Regulation of sleep and metabolic homeostasis is critical to an animal's survival and under stringent evolutionary pressure. Animals display remarkable diversity in sleep and metabolic phenotypes; however, an understanding of the ecological forces that select for, and maintain, these phenotypic differences remains poorly understood. The fruit fly, Drosophila melanogaster, is a powerful model for investigating the genetic regulation of sleep and metabolic function, and screening in inbred fly lines has led to the identification of novel genetic regulators of sleep. Nevertheless, little is known about the contributions of naturally occurring genetic differences to sleep, metabolic phenotypes, and their relationship with geographic or environmental gradients. Here, we quantified sleep and metabolic phenotypes in 24 D. melanogaster populations collected from diverse geographic localities. These studies reveal remarkable variation in sleep, starvation resistance, and energy stores. We found that increased sleep duration is associated with proximity to the equator and elevated average annual temperature, suggesting that environmental gradients strongly influence natural variation in sleep. Further, we found variation in metabolic regulation of sleep to be associated with free glucose levels, while starvation resistance associates with glycogen and triglyceride stores. Taken together, these findings reveal robust naturally occurring variation in sleep and metabolic traits in D. melanogaster, providing a model to investigate how evolutionary and ecological history modulate these complex traits.

Metabolic state is a potent modulator of sleep and circadian behavior, and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Across phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes.

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17Metabolic state is a potent modulator of sleep and circadian behavior and animals acutely 18 modulate their sleep in accordance with internal energy stores and food availability. Across 19 phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and 20 behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a 21 critical regulator of complex behaviors, but little is known about the genes that function within 22 the fat body to regulate sleep. To identify molecular factors functioning in the periphery to 23 regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. 24We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly 25 conserved gene involved the biosynthesis of purines, reduces sleep and energy stores. Flies 26 heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially 27 rescued by restoring Ade2 to the fat body. Targeted knockdown of Ade2 in the fat body does not 28 alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific 29 role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions 30 within the fat body to promote both sleep and energy storage, providing a functional link 31 between these processes. 32 20 /+) (Fig 2A,B). However, fat body expression did not fully rescue sleep, as rescue flies slept 131 less than control flies harboring CG-GAL4 or UAS-Ade2 transgenes alone. Therefore, 132 restoration of Ade2 to the fat body partially restores sleep to Ade2 3-20 mutant flies. Similarly, 133restoring Ade2 within the fat body of flies heterozygous for the Ade2 1-6 mutation (CG-134 GAL4>UAS-Ade2; Ade2 1-6 /+) partially restores sleep, with rescue flies sleeping significantly 135 more than UAS-Ade2; Ade2 3-20 /+ heterozygous flies, but less than flies harboring CG-GAL4 136 transgene alone (Fig 2C,D). Expression of Ade2 in the fat body of flies heterozygous for Ade2 3-137 20 or Ade2 1-6 rescued both average sleep bout length during nighttime and sleep bout number 138

19Regulation of sleep and metabolic homeostasis are critical to an animal's survival and under 20 stringent evolutionary pressure. Animals display remarkable diversity in sleep and metabolic 21 phenotypes; however, an understanding of the ecological forces that select for, and maintain, 28populations collected from unique geographic localities. These studies reveal remarkable 29 diversity in sleep, starvation resistance, and energy stores. We found that increased sleep 30 duration is strongly associated with proximity to the equator and elevated average annual 31 temperature, suggesting that environmental gradients strongly influence natural variation in 32 sleep. Further, we found variation in metabolic regulation of sleep to be associated with free 33 glucose levels, while starvation resistance associates with glycogen and triglyceride stores. 34Taken together, these findings reveal robust naturally occurring variation in sleep and metabolic 35 traits in D. melanogaster and suggest that distance from the equator and median temperature is 36 a significant evolutionary factor in sleep regulation and architecture. 50Determining the relationship between sleep and metabolic function, and how evolution shapes 51 these processes, is critical for understanding the function of sleep and basis for sleep differences 52 between and within species. 53The fruit fly Drosophila melanogaster presents a powerful model for investigating genetic 54 interactions between sleep and metabolic processes (Erion et al., 2012; Yurgel et al., 2014 (Shaw et al., 2000). Flies acutely modulate their sleep in accordance with 59 nutrient availability, and starvation potently inhibits sleep and initiates foraging, thereby 60 providing a system to investigate the relationship between sleep and metabolic regulation 61 (Keene et al., 2010; Lee and Park, 2004; Linford et al., 2012 (Cirelli et al., 2005; Koh et al., 2008; Rogulja and Young, 2012), naturally occurring 68 variation has also been leveraged to identify the genetic architecture regulating these processes 69 (Harbison et al., 2009a(Harbison et al., , 2013 95San Diego) with stock numbers provided in Table 1. Flies were reared and maintained on a 12:12 96 light-dark cycle in humidified incubators at 25°C and 65% humidity (Percival Scientific, Perry, IA). 97Unless otherwise noted, all flies were maintained and tested on standard cornmeal/agar 98 medium. 100 | Behavioral Analysis 101Mated female flies aged 3-5 days were briefly anesthetized using CO 2 and then individually 102 placed into plastic tubes containing standard food. Flies were then acclimated to these 103 conditions for at least 24hrs prior to testing. Fly activity was monitored using DAM2 Drosophila 124were assigned a positive value, while measurements to the west were assigned a negative value. 125Since the altitude measurements were not normally distributed, the Log 10 of altitude was used. BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder fo...