Effects of an electric field on sleep quality and life span mediated by ultraviolet (UV)-A/blue light photoreceptor CRYPTOCHROME in Drosophila

Although electric fields (EF) exert beneficial effects on animal wound healing, differentiation, cancers and rheumatoid arthritis, the molecular mechanisms of these effects have remained unclear about a half century. Therefore, we aimed to elucidate the molecular mechanisms underlying EF effects in Drosophila melanogaster as a genetic animal model. Here we show that the sleep quality of wild type (WT) flies was improved by exposure to a 50-Hz (35 kV/m) constant electric field during the day time, but not during the night time. The effect was undetectable in cryptochrome mutant (cryb) flies. Exposure to a 50-Hz electric field under low nutrient conditions elongated the lifespan of male and female WT flies by ~ 18%, but not of several cry mutants and cry RNAi strains. Metabolome analysis indicated that the adenosine triphosphate (ATP) content was higher in intact WT than cry gene mutant strains exposed to an electric field. A putative magnetoreceptor protein and UV-A/blue light photoreceptor, CRYPTOCHROME (CRY) is involved in electric field (EF) receptors in animals. The present findings constitute hitherto unknown genetic evidence of a CRY-based system that is electric field sensitive in animals.


Results
Daytime exposure to an electric field improves sleep quality.. We assayed sleep in Drosophila to determine the effects of a 50-Hz EF on health. The sleep quality of WT flies exposed to a 12-h EF during the day (ZT 0-12) or night (ZT 13-24) differed (Fig. 1a). A pilot study showed that exposure to EF during the day decreased sleep bout number during the night, but increased the sleep bout length and total sleep in WT flies (Fig. 1b). These results suggest that sleep fragmentation was avoided after daytime EF exposure.
We explored this possibility by evaluating sleep in a large number of WT and cryptochrome mutant flies. The results of avoidance of sleep fragmentation as those in the WT were the same (avoidance of sleep fragmentation) in the WT but not in a cryptochrome mutant, cry b (Fig. 1c, lower panels). The differences between sham control flies and EF exposed flies were significant (Fig. 1c, upper panels). We introduced Gal4/UAS system to drive cry gene in cry mutant flies by using cry-driver. Although the parents strains (cry-Gal4; cry 01 and UAS-cry; cry 01 ) did not exhibit the sleep improvement effect by EF exposure, cry gene driven strain (cry-Gal4/UAS-cry; cry 01 ) partially rescued the sleep improvement by EF exposure (Fig. 1d). We concluded that daytime exposure to a 50-Hz EF improved sleep quality via regulating the cryptochrome gene.
Exposure to EF prolongs average life span under low nutrients. We maintained flies under low nutritional status and then exposed them to EF to determine its effects on lifespan. The average lifespan of WT flies exposed to EF was significantly increased ~ 18% compared with that of sham-treated control flies (Fig. 2). These results showed that exposure to an EF significantly increases the average lifespan of Drosophila under low nutritional conditions. Exposure EF increases lifespan of Drosophila under starvation.. We compared the lifespans of starved male and female WT flies exposed to EF to determine whether the longevity effect would persist. The average lifespan of both sexes were significantly increased by ~ 20% in these flies compared with sham-treated flies (Fig. 3a). These results showed that EF increased the lifespan of WT flies under starvation and in low nutritional conditions.
To confirm that cryptochrome is involved in the lifespan elongation mechanism as observed in sleep, we prepared the cryptochrome mutants, cry b , cry 01 , and cry 03 (Fig. S1). Interestingly, EF exposure did not alter the lifespan under starvation in any of these mutant lines (Fig. 3b).
We also assessed the EF effect on the lifespan of RNAi mutant flies under starvation (Fig. 3c). Although EF significantly elongated the average lifespan of the parental lines, that of the RNAi cryptochrome mutant flies did not differ between sham and EF exposure. When cry gene was driven in cry mutant fly by using cry-driver, EF effect on lifespan elongation was rescued (Fig. 3d). These data indicated that cryptochrome is involved in lifespan elongation caused by EF exposure. Furthermore, we prepared cry b strain outcrossed 7times with wild type (Canton S) fly to compare lifetime under EF exposed with the homogenous genetic background (Fig. 3e). Predictably, EF exposure elongated lifespan of wild type flies but not that of cry mutant with the same background.

Metabolome analysis showed increased ATP levels in flies.
Although we showed that the cryptochrome gene is involved in sleep and the lifespan of flies, the involved metabolic pathways remained unknown. We compared the metabolomes of WT and cry b mutant flies with or without EF exposure to determine whether EF induces differences in metabolites. Figure 4a shows many differences between control and EF-exposed flies. The difference between Oregon R and cry b mutant flies might be explained by the genetic backgrounds of the parent flies. Sleep quality was significantly improved by daytime EF exposure. (a) Assessment of EF effects on sleep in WT Drosophila (Oregon R). Nighttime sleep was analyzed after daytime EF (DEF) or nighttime EF (NEF) exposure to EF (AC35 kV/m, 50-Hz) for 12 h. (b) Comparison of DEF and NEF exposure (shown above) on nighttime sleep between WT Drosophila on day 2. Averaged number of sleep bout, averaged length of sleep bout, and total sleep were calculated from locomotor activity data as reported by Ito et al. 25 . Error bars indicate standard deviation. Small number and long length of sleep bout indicates that sleep fragmentation was prevented. Sleep bouts, duration and total sleep were determined in sham control flies (−). DEF exposure decreased sleep bout number, and increased sleep bout length and total sleep time in Oregon-R. There is a possibility that DEF exposure improve sleep quality. (c) Effects of daytime EF exposure on nighttime sleep in clock mutant cry b and WT (Oregon R) flies. Two days after EF exposure, we determined sleep analysis as sum of four independent experiments. Number (left), duration (middle) of sleep bouts, and sleep amount (right) to compare EF exposed flies (+) and sham controlled flies (−). Statistical data by Student's t-test are expressed as mean ± SD. *represents significant differences (p < 0.05). Daytime EF significantly decreased sleep bout number, and increased bout length and total sleep in WT, but did not significantly affect these in cry mutant flies. EF electric field, WT wild type. (d) EF dependent sleep quality improvement in cry 01 mutant flies was rescued by cry promoter driven CRY expression. These three strains with mutated endogenous cry gene (cry 01 ) were used for sleep analysis. Each sleep analysis data of UAS-cry; cry 01 (EF, n = 9; sham, n = 8) and cry-Gal4; cry 01 (EF, n = 17; sham, n = 19) indicated EF exposure is not affect to the sleep quality improvement. But in the cry rescued strain, total sleep amount was significantly increased (p < 0.05, t-test). This data suggests a possibility that EF exposure improved sleep quality of flies through rescued cry gene expression (EF, n = 21; sham, n = 21). www.nature.com/scientificreports/ Exposure to EF significantly prolonged average lifespan of WT flies maintained under low nutrient food conditions. Evaluation of senescence status in EF exposed flies (n = 89) versus shame flies (n = 85). Flies under low nutritional status were exposed to 50-Hz EF. This experiment was repeated 3 times. p < 0.001, logrank test. EF electric field, WT wild type (Oregon R). www.nature.com/scientificreports/ (a) The average lifespan of wild type flies (Oregon R) with both of sex was ~ 20% prolonged significantly by 50 Hz EF exposure that started at 2-3 days after eclosion. Starvation was in 1% agarose (that maintained humidity). Upper panel shows the survival curves of male EF exposed flies (n = 60) and male shame controlled flies (n = 59). p < 0.001, log-rank test. Lower panel shows the survival curves of female EF exposed flies (n = 58) and female shame control flies (n = 60 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4b shows an increased ratio of metabolites in EF-compared with sham-treated flies. The graph is arranged in decreased order for wild type flies (blue) and cry b mutant flies (red). The difference between control and EF-exposed WT was remarkably compared with the cry b mutant. Table 1 summarizes the data. The www.nature.com/scientificreports/ abundance of ATP was 5.99-fold higher in EF-than sham-treated flies, but that in cry b mutant flies was ~ 1.2-fold higher (Fig. 4b, Table 1). To confirm metabolome data, ATP contents after EF exposure was measured by using the luciferase kit (Supplemental Fig. 2). These data indicated that EF exposure significantly increased ATP level and several nucleic acid metabolism in whole fly.

Discussion
Exposure to 50 Hz of EF (35 kV/m, constant) during the daytime improved sleep quality in WT flies, whereas nighttime exposure did not. Interestingly, this effect did not observed in cry b mutant flies. The fact that the number of sleep bouts decreased but the sleep bout length and total sleep time increased with daytime EF exposure suggests that sleep fragmentation was avoided and the duration of each bout of sleep was increased. This phenomenon is considered to be good marker of sleep quality and health in medical science. Exposure to 50 Hz EF at night did not exert such effects. As one of the possibilities, the difference might be explained by the different sensitivity of CRY during day and night. Though cry mRNA expression oscillates with the circadian rhythms, CRY protein does not cycle and simply accumulated in darkness. The protein might be oscillating in LD for light induce CRY degradation. One of possibility is that CRY may be stabilized by EF exposure similarly with magnetic field 8 . Further research is needed to clarify such possibility. We also found that wild type fly placed in 50 Hz Electric Field elongated average lifespan about 18%. Such lifespan elongation effect was not observed by using several lines including cry gene mutant strains and cry RNAi strain. CRYPTOCHROMEs (CRYs) are structurally related to ultraviolet (UV)/blue-sensitive DNA repair enzymes called photolyases but lack the ability to repair pyrimidine dimers generated by UV exposure. The cry gene was originally identified as a blue light photoreceptor in plants 16,17 . The role of CRY in Drosophila is cell-autonomous synchronization and the entrainment of circadian clocks 18 . However, CRY plays a key role in magnetic field detection (8 ~ 14). Here we show that the putative magnetoreceptor protein and UV-A/blue light photoreceptor, CRY is deeply involved in EF receptor in animals. CRY1 and CRY2 have circadian clock functions and may detect magnetic fields in various animals 19 .
Metabolome comparisons between intact WT and cry gene mutant strains after exposure to EF indicated that ATP was 5.99 fold more abundant in the WT. Daytime exposure to a 50 Hz EF might activate cellular activities or motility in flies because ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities 20 . ATP is produced in mitochondria. Interestingly, Alex et al. reported that RNAi of cry gene brings degenerated mitochondria in fly heart by coherence microscopy observation 21 . The paper suggests that cry might be involved in mitochondria development and function in Drosophila. ATP is produced in the mitochondrial electron transport chain and EF exposure may be involved in the supplier for electron in this system. Quantum biological analysis of electron donor/acceptor might be required for further explanation.
We recognize that this work for EF is different from magnetic field because the magnetic field produced from our device is very weak (Approximately 0.0007 Gauss) to the level on earth (Geomagnetism is 0.5 Gauss). The present findings provide the first genetic evidence of a CRY-based EF sensitive system in animals. Electric field exposure. We created a uniform EF by transforming a 50-Hz alternating current at 35 kV/m using a Healthtron HEF-P3500 (Hakuju Institute for Health Science Co., Ltd., Tokyo, Japan) to deliver EF. Flies and medium were placed in vials or tubes and placed 3-5 cm apart between the electrodes of the Healthtron for EF exposure.

Flies. The
Assays of sleep behavior. Male flies were individually placed in glass tubes (inner diameter, 3 mm) containing 5% sucrose and 2% agarose and exposed to EF. The tubes were then transferred to a Drosophila Activity Monitoring (DAM) system (TriKinetics Inc., Waltham, MA, USA) and locomotor activity was measured in 1-min bins. Sleep was defined as > 5 min of consolidated inactivity 24,25 . Metabolome analysis. Two days after eclosion, male Oregon R or cry b flies were exposed to EF for 48 h at DD in starved condition, then whole body flies were stored (30-40 mg batches) frozen in liquid nitrogen. Metabolites were extracted and metabolomes were measured at Human Metabolome Technologies Inc. (HMT, Tsuruoka, Japan. For CE-TOFMS analysis, 1500 μL of 50% acetonitrile (v/v) was added to Drosophila samples and blended under cooling using a BMS-M10N21 homogenizer (Bio Medical Science Inc., Tokyo, Japan) at 1500 rpm, 120 s × 5 times). Homogenates were separated by centrifugation at 2300×g for 5 min at 4 °C and the upper aqueous layer was centrifugally passed through Millipore 5 kDa cut-of filter (Ultrafree MC-PLHCC, HMT; Millipore Sigma Co., Ltd., Burlington, MA, USA) at 9100×g for 120 min at 4 °C to remove macromolecules. The filtrate was then concentrated by centrifugation and reconstituted in 100 μL of Milli-Q water before CE-TOFMS analysis.
Cationic metabolites were analyzed using a fused silica capillary (50 μm i. www.nature.com/scientificreports/ at a pressure of 50 mbar for 10 s, and the applied voltage was 27 kV. Samples were assessed by electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode, at a capillary voltage of 4000 V. Spectra were obtained between m/z 50 and 1000. Anionic metabolites were analyzed using a fused silica capillary (50 μm i.d. × 80 cm), with the anionic electrophoresis buffer H3302-1021 (Human Metabolome Technologies) as the electrolyte. Samples were injected at a pressure of 50 mbar for 25 s, and the applied voltage was 30 kV. Samples were assessed by ESI-MS in the negativne ion mode, and the capillary voltage was 3500 V. Spectra were obtained between m/z 50 and 1000.