Reduced and fragmented sleep in D.mojavensis L2 across temperature conditions

(A-D) D.melanogaster (CS, grey, n=42) and D.mojavensis (Baja, light blue, n=26; Wrigleyi, cyan, n=19; Moja, dark blue, n=21) were reared at a uniform 25°C, and L2 sleep was measured over a three-hour interval at 25°C. (A) Total sleep duration. (B) Sleep bout number. (C) Sleep bout length. (D) Waking activity. (A-D) One-way ANOVAs followed by Dunnett’s multiple comparisons test (**** p<0.0001, * p<0.05, n.s. p>0.05). (E-H) D.melanogaster (CS, grey, n=49) and D.mojavensis (Baja, light blue, n=31; Wrigleyi, cyan, n=23; Moja, dark blue, n=34) were raised at their optimal temperatures (D.melanogaster, 25°C; D.mojavensis, 29°C), and L2 sleep was measured over a three-hour interval at 25°C. (E) Total sleep duration. (F) Sleep bout number. (G) Sleep bout length. (H) Waking activity. (E-H) One-way ANOVAs followed by Dunnett’s multiple comparisons test (**** p<0.0001, * p<0.05, n.s. p>0.05). (I-L) D.melanogaster (CS, grey, n=77) and D.mojavensis (Baja, light blue, n=28; Wrigleyi, cyan, n=22; Moja, dark blue, n=28) were raised at their optimal temperatures, and L2 sleep was measured over a three-hour interval at 29°C. (I) Total sleep duration. (J) Sleep bout number. (K) Sleep bout length. (L) Waking activity. (I-L) One-way ANOVAs followed by Dunnett’s multiple comparisons test (**** p<0.0001, ** p<0.01, * p<0.05, n.s. p>0.05).

Homeostatic sleep regulation and higher arousal threshold in D.mojavensis L2

(A-D) D.melanogaster (CS, grey) and D.mojavensis (Baja, light blue) L2 were subjected to 3 hours of sleep deprivation (SD) via repetitive light pulses (Baseline) followed by a 3-hour recovery period (Rebound) (CS Baseline Ctrl, n=27; CS Baseline Dep, n=25; CS Rebound Ctrl, n=27; CS Rebound Dep, n=25; Baja Baseline Ctrl, n=30; Baja Baseline Dep, n=29; Baja Rebound Ctrl, n=30; Baja Rebound Dep, n=29). Quantifications of (A) total sleep duration, (B) sleep bout number, (C) Sleep bout length, and (D) waking activity demonstrate intact sleep homeostasis in D.mojavensis L2. (A-D) One-way ANOVAs followed by Sidak’s multiple comparisons test (**** p<0.0001, * p<0.05, n.s. p>0.05). (E) D.melanogaster (CS, grey, n=4 replicates) and D.mojavensis (Baja, light blue, n=5 replicates) L2 were exposed to repetitive low-intensity (1 Amp) light pulses over a 2-hour testing window. The percentage of L2 aroused upon stimulation was measured, and activity levels before (Pre-Sti) and during (Sti) stimulation were recorded to assess baseline light responsiveness (CS Pre-Sti, n=30; CS Sti, n=30; Baja Pre-Sti, n=21; Baja Sti, n=21). (E) Arousal threshold comparison: unpaired two-tailed Student’s t-test (* p<0.05). Activity quantification: one-way ANOVAs followed by Sidak’s multiple comparisons test (**** p<0.0001, n.s. p>0.05). (F) D.melanogaster (CS, grey, n=3 replicates) and D.mojavensis (Baja, light blue, n=3 replicates) L2 were exposed to repetitive high-intensity (5 Amp) light pulses over a 2-hour testing window. L2 arousal and activity were quantified as in (E) (CS Pre-Sti, n=88; CS Sti, n=88; Baja Pre-Sti, n=68; Baja Sti, n=68). (F) Arousal threshold comparison: unpaired two-tailed Student’s t-test (* p<0.05). Activity quantification: one-way ANOVAs followed by Sidak’s multiple comparisons test (**** p<0.0001, n.s. p>0.05).

Enhanced larval feeding and developmental energy storage

(A-C) D.melanogaster (CS, grey) and D.mojavensis (Baja, light blue) L2 were assayed for food intake using a dye-based absorbance assay. (A) Under matched sample sizes (Matched N) (CS, n=5 replicates; Baja, n=5 replicates). Unpaired two-tailed Student’s t-test (* p<0.05). (B) Body weight measurements across life stages (CS, n=5 replicates for each stage; Baja, n=5 replicates for each stage). Unpaired two-tailed Student’s t-test (*** p<0.001) (C) Under weight-matched conditions (Matched Weight) (CS, n=5 replicates; Baja, n=5 replicates). Unpaired two-tailed Student’s t-test (*** p<0.001). (D-G) D.melanogaster (CS, grey, n=17) and D.mojavensis (Baja, light blue, n=27) L3 were analyzed for sleep. (D) Total sleep duration. (E) Sleep bout number. (F) Sleep bout length. (G) Waking activity. (D-G) Unpaired two-tailed Student’s t-test (**** p<0.0001, *** p<0.001, * p<0.05). (H) D.melanogaster (CS, grey, n=5 replicates) and D.mojavensis (Baja, light blue, n=5 replicates) L3 were analyzed for feeding. Unpaired two-tailed Student’s t-test (**** p<0.0001). (I-K) D.melanogaster (CS, grey) and D.mojavensis (Baja, light blue) adults were analyzed for feeding. (I) The total number of individual food interactions over 24h was significantly elevated in adult CS females relative to Baja. Welch’s t-test t=11.57, p<0.0001. (J) The mean duration of feeding events that include consecutive licks was prolonged in Baja adults compared to CS. Welch’s t-test t=4.164, p=0.0003. (K) The total number of feeding events per day was increased in CS adult females relative to Baja. Welch’s t-test t=8.608, p<0.0001. n=15-16 flies/group for (I-K). (L) Whole-body triglyceride (TAG) concentrations across development (L2, n=25/group; L3, n=25/group; pupae, n=5/group; adults, n=5/group). TAG levels were comparable at the L2 stage but became significantly higher in D.mojavensis from L3 onward, indicating increased energy storage initiated in later larval stages and sustained into adulthood. Unpaired two-tailed Student’s t-test (*** p<0.001).

D.mojavensis L2 exhibit differential behavioral responses to starvation and enhanced survival.

(A-D) D.melanogaster (CS, grey, n=21 for “Food”, and n=24 for “No Food”) and D.mojavensis (Baja, light blue, n=20 for “Food), and n=39 for “No Food”) L3 were analyzed for sleep under starvation stresses. (A) Total sleep duration. (B) Sleep bout number. (C) Sleep bout length. (D) Waking activity. (A-D) One-way ANOVAs followed by Tukey’s multiple comparisons test (**** p<0.0001, *** p<0.001, ** p<0.01). (E-F) Survival curves of D.melanogaster (CS, grey) and D.mojavensis (Baja, light blue) L2 larvae under starvation conditions. (E) Probability of survival at 25°C. D.mojavensis L2 (Baja, n=14) show significantly longer survival compared to D.melanogaster L2 (CS, n=14) under starvation. (F) Probability of survival at 25°C. D.mojavensis L2 (Baja, n=14) show significantly longer survival compared to D.melanogaster L2 (CS, n=14) under starvation. (E-F) Simple survival analysis (Kaplan-Meier) followed by Log-rank (Mantel-Cox) test (**** p<0.0001).

Experimental design schematic and developmental timing synchronization across species

(A) Schematic of the experimental design. Embryos were collected from crosses of 40♀ female × 20♂ male flies and reared on agar-yeast plates under an 8:8 light-dark (LD) cycle. Larvae were aged to the second instar larval stage (L2) over three days, and freshly molted L2 were collected for subsequent behavioral assays. (B) Mean developmental time (hours from embryo to L2) for D.melanogaster (CS, grey, n=20) and three D.mojavensis subspecies (Baja, light blue, n=20; Wrigleyi, cyan, n=20; Mojavensis, dark blue, n=20) at 25°C and 29°C. Created with BioRender.com.

Reduced and fragmented sleep in D.mojavensis L2 persists on natural cactus-based food across generations.

(A-D) D.melanogaster (CS, grey, n=27) and D.mojavensis (Baja, light blue, n=26) P0 generation were raised on banana-Opuntia cactus medium. L2 sleep was measured over a 3-hour interval in the F1 generation. (A) Total sleep duration. (B) Sleep bout number. (C) Sleep bout length. (D) Waking activity. (A-D) Unpaired two-tailed Student’s t-test (**** p<0.0001, n.s. p>0.05). (E-H) D.melanogaster (CS, grey, n=27) and D.mojavensis (Baja, light blue, n=34) P0 generation were raised on banana-Opuntia cactus medium. L2 sleep was measured over a 3-hour interval in the F5 generation (after five generations on the cactus diet) to minimize laboratory adaptation epigenetic effects. (E) Total sleep duration. (F) Sleep bout number. (G) Sleep bout length. (H) Waking activity. (E-H) Unpaired two-tailed Student’s t-test (**** p<0.0001, *** p <0.001).

L2 sleep reduction and fragmentation are conserved in a closely related desert species.

(A-D) D.melanogaster (CS, grey, n=58) and D.arizonae (green, n=37) L2 were monitored for 3 hours to assess sleep. (A) Total sleep duration. (B) Sleep bout number. (C) Sleep bout length. (D) Waking activity. (A-D) Unpaired two-tailed Student’s t-test (**** p<0.0001).