DOI: 10.5061/dryad.w9ghx3fxw
title: Elevated sleep quota in a stress-resilient Drosophila species

description: Sleep is broadly conserved across the animal kingdom but can vary widely
 between species. It is currently unclear which selective pressures and
 regulatory mechanisms influence differences in sleep between species. The
 fruit fly Drosophilamelanogaster has become a successful model system for
 examining sleep regulation and function, but little is known about the
 sleep patterns in many related fly species.  Here, we find that
 fly species with adaptations to extreme desert environments, including D.
 mojavensis, exhibit strong increases in baseline sleep compared to D.
 melanogaster. Long-sleeping D. mojavensis show intact homeostasis,
 indicating that desert flies carry an elevated drive for sleep. In
 addition, D. mojavensis exhibit altered abundance or distribution of
 several sleep/wake related neuromodulators and neuropeptides that are
 consistent with their reduced locomotor activity and increased sleep.
 Finally, we find that in a nutrient-deprived environment, the sleep
 patterns of individual D. mojavensis are strongly correlated with their
 survival time and that disrupting sleep via constant light stimulation
 renders D. mojavensis more sensitive to starvation. Our results
 demonstrate that D. mojavensis is a novel model for studying organisms
 with high sleep drive, and for exploring sleep strategies that provide
 resilience in extreme environments.
description: <em>Behavior</em> 4-8 day old
 female flies were housed individually in borosilicate glass tubes (65mm
 length, 5mm diameter) containing fly food coated with paraffin wax at one
 end and a foam plug in the other. Locomotor activity was recorded using
 DAM5M or DAM5H multibeam <em>Drosophila </em>Activity Monitors
 from Trikinetics Inc. (Waltham MA, USA) and sleep was analyzed in Matlab
 (MathWorks Inc) with the SCAMP script package<sup>90</sup>.
 Locomotor activity was measured as the number of movements between beams
 per one-minute bins. Periods of sleep were defined by at least 5 minutes
 with no change in position within the multibeam activity monitors. Sleep
 time courses display 30-min time bins. <em>Sleep
 Deprivation and Arousability </em> Sleep
 deprivations were performed mechanically by mounting DAM5M activity
 monitors onto platform vortexers (VWR 58816-115). Individual tubes were
 plugged with food at one end and 3D-printed PLA plastic caps at the other.
 Monitors were vortexed at an intensity of 2.5g for 3-second pulses every
 minute through the duration of the 12-hour dark period. Arousability was
 tested in a darkened incubator with 60 seconds of blue light (luminance
 0.048 Lv) every hour for 24 hours following sleep deprivation.
 <em>Food- and Water- Deprivation
 Assays</em> All flies were put in DAM5H activity
 monitors on standard food for baseline recording. After 2-3 days, control
 flies were transferred to tubes containing fresh food, food-deprived flies
 to tubes containing a 1% agar gel, and food-and-water-deprived flies to
 empty tubes plugged with foam at both ends. Flies immobile for at least 24
 hours were defined as dead and data subsequent to their last full day
 alive was removed from sleep analysis.
 <em>Pharmacological Microinjections</em>
 4-8 day old female flies were loaded into behavior tubes and
 monitored in DAM5M Activity Monitors to obtain baseline sleep and
 locomotor activity under 12h light: 12h dark (25˚C). After 1-2 days of
 baseline in DAM5M monitors, flies housed in borosilicate tubes were placed
 on ice for anesthetization prior to injection using Drummond Nanoject II.
 For injection of exogenous neuromodulators, the anteriormost ocelli of
 <em>D. mojavensis baja </em>were injected with 18.4nl of
 20mg/mL of Octopamine (Sigma-Aldrich, Catalog # O0250). For each round of injections, new OA is solubilized using Schneider’s <em>Drosophila</em> Medium with L-Glutamine (Genesee Scientific, Catalog # 25-515). Following each individual injection, flies are returned back into individual borosilicate tubes, and placed in respective DAM5M Activity Monitors to continue sleep and activity surveillance for &gt;48h. <em>Neurochemical Quantifications</em> Sample preparation protocol Fly brain samples were stored at -80°C then treated with 99.9/1 Water/Formic Acid. An internal standard (IS) of each targeted compound was added to every sample to account for compound loss during sample processing. The samples are vortexed, homogenized for 30 sec in a bead beater using 2.0 mm zirconia beads, and centrifuged at 16.000xg for 5 min. The supernatant is transferred to new microcentrifuge test tubes and dried in a vacuum concentrator. The samples are reconstituted in 40 µl of water, vortexed, and centrifuged. The supernatant is transferred to HPLC vials and 10 µl is injected to an HPLC - triple quadrupole mass spectrometer system for analysis. Liquid Chromatography-Tandem Mass Spectrometry LC-MS A targeted LC-MS/MS assay was developed for each compound using the multiple reaction monitoring (MRM) acquisition method on a triple quadrupole mass spectrometer (6460, Agilent Technologies) coupled to an HPLC system (1290 Infinity, Agilent Technologies) with an analytical reversed phase column (GL Sciences, Phenyl 2 µm 150 x 2.1 mm UP). The HPLC method utilized a mobile phase constituted of solvent A (100/0.1, v/v, Water/Formic Acid) and solvent B (100/0.1, v/v, Acetonitrile/Formic Acid) and a gradient was used for the elution of the compounds (min/%B: 0/0, 10/0, 25/75, 27/0, 35/0). The mass spectrometer was operated in positive ion mode and fragment ions originating from each compound was monitored at specific LC retention times to ensure specificity and accurate quantification in the complex biological samples (Octopamine OA 159-136, Histamine HA 112-95, Dopamine DA 154-137, Serotonin 5HT 177-160). The standard curve was made by plotting the known concentration for each analyte of interest (CDN Isotopes) against the ratio of measured chromatographic peak areas corresponding to the analyte over that of the labeled standards. The trendline equation was then used to calculate the absolute concentrations of each compound in fly brain tissue. <strong>QUANTIFICATION AND STATISTICAL ANALYSIS</strong> <em>Statistical Analysis</em> Statistical tests were completed as described in the figure legends using Prism 9 (GraphPad Software, Boston MA, USA). Statistical comparisons primarily consist of one- or two-way ANOVAs followed by pairwise Holm-Sidak’s multiple comparisons test when experiments include at least three experimental groups or two-tailed Student’s T-test for experiments that include two groups; specific tests used are described in each figure legend. All data figures pool individual data points from at least two independent replicates. 
description: # Elevated sleep quota in a stress-resilient Drosophila species
 [https://doi.org/10.5061/dryad.w9ghx3fxw](https://doi.org/10.5061/dryad.w9ghx3fxw) Dataset contains underlying data for main and supplemental figures from manuscript. Included files: 1. Source data for Figure 1.xls 1. Baseline sleep time course data (Figure 1A), total day and night sleep (Figure 1B; values in minutes of sleep), P(wake) (Figure 1C), P(doze) (Figure 1D), and counts/waking minute (Figure 1E) for females from D. melanogaster and D. mojavensis stocks. Columns represent data from individual flies with stock label in Row 1. Rows represent 30-min time bins for Figure 1A, or day and night for Figures 1B-E. 2. Length of individual sleep bouts during the day (Figure 1F) and night (1G) from D. melanogaster and D. mojavensis stocks. 3. Daily sleep amount for D. melanogaster and D. mojavensis stocks using different time durations for minimum sleep bout length (Figure 1H; values in minutes of sleep). 4. Sleep time course for D. moj. moj. individuals across a 7-day experiment. Columns represent data from individual flies, rows show sleep in 30-min time bins (Figure 1I-J). 2. Source data for Figure 2.xls 1. Sleep time course (Figure 2A, left) and daily sleep total (Figure 2A, right; values in minutes of sleep) for three stocks of D. melanogaster 2. Sleep time course (Figure 2B, left) and daily sleep total (Figure 2B, right; values in minutes of sleep) for D. melanogaster and D. mojavensis stocks fed Banana-Cactus media 3. Sleep time course (Figure 2C, left) and daily sleep total (Figure 2C, right; values in minutes of sleep) for stocks of D. melanogaster and 4 species of desert-adapted flies (D. arizonae, D. buzzatii, D. mulleri & D. mojavensis) 4. Daily sleep totals for D. melanogaster and D. mojavensis housed at different constant temperatures from 25˚C to 31˚C (Figure 2D; values in minutes of sleep) 5. Absolute and relative sleep time courses for D. melanogaster and D. mojavensis flies exposed to a daily temperature ramp from 25˚C to 35˚C (Figures 2E & 2F) 3. Source data for Figure 3.xls 1. Sleep time course for D. moj. moj (Figure 3A) and D. moj baja (Figure 3B) for flies during one day of baseline, one day with overnight vortex sleep deprivation, and one recovery day (SD group) along with one group re-plotting the baseline sleep timecourse for visual comparison (Baseline replotted). Columns represent data from individual flies; rows are sleep in 30-min bins. 2. % of sleep recovered from D. melanogaster and D. mojavensis stocks after overnight vortex sleep deprivation (Figure 3C). 3. % of flies awakened (Figure 3D) and Latency to sleep (Figure 3E) for D. moj moj flies that were exposed to hourly blue LED stimulation. Columns show experimental group (Control, SD, SD+24h recovery), rows represent mean group responses for individual stimulus trials. 4. Sleep time course for D. moj. moj (Figure 3F) and D. moj. baja (Figure 3G) for flies during on one day of baseline, one day with overnight blue LED sleep deprivation, and one recovery day (Blue LED SD group) along with one group that did not receive blue LED stimulation  (Control group). Columns represent data from individual flies; rows are sleep in 30-min bins. 5. % of sleep lost for D. moj moj and D. moj baja during overnight blue LED stimulation (Figure 3H). Columns show D. moj subspecies, rows show individual fly responses. 6. Daily sleep totals for experimental groups from Figure 3G and 3H (Figure 3I; values in minutes of sleep). Columns represent individual flies, rows show each day of experiment. 7. Sleep time course for D. moj moj (Figure 3J) and D. moj baja (Figure 3K) for flies housed in 12h:12h LD followed by 2-days in constant darkness. Daily sleep totals across the experiment in Figure 3L (values in minutes of sleep).  4. Source data for Figure 4.xls 1. Relative neurochemical abundance from D. melanogaster and D. mojavensis for 5HT (Figure 4A), OA (Figure 4B), DA (Figure 4C), and HA (Figure 4D). Values are normalized to mean from Canton-S D. melanogaster abundance for each assay. 2. Sleep timecourse (Figure 4K) for flies that received microinjections of octopamine or vehicle control. Total sleep and activity counts in 24h following injection in Figure L (values in minutes of sleep) & M. 5. Source data for Figure 6.xls 1. Survival timecourse for D. melanogaster (Cs) and D. mojavensis (moj and baja) during food deprivation (Figure 6A) or food and water deprivation (Figure 6B). 2. Daily sleep totals for D. moj baja (Figure 6C) and D. moj baja (Figure 6D) for flies that are either fed on standard fly media, food deprived, or food and water deprived. Values shown in minutes of sleep. 3. Survival times for D. moj baja flies that were either housed in 12h:12h LD or constant blue light during food deprivation (Figure 6E) 4. Scatterplot data for total sleep (values in minutes of sleep) vs survival time for individual D. moj baja flies during food deprivation (Figure 6F). 5. Survival times for D. moj baja flies that were either housed in 12h:12h LD or constant blue light during food and water deprivation (Figure 6G) 6. Scatterplot data for total sleep (values in minutes of sleep) vs survival time for individual D. moj baja flies during food and water deprivation (Figure 6H). 7. Survival times for D. moj moj flies that were either housed in 12h:12h LD or constant blue light during food deprivation (Figure 6I). 8. Daily sleep times for D. moj moj flies housed in 12h:12h LD or constant blue light during food deprivation (Figure 6J; values in minutes of sleep). 9. Survival times for responding vs non-responding D. moj moj flies housed in constant blue light during food deprivation (Figure 6K). 10. Scatterplot data for total sleep (values in minutes of sleep) vs survival time for individual D. moj moj flies during food and water deprivation (Figure 6L). 11. Survival times for D. moj moj flies that were either housed in 12h:12h LD or constant blue light during food and water deprivation (Figure 6M). 12. Daily sleep times for D. moj moj flies housed in 12h:12h LD or constant blue light during food and water deprivation (Figure 6N; values in minutes of sleep). 13. Survival times for responding vs non-responding D. moj moj flies housed in constant blue light during food and water deprivation (Figure 6O). 14. Scatterplot data for total sleep (values in minutes of sleep) vs survival time for individual D. moj moj flies during food and water deprivation (Figure 6P). 6. Source data for Figure S1.xls  1. Baseline sleep time course data (Figure S1A), total day and night sleep (Figure S1B; values in minutes of sleep), P(wake) (Figure S1C), P(doze) (Figure 1SD), and counts/waking minute (Figure S1E) for males from D. melanogaster and D. mojavensis stocks. Columns represent data from individual flies with stock label in Row 1. Rows represent 30-min time bins for Figure 1A, or day and night for Figures 1B-E. 7. Source data for Figure S3.xls 1. Sleep time course for D. melanogaster (Cs) and D. mojavensis females while flies were housed at 25˚C for one day followed by three days at 27˚C (Figure S3A), 29˚C (Figure S3B), or 31˚C (Figure S3C). Columns represent data from individual flies, rows show sleep time/30-min bin. 8. Source data for Figure S4.xls 1. P(wake) values for individual D. moj moj (Figure S4A) or D. moj baja (Figure S4B) during the day and night on a baseline day and recovery day after 12h overnight vortex sleep deprivation. 2. Activity counts/waking minute values for individual D. moj moj (Figure S4C) or D. moj baja (Figure S4D) during the day and night on a baseline day and recovery day after 12h overnight vortex sleep deprivation. 3. Sleep timecourse for D. moj moj (Figure S4E) or D. moj baja (Figure S4F) for flies during two days of recovery after 12h overnight vortex sleep deprivation (Recovery days (after overnight vortex SD) group) or replotting of sleep time from baseline days for visual comparison (Baseline replotted group). 9. Source data for Figure S6.xls 1. Survival times for fed D. moj moj and D. moj baja flies housed either in 12h:12h LD or constant blue light (Figure S6A). 2. Daily sleep time (values in minutes of sleep) for D. moj baja flies housed in either 12h:12h LD or constant blue light during food deprivation (Figure S6B) or food and water deprivation (Figure S6C).  3. Scatter plot data for D. moj baja flies during food deprivation: Daily activity counts vs Survival time (hrs) (Figure S6D); Activity counts/waking minute vs Survival time (hrs) (Figure S6E); Daily sleep (values in mins of sleep) vs Daily activity counts (Figure S6F). 4. Scatter plot data for D. moj baja flies during food and water deprivation: Daily activity counts vs Survival time (hrs) (Figure S6G); Activity counts/waking minute vs Survival time (hrs) (Figure S6H); Daily sleep (values in mins of sleep) vs Daily activity counts (Figure S6I). 5. Scatter plot data for D. moj moj flies during food deprivation: Daily activity counts vs Survival time (hrs) (Figure S6J); Activity counts/waking minute vs Survival time (hrs) (Figure S6K); Daily sleep (values in mins of sleep) vs Daily activity counts (Figure S6L). 6. Scatter plot data for D. moj moj flies during food and water deprivation: Daily activity counts vs Survival time (hrs) (Figure S6M); Activity counts/waking minute vs Survival time (hrs) (Figure S6N); Daily sleep (values in mins of sleep) vs Daily activity counts (Figure S6O).