DOI: 10.5061/dryad.683b46g
title: Short neuropeptide F regulates the starvation mediated enhanced locomotor activity in Drosophila

description: 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. 
description: 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 70oC 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.