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.

Behavior

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 Drosophila Activity Monitors from Trikinetics Inc. (Waltham MA, USA) and sleep was analyzed in Matlab (MathWorks Inc) with the SCAMP script package⁹⁰. 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.

Sleep Deprivation and Arousability

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.

Food- and Water- Deprivation Assays

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.

Pharmacological Microinjections

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 D. mojavensis baja 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 Drosophila 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 >48h.

Neurochemical Quantifications

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.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical Analysis

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.