A short photoperiod alters brain metabolism and cold resistance in Drosophila melanogaster

A short photoperiod alters brain metabolism and cold resistance in Drosophila melanogaster | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A short photoperiod alters brain metabolism and cold resistance in Drosophila melanogaster Madhura Sapre, Anna Hovhanyan, Werner Schmitz, Peter Deppisch, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6800169/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract To survive, animals need to prepare for winter in advance, and this process begins in the brain in response to the shortening of the photoperiod in fall. Here, we demonstrate that exposing adult flies for just 14 days to a short photoperiod at a constant temperature of 20°C increases their cold resistance and dramatically alters brain metabolism. Such flies have significantly lower levels of monosaccharides, and a lower ATP/AMP ratio in their brains than flies exposed to a long photoperiod, despite being less active and eating more. The levels of storage and structural lipids (triacylglycerols and phospholipids) as well as the number of lipid droplets in the brain increase, suggesting the utilization of glucose for the synthesis of lipids via the citrate shuttle. In addition, during short days, the ratio between the reduced and oxidized forms of glutathione increase, as do detoxification processes and autophagy. This suggests that the brain of short-term flies is less sensitive to oxidative stress and neurodegeneration, which is essential for survival throughout the winter. Overall, our results show that exposure to a short photoperiod has significant metabolic and physiological consequences in the fly brain that serve to prepare for the coming winter. Biological sciences/Molecular biology Biological sciences/Neuroscience LC-MS based metabolomics lipids polar metabolites photoperiod feeding activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Organisms living in temperate zones are exposed to strong seasonal changes to which they must adapt. Winter poses a problem for organisms for two key reasons: energy and water. Food sources become scarce, humidity decreases, and temperatures fall below the optimal range, which means that the cellular functions that provide energy falter and eventually fail. This is particularly relevant for small organisms such as insects, which are unable to maintain a constant body temperature. When internal temperatures fall below freezing, the water that makes up at least 70 percent of animal cells freezes, and the organism dies. To avoid suboptimal internal temperatures and dehydration, animals can migrate to warmer regions, enter a dormant state with reduced energy expenditure, and develop resistance to cold and dryness 1 . To survive, animals must adjust their metabolism long before winter sets in. Those who do not prepare for winter in time will have to deal with severe fitness disadvantages. But how do they recognize that winter is coming? Temperature can fluctuate greatly and is therefore no reliable indicator of the coming season. The most robust cue for assessing the onset of winter is the shortening of day length (photoperiod) in the fall. Therefore, all animals have mechanisms in their brains to measure photoperiod, which communicate with the endocrine system and initiate physiological and metabolic changes when it falls below a critical level, even if ambient temperatures are still pleasant. These mechanisms are called photoperiodic responses and have been well-studied in several mammals and insects 2 , 3 . They need an internal circadian clock as a reference for measuring the length of the day 4 . The fly Drosophila melanogaster has been successfully used as a model to understand the role of the circadian clock in photoperiodic responses 5 – 8 . Light and the circadian clock are known to affect metabolite levels in the body and head of fruit flies 9 , 10 . However, nothing is known about the metabolic responses of the brain to such changes. This is surprising, since the brain contains the master circadian clock, processes light information, measures photoperiod and initiates seasonal changes in behavior and in the neuroendocrine system 11 , 12 . In addition, the brain is one of the most metabolically expensive organs in the body, and this is true for all animals 13 . As an example, in humans, the brain makes up about 2% of body mass but consumes 20% of total energy and this applies during wakefulness and sleep. Consequently, animals have evolved efficient ways to reduce the energy costs of their brain during conditions of food scarcity such as the winter. Some moles even shrink the size of their brain in winter and regrow it in spring 14 . Such extreme effects are extremely unlikely in fruit flies, which only survive the winter once in a dormant state before producing the next generation the following spring. Nevertheless, significant metabolic adaptations to photoperiod shortening are expected. An earlier study showed that flies already increase their cold resistance when reared in short-day conditions compared to flies reared in long-day conditions at the same temperature 5 , 15 . Since Drosophila flies overwinter as adults and photoperiod is also measured in the adult stage, we investigated whether exposing adult flies to short-day conditions is sufficient to cause metabolic changes in the brain. We found that the exposure of adult flies to a short photoperiod is sufficient to increase their cold resistance and to change their brain metabolism, while the metabolism in the whole head and body remain unchanged. In their brains, flies appear to metabolize sugar to synthesize the storage lipids triacylglycerols and structural phospholipids. They increase polyamine levels which enhance autophagy and may extend lifespan, and they appear to become more resistant to cold and oxidative stress. All these changes can be regarded as preparation for the coming winter. Material and Methods Fly rearing and adult maintenance Wild-type Drosophila melanogaster flies of the strain CantonS (WT cs ) were reared under 12h:12h light:dark cycles (LD12:12) at 25 ± 0.2°C and 60 ± 2% relative humidity on Drosophila food consisting of 0.8% agar, 2.2% sugar beet syrup, 8.0% malt extract, 1.8% yeast, 1.0% soy flour, 8.0% corn flour, and 0.3% hydroxybenzoic acid. After eclosion, adult flies were transferred to new fly vials containing the same food and exposed for 14 days either to long (LD16:8) or short (LD8:16) photoperiods, while temperature was kept constant at 20 °± 0.2°C. In the following experiments, the so treated flies are called long- and short-day flies, respectively. All experiments were performed with male flies. Locomotor activity recordings The locomotor activity of 32 flies each was recorded under long (LD16:8) and short (LD8:16) photoperiods in the Drosophila Activity Monitor (DAM)-System of TriKinetics (Waltham, MA, USA). Flies were transferred into 5 mm tubes containing food (4% sucrose and 2% agar in water) using CO 2 anesthesia. Activity was monitored by detecting the disruptions in an infrared light beam at 1-minute intervals. The activity was tracked over a period of about 9 days at 20°C with 60% relative humidity. Light intensity during the experiments was set to 100 lux. Data analysis To reveal the activity pattern of the flies, activity data were plotted as individual and average actograms using the ImageJ plug-in ActogramJ 16 . Furthermore, individual and average activity profiles were calculated for the two fly groups and overall daily activity levels were determined as described in Schlichting and Helfrich-Förster 17 for average actograms and average activity profiles see Figure S1 ). Capillary feeding (CAFE) assay Feeding was assessed with the CAFÉ assay by two methods. First, food consumption was measured for 2 hours in groups of 15 flies, and second, in individual flies for 24 hours. For the first assay 20 groups of flies consisting of 15 long- and short-day flies, each, were transferred to vials without food on day 14, directly after lights-on. After 2 hours of starvation, the fly vials were placed at an angle of 45° and two microcapillary pipettes were inserted, one containing 5 µl of normal tap water and the other 5 µl of 5% sucrose solution with blue food dye. The flies could choose freely between the two capillaries. Every 20 minutes, we recorded how much liquid they had consumed and after 2 hours, the total consumed food was determined for all 40 fly groups (in total 300 long- and short-day flies, respectively). An empty vial containing only the two capillaries served as a control for evaporation. For the second assay, individual flies were confined to 2 ml Eppendorf cups. The top of each cup contained a hole, in which a capillary containing 5 µl of 5% sucrose solution was inserted. There were two more holes on the sides of the tubes for aeration. After 24 hours the amount of food consumed by each fly was determined. Like before, an empty vial containing only the capillary was used as a control for evaporation. Data analysis At first, the data were checked for a normal distribution (Kolmogorov-Smirnov test; p < 0.008, Shapiro-Wilk test; p < 0.03). Since the data deviated significantly from normality, the non-parametric Mann-Whitney-U test was applied for checking statistically significant differences. Cold tolerance assay Cold tolerance was measured by recovery from chill coma (David et al., 1998; Macdonald et al., 2004). Flies from twenty vials, containing 10 long- and short-day flies, each, were transferred to empty fly vials equipped only with water-moistened filter paper. The vials were then stored in darkness on crushed ice at 0°C for 24 hours. After cold exposure, the flies were transferred to 25°C for recovery. They were immediately individually placed into glass tubes of the DAM TriKinetics system to monitor their activity. The percentage of flies that resumed activity indicating survival of cold exposure was determined. Furthermore, the time passing until the first movement was measured in flies surviving the treatment Lipid analysis For the body and head sampling, long- and short-day flies were shock frozen in liquid nitrogen (shortly after lights-on) and stored at -80°C. Fly heads were separated from the body on ice and three fly heads, and three fly bodies were pooled for each sample. For the brain sampling, long- and short-day flies were quickly killed by submersion in 4% paraformaldehyde (shortly after lights-on). After 1h of fixation, the brains of the flies were dissected in phosphate buffered saline and transferred to methanol. 10 brains were pooled for each sample and stored at -80 o C until analysis. Blight-Dyer extraction was performed as published in Mueller et al. (2015) and lipid profiling of the organic phase with LC-MS was performed in accordance with Schäbler et al. 10 . For lipid fingerprinting, the acquired data used for lipid profiling were pre-processed with Progenesis QI (version 1.0, Waters) and MetaboAnalyst 6.0 was used for statistical analysis 18 . Graphs were generated with R (v4.2.1) via Rstudio (2022-06-23) using ggplot2 package. Targeted analysis of water-soluble primary metabolites Water-soluble metabolites from 10 pooled Drosophila melanogaster brains were extracted with 500 µl ice-cold MeOH/H 2 O (80/20, v/v) containing 0.04 µM lamivudine and 4 µM each of D 2 -glucose, D 4 -succinate, D 5 -glycine and 15 N-glutamate (Sigma-Aldrich, St. Louis, USA). After centrifugation, the resulting supernatants were evaporated in a rotary evaporator (Savant, Thermo Fisher Scientific, Waltham, USA). Dry sample extracts were redissolved in 50 µl 5 mM NH 4 OAc in CH 3 CN / H 2 O (50/50, v/v). 20 µl supernatant was transferred to LC-vials. Metabolites were analyzed by LC-MS using the following settings: For LC-MS analysis 3 µl of each sample was applied to a SeQuant ZIC-cHILIC (3 µm particles, 100 × 2.1 mm) (Merck, Darmstadt, Germany). Metabolites were separated with Solvent A, consisting of 5 mM NH 4 OAc in CH 3 CN/H 2 O (40/60, v/v) and solvent B consisting of 5 mM NH 4 OAc in CH 3 CN/H 2 O (95/5, v/v) at a flow rate of 200 µl/min at 45°C by LC using a DIONEX Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany). A linear gradient starting after 2 min with 100% solvent B decreasing to 10% solvent B within 23 min, followed by 16 min 10% solvent B and a linear increase to 100% solvent B in 2 min was applied. Recalibration of the column was achieved by 7 min prerun with 100% solvent B before each injection. All MS-analyses were performed on a high-resolution Q Exactive mass spectrometer equipped with a HESI probe (Thermo Fisher Scientific, Bremen, Germany) in alternating positive and negative full MS mode with a scan range of 69.0–1000 m/z at 70K resolution and the following ESI source parameters: sheath gas: 30, auxiliary gas: 1, sweep gas: 0, aux gas heater temperature: 120°C, spray voltage: 3 kV, capillary temperature: 320°C, S-lens RF level: 50. XIC generation and signal quantitation was performed using TraceFinder™ V 5.1 (Thermo Fisher Scientific, Bremen, Germany) integrating peaks which corresponded to the calculated monoisotopic metabolite masses (MIM +/- H + ± 2 mMU). All analyses were performed in six independent replicates. Fluorescent immunohistochemistry and microscopy for lipid droplets Immunohistochemistry was performed on male flies entrained to long and short photoperiods for 14 days, following the protocol of Schubert et al. 19 , to quantify the number and area of lipid droplets in the brain using BODIPY493/503 (Invitrogen, Cat. No: D3922). Flies were fixed in 4% paraformaldehyde in PBS with 0.5% Triton X-100 (PBST) for 3.5 hours. After three PBS washes, flies were dissected, and the brains were washed twice with PBST. Brains were then incubated overnight at 4°C with BODIPY493/503, followed by six PBST washes. Finally, brains were mounted on slides using Vectashield (Vector Laboratories, Burlingame, CA, USA) with ~ 150 µm spacers. The images were acquired with a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with hybrid detectors, photon multiplier tubed and a white laser for excitation. We used 20-fold glycerol immersion objective (HC PL APO, Leica Microsystems, Wetzlar Germany) and obtained confocal stacks with 2 µm z-step size and 1024 × 512 pixels. The obtained images were analyzed with Fiji ImageJ 20 . Lipid droplet analysis was performed on the central brain region only. A maximum Z-projection was generated, and noise was reduced using the Despeckle function. Manual thresholding was applied, followed by Watershed to separate overlapping droplets. The Analyze Particles function in ImageJ was used to quantify lipid droplet number and total area. Approximately 13 brains were analyzed per condition. Results Flies are less active but eat slightly more under short days Our locomotor activity recordings showed that the flies were significantly less active under short days than under long days (Fig. 1 a, S1). This suggests that they would need less energy in short days and consequently should eat less. However, this was not the case. Our capillary feeder assay (CAFE) revealed that short-day flies of the same age ate even slightly more than long-day flies over a two-hour duration, and our 24 h feeding assay confirmed significantly higher food intake (Fig. 1 b). This suggests that the flies use the extra energy uptake to adapt their metabolism to the coming winter. Short days during adulthood increase cold tolerance of the flies Previous results have revealed that flies reared in short-day conditions increase their cold resistance when compared to flies reared in long-day conditions at the same temperature 5 . Here, we aimed to determine whether a 14-day exposure period to short days during adulthood is enough to increase cold tolerance. We adopted a simple assay to measure cold tolerance called chill coma recovery, in which flies are exposed for a certain period to cold stress (around 0°C). Flies are then transferred to room temperature and the percentage of surviving flies, and their recovery time are measured 21 , 22 . Cold-adapted flies should perform a better recovery from chill coma. Indeed, we found that a significantly higher percentage of short-day flies survived an exposure of 24 h to 0°C (Fig. 1 c). We also measured recovery time of the surviving flies by measuring their start of locomotor activity in Drosophila activity monitors. We found that the flies needed about 4 h to move again and that this time was not significantly different in short- and long-day flies. Short days alter lipid levels in the brain but not in the body and the head Higher levels of lipids, particularly triacylglycerols (TAG), have been found in cold-adapted whole flies 23 . Therefore, we first analyzed lipids in the head and body of the fly. Three fly heads and three fly bodies were pooled as one sample, and five replicates were analyzed by LC-MS coupled to electrospray ionization in positive mode. First, we performed a lipid fingerprinting procedure, where all aligned peaks were taken into account to filter out lipid features with significantly different levels. A total of 1337 lipid features (Table S1 ) were detected in the body (abdomen and thorax) and 1050 in the head (Table S2). Principal component analysis did not show a clear separation between long and short-day flies (Fig. 2a, d), nor did cluster analysis show a clear grouping (Fig. 2b, e). Next, we profiled energy storage lipids (TAGs) and structural lipids (phosphoethanolamines (PEs) and phophoglycerocholines (PCs)) according to Schäbler et al. 10 . The levels of TAGs, PEs and PCs in the body (Fig. 2c) and head (Fig. 2f) were not significantly different between short- and long-day flies. Therefore, we conclude that 14 short days are not sufficient to alter lipid levels in the body and the entire head. Since the photoperiod is first detected in the brain, which then signals the fat tissue in the head and body to trigger changes, our next step was to analyze the lipidome in isolated brains. Ten brains were pooled for one sample, and six replicates were measured by LC-MS using positive and negative electrospray ionization. First, we applied a metabolite fingerprinting approach to the annotated 875 lipid features (Fig. S2, Table S3). Remarkably, principal component analysis revealed a clear separation between the six long-day and short-day brain samples (Fig. 2g). In addition, unsupervised clustering revealed strong similarities between the six replicates, except for one long-day sample, which was grouped with the short-day samples (Fig. 2h). These results reflect clear differences between the brain lipidome of long- and short-day flies. Finally, lipid profiling showed that the total levels of TAGs, PEs and PCs were significantly higher in the brains of short-day flies compared to those of long-day flies (Fig. 2i). To find out which lipid species differ, we used the lipid dataset of 24 and profiled all TAGs, PEs, PCs, ceramidophosphoethanolamines (CerPE), alkyl-acyl-glycerophosphoethanolamines (PEO), phosphoniositols (PI) and phosphoserines (PS) detected in the brains (Fig. 3 ). We identified 23 energy storage lipids (TAG species), out of which 22 were significantly higher with a fold change of around 1.5 in short-day compared to long-day brains (Fig. 3 a). Of structural lipids, we identified 5 PSs, 1 PI, 5 PEOs, 15 PEs, 13 PCs and 2 CerPEs. Except for three species, all structural lipids appeared to have higher levels under short days, and this turned out to be significant for the CerPEs, most PEs, PCs and for one PEO species (Fig. 3 b). Lipid profiling results showed that the levels of major triacylglycerols and phospholipids were between 10–80% higher in the brains of short-day flies compared to long day flies. This explains well why the principal component analyses separated short-day and long-day samples (Fig. 2g). The fluidity of the membrane can adapt to changes in environmental temperature, thereby maintaining membrane function homeostasis. One parameter that describes membrane fluidity is the double bond index (DBI), that indicates the average number of double bonds in esterified fatty acids 25 . We did not detect any significant differences in the double bond index of TAGs (long day: 0.403 ± 0.008, short day: 0.411 ± 0.006, p = 1.00), PEs (long day: 1.324 ± 0.007, short day: 1.320 ± 0.006, p = 1.00) or PCs (long day: 1.088 ± 0.008, short day: 1.081 ± 0.013, p = 1.01). Our results suggest that a shortened photoperiod is insufficient to alter the number of double bonds in the esterified fatty acids of energy storage and structural lipids in the brains of Drosophila melanogaster . Our results indicate that photoperiod affects the lipidome in the brain, but not in the fly head and body. Furthermore, 14 short days led to an increase in the levels of energy storage lipids (TAGs) and structural lipids (PEs, PCs, CerPEs, PEOs) in the brain. Short days alter the level of water-soluble metabolites involved in brain energy metabolism To understand which metabolic pathway potentially supports the increasing lipid levels, we compared the water-soluble metabolites in the brains of long- and short-day flies. By LC-MS, we identified 151 metabolites out of which approximately 65 metabolites were significantly different between the two conditions (Table S4; Fig. S3). We observed lower levels in monosaccharides (sugars) in short-day flies as compared to long-day flies (Fig. 4 b). Since this reduction in sugar levels is not caused by decreased feeding (Fig. 1 b), it likely results from increased breakdown of glucose via glycolysis. This glycolytic process might supply energy in the form of ATP or generate acetyl-CoA which can be further used for the synthesis of lipids and other metabolites. Indeed, we found that the glycolysis intermediates phosphoglycerate and phosphoenolpyruvate were lower in short day flies, while pyruvate levels were not significantly reduced (Fig. 4 c). This could speak for a rapid conversion of glucose to pyruvate in glycolysis (Fig. 4 a) and a subsequent utilization of pyruvate for the synthesis of acetyl-CoA. Notably, ATP/AMP ratios were lower in short-day brains (Fig. 5 a) suggesting that the ATP produced in glycolysis was used up for anabolic processes (e.g., ATP citrate lyase for lipid synthesis). Acetyl-CoA, produced by mitochondrial pyruvate dehydrogenase, enters the tricarboxylic acid (TCA) cycle via citrate synthase, transferring the acetyl moiety to oxaloacetate, thereby generating citrate. We detected 8 TCA cycle intermediates and indeed found a slight increase only in citrate and oxaloacetate in short-day flies (Fig. 4 d). On the other hand, the amounts of oxaloacetate precursors (malate and fumarate), as well as products, generated from citrate within the TCA cycle (a-ketoglutarate and succinate) did not significantly increase, suggesting that citrate is not being used for gaining energy in the TCA cycle, but instead is shuttled from mitochondria to the cytosol for the synthesis of other compounds such as fatty acids (Figs. 3 , 4 ). As already mentioned, the ratio between ATP and AMP was lower in short-day brains (Fig. 5 a) suggesting that the energy gained in glycolysis is immediately consumed for anabolic processes. In addition, the ratio between the reduced and oxidized forms of glutathione (GSH/GSSG) was higher in the brains of short-day flies compared to those of long-day flies (Fig. 5 a). The nucleotide analysis revealed lower levels of several purines and pyrimidines in short-day as compared to long-day flies (Fig. 5 b). In particular, the mono- and di-phosphates of the purine nucleotides inosine, adenosine and guanosine (IMP, AMP, ADP, GMP and GDP) and the pyrimidine nucleotides uracil and cytosine (UMP, UDP, and CMP) were lower under short days (Fig. 5 b). In addition, purine degradation appears to be enhanced under short days, since the end products of purine breakdown, uric acid and xanthine are significantly elevated under such conditions (Fig. 5 b). We also found slightly higher arginine levels in the brains of short-day flies (Fig. 5 c). Arginine is produced in the urea cycle that serves the detoxification of ammonia during the breakdown of amino acids in the mitochondria. Otherwise, most amino acid levels were comparable between short and long days. Only the amino acids proline, glutamine and methionine were slightly but significantly lower under short-day conditions (Fig. 5 c). Overall, our results suggest increased degradation of nucleotides and a minor influence on amino acid levels under short days. Another interesting finding of our analysis was the increase in polyamines levels (putrescine, spermidine) in short days (Fig. 5 d). Polyamines are low-molecular‐mass, ubiquitous polycations that are well documented for combating senescence and stress, a role that is attributed to them for their cell‐membrane‐stabilizing, free‐radical–scavenging, and acid‐neutralizing properties 26 . In plants, polyamines are known as cryoprotectants that increase the accumulation of osmolytes, regulate redox homeostasis, stabilize membranes, and regulate gene expression to survive cold stress 27 , 28 . It is not known whether polyamines have also cryoprotective functions in animals, but if so, this can explain the higher chill coma survival rate of short-day flies. In animals, the crucial role of polyamines in autophagy, which removes unnecessary components from the brain, reduces neurodegeneration, and extends lifespan, is well documented 29 . Flies may need all of this to survive the winter. Short days increase the number of lipid droplets in the brain Lipid droplets are intracellular organelles responsible for energy storage, consisting of a hydrophobic core—primarily triacylglycerols (TAGs)—enclosed in a phospholipid monolayer coated with proteins. The increase in both storage lipids (TAGs) and structural lipids like PEs, PCs and PSs that we observed (Fig. 2i) suggest a potential impact of photoperiod on the abundance of lipid droplets in the fly brain. To investigate this, we stained and quantified the number of lipid droplets in the brains of short- and long-day flies. Lipid droplets were predominantly localized to the cell bodies of the central brain and to a lesser extent in the optic lobes (Fig. 6 a). We, therefore, focused our analysis on the central brain, and quantified the number of lipid droplets present. We found that short-day flies exhibited a significantly higher number of lipid droplets (Fig. 6 b). In summary, our results suggest that day length can significantly modulate metabolic pathways in the brain. As a main effect, sugars are degraded under short days, most probably for the synthesis of fatty acids. In addition, it appears that ammonia detoxification, autophagy and resistance to oxidative stress are increased under short-day conditions, which might increase cold resistance and lengthen lifespan under short-day conditions. All of this can be seen as preparation of the brain for the coming winter. Discussion Photoperiodic responses are known in many organisms living in temperate zones and exposed to strong seasonal changes. Preparing in advance to the coming winter is essential for survival, which is why organisms change their physiology and metabolism in response to the shortening of the photoperiod in autumn. Adult fruit flies have a lifespan of few weeks in summer, but in autumn their lifespan increases dramatically so that they survive the entire winter without food in a dormant state. To make this possible, they increase their cold and overall stress resistance, fill their lipid stores and lower their general metabolism. These changes happen in response to the decreasing photoperiod and temperature in autumn 30 . Although fruit flies of the species Drosophila melanogaster cannot be regarded as typical photoperiodic animals, they clearly respond to a shortening of daylength in autumn 6 , 31 . Previous studies showed that D. melanogaster reared for one generation under short photoperiods but at a constant temperature of 20°C, increase their cold resistance 5 , 15 . Here, we report that in adult flies, 14 days of exposure to short days (8 h of light and 16 h of darkness) at a temperature of 20°C is already sufficient to increase cold resistance. Furthermore, we show that this exposure significantly alters lipid metabolism in the brain, but not in the head and body. This proves that the brain is the first organ to respond to seasonal changes and confirms that it is the place where the length of the day is measured with the help of the circadian clock in mammals and insects 11 , 32 . As soon as the daylength falls below a certain threshold, this information is transmitted via several pathways to the neuroendocrine system of the brain, which alters neurohormonal signaling to the body (Helfrich-Förster, 2024). Another reason why metabolic changes are first observed in the brain is probably its high energy requirements 33 , 34 . The brain requires a lot of energy even when the animal is resting, sleeping or hibernating, while the skeletal muscles have almost no energy requirements at these times. Therefore, this energy requirement must be met first. Preparation for the winter needs energy Our results show that flies eat more under short-day conditions but are less active than under long-days, suggesting that they use excess energy for anabolic processes. This is in line with previous studies that have measured the oxygen consumption of adult short-day and long-day flies. Flies that were kept under short-day conditions (8 hours of light and 16 hours of darkness) for three days and flies that had already been reared under short-day conditions consumed significantly more oxygen than flies kept under long-day conditions during the same period 35 , 36 . Flies appear to use excess energy for lipid synthesis. Short-day flies appear to increase the levels of all major lipid classes in their brains at the expense of nucleotide and protein synthes 1 is. We propose the following metabolic shift under short days: glycolytic and pentose phosphate pathway intermediates decrease, along with most TCA cycle metabolites—except for citrate and oxaloacetate. Due to the decreased levels of ribose, de novo nucleotide biosynthesis might be low under short-day conditions as visible in low levels of nucleotide intermediates. But we also detected high levels of xanthine and uric acid, the products of purine degradation in short-day flies, indicating a shift toward catabolic purine turnover. Based on our observations, we suggest that under short photoperiod, acetyl-CoA and ATP generated through glycolysis are utilized by mitochondrial TCA cycle to produce citrate, which is subsequently transported via the citrate shuttle to the cytosol for fatty acid synthesis (Fig. 7 ). Remarkably, all major lipid classes including storage (TAGs) and membrane lipids (PEs, PCs, PEOs, PSs, CerPEs) are elevated in the brain of short-day fly brains. While the synthesis and accumulation of storage lipids (TAGs) can be understood as immediate preparation for winter, the synthesis of membrane lipids appears less clear – at least at the first glance. Lipids are stored in droplets surrounded by a phospholipid monolayer. In short-day flies, there was a notable increase in lipid droplet accumulation, particularly in the central brain. This rise in lipid droplets may also contribute to an overall increase in membrane phospholipids. Additionally, these phospholipids also contribute to apoptosis- particularly, phosphatidylserine (PS) which enables the fusion of exocytotic vesicles with plasma membrane and serves as a signal for the recognition and clearance of apoptotic cells. In Drosophila , PE is a key phospholipid essential for maintaining the stability of the mitochondrial inner membrane. It is synthesized from PCs and PSs by the genes Pss and Pisd . Mutations in Pss lead to neurodegeneration, mitochondrial dysfunction and elevated reactive oxygen species (ROS) levels 37 . According to these findings, we assume that the elevated levels of PSs, PCs, and PEs under short days, might help in the maintenance of mitochondrial integrity, reduce oxidative stress, and support normal cellular function. Short days appear to reduce oxidative stress and increase detoxification and neuroprotection Since short-day flies have to survive the entire winter, they need to protect their brains from neurodegeneration. Oxidative stress, characterized by an imbalance between ROS and antioxidant defenses, plays a significant role in the progression of neurodegeneration. The brain is one of organs especially vulnerable to the effects of ROS because of its high oxygen demand and its abundance of peroxidation-susceptible lipid cells. In humans, previous studies have demonstrated that oxidative stress plays a central role in a common pathophysiology of neurodegenerative diseases such as Alzheimer's and Parkinson's disease 38 , 39 . We found a high ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG) in the brains of short-day flies. GSH is a key antioxidant that enables a high degree of antioxidant defense minimizing oxidative damage. Another antioxidant is uric acid, the product of purine metabolism, which can scavenge ROS and is also elevated in short-day flies. GSH and uric acid may work together to mitigate damage caused by oxidative stress. In addition, we detected elevated levels of polyamines in short-day flies. Spermidine is a natural polyamine that induces autophagy, an evolutionarily conserved recycling process of waste products in cells in eukaryotes that prolongs the lifespan of all animals studied to date, including Drosophila 40 . Overall, our study suggests that photoperiod alone is sufficient to initiate the physiological changes necessary for seasonal adaptation, while temperature may amplify these responses. We propose a model in which short-day flies utilize metabolic resources specifically for energy production and storage in the brain. Future studies involving stable isotope-labelling experiments are needed to validate this model. Declarations Competing Interests The authors declare no competing interests. Funding This work was supported by the German Research Foundation (DFG) (grant numbers: FO207/15 − 2 and Me 4966/1–2). Open Access funding was enabled and organized by Project DEAL. Author Contribution M.S. and A.H. dissected and prepared the flies for the mass spectrometric measurements and performed data mining of the lipid analysis under supervision of A.F. M.S, A.H. and J.G. performed the feeding, M.S. the locomotor activity and P.D. the chill coma experiments. W.S. performed the mass spectrometric measurements and data mining for the water-soluble components. M.S., A.H. and P.M. designed the study and A.F. and C.H.-F. supervised the experiments. M.J.M. gave valuable advice. M.S. and C.H.-F composed the figures, and A.F. and C.H.-F. wrote the paper with help of the others. Acknowledgement Lipid analysis was performed by the Metabolomics Core Unit at the University of Würzburg. We are grateful to Barbara Mühlbauer, Maria Lesch and Markus Krischke for excellent technical assistance, Martin Eilers and Thomas Raabe for providing lab space and technical support, and the members at the Neurobiology and Genetics for fruitful discussions. 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E., Rhie, S. J. & Yoon, S. The Role of Oxidative Stress in Neurodegenerative Diseases. Experimental Neurobiology 24 , 325–340 (2015). Hofer, S. J. et al. Mechanisms of spermidine-induced autophagy and geroprotection. Nat Aging 2 , 1112–1129 (2022). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6800169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475906459,"identity":"36875930-e1fd-410f-a697-d2796c3d1f10","order_by":0,"name":"Madhura Sapre","email":"","orcid":"","institution":"Theodor-Boveri-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Madhura","middleName":"","lastName":"Sapre","suffix":""},{"id":475906460,"identity":"e8ef214f-c448-4d43-bbb7-de3e5548b263","order_by":1,"name":"Anna Hovhanyan","email":"","orcid":"","institution":"Theodor-Bover-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Hovhanyan","suffix":""},{"id":475906461,"identity":"a77395a1-510b-45db-ae7d-5e5c58762fac","order_by":2,"name":"Werner Schmitz","email":"","orcid":"","institution":"Theodor-Boveri-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Werner","middleName":"","lastName":"Schmitz","suffix":""},{"id":475906462,"identity":"0108200f-af39-4965-98ae-1294aa8f81bf","order_by":3,"name":"Peter Deppisch","email":"","orcid":"","institution":"Theodor-Boveri-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Deppisch","suffix":""},{"id":475906463,"identity":"f7437d20-c538-493f-8e1b-e212019f13fd","order_by":4,"name":"Jayati Gera","email":"","orcid":"","institution":"Theodor-Boveri-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Jayati","middleName":"","lastName":"Gera","suffix":""},{"id":475906464,"identity":"91a14d97-ccd9-410a-a95d-a949c4cfc7a8","order_by":5,"name":"Martin J Mueller","email":"","orcid":"","institution":"Julius-von-Sachs-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"J","lastName":"Mueller","suffix":""},{"id":475906465,"identity":"9b630536-f5d0-4d72-b7d3-6e07bc2271f9","order_by":6,"name":"Pamela Menegazzi","email":"","orcid":"","institution":"Theodor-Boveri-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Pamela","middleName":"","lastName":"Menegazzi","suffix":""},{"id":475906466,"identity":"965400ac-1008-435d-bdab-192fda1019f7","order_by":7,"name":"Agnes Fekete","email":"","orcid":"","institution":"Julius-von-Sachs-Institute, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Agnes","middleName":"","lastName":"Fekete","suffix":""},{"id":475906467,"identity":"ab721b26-fc43-426e-8dfc-a8a4af80468b","order_by":8,"name":"Charlotte Helfrich-Förster","email":"data:image/png;base64,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","orcid":"","institution":"Theodor-Boveri-Institute, University of Würzburg","correspondingAuthor":true,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Helfrich-Förster","suffix":""}],"badges":[],"createdAt":"2025-06-02 08:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6800169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6800169/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-22793-7","type":"published","date":"2025-10-07T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85551088,"identity":"1f38bddc-b36d-4e3d-8563-54485f047c1c","added_by":"auto","created_at":"2025-06-27 09:48:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17116,"visible":true,"origin":"","legend":"\u003cp\u003eDaily activity, food intake and cold tolerance of long- and short-day flies. \u003cstrong\u003ea\u003c/strong\u003e Mean daily activity of ~30 flies recorded by infrared light beam crosses in TriKinetics monitors (top; details see Fig. S1). \u003cstrong\u003eb\u003c/strong\u003eAmount of food consumed by 20 groups of 15 flies during a time span of 2 hours (left) and single flies throughout 24 h (right) in the capillary feeding (CAFE) assay. \u003cstrong\u003ec\u003c/strong\u003e Percentage of flies surviving chill coma (left) and time the surviving flies took for recovery (right). The experiment was repeated 20 times with 10 flies each. The time to recover was calculated for all surviving flies. The number of individually tested flies or fly groups is indicated in the bottom of the diagrams. Asterisks indicate significance (*: p\u0026lt;0.05, **: p=0.01, ***: p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/746c5452f58fec49b3cb5576.png"},{"id":85551091,"identity":"5ff968a0-cf00-48a8-bcdd-dbd7352be9d9","added_by":"auto","created_at":"2025-06-27 09:48:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147712,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis, clustering and lipid profiling of bodies, heads and brains of long- and short-day flies. Organic phases of all samples (three \u003cem\u003eDrosophila\u003c/em\u003e bodies and heads and 10 brains were pooled) were analyzed by LC-MS and all detectable lipid features were investigated. The results for long-day flies are shown in white or light gray, and those for short-day flies are shown in black or dark gray. Principal component analysis (a, d, g) and clustering (b, e, h) showed a clear separation of long- and short-day fly samples in brains but not in bodies and heads. The levels of triacylglycerols (TAGs), phosphoethanolamines (PEs), and phosphoglycerocholines (PCs) were determined by lipid profiling (c, f, i) and showed higher levels in the brain of short-day flies compared to long-day flies, but not in the body and head. PC1, PC2: first and second principal components (percent variance in parenthesis), Au: arbitrary unit. The number of replicates was 5 for heads and bodies and 6 for brains. Asterisks indicate significance (*: p\u0026lt;0.05, **: p=0.01, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/24c64afa6ff0e2f5d5d04c34.png"},{"id":85551789,"identity":"26e125b8-c737-4694-ad1a-a29e0fe510cc","added_by":"auto","created_at":"2025-06-27 09:56:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52215,"visible":true,"origin":"","legend":"\u003cp\u003eLog2 fold changes in the levels of identified energy storage lipids (a) and structural lipids (b) in the brains of short-day flies in relation to long-day flies (the values in long-day flies were set to 0). Lipid species are defined according to lipid class and acyl chains: the letters indicate the lipid class; the first number is the number of acyl carbons and the last number the number of acyl double bonds. Fold changes are calculated by dividing brain levels averaged from the six replicates in short-day flies by long-day flies. TAG: triacylglycerol, PS: phosphoserine, PEO alkyl-acyl-glycerophosphoethanolamine, PE: phosphoethanolamine, PC: phosphoglycerocholine, CerPE: ceramidophosphoethanolamine. The number of replicates was 5 for heads and bodies and 6 for brains. Asterisks indicate significance (*: p\u0026lt;0.05, **: p=0.01, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/5c9f9c5d0da1132e7b5b4c3c.png"},{"id":85551090,"identity":"f511af9a-cefc-4c47-b953-b1f856a86044","added_by":"auto","created_at":"2025-06-27 09:48:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73492,"visible":true,"origin":"","legend":"\u003cp\u003elog2 fold changes in the levels of (b) sugars (c) Glycolysis intermediates (d) TCA cycle. Details see text. Asterisks indicate significance (*: p\u0026lt;0.05, **: p=0.01, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/50a103aaf3bcc6385d2ccc3f.png"},{"id":85551102,"identity":"d7d6e313-ef3c-471f-b522-56fdaac90558","added_by":"auto","created_at":"2025-06-27 09:48:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54981,"visible":true,"origin":"","legend":"\u003cp\u003eRatio of ATP/AMP and reduced/oxidized forms of glutathione (GSH/GSSG) (a), and log2 fold changes in the levels of nucleotides (b), amino acids (c) and polyamines (d). Labeling as in Figure 4. Details see text. (*: p\u0026lt;0.05, **: p=0.01, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/38c19d2bc5fdb56f6c21979e.png"},{"id":85551793,"identity":"4521df7a-f328-41b3-8c09-a77f8db12fa3","added_by":"auto","created_at":"2025-06-27 09:56:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":58446,"visible":true,"origin":"","legend":"\u003cp\u003eLipid droplets in the brain of long- and short-day flies. \u003cstrong\u003ea\u003c/strong\u003eOverlay of 40 confocal stacks of the anterior brain stained with “BODIPY493/503” for lipid droplets. AL: antennal lobes; OL optic lobes. \u003cstrong\u003eb\u003c/strong\u003eNumber of lipid droplets in the central brain of long- and short-day flies. *: p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/ac26a61cf985efc3621d73d6.png"},{"id":85551097,"identity":"f614ec2b-0171-4e57-b3a4-21ffc7b77c59","added_by":"auto","created_at":"2025-06-27 09:48:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16289,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model: Metabolic substrates from glycolysis, pentose-phosphate pathway and TCA cycle are utilized for lipid synthesis via citrate shuttle\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/d09bc8febed73800c028a60b.png"},{"id":93419631,"identity":"1a712415-9cf4-454f-adf4-a4b79b60eaaa","added_by":"auto","created_at":"2025-10-13 16:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1578970,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/60c6d479-0b3c-4671-ac3f-d83af4a87627.pdf"},{"id":85551094,"identity":"d01445b5-9d94-47d8-8d53-90bc1aa70191","added_by":"auto","created_at":"2025-06-27 09:48:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":880365,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6800169/v1/df683dbf5cac0ce8b1759846.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A short photoperiod alters brain metabolism and cold resistance in Drosophila melanogaster","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOrganisms living in temperate zones are exposed to strong seasonal changes to which they must adapt. Winter poses a problem for organisms for two key reasons: energy and water. Food sources become scarce, humidity decreases, and temperatures fall below the optimal range, which means that the cellular functions that provide energy falter and eventually fail. This is particularly relevant for small organisms such as insects, which are unable to maintain a constant body temperature. When internal temperatures fall below freezing, the water that makes up at least 70 percent of animal cells freezes, and the organism dies. To avoid suboptimal internal temperatures and dehydration, animals can migrate to warmer regions, enter a dormant state with reduced energy expenditure, and develop resistance to cold and dryness \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo survive, animals must adjust their metabolism long before winter sets in. Those who do not prepare for winter in time will have to deal with severe fitness disadvantages. But how do they recognize that winter is coming? Temperature can fluctuate greatly and is therefore no reliable indicator of the coming season. The most robust cue for assessing the onset of winter is the shortening of day length (photoperiod) in the fall. Therefore, all animals have mechanisms in their brains to measure photoperiod, which communicate with the endocrine system and initiate physiological and metabolic changes when it falls below a critical level, even if ambient temperatures are still pleasant. These mechanisms are called photoperiodic responses and have been well-studied in several mammals and insects \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. They need an internal circadian clock as a reference for measuring the length of the day \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe fly \u003cem\u003eDrosophila melanogaster\u003c/em\u003e has been successfully used as a model to understand the role of the circadian clock in photoperiodic responses \u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Light and the circadian clock are known to affect metabolite levels in the body and head of fruit flies \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, nothing is known about the metabolic responses of the brain to such changes. This is surprising, since the brain contains the master circadian clock, processes light information, measures photoperiod and initiates seasonal changes in behavior and in the neuroendocrine system \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In addition, the brain is one of the most metabolically expensive organs in the body, and this is true for all animals \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. As an example, in humans, the brain makes up about 2% of body mass but consumes 20% of total energy and this applies during wakefulness and sleep. Consequently, animals have evolved efficient ways to reduce the energy costs of their brain during conditions of food scarcity such as the winter. Some moles even shrink the size of their brain in winter and regrow it in spring \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Such extreme effects are extremely unlikely in fruit flies, which only survive the winter once in a dormant state before producing the next generation the following spring. Nevertheless, significant metabolic adaptations to photoperiod shortening are expected. An earlier study showed that flies already increase their cold resistance when reared in short-day conditions compared to flies reared in long-day conditions at the same temperature \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Since \u003cem\u003eDrosophila\u003c/em\u003e flies overwinter as adults and photoperiod is also measured in the adult stage, we investigated whether exposing adult flies to short-day conditions is sufficient to cause metabolic changes in the brain.\u003c/p\u003e \u003cp\u003eWe found that the exposure of adult flies to a short photoperiod is sufficient to increase their cold resistance and to change their brain metabolism, while the metabolism in the whole head and body remain unchanged. In their brains, flies appear to metabolize sugar to synthesize the storage lipids triacylglycerols and structural phospholipids. They increase polyamine levels which enhance autophagy and may extend lifespan, and they appear to become more resistant to cold and oxidative stress. All these changes can be regarded as preparation for the coming winter.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFly rearing and adult maintenance\u003c/h2\u003e \u003cp\u003eWild-type \u003cem\u003eDrosophila melanogaster\u003c/em\u003e flies of the strain \u003cem\u003eCantonS\u003c/em\u003e (WT\u003csub\u003ecs\u003c/sub\u003e) were reared under 12h:12h light:dark cycles (LD12:12) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;2% relative humidity on \u003cem\u003eDrosophila\u003c/em\u003e food consisting of 0.8% agar, 2.2% sugar beet syrup, 8.0% malt extract, 1.8% yeast, 1.0% soy flour, 8.0% corn flour, and 0.3% hydroxybenzoic acid. After eclosion, adult flies were transferred to new fly vials containing the same food and exposed for 14 days either to long (LD16:8) or short (LD8:16) photoperiods, while temperature was kept constant at 20 \u0026deg;\u0026plusmn; 0.2\u0026deg;C. In the following experiments, the so treated flies are called long- and short-day flies, respectively. All experiments were performed with male flies.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLocomotor activity recordings\u003c/h3\u003e\n\u003cp\u003eThe locomotor activity of 32 flies each was recorded under long (LD16:8) and short (LD8:16) photoperiods in the \u003cem\u003eDrosophila\u003c/em\u003e Activity Monitor (DAM)-System of TriKinetics (Waltham, MA, USA). Flies were transferred into 5 mm tubes containing food (4% sucrose and 2% agar in water) using CO\u003csub\u003e2\u003c/sub\u003e anesthesia. Activity was monitored by detecting the disruptions in an infrared light beam at 1-minute intervals. The activity was tracked over a period of about 9 days at 20\u0026deg;C with 60% relative humidity. Light intensity during the experiments was set to 100 lux.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eTo reveal the activity pattern of the flies, activity data were plotted as individual and average actograms using the ImageJ plug-in ActogramJ \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, individual and average activity profiles were calculated for the two fly groups and overall daily activity levels were determined as described in Schlichting and Helfrich-F\u0026ouml;rster \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e for average actograms and average activity profiles see Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCapillary feeding (CAFE) assay\u003c/h3\u003e\n\u003cp\u003eFeeding was assessed with the CAF\u0026Eacute; assay by two methods. First, food consumption was measured for 2 hours in groups of 15 flies, and second, in individual flies for 24 hours. For the first assay 20 groups of flies consisting of 15 long- and short-day flies, each, were transferred to vials without food on day 14, directly after lights-on. After 2 hours of starvation, the fly vials were placed at an angle of 45\u0026deg; and two microcapillary pipettes were inserted, one containing 5 \u0026micro;l of normal tap water and the other 5 \u0026micro;l of 5% sucrose solution with blue food dye. The flies could choose freely between the two capillaries. Every 20 minutes, we recorded how much liquid they had consumed and after 2 hours, the total consumed food was determined for all 40 fly groups (in total 300 long- and short-day flies, respectively). An empty vial containing only the two capillaries served as a control for evaporation.\u003c/p\u003e \u003cp\u003eFor the second assay, individual flies were confined to 2 ml Eppendorf cups. The top of each cup contained a hole, in which a capillary containing 5 \u0026micro;l of 5% sucrose solution was inserted. There were two more holes on the sides of the tubes for aeration. After 24 hours the amount of food consumed by each fly was determined. Like before, an empty vial containing only the capillary was used as a control for evaporation.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAt first, the data were checked for a normal distribution (Kolmogorov-Smirnov test; p\u0026thinsp;\u0026lt;\u0026thinsp;0.008, Shapiro-Wilk test; p\u0026thinsp;\u0026lt;\u0026thinsp;0.03). Since the data deviated significantly from normality, the non-parametric Mann-Whitney-U test was applied for checking statistically significant differences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCold tolerance assay\u003c/h2\u003e \u003cp\u003eCold tolerance was measured by recovery from chill coma (David et al., 1998; Macdonald et al., 2004). Flies from twenty vials, containing 10 long- and short-day flies, each, were transferred to empty fly vials equipped only with water-moistened filter paper. The vials were then stored in darkness on crushed ice at 0\u0026deg;C for 24 hours. After cold exposure, the flies were transferred to 25\u0026deg;C for recovery. They were immediately individually placed into glass tubes of the DAM TriKinetics system to monitor their activity. The percentage of flies that resumed activity indicating survival of cold exposure was determined. Furthermore, the time passing until the first movement was measured in flies surviving the treatment\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLipid analysis\u003c/h3\u003e\n\u003cp\u003eFor the body and head sampling, long- and short-day flies were shock frozen in liquid nitrogen (shortly after lights-on) and stored at -80\u0026deg;C. Fly heads were separated from the body on ice and three fly heads, and three fly bodies were pooled for each sample. For the brain sampling, long- and short-day flies were quickly killed by submersion in 4% paraformaldehyde (shortly after lights-on). After 1h of fixation, the brains of the flies were dissected in phosphate buffered saline and transferred to methanol. 10 brains were pooled for each sample and stored at -80 \u003csup\u003eo\u003c/sup\u003eC until analysis. Blight-Dyer extraction was performed as published in Mueller et al. (2015) and lipid profiling of the organic phase with LC-MS was performed in accordance with Sch\u0026auml;bler et al. \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. For lipid fingerprinting, the acquired data used for lipid profiling were pre-processed with Progenesis QI (version 1.0, Waters) and MetaboAnalyst 6.0 was used for statistical analysis \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Graphs were generated with R (v4.2.1) via Rstudio (2022-06-23) using ggplot2 package.\u003c/p\u003e\n\u003ch3\u003eTargeted analysis of water-soluble primary metabolites\u003c/h3\u003e\n\u003cp\u003eWater-soluble metabolites from 10 pooled \u003cem\u003eDrosophila melanogaster\u003c/em\u003e brains were extracted with 500 \u0026micro;l ice-cold MeOH/H\u003csub\u003e2\u003c/sub\u003eO (80/20, v/v) containing 0.04 \u0026micro;M lamivudine and 4 \u0026micro;M each of D\u003csub\u003e2\u003c/sub\u003e-glucose, D\u003csub\u003e4\u003c/sub\u003e-succinate, D\u003csub\u003e5\u003c/sub\u003e-glycine and \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN-glutamate (Sigma-Aldrich, St. Louis, USA). After centrifugation, the resulting supernatants were evaporated in a rotary evaporator (Savant, Thermo Fisher Scientific, Waltham, USA). Dry sample extracts were redissolved in 50 \u0026micro;l 5 mM NH\u003csub\u003e4\u003c/sub\u003eOAc in CH\u003csub\u003e3\u003c/sub\u003eCN / H\u003csub\u003e2\u003c/sub\u003eO (50/50, v/v). 20 \u0026micro;l supernatant was transferred to LC-vials. Metabolites were analyzed by LC-MS using the following settings: For LC-MS analysis 3 \u0026micro;l of each sample was applied to a SeQuant ZIC-cHILIC (3 \u0026micro;m particles, 100 \u0026times; 2.1 mm) (Merck, Darmstadt, Germany). Metabolites were separated with Solvent A, consisting of 5 mM NH\u003csub\u003e4\u003c/sub\u003eOAc in CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO (40/60, v/v) and solvent B consisting of 5 mM NH\u003csub\u003e4\u003c/sub\u003eOAc in CH\u003csub\u003e3\u003c/sub\u003eCN/H\u003csub\u003e2\u003c/sub\u003eO (95/5, v/v) at a flow rate of 200 \u0026micro;l/min at 45\u0026deg;C by LC using a DIONEX Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany). A linear gradient starting after 2 min with 100% solvent B decreasing to 10% solvent B within 23 min, followed by 16 min 10% solvent B and a linear increase to 100% solvent B in 2 min was applied. Recalibration of the column was achieved by 7 min prerun with 100% solvent B before each injection.\u003c/p\u003e \u003cp\u003eAll MS-analyses were performed on a high-resolution Q Exactive mass spectrometer equipped with a HESI probe (Thermo Fisher Scientific, Bremen, Germany) in alternating positive and negative full MS mode with a scan range of 69.0\u0026ndash;1000 m/z at 70K resolution and the following ESI source parameters: sheath gas: 30, auxiliary gas: 1, sweep gas: 0, aux gas heater temperature: 120\u0026deg;C, spray voltage: 3 kV, capillary temperature: 320\u0026deg;C, S-lens RF level: 50. XIC generation and signal quantitation was performed using TraceFinder\u0026trade; V 5.1 (Thermo Fisher Scientific, Bremen, Germany) integrating peaks which corresponded to the calculated monoisotopic metabolite masses (MIM +/- H\u003csup\u003e+\u003c/sup\u003e \u0026plusmn; 2 mMU). All analyses were performed in six independent replicates.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent immunohistochemistry and microscopy for lipid droplets\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was performed on male flies entrained to long and short photoperiods for 14 days, following the protocol of Schubert et al. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, to quantify the number and area of lipid droplets in the brain using BODIPY493/503 (Invitrogen, Cat. No: D3922). Flies were fixed in 4% paraformaldehyde in PBS with 0.5% Triton X-100 (PBST) for 3.5 hours. After three PBS washes, flies were dissected, and the brains were washed twice with PBST. Brains were then incubated overnight at 4\u0026deg;C with BODIPY493/503, followed by six PBST washes. Finally, brains were mounted on slides using Vectashield (Vector Laboratories, Burlingame, CA, USA) with ~\u0026thinsp;150 \u0026micro;m spacers. The images were acquired with a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with hybrid detectors, photon multiplier tubed and a white laser for excitation. We used 20-fold glycerol immersion objective (HC PL APO, Leica Microsystems, Wetzlar Germany) and obtained confocal stacks with 2 \u0026micro;m z-step size and 1024 \u0026times; 512 pixels. The obtained images were analyzed with Fiji ImageJ \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLipid droplet analysis was performed on the central brain region only. A maximum Z-projection was generated, and noise was reduced using the Despeckle function. Manual thresholding was applied, followed by Watershed to separate overlapping droplets. The Analyze Particles function in ImageJ was used to quantify lipid droplet number and total area. Approximately 13 brains were analyzed per condition.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlies are less active but eat slightly more under short days\u003c/h2\u003e \u003cp\u003eOur locomotor activity recordings showed that the flies were significantly less active under short days than under long days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, S1). This suggests that they would need less energy in short days and consequently should eat less. However, this was not the case. Our capillary feeder assay (CAFE) revealed that short-day flies of the same age ate even slightly more than long-day flies over a two-hour duration, and our 24 h feeding assay confirmed significantly higher food intake (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This suggests that the flies use the extra energy uptake to adapt their metabolism to the coming winter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eShort days during adulthood increase cold tolerance of the flies\u003c/h2\u003e \u003cp\u003ePrevious results have revealed that flies reared in short-day conditions increase their cold resistance when compared to flies reared in long-day conditions at the same temperature \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Here, we aimed to determine whether a 14-day exposure period to short days during adulthood is enough to increase cold tolerance.\u003c/p\u003e \u003cp\u003eWe adopted a simple assay to measure cold tolerance called chill coma recovery, in which flies are exposed for a certain period to cold stress (around 0\u0026deg;C). Flies are then transferred to room temperature and the percentage of surviving flies, and their recovery time are measured \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Cold-adapted flies should perform a better recovery from chill coma. Indeed, we found that a significantly higher percentage of short-day flies survived an exposure of 24 h to 0\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We also measured recovery time of the surviving flies by measuring their start of locomotor activity in \u003cem\u003eDrosophila\u003c/em\u003e activity monitors. We found that the flies needed about 4 h to move again and that this time was not significantly different in short- and long-day flies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eShort days alter lipid levels in the brain but not in the body and the head\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHigher levels of lipids, particularly triacylglycerols (TAG), have been found in cold-adapted whole flies \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, we first analyzed lipids in the head and body of the fly. Three fly heads and three fly bodies were pooled as one sample, and five replicates were analyzed by LC-MS coupled to electrospray ionization in positive mode. First, we performed a lipid fingerprinting procedure, where all aligned peaks were taken into account to filter out lipid features with significantly different levels. A total of 1337 lipid features (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were detected in the body (abdomen and thorax) and 1050 in the head (Table S2). Principal component analysis did not show a clear separation between long and short-day flies (Fig.\u0026nbsp;2a, d), nor did cluster analysis show a clear grouping (Fig.\u0026nbsp;2b, e). Next, we profiled energy storage lipids (TAGs) and structural lipids (phosphoethanolamines (PEs) and phophoglycerocholines (PCs)) according to Sch\u0026auml;bler et al. \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The levels of TAGs, PEs and PCs in the body (Fig.\u0026nbsp;2c) and head (Fig.\u0026nbsp;2f) were not significantly different between short- and long-day flies. Therefore, we conclude that 14 short days are not sufficient to alter lipid levels in the body and the entire head.\u003c/p\u003e \u003cp\u003eSince the photoperiod is first detected in the brain, which then signals the fat tissue in the head and body to trigger changes, our next step was to analyze the lipidome in isolated brains. Ten brains were pooled for one sample, and six replicates were measured by LC-MS using positive and negative electrospray ionization. First, we applied a metabolite fingerprinting approach to the annotated 875 lipid features (Fig. S2, Table S3). Remarkably, principal component analysis revealed a clear separation between the six long-day and short-day brain samples (Fig.\u0026nbsp;2g). In addition, unsupervised clustering revealed strong similarities between the six replicates, except for one long-day sample, which was grouped with the short-day samples (Fig.\u0026nbsp;2h). These results reflect clear differences between the brain lipidome of long- and short-day flies. Finally, lipid profiling showed that the total levels of TAGs, PEs and PCs were significantly higher in the brains of short-day flies compared to those of long-day flies (Fig. 2i).\u003c/p\u003e \u003cp\u003eTo find out which lipid species differ, we used the lipid dataset of \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and profiled all TAGs, PEs, PCs, ceramidophosphoethanolamines (CerPE), alkyl-acyl-glycerophosphoethanolamines (PEO), phosphoniositols (PI) and phosphoserines (PS) detected in the brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We identified 23 energy storage lipids (TAG species), out of which 22 were significantly higher with a fold change of around 1.5 in short-day compared to long-day brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Of structural lipids, we identified 5 PSs, 1 PI, 5 PEOs, 15 PEs, 13 PCs and 2 CerPEs. Except for three species, all structural lipids appeared to have higher levels under short days, and this turned out to be significant for the CerPEs, most PEs, PCs and for one PEO species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Lipid profiling results showed that the levels of major triacylglycerols and phospholipids were between 10\u0026ndash;80% higher in the brains of short-day flies compared to long day flies. This explains well why the principal component analyses separated short-day and long-day samples (Fig.\u0026nbsp;2g).\u003c/p\u003e \u003cp\u003eThe fluidity of the membrane can adapt to changes in environmental temperature, thereby maintaining membrane function homeostasis. One parameter that describes membrane fluidity is the double bond index (DBI), that indicates the average number of double bonds in esterified fatty acids \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We did not detect any significant differences in the double bond index of TAGs (long day: 0.403\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008, short day: 0.411\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006, p\u0026thinsp;=\u0026thinsp;1.00), PEs (long day: 1.324\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007, short day: 1.320\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006, p\u0026thinsp;=\u0026thinsp;1.00) or PCs (long day: 1.088\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008, short day: 1.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013, p\u0026thinsp;=\u0026thinsp;1.01). Our results suggest that a shortened photoperiod is insufficient to alter the number of double bonds in the esterified fatty acids of energy storage and structural lipids in the brains of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eOur results indicate that photoperiod affects the lipidome in the brain, but not in the fly head and body. Furthermore, 14 short days led to an increase in the levels of energy storage lipids (TAGs) and structural lipids (PEs, PCs, CerPEs, PEOs) in the brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eShort days alter the level of water-soluble metabolites involved in brain energy metabolism\u003c/h2\u003e \u003cp\u003eTo understand which metabolic pathway potentially supports the increasing lipid levels, we compared the water-soluble metabolites in the brains of long- and short-day flies. By LC-MS, we identified 151 metabolites out of which approximately 65 metabolites were significantly different between the two conditions (Table S4; Fig. S3).\u003c/p\u003e \u003cp\u003eWe observed lower levels in monosaccharides (sugars) in short-day flies as compared to long-day flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Since this reduction in sugar levels is not caused by decreased feeding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), it likely results from increased breakdown of glucose via glycolysis. This glycolytic process might supply energy in the form of ATP or generate acetyl-CoA which can be further used for the synthesis of lipids and other metabolites. Indeed, we found that the glycolysis intermediates phosphoglycerate and phosphoenolpyruvate were lower in short day flies, while pyruvate levels were not significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This could speak for a rapid conversion of glucose to pyruvate in glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and a subsequent utilization of pyruvate for the synthesis of acetyl-CoA. Notably, ATP/AMP ratios were lower in short-day brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) suggesting that the ATP produced in glycolysis was used up for anabolic processes (e.g., ATP citrate lyase for lipid synthesis).\u003c/p\u003e \u003cp\u003eAcetyl-CoA, produced by mitochondrial pyruvate dehydrogenase, enters the tricarboxylic acid (TCA) cycle via citrate synthase, transferring the acetyl moiety to oxaloacetate, thereby generating citrate. We detected 8 TCA cycle intermediates and indeed found a slight increase only in citrate and oxaloacetate in short-day flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). On the other hand, the amounts of oxaloacetate precursors (malate and fumarate), as well as products, generated from citrate within the TCA cycle (a-ketoglutarate and succinate) did not significantly increase, suggesting that citrate is not being used for gaining energy in the TCA cycle, but instead is shuttled from mitochondria to the cytosol for the synthesis of other compounds such as fatty acids (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs already mentioned, the ratio between ATP and AMP was lower in short-day brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) suggesting that the energy gained in glycolysis is immediately consumed for anabolic processes. In addition, the ratio between the reduced and oxidized forms of glutathione (GSH/GSSG) was higher in the brains of short-day flies compared to those of long-day flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe nucleotide analysis revealed lower levels of several purines and pyrimidines in short-day as compared to long-day flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In particular, the mono- and di-phosphates of the purine nucleotides inosine, adenosine and guanosine (IMP, AMP, ADP, GMP and GDP) and the pyrimidine nucleotides uracil and cytosine (UMP, UDP, and CMP) were lower under short days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In addition, purine degradation appears to be enhanced under short days, since the end products of purine breakdown, uric acid and xanthine are significantly elevated under such conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). We also found slightly higher arginine levels in the brains of short-day flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Arginine is produced in the urea cycle that serves the detoxification of ammonia during the breakdown of amino acids in the mitochondria. Otherwise, most amino acid levels were comparable between short and long days. Only the amino acids proline, glutamine and methionine were slightly but significantly lower under short-day conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Overall, our results suggest increased degradation of nucleotides and a minor influence on amino acid levels under short days.\u003c/p\u003e \u003cp\u003eAnother interesting finding of our analysis was the increase in polyamines levels (putrescine, spermidine) in short days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Polyamines are low-molecular‐mass, ubiquitous polycations that are well documented for combating senescence and stress, a role that is attributed to them for their cell‐membrane‐stabilizing, free‐radical\u0026ndash;scavenging, and acid‐neutralizing properties \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In plants, polyamines are known as cryoprotectants that increase the accumulation of osmolytes, regulate redox homeostasis, stabilize membranes, and regulate gene expression to survive cold stress \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. It is not known whether polyamines have also cryoprotective functions in animals, but if so, this can explain the higher chill coma survival rate of short-day flies. In animals, the crucial role of polyamines in autophagy, which removes unnecessary components from the brain, reduces neurodegeneration, and extends lifespan, is well documented \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Flies may need all of this to survive the winter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eShort days increase the number of lipid droplets in the brain\u003c/h2\u003e \u003cp\u003eLipid droplets are intracellular organelles responsible for energy storage, consisting of a hydrophobic core\u0026mdash;primarily triacylglycerols (TAGs)\u0026mdash;enclosed in a phospholipid monolayer coated with proteins. The increase in both storage lipids (TAGs) and structural lipids like PEs, PCs and PSs that we observed (Fig.\u0026nbsp;2i) suggest a potential impact of photoperiod on the abundance of lipid droplets in the fly brain. To investigate this, we stained and quantified the number of lipid droplets in the brains of short- and long-day flies. Lipid droplets were predominantly localized to the cell bodies of the central brain and to a lesser extent in the optic lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We, therefore, focused our analysis on the central brain, and quantified the number of lipid droplets present. We found that short-day flies exhibited a significantly higher number of lipid droplets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, our results suggest that day length can significantly modulate metabolic pathways in the brain. As a main effect, sugars are degraded under short days, most probably for the synthesis of fatty acids. In addition, it appears that ammonia detoxification, autophagy and resistance to oxidative stress are increased under short-day conditions, which might increase cold resistance and lengthen lifespan under short-day conditions. All of this can be seen as preparation of the brain for the coming winter.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePhotoperiodic responses are known in many organisms living in temperate zones and exposed to strong seasonal changes. Preparing in advance to the coming winter is essential for survival, which is why organisms change their physiology and metabolism in response to the shortening of the photoperiod in autumn. Adult fruit flies have a lifespan of few weeks in summer, but in autumn their lifespan increases dramatically so that they survive the entire winter without food in a dormant state. To make this possible, they increase their cold and overall stress resistance, fill their lipid stores and lower their general metabolism. These changes happen in response to the decreasing photoperiod and temperature in autumn \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Although fruit flies of the species \u003cem\u003eDrosophila melanogaster\u003c/em\u003e cannot be regarded as typical photoperiodic animals, they clearly respond to a shortening of daylength in autumn \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Previous studies showed that \u003cem\u003eD. melanogaster\u003c/em\u003e reared for one generation under short photoperiods but at a constant temperature of 20\u0026deg;C, increase their cold resistance \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Here, we report that in adult flies, 14 days of exposure to short days (8 h of light and 16 h of darkness) at a temperature of 20\u0026deg;C is already sufficient to increase cold resistance.\u003c/p\u003e \u003cp\u003eFurthermore, we show that this exposure significantly alters lipid metabolism in the brain, but not in the head and body. This proves that the brain is the first organ to respond to seasonal changes and confirms that it is the place where the length of the day is measured with the help of the circadian clock in mammals and insects \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. As soon as the daylength falls below a certain threshold, this information is transmitted via several pathways to the neuroendocrine system of the brain, which alters neurohormonal signaling to the body (Helfrich-F\u0026ouml;rster, 2024). Another reason why metabolic changes are first observed in the brain is probably its high energy requirements \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The brain requires a lot of energy even when the animal is resting, sleeping or hibernating, while the skeletal muscles have almost no energy requirements at these times. Therefore, this energy requirement must be met first.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePreparation for the winter needs energy\u003c/h2\u003e \u003cp\u003eOur results show that flies eat more under short-day conditions but are less active than under long-days, suggesting that they use excess energy for anabolic processes. This is in line with previous studies that have measured the oxygen consumption of adult short-day and long-day flies. Flies that were kept under short-day conditions (8 hours of light and 16 hours of darkness) for three days and flies that had already been reared under short-day conditions consumed significantly more oxygen than flies kept under long-day conditions during the same period \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlies appear to use excess energy for lipid synthesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eShort-day flies appear to increase the levels of all major lipid classes in their brains at the expense of nucleotide and protein synthes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eis. We propose the following metabolic shift under short days: glycolytic and pentose phosphate pathway intermediates decrease, along with most TCA cycle metabolites\u0026mdash;except for citrate and oxaloacetate. Due to the decreased levels of ribose, \u003cem\u003ede novo\u003c/em\u003e nucleotide biosynthesis might be low under short-day conditions as visible in low levels of nucleotide intermediates. But we also detected high levels of xanthine and uric acid, the products of purine degradation in short-day flies, indicating a shift toward catabolic purine turnover. Based on our observations, we suggest that under short photoperiod, acetyl-CoA and ATP generated through glycolysis are utilized by mitochondrial TCA cycle to produce citrate, which is subsequently transported via the citrate shuttle to the cytosol for fatty acid synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRemarkably, all major lipid classes including storage (TAGs) and membrane lipids (PEs, PCs, PEOs, PSs, CerPEs) are elevated in the brain of short-day fly brains. While the synthesis and accumulation of storage lipids (TAGs) can be understood as immediate preparation for winter, the synthesis of membrane lipids appears less clear \u0026ndash; at least at the first glance. Lipids are stored in droplets surrounded by a phospholipid monolayer. In short-day flies, there was a notable increase in lipid droplet accumulation, particularly in the central brain. This rise in lipid droplets may also contribute to an overall increase in membrane phospholipids. Additionally, these phospholipids also contribute to apoptosis- particularly, phosphatidylserine (PS) which enables the fusion of exocytotic vesicles with plasma membrane and serves as a signal for the recognition and clearance of apoptotic cells. In \u003cem\u003eDrosophila\u003c/em\u003e, PE is a key phospholipid essential for maintaining the stability of the mitochondrial inner membrane. It is synthesized from PCs and PSs by the genes \u003cem\u003ePss\u003c/em\u003e and \u003cem\u003ePisd\u003c/em\u003e. Mutations in \u003cem\u003ePss\u003c/em\u003e lead to neurodegeneration, mitochondrial dysfunction and elevated reactive oxygen species (ROS) levels \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. According to these findings, we assume that the elevated levels of PSs, PCs, and PEs under short days, might help in the maintenance of mitochondrial integrity, reduce oxidative stress, and support normal cellular function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eShort days appear to reduce oxidative stress and increase detoxification and neuroprotection\u003c/h2\u003e \u003cp\u003eSince short-day flies have to survive the entire winter, they need to protect their brains from neurodegeneration. Oxidative stress, characterized by an imbalance between ROS and antioxidant defenses, plays a significant role in the progression of neurodegeneration. The brain is one of organs especially vulnerable to the effects of ROS because of its high oxygen demand and its abundance of peroxidation-susceptible lipid cells. In humans, previous studies have demonstrated that oxidative stress plays a central role in a common pathophysiology of neurodegenerative diseases such as Alzheimer's and Parkinson's disease \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. We found a high ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG) in the brains of short-day flies. GSH is a key antioxidant that enables a high degree of antioxidant defense minimizing oxidative damage. Another antioxidant is uric acid, the product of purine metabolism, which can scavenge ROS and is also elevated in short-day flies. GSH and uric acid may work together to mitigate damage caused by oxidative stress. In addition, we detected elevated levels of polyamines in short-day flies. Spermidine is a natural polyamine that induces autophagy, an evolutionarily conserved recycling process of waste products in cells in eukaryotes that prolongs the lifespan of all animals studied to date, including \u003cem\u003eDrosophila\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOverall, our study suggests that photoperiod alone is sufficient to initiate the physiological changes necessary for seasonal adaptation, while temperature may amplify these responses. We propose a model in which short-day flies utilize metabolic resources specifically for energy production and storage in the brain. Future studies involving stable isotope-labelling experiments are needed to validate this model.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the German Research Foundation (DFG) (grant numbers: FO207/15\u0026thinsp;\u0026minus;\u0026thinsp;2 and Me 4966/1\u0026ndash;2). Open Access funding was enabled and organized by Project DEAL.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.S. and A.H. dissected and prepared the flies for the mass spectrometric measurements and performed data mining of the lipid analysis under supervision of A.F. M.S, A.H. and J.G. performed the feeding, M.S. the locomotor activity and P.D. the chill coma experiments. W.S. performed the mass spectrometric measurements and data mining for the water-soluble components. M.S., A.H. and P.M. designed the study and A.F. and C.H.-F. supervised the experiments. M.J.M. gave valuable advice. M.S. and C.H.-F composed the figures, and A.F. and C.H.-F. wrote the paper with help of the others.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eLipid analysis was performed by the Metabolomics Core Unit at the University of W\u0026uuml;rzburg. We are grateful to Barbara M\u0026uuml;hlbauer, Maria Lesch and Markus Krischke for excellent technical assistance, Martin Eilers and Thomas Raabe for providing lab space and technical support, and the members at the Neurobiology and Genetics for fruitful discussions.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll metabolomic data generated or analyzed during this study are included in this published article (and its Supplementary Information files).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAuteri, G. G. A conceptual framework to integrate cold-survival strategies: torpor, resistance and seasonal migration. \u003cem\u003eBiol Lett\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 20220050 (2022).\u003c/li\u003e\n\u003cli\u003eSaunders, D. Photoperiodism in Insects and Other Animals. in \u003cem\u003ePhotobiology: The Science of Life and Light\u003c/em\u003e (ed. Bj\u0026ouml;rn, L. 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J. \u003cem\u003eet al.\u003c/em\u003e Mechanisms of spermidine-induced autophagy and geroprotection. \u003cem\u003eNat Aging\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 1112\u0026ndash;1129 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LC-MS based metabolomics, lipids, polar metabolites, photoperiod, feeding, activity","lastPublishedDoi":"10.21203/rs.3.rs-6800169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6800169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo survive, animals need to prepare for winter in advance, and this process begins in the brain in response to the shortening of the photoperiod in fall. Here, we demonstrate that exposing adult flies for just 14 days to a short photoperiod at a constant temperature of 20\u0026deg;C increases their cold resistance and dramatically alters brain metabolism. Such flies have significantly lower levels of monosaccharides, and a lower ATP/AMP ratio in their brains than flies exposed to a long photoperiod, despite being less active and eating more. The levels of storage and structural lipids (triacylglycerols and phospholipids) as well as the number of lipid droplets in the brain increase, suggesting the utilization of glucose for the synthesis of lipids via the citrate shuttle. In addition, during short days, the ratio between the reduced and oxidized forms of glutathione increase, as do detoxification processes and autophagy. This suggests that the brain of short-term flies is less sensitive to oxidative stress and neurodegeneration, which is essential for survival throughout the winter. Overall, our results show that exposure to a short photoperiod has significant metabolic and physiological consequences in the fly brain that serve to prepare for the coming winter.\u003c/p\u003e","manuscriptTitle":"A short photoperiod alters brain metabolism and cold resistance in Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 09:48:39","doi":"10.21203/rs.3.rs-6800169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T11:27:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T18:30:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178425920923183106589021499297177791484","date":"2025-06-29T20:06:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46666271365690991519142512318217928858","date":"2025-06-24T17:41:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T16:32:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-05T07:00:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-04T08:45:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-02T08:20:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a9432854-a443-44d0-812a-11b8e6a7e370","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50536091,"name":"Biological sciences/Molecular biology"},{"id":50536092,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-10-13T15:59:48+00:00","versionOfRecord":{"articleIdentity":"rs-6800169","link":"https://doi.org/10.1038/s41598-025-22793-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-07 15:57:14","publishedOnDateReadable":"October 7th, 2025"},"versionCreatedAt":"2025-06-27 09:48:39","video":"","vorDoi":"10.1038/s41598-025-22793-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22793-7","workflowStages":[]},"version":"v1","identity":"rs-6800169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6800169","identity":"rs-6800169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}