The circadian clock regulates various behavioral, metabolic and physiological processes to occur at the most suitable time of the day. Internal energy stores and nutrient availability modulates the most apparent circadian clock mediated locmotor activity rhythm in Drosophila. Although previous studies unraveled the role of circadian clock in metabolism and activity rest rhythm, the precise pathway through which the circadian neuropeptidergic signaling regulates internal energy storage and the starvation-mediated increase in activity resembling foraging remains largely unclear.  This study was aimed to elucidate the role of circadian neuropeptide, short neuropeptide F (sNPF) in triglyceride metabolism, starvation resistance and starvation-mediated increased locomotor activity in Drosophila.  The results showed that snpf transcripts exhibits significant rhythmicity in wild type flies under 12:12 hour light-dark cycles (LD) and constant darkness (DD) whereas snpf transcript level in period null flies did not exhibit any significant rhythmicity under LD.  Knockdown of sNPF in circadian clock neurons reduced the triglyceride level, starvation resistance and increased the starvation-mediated hyperactivity response after 24 hour of starvation.  Further studies showed that knock down of sNPF receptors (sNPFR) expressed in insulin producing cells (IPC) increased the starvation resistance and reduced starvation-induced hyperactivity response after 24 hour of starvation. Collectively, our results suggest that transcriptional oscillation of snpf mRNA is endogenously controlled by the circadian clock and elucidate the role of sNPF in modulating locomotor activity in accordance with the nutrient availability in Drosophila. 

Figure 1 Raw data

To assess the mRNA transcription profiles of snpf, three replicates were used for each time point.  Each replicate contained 30 heads from w¹¹¹⁸flies.  Total RNA was extracted using QIAGEN RNAeasy Plus Mini kit; DNAase digestion was performed using QIAGEN RNase–free DNase.  cDNA was synthesized using SuperScript™ III First-Strand Synthesis System (Invitrogen, Cat. No. 18080051) and real-time PCR was performed using Bio-Rad CFX96TM with the cDNA template, Power SYBR® Green PCR Master Mix (Cat. # 4368702) from ThermoFisher Scientific to check temporal transcription profile of snpf.  mRNA levels were measured under LD cycle and constant darkness (DD).  The molecular oscillation of mRNA levels was determined by normalizing the mRNA level of the gene of interest with the mRNA level of rp49 at each time point.  Cosinor analysis was implemented in MATLAB-R2016a to test for rhythmicity, to estimate the peak phase and the amplitude (peak/trough ratio) of relative mRNA expression of snpf.

Figure 2 and 3 Raw data

For activity/rest assay, freshly emerged male flies were pre-entrained for two days in the LD cycle at 25 ^oC and a humidity of 75 ± 5% inside the incubator (MIR-154, Panasonic, Japan).  Two-day-old virgin males were individually introduced into activity tubes.  Flies were provided with standard cornmeal medium and their activity/rest behavior was recorded using Drosophila Activity Monitors (DAM, Trikinetics, USA).  Following acclimatization in activity tubes for 24 hours, flies were transferred to 1% agar medium or to standard cornmeal medium in glass tubes at ZT12 (during the onset of dark phase) to provide starved or fed conditions, respectively, and locomotor activity was recorded for 36 hours at 25 ^oC.  Each experiment was performed with 28-32 flies for each genotype and DAM readings were collected in every 1-minute interval and the activity counts/ hour is plotted in the locomotor activity waveform.  Flies died within 24 hour of starvation were excluded and flies survived for 24-36 hours of starvation were used for the analysis. Change in activity under starvation was calculated as [(Activity under starvation-Activity under fed) /Activity under fed condition] x 100.  Separate sets of flies were used for recording the locomotor activity under fed and starved flies condition.  Hence the change in activity under starvation condition was calculated as  [(Activity of individual fly under starvation-Average activity of flies under fed) / Average activity of flies under fed] x 100. The waveform of activity/rest rhythm, day time activity, nighttime activity, change in locomotor activity under starvation condition were analyzed by using ANOVA followed by Tukey’s post hoc multiple comparisons. 

Triglyceride assay

This assay comprised of three replicates and each replicate containing five male flies under fed condition was homogenized in homogenization buffer (0.05% Tween 20).  The homogenate was incubated at 70^oC for 5 minutes to inactivate the enzymes and centrifuged at 14000 rpm for 3 minutes and the supernatant was collected into a sample tube.  Sigma serum triglyceride determination kit TR0100 was used to estimate the triglyceride level in the sample by measuring absorbance at 540 nm using TECAN Infinite M200 pro-multimode plate reader.  The triglyceride content was normalized to the total protein content of the flies estimated by Quick Start™ Bradford 1X Dye Reagent (BioRad, Cat. # 500-0205).  Freshly emerged flies feed less and possess larval triglyceride energy storage.  Larval fat cells persist only up to about two days and are replaced by adult fat cells.  Hence assays were carried out at ZT 12 in freshly emerged (Day 0), two day old (Day 2) and five day old (Day 5) flies to estimate the triglyceride levels during both the development and in adult flies.  TGA levels was analyzed by Student’s t-test.  

Figure S1 Raw data 

Starvation resistance Assay

Six replicates with each vial containing 10-15 flies were used for each genotype.  Five day old male flies were kept in the 1% agar medium vials. The number of dead flies was recorded at every two-hour interval until all the flies died. Log-rank test was used to compare and analyze the survivorship curves under starvation.