to support this\nbroader context.\nComment:\nTo conclude, I am convinced that anemones (and jellyfish) have a sleep state, and this alone is a\nnice contribution. However, I am less convinced that the DNA-damage sleep hypothesis has been\nso clearly demonstrated in these animals based on the current data presented.\nResponse:\nWe believe that our detailed responses, corresponding text modifications, and the additional\nexperiments—namely: (1) the effect of the revised sleep deprivation protocol on sleep and DNA\ndamage; (2) melatonin treatments administered during both day and night; and (3) continuous\nquantification of DNA damage and sleep following UV and etoposide treatments—collectively\nstrengthen the manuscript. These new data provide compelling evidence for a bi-directional\nrelationship between DNA damage and sleep in both the diurnal C. andromeda and the\ncrepuscular N. vectensis.\nAdditional and minor comments:\nComment:\nFigure 2—The arousal threshold data, circadian and clock mutant data are all very good. The\nSleep deprivation data is good but a few additional controls would be useful. For example, the\nrebound is shown after depriving the animals of the daytime siesta, but the sleep traces show a\nlot of sleep in the latter half of the night as well—even more in fact. Showing SD for this\nwindow would also be good, as in other species like Drosophila is not totally clear that the siesta\nand night sleep are identically regulated. Perhaps more important is to use the SD method at a time of\nrelative wakefulness and control for rebound due to physical exhaustion of being kept awake, a\nstandard test in the field.\nResponse:\nAs recommended by the reviewer, we revised the sleep deprivation (SD) method and applied it\nduring both the sleep period (ZT20–ZT4) and a period of relative wakefulness (ZT8–ZT16). A clear\nsleep rebound was observed only following SD administered during the sleep period, indicating\nthat sleep loss must occur during the natural rest phase to elicit a homeostatic response.\nThese findings are described in the Results section (Page 05, Line 159): “To test whether\nhomeostatic process regulates sleep, we sleep-deprived the N. vectensis for 8h during sleep\nperiod (ZT20-ZT4) using gradual and gentle water exchange once per hour (Extended Data Movie\n4). Following SD, sleep-deprived animals slept 1.24-fold more than control animals (Fig. 2t, u),\nindicating a homeostatic sleep drive. In contrast, control SD during the activity period (ZT8-ZT16)\ndid not induce a sleep rebound (Fig. 2v, w).” The SD protocol is described in the methods section\n(page 22, line 634): “In SD experiments, the animals were stimulated by gentle water exchange\nonce per hour during their peak of sleep period (ZT20-ZT4) and peak of activity period (ZT8-ZT16;\nExtended Data Movie 4) and sleep was monitored before and after the SD.” The SD data is\npresented in Fig. 2t-w\nComment:\nFigure 2—the SD method uses light, which may be altering circadian clock function and timing as\nwell as sleep deprivation. Either a non-light method for SD should be used, or show that clocks\naren’t shifted by the light pulses.\nResponse:\nAs requested by the reviewer, and to minimize potential effects on the circadian clock, we\nemployed a non-light-based method for sleep deprivation—specifically, gradual and gentle water\nexchange.\nComment:\nFigure 2- methods; the details for the light stimulation SD are not provided in the methods as far\nas I can see, including light levels, etc.\nResponse:\nIn the revised manuscript, we no longer use light-based stimulation for sleep deprivation. Instead,\nwe implemented a protocol involving gentle, gradual water exchange once per hour. This method\nis described in the Methods section and demonstrated in Extended Data Movie 4.\nThe following text has been added to the Methods section page 22, line 634: “In SD experiments,\nthe animals were stimulated by gentle water exchange once per hour during their peak of sleep\nperiod (ZT20-ZT4) and peak of activity period (ZT8-ZT16; Extended Data Movie 4) and sleep was\nmonitored before and after the SD.”\nComment:\nFigure 2t—if light is being used to sleep deprive, it isn’t clear to me why there is a gap in the\ndata—it would be better to track the animals during the SD to see exactly how much sleep is lost.\nI can understand why there is a gap in Figure 1, where the SD is done physically that will interfere\nwith the tracking, but light should be straightforward to track.\nResponse:\nThank you for the feedback. During sleep deprivation, whether using the previous light-based\nprotocol or the revised method involving gentle hourly water exchange, animals were removed\nfrom the imaging setup, making it impossible to continuously track their sleep behavior during the\nSD period (Extended Data Movie 4).\nComment:\nFigure 2m—defining the windows of analysis across the dark-light transition (ZT18-6 and 6-18) is\na bit odd, I would just break it down into night 2nd half and day first half to be a little clearer.\nResponse:\nWe appreciate the reviewer’s suggestion and agree that the ZT18-6 and 6-18 is rather\nunconventional. To maintain consistency with the 12-hour time windows commonly used under\nlight or dark conditions (e.g., Fig. 1l), we identified the sleep and wake periods of N. vectensis\nunder LD conditions (Fig. 2l, Fig. 4b) and defined the 12-hour analysis windows accordingly.\nAlthough we acknowledge that this division (ZT18–6 for sleep, ZT6–18 for wake) is\nunconventional, it was necessary given the crepuscular nature of the animals and permits direct\ncross-species comparison.\nFollowing the reviewer’s suggestion, we expanded the explanation for this choice in the results\npage 4, line 121: “We analyzed 6h bins (Extended Data Fig. 4) but chose 12h windows (ZT18-6\nand ZT6-18) because they encompass the full crepuscular (i.e., active at dusk) behavioral pattern\nand align with the 12h light/dark periods used for C. andromeda (Fig. 1m), facilitating cross-\nspecies statistics.”. The data is presented Extended data Fig.4:\nComment:\nFigure 3J—100 mM melatonin must be a typo, and it is listed as 100uM in the methods (and that\ncould still be considered high). To convince that melatonin plays no role in the sleep of the\nanemone, adding melatonin at different timepoints will be critical (see main comments).\nResponse:\nGood catch—thank you. This typo error was corrected. We used 100 µM melatonin, consistent\nwith concentrations previously used in C. xamanacha15. As explained above and in line with the\nreviewer’s recommendation, we treated the animals with melatonin at different time points to\nassess its effects during both their active and sleep phases.\nComment:\nThe term “sleep quality” is too loaded a term—it implies something about a “good” night or a “bad”\nnight of sleep but it is impossible to say that fragmented sleep or consolidated sleep is of more or\nlesser quality without showing effects on performance the next day. I would recommend “sleep\nstructure” or “sleep architecture” or similar when looking at sleep bout counts and lengths.\nResponse:\nAs recommended by the reviewer, we replaced the term “sleep quality” with “sleep architecture”\nthroughout the manuscript.\nComment:\nFigure 4: The SD experiment for jellyfish is from ZT17-23, but for N vectensis is ZT0-8. ZT0-8\ndoes not quite line up with maximal sleep in this species ZT18-24 or ZT18-6 makes better\nalignment. Figure 4A, showing the rolling 12 hour pervious sleep time is misleading on this – the\nfigure 4b data is high already at ZT0 and peaking at ZT6, but that means the sleep is maximal in\nthe PREVIOUS 12 hours, which is when the SD should be done for testing the effect of SD on\nsleep. Plotting it this way is visually confusing compared to Figure 2L, where the sleep timing is\neasier to appreciate.\nResponse:\nIn accordance with the reviewer’s suggestion, we sleep-deprived N. vectensis during its peak\nsleep period (ZT20–ZT3), which resulted in a clear sleep rebound. To help readers better\nunderstand the rationale behind the selected SD time window, we plotted the sleep time data\nabove the sleep accumulation data in Fig. 4. The rationale is explained in the results section page\n07, line 200: “We hypothesized that DNA damage would peak and decline following extended\nwake and sleep, respectively. To identify the time points of maximal and minimal sleep pressure,\nwe calculated the average sleep time over the past 12h that preceded each time point.” The sleep\ndeprivation period is indicated by a red dashed line in Fig.4a and b:\nComment:\nFigure 4: If sleep is really important for repairing the DNA damage, then why does it not recover\nin the anaemone after SD from ZT0-8? It is confusing because they sleep more from 12-17 then\nthey even do during the “SD” ZT0-8 window, so if this were all about sleep driving the recovery,\nthen wouldn’t we expect it to be recovered? Figure 3 shows after SD, the anemones sleep more,\nso this should have resolved the foci if the interpretation were correct. This seems counter to the\nconclusion of line 180-183: “These experiments show that DNA damage increased during\nwakefulness and decreased during sleep in either diurnal or crepuscular cnidarians. Furthermore,\nthese findings suggest that alleviating the levels of DNA damage, which accumulated during\nwakefulness, is an evolutionarily conserved cellular role of sleep.\nResponse:\nAs addressed above, a revised sleep deprivation (SD) protocol was applied during the sleep\nperiod (ZT20–ZT3). This protocol led to an increase in DNA damage and induced a sleep rebound,\nwhich was subsequently followed by a reduction in DNA damage levels.\nComment:\nFigure 4: The data is not showing independent Ns…. The legend says n=291 cells, but how many\nanimals? One cannot use a cell in the same animal as an independent measure for the stats\nunless you control for them as dependent on each other. What stats are being done in Figure 4?\nThe legend doesn’t say. It says Tukey, but that is a post-hoc test. It also lists a lot of\ncross comparisons, how is this being controlled for multiple comparisons? There are really two-factors,\nSD/no SD and time, so a 2-factor analysis design would be clearer.\nResponse:\nThe analysis presented in Figure 4i–j focuses on a cellular phenotype rather than a behavioral\nphenotype. As explained above, individual cells are treated as the experimental units in this\nanalysis. As requested by the reviewer we added statistical details and sample sizes in the\ncorresponding figure legend and in the methods section. The revised legend now specifies the full\nstatistical workflow of two-way ANOVA (treatment × time) followed by Tukey HSD, corrected for\nmultiple comparisons.\nComment:\nFigure 5: the post-UV sleep data is too summarized. IT would be better to see the time course of\nthe behavior afterwards. Also, the recovery on the following day—sleep is being driven so high\nafter UV are you sure they aren’t permanently harmed, i.e. sleeping a lot but actually damaged?\nResponse:\nAs addressed above, and in response to the reviewer’s suggestion, we performed a time-course\nanalysis of behavior before and after UV irradiation. In parallel, we quantified DNA damage at\nthree time points: before UV exposure, immediately after irradiation, and following the recovery\nsleep period. The animals remained viable and fully recovered from the UV treatment. These\nresults are presented in Fig. 5.\nComment:\n5e and 5f are not presented in the same manner, one show ZT8-12 and ZT12-0, the other says\nPre: Post, but over what windows constitute “pre” and what consistutes “post”? The legend says\n1hr pre and 1 hr post, but that seems like too short a time? What would be better would be to\ninduce UV damage, see how long the damage takes to be resolved and correlate that timecourse\nwith the sleep timecourses— i.e. once the damage is resolved, does sleep end?\nResponse:\nWe have corrected the terminology and now refer to the sampling time points using ZT values.\nThese updates are reflected in Fig. d–f.\nComment:\nAnother method to induce DNA damage? These species are quite sensitive behaviorally to UV\nlight directly, for example the anemone in the larval state are induced by UV into plaktonic\nswimming (Lilly et al 2024). Can you distinguish between these two models UV◊DNA damage◊\nsleep vs. UV◊ sleep (independent of the DNA damage?\nResponse:\nYes, we used an additional method to induce DNA damage. As explained above, and to support\nthe role of UV-mediated DNA damage specifically—rather than alternative pathways—we treated\nanimals with etoposide, a well-established inducer of DNA damage via topoisomerase II inhibition.\nEtoposide treatment also led to increased sleep, consistent with a DNA damage-mediated sleep\nresponse. Notably, in both the UV and etoposide treatments, DNA damage levels decreased\nfollowing the sleep rebound period (Fig. 5), further strengthening our conclusion that sleep\nfacilitates the reduction of DNA damage.\nNonetheless, we do not exclude the involvement of additional mechanisms. This point is\nacknowledged in the Results section. These results are described in the Results section (Page\n08, Line 234: “These results suggest that UV-mediated induction of DNA damage increases sleep\npressure. Since UVB may also affect the behavior of cnidarians in alternative pathways46, we\ntreated N. vectensis with the mutagenic compound Etoposide47. While UVB induces oxidative\nstress and damage to DNA bases, Etoposide inhibits the topoisomerase II enzyme and stabilizes\nDNA double strand break formation. Similar to the effect of UV, Etoposide treatment abruptly\nincreased the number of γH2AX foci (Fig. 5c, f). The animals then increased sleep during the\nfollowing day and night (Fig. 5k, l), and DNA damaged was reduced (Fig. 5c, f). These results\nshow that elevated DNA damage can promote sleep in early divergent lineage.” The results of the\netoposide treatment are presented in Fig. 5c, f, k, l. And we cite Lilly et al., 2024 to support this\nbroader context.\nComment:\nThe evolutionary claim seems too strong: “Altogether, these inter-species comparative findings\nsuggest that environmental stressors, such as UV radiation, which can induce DNA damage and\ncellular stress, drove the evolutionary origin of sleep in the day or crepuscular active cnidarians.”\nWhat exactly is the evidence that this was the “driver” of evolution? Showing that DNA damage\ninduces sleep and sleep resolves DNA damage in these species isn’t quite the same as saying\nthat this relationship is what “drives” evolution of sleep.\nResponse:\nWe have moderated this hypothesis in the revised manuscript. The updated text now reads: (page\n11, line 350): “Moreover, considering that the interplay between the circadian clock and genomic\nstability has been shown in non-neuronal mammalian cultured cells, fungi and cyanobacteria78–81,\nsleep-like states may have preceded the evolution of neurons, originally evolving to support\nsynchronized and consolidate periods of cellular maintenance.”\nReferences\n1. Mascetti, G. G. Unihemispheric sleep and asymmetrical sleep: behavioral,\nneurophysiological, and functional perspectives. Nat Sci Sleep 8, 221–238 (2016).\n2. Kim, P. et al. Melatonin’s role in the timing of sleep onset is conserved in nocturnal mice.\nNPJ Biol Timing Sleep 1, 13 (2024).\n3. Omond, S. et al. Inactivity Is Nycthemeral, Endogenously Generated, Homeostatically\nRegulated, and Melatonin Modulated in a Free-Living Platyhelminth Flatworm. Sleep 40,\n(2017).\n4. Pregueiro, A. M., Liu, Q., Baker, C. L., Dunlap, J. C. & Loros, J. J. The Neurospora\nCheckpoint Kinase 2: A Regulatory Link Between the Circadian and Cell Cycles. Science 313,\n644–649 (2006).\n5. Liao, Y. & Rust, M. J. The circadian clock ensures successful DNA replication in\ncyanobacteria. Proceedings of the National Academy of Sciences of the United States of America\n118, (2021).\n6. Gamsby, J. J., Loros, J. J. & Dunlap, J. C. A phylogenetically conserved DNA damage\nresponse resets the circadian clock. J Biol Rhythms 24, 193–202 (2009).\n7. Oklejewicz, M. et al. Phase Resetting of the Mammalian Circadian Clock by DNA Damage.\nCurrent Biology 18, 286–291 (2008).\n8. Durieux, D. M. et al. Benthic jellyfish act as suction pumps to facilitate release of interstitial\nporewater. Sci Rep 13, 3770 (2023).\n9. Appelbaum, L. et al. Sleep-wake regulation and hypocretin-melatonin interaction in\nzebrafish. Proc Natl Acad Sci U S A 106, 21942–21947 (2009).\n10. Zada, D., Bronshtein, I., Lerer-Goldshtein, T., Garini, Y. & Appelbaum, L. Sleep increases\nchromosome dynamics to enable reduction of accumulating DNA damage in single neurons. Nat\nCommun 10, 895 (2019).\n11. Zada, D. et al. Parp1 promotes sleep, which enhances DNA repair in neurons. Mol Cell\n81, 4979-4993.e7 (2021).\n12. Noubissi, F. K. et al. Detection and quantification of γ-H2AX using a dissociation enhanced\nlanthanide fluorescence immunoassay. Sci Rep 11, 8945 (2021).\n13. Sprecher, S. G. Neural Cell Type Diversity in Cnidaria. Front Neurosci 16, 909400 (2022).\n14. Bellesi, M., Bushey, D., Chini, M., Tononi, G. & Cirelli, C. Contribution of sleep to the repair\nof neuronal DNA double-strand breaks: evidence from flies and mice. Sci Rep 6, 36804 (2016).\n15. Nath, R. D. et al. The Jellyfish Cassiopea Exhibits a Sleep-like State. Curr Biol 27, 2984-\n2990.e3 (2017)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have effectively addressed my concerns. I have nothing new to add.", "reviewer_2": "The revised manuscript has addressed many of the concerns raised in the previous round, and I acknowledge a clear\noverall improvement in both clarity and scientific rigor. In particular, the experimental validation of sleep phenomena in C.\nandromeda and N. vectensis appears robust and convincing. The study adds valuable insights into sleep regulation in\ncnidarians and constitutes a meaningful contribution to the field.\nHowever, regarding the central claim expressed in the title—namely, that \"DNA damage is a sleep driver in ancient\ncnidarians\"—I believe further caution and refinement are required. Specifically, this claim demands greater precision and\nstrength in both experimental design and data analysis. I outline below several key issues that I recommend the authors\naddress in the current revision.\n1. Y-axis scaling and quantification in Figure 4i,j\nThe vertical axis in Figure 4i,j lacks sufficient explanation, making it difficult to interpret. If the same quantification method\n(i.e., puncta/cell) as in Figure 5d–f was used, then I suggest that the graphical representation should be standardized across\nfigures for consistency.\nConversely, if a different quantification method was employed, then the authors should explain why the approach used in\nFigure 5 was not applied here. In that case, the rationale for using an alternative method should be clearly justified from a\nscientific standpoint. Overall, the quantification approach should be unified where possible.\n2. Statistical annotation (Figure 4i,j and other figures)\nThe notation \"different lowercase letters above the bars indicate statistical significance at p < 0.05, Tukey HSD, two-tailed;\nFWER controlled\" is vague and difficult to interpret. I strongly recommend that the authors explicitly state which groups were\ncompared and the nature of the statistically significant differences.\nSimilar statistical annotations appear in other figures, including supplementary figures. A more intuitive and consistent\npresentation would enhance interpretability across the manuscript.\n3. Relationship between sleep pressure and DNA damage\nThe current manuscript presents compelling evidence that DNA damage accumulates during wakefulness and is reduced\nduring sleep under light–dark conditions. It also demonstrates that short-term sleep deprivation (SD) induces DNA damage,\nsupporting the hypothesis that sleep contributes to DNA repair. These are important findings.\nHowever, to convincingly support the central claim that \"DNA damage is a sleep driver,\" I believe further experimentation is\nneeded. Specifically, I encourage the authors to explore whether prolonged sleep deprivation—e.g., over 24 hours or\nmultiple days—leads to a measurable accumulation of DNA damage. This would provide critical support for the proposed\ncausal relationship between sleep pressure and DNA damage.\nFurthermore, the authors have shown that melatonin induces sleep during the active phase but has no effect on sleep\nduration during the sleep phase. This feature could be leveraged experimentally. For example:\nDoes melatonin-induced sleep during the active phase reduce DNA damage?\nDoes melatonin administration during the sleep phase alter DNA damage independently of sleep induction?\nThese complementary experiments would allow the authors to distinguish between the antioxidative effects of melatonin and\nthe role of sleep per se in promoting genomic stability. I believe that these additions would significantly strengthen the\nmanuscript and make the central claim more persuasive.\n4. Number of animals used in DNA damage assays\nWhile the number of cells analyzed appears sufficient, the number of animals examined seems relatively low. Given that\nsleep-related behavioral metrics (e.g., bout duration, total sleep time) were calculated from a large number of animals, the\nDNA damage experiments should ideally match that level of biological replication.\nTo ensure comparability and statistical robustness, I recommend increasing the number of animals used in the DNA damage\nassays to align with those used in the sleep behavior analyses.", "reviewer_3": "The authors have mostly addressed the reviewers’ comments and my concerns, including with important new experiments\nthat greatly improve the manuscript. With one very important exception that must be resolved before I could consider\nacceptance of this paper.\nIn my original review, I raised an issue of statistical pseudoreplication that comes from treating neurons within the same\nanimal as “independent”. Their response was not to correct this but instead claim that they “focus on a cellular phenotype”\nand that “each neuron independently accumulates and repairs DNA damage and is therefore an individual experimental\nunit.” I very strongly disagree with this claim, on scientific first principles. In support, the authors state this is an established\nmethod by citing their own papers! Even if this is something that was done in other papers, it doesn’t make the use of this\napproach in this paper right; it just means that those other papers are also doing this analysis incorrectly.\nA few simple thought experiments can illustrate the point that neurons in the same animal are not, and cannot, be treated as\nstatistically independent. First, consider an experiment where you expose some hydra to UV to see if DNA damage is\nresolved by sleep. At the first timepoint post UV, you grab a single animal that happened to be closer to the UV source for\nthe duration of the experiment, and then you count DNA damage foci in 100s of neurons in that single animal. Now, on the\nsecond timepoint after sleep, you grab a single hydra that happened to stay far from the UV source (darn chance!), and count\n100s of neurons. Is the n=100s for each group, or is the n=1? If you use n=100, even a very tiny effect, possibly caused by\ndifferences in UV exposure alone, will have some very small p-value, and you will rejoice that your experiment “worked”. But\nif you use n=1, you will correctly surmise that the n is too small to draw a meaningful conclusion and you won’t be misled by\na chance result.\nEven though the idea that n=100 can come from one animal already makes the point pretty obvious, maybe one can try to\nargue that all the animals must get the same UV. Consider a second thought experiment then, in which a single neuron in an\nanimal gets zapped by UV that generates some DNA damage that causes it to become hyperactive. This single neuron then\ncauses downstream neurons to become active as well, which we know can trigger DNA damage in those cells. You see?\nThe DNA damage accumulating in neurons doesn’t happen independently of the other neurons in that animal, so they\ncannot be treated as independent. To do so inflates artificially the n, which for statistical purposes must be independent of\neach other. This is why in Figure 4 and 5, the authors report such small p-values, despite a fairly variable set of data—the n\nis inflated.\nThis fundamental misunderstanding does make me worried about how other analyses were done in the paper, but I don’t\nsee anything clearly wrong in other figures."}, "author_response": "We appreciate your kind words and your support for our manuscript.\nComment:\nHowever, regarding the central claim expressed in the title—namely, that \"DNA\ndamage is a sleep driver in ancient cnidarians\"—I believe further caution and\nrefinement are required.\nResponse:\nAs suggested by the reviewer, we conducted additional experiments (detailed below)\nthat further support our central claim. In addition, we refined the title, which now reads:\n“DNA damage modulates sleep drive in basal cnidarians with divergent chronotypes.”\nComment:\nSpecifically, this claim demands greater precision and strength in both experimental\ndesign and data analysis. I outline below several key issues that I recommend the\nauthors address in the current revision.\n1. Y-axis scaling and quantification in Figure 4i,j.\nThe vertical axis in Figure 4i,j lacks sufficient explanation, making it difficult to interpret.\nIf the same quantification method (i.e., puncta/cell) as in Figure 5d–f was used, then I\nsuggest that the graphical representation should be standardized across figures for\nconsistency.\nConversely, if a different quantification method was employed, then the authors should\nexplain why the approach used in Figure 5 was not applied here. In that case, the\nrationale for using an alternative method should be clearly justified from a\nscientific standpoint. Overall, the quantification approach should be unified where possible.\nResponse:\nA unified quantification method (puncta per cell) was applied in both Figures 4 and 5.\nFollowing the reviewer’s suggestion, we standardized the Y-axis labeling across\nfigures to “Normalized number of γH2AX foci (a.u.).” The updated charts are now\npresented in Figures 4 and 5, and the corresponding legends have been revised\naccordingly.\nComment:\n2. Statistical annotation (Figure 4i,j and other figures).\nThe notation \"different lowercase letters above the bars indicate statistical significance\nat p < 0.05, Tukey HSD, two-tailed; FWER controlled\" is vague and difficult to interpret.\nI strongly recommend that the authors explicitly state which groups were compared\nand the nature of the statistically significant differences.\nSimilar statistical annotations appear in other figures, including supplementary figures.\nA more intuitive and consistent presentation would enhance interpretability across the\nmanuscript.\nResponse:\nAs recommended by the reviewer, we clarified the statistical representation in the\nrevised manuscript. Rather than applying Tukey HSD to all pairwise group\ncombinations, we now use Mann–Whitney tests to compare only the groups of interest.\nThis approach simplifies the result presentation, directly addresses the experimental\nquestion, and aligns the analysis with the methods used elsewhere in the paper.\nImportantly, the revised statistical analysis did not alter the conclusions. Furthermore,\nthe updated approach highlights the specific comparisons of interest, and the nature\nof statistically significant differences is explicitly stated in all figure legends. These\nchanges have been implemented across both the main and supplementary figures.\nComment:\n3. Relationship between sleep pressure and DNA damage.\nThe current manuscript presents compelling evidence that DNA damage accumulates\nduring wakefulness and is reduced during sleep under light–dark conditions. It also\ndemonstrates that short-term sleep deprivation (SD) induces DNA damage, supporting\nthe hypothesis that sleep contributes to DNA repair. These are important findings.\nResponse:\nThank you for the supportive comment and the summary of one of the key conclusions\nof the manuscript.\nComment:\nHowever, to convincingly support the central claim that \"DNA damage is a sleep driver,\"\nI believe further experimentation is needed. Specifically, I encourage the authors to\nexplore whether prolonged sleep deprivation-e.g., over 24 hours or multiple days-leads\nto a measurable accumulation of DNA damage. This would provide critical support for\nthe proposed causal relationship between sleep pressure and DNA damage.\nResponse:\nWe calibrated the timing and duration of sleep deprivation (SD) and determined that 6\nhours (ZT17–ZT23, Fig. 1o) and 8 hours (ZT20–ZT4, Fig. 2t) represent optimal SD\nperiods. These conditions consistently caused a robust homeostatic sleep response\n(Fig. 1o, p; Fig. 2t, u) and elevated DNA damage levels (Fig. 4k, l). Our empirical tests\nshowed that extending SD beyond 24 hours increased the likelihood of non-sleep-\nspecific stress responses. In both Cassiopea and Nematostella, prolonged SD led to\ntissue degradation and disintegration. Similar confounding effects of extended SD\nhave been documented in other animal models (Vaccaro et al., 2020; Sang et al., 2023;\nWu et al., 2023), complicating the interpretation of sleep-specific mechanisms.\nAccordingly, we adopted an SD protocol optimized for both efficacy and animal viability,\nproviding a physiologically relevant framework for investigating the role of sleep in\nregulating DNA damage.\nComment:\nFurthermore, the authors have shown that melatonin induces sleep during the active\nphase but has no effect on sleep duration during the sleep phase. This feature could\nbe leveraged experimentally. For example:\nDoes melatonin-induced sleep during the active phase reduce DNA damage?\nDoes melatonin administration during the sleep phase alter DNA damage\nindependently of sleep induction?\nThese complementary experiments would allow the authors to distinguish between the\nantioxidative effects of melatonin and the role of sleep per se in promoting genomic\nstability. I believe that these additions would significantly strengthen the manuscript\nand make the central claim more persuasive.\nResponse:\nWe agree with the reviewer that testing the effect of melatonin-induced sleep could\nstrengthen one of the manuscript’s conclusions. Following this recommendation, we\nexamined the effect of melatonin on DNA damage during the active period in both C.\nandromeda (ZT13; Fig. 5d, i) and N. vectensis (ZT3; Fig. 5e, j). Melatonin treatment\nreduced DNA damage levels in both species, consistent with its sleep-promoting effect\n(Fig. 3m, p). These results further support the conclusion that sleep alleviates DNA\ndamage in both cnidarians. The additional melatonin experiments are presented in Fig.\n5d, e, i, j and are described in the Results and Methods sections of the revised\nmanuscript. Potential additional effects of melatonin as an antioxidant are discussed\nin the Discussion section (starting at line 301).\nComment:\n4. Number of animals used in DNA damage assays.\nWhile the number of cells analyzed appears sufficient, the number of animals\nexamined seems relatively low. Given that sleep-related behavioral metrics (e.g., bout\nduration, total sleep time) were calculated from a large number of animals, the\nDNA damage experiments should ideally match that level of biological replication.\nTo ensure comparability and statistical robustness, I recommend increasing the\nnumber of animals used in the DNA damage assays to align with those used in the\nsleep behavior analyses.\nResponse:\nAs recommended by the reviewer, in the revised version of the manuscript, we\nrepeated all DNA damage quantification experiments and increased the animal sample\nsize to match the level of biological replication used in the behavioral assays. While\nthe additional data improved statistical robustness, it did not change the conclusions\nof the previous version. The new data sets are incorporated into Figs. 4 and 5, with the\nnumber of animals indicated in the corresponding figure legends. The Results and\nMethods sections have been revised accordingly.\nReviewer #3 (Remarks to the Author):\nThe authors have mostly addressed the reviewers’ comments and my concerns,\nincluding with important new experiments that greatly improve the manuscript.\nResponse:\nWe thank the reviewer for the kind and supportive comments.\nComment:\nWith one very important exception that must be resolved before I could consider\nacceptance of this paper.\nIn my original review, I raised an issue of statistical pseudoreplication that comes from\ntreating neurons within the same animal as “independent”. Their response was not to\ncorrect this but instead claim that they “focus on a cellular phenotype” and that “each\nneuron independently accumulates and repairs DNA damage and is therefore an\nindividual experimental unit.” I very strongly disagree with this claim, on scientific first\nprinciples. In support, the authors state this is an established method by citing their\nown papers! Even if this is something that was done in other papers, it doesn’t make\nthe use of this approach in this paper right; it just means that those other papers are\nalso doing this analysis incorrectly.\nA few simple thought experiments can illustrate the point that neurons in the same\nanimal are not, and cannot, be treated as statistically independent. First, consider an\nexperiment where you expose some hydra to UV to see if DNA damage is resolved by\nsleep. At the first timepoint post UV, you grab a single animal that happened to be\ncloser to the UV source for the duration of the experiment, and then you count DNA\ndamage foci in 100s of neurons in that single animal. Now, on the second timepoint\nafter sleep, you grab a single hydra that happened to stay far from the UV source (darn\nchance!), and count 100s of neurons. Is the n=100s for each group, or is the n=1? If\nyou use n=100, even a very tiny effect, possibly caused by differences in UV exposure\nalone, will have some very small p-value, and you will rejoice that your experiment\n“worked”. But if you use n=1, you will correctly surmise that the n is too small to draw\na meaningful conclusion and you won’t be misled by a chance result.\nEven though the idea that n=100 can come from one animal already makes the point\npretty obvious, maybe one can try to argue that all the animals must get the same UV.\nConsider a second thought experiment then, in which a single neuron in an animal\ngets zapped by UV that generates some DNA damage that causes it to become\nhyperactive. This single neuron then causes downstream neurons to become active\nas well, which we know can trigger DNA damage in those cells. You see? The DNA\ndamage accumulating in neurons doesn’t happen independently of the other neurons\nin that animal, so they cannot be treated as independent. To do so inflates artificially\nthe n, which for statistical purposes must be independent of each other. This is why in\nFigure 4 and 5, the authors report such small p-values, despite a fairly variable set of\ndata—the n is inflated.\nThis fundamental misunderstanding does make me worried about how other analyses\nwere done in the paper, but I don’t see anything clearly wrong in other figures.\nResponse:\nWe appreciate the reviewer’s concern and agree that the animal is an ideal\nexperimental unit (n). Nonetheless, in molecular neuroscience, particularly\nneurophysiology, it is standard to consider individual cells as experimental units,\nespecially when studying DNA damage, neuronal activity, and other cell-specific\nprocesses. Neurons are highly heterogeneous in both activity and genetic identity\n(e.g., excitatory glutamatergic vs. inhibitory GABAergic) and are therefore frequently\ntreated as independent units. The reviewer suggested that “DNA damage\naccumulating in neurons doesn’t happen independently of the other neurons.”\nHowever, neuronal responses are heterogeneous within a single animal: some cells\nare excitatory, others inhibitory, and their activity is differentially modulated.\nConsequently, DNA damage does not accumulate uniformly; it may increase in active\ncells while remaining lower in inhibited ones. Moreover, neuronal activity is only one of\nseveral contributing factors (e.g., ROS, enzymatic errors, chromatin dynamics,\nradiation). Our data, consistent with the broader literature, support that DNA damage\naccumulation is cell-specific. If neurons responded uniformly, homogeneous levels\nwould be expected across cells, which is not observed.\nHowever, in response to the reviewer’s recommendation, we repeated all experiments\nshown in Figs. 4 and 5, increased the number of animals analyzed, and re-analyzed\nthe data statistically. In N. vectensis, the statistical unit is now animals (n=animals). In\nthe larger C. andromeda, which has a long life cycle and challenging reproduction, the\nstatistical unit is the ganglion, a morphologically and functionally distinct pacemaker\nunit with independent activity patterns (Lyndby et al., 2023, PMID: 37752841;\nAbrams et al., 2025, PMID: 40658847). In both species, DNA damage was quantified across\ndozens of cells per unit. The numbers of ganglia and animals are explicitly reported in\nthe figure legends. As anticipated, the additional data and revised analysis did not alter\nthe conclusions of the original manuscript.\nFinally, to further strengthen our findings, we conducted additional experiments\nshowing that melatonin treatment during the active period promoted sleep (Fig. 3m–o,\np–r) and reduced DNA damage levels (Fig. 5d, i; e, j) in both species."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have effectively addressed my concerns. I have nothing new to add.", "reviewer_2": "The authors have responded thoughtfully and comprehensively to the points I raised in my reviews. Their additional\nexperiments and revisions have successfully reinforced the manuscript's main conclusions. Furthermore, the revised title\nnow better represents the content and focus of the study.\nI have no additional comments or concerns. I support publication of this manuscript as currently revised.", "reviewer_3": "The authors have addressed all of my concerns."}, "author_response": "We thank you very much for supporting our paper.\nWe appreciate your kind words and your support for our manuscript."}]

Decision letter