Of Biology, Iowa City, IA 52242]110. Photos were obtained using a Zeiss LSM 710 Confocal Microscope and images have been analyzed applying FIJI software111. NPY Y1 receptor Antagonist Accession Normally 50 CNSs were mounted for observation and 1 representative image per genotype is depicted in figures. CNSs from male and female larvae were scored collectively. General molecular biology. gDNA was extracted as previously described26,112. Briefly, one or two flies have been macerated using pellet pestles and homogenized in one hundred l DNA extraction buffer (1 M Tris-HCl at pH eight.two, 0.five M EDTA, five M NaCl). Then, we added 1 l proteinase K (final concentration of 400 g/mL), and incubated the mixture at 37 for 1 h, followed by 95 for 5 min, to inactivate the protease. RNA was extracted employing either the Direct-zol RNA MiniPrep kit (Zymo Analysis), Higher Pure RNA Tissue Kit (Roche) or NZY Total RNA isolation kit (NZYtech), following the manufacturer’s guidelines. The material made use of for the qRT CR experiments described in Figs. 2, 6j, and 7n have been obtained from 1-5 staged animals, depending on the experiment, and was macerated employing pellet pestles and homogenized in 800 l of TRI Reagent or NZYol and centrifuged at 12,000 g for 1 min, to decrease tissue debris. Immediately after the centrifugation, half volume of absolute ethanol was added towards the supernatant and mixed well. Then, the sample was loaded inside a binding column in the RNA extraction kit. An additional DNAse therapy (Turbo DNA-free kit, Ambion, Life Technologies) was performed to reduce gDNA contamination. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT uantitative PCR (Thermo Scientific) or NZY First-Strand cDNA Synthesis Kit, following manufacturer’s guidelines.NATURE COMMUNICATIONS | (2021)12:3328 | https://doi.org/10.1038/s41467-021-23218-5 | www.nature.com/naturecommunicationsARTICLENATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23218-In situ hybridization probes, PCR, and qRT-PCR primers are described in their respective sections under and in Supplementary Table 1. Briefly, their specificity was tested applying Primer BLAST or Primer3. Primers and MT1 Agonist supplier probes for Ceratitis capitata were obtained from InsectBase http://www.insect-genome.com/ [Whole genome assembly of Mediterranean fruit fly (Ceratitis capitata) as part of the BCMHGSC i5k Pilot Project; ref. 113]. C. capitata ilp8 (cilp8) corresponds to uncharacterized protein LOC101461861 [Ceratitis capitata], NCBI Reference Sequence: XP_004525593.1, Gene ID GI: 498965474. C. capitata Rp49 (cRp49) corresponds to LOC101451559 60 S ribosomal protein L32 [Ceratitis capitata], NCBI Reference Sequence: XP_004517954.1, Gene ID: 101451559. 20HE therapy. dilp8ag52 flies were left to lay eggs for two h on apple plates. 20 to 30 larvae were transferred to vials with standard meals at 48 h following egg laying. Larvae were then collected at 96 h soon after egg laying, washed in PBS, and also the carcass was dissected from the rest on the larva tissue in Schneider Medium (Gibco – cat. #21720-024). Two carcasses have been incubated for each therapy in a 24-well dish. The carcasses have been incubated in Schneider medium for 1 h with oxygenation by agitation (250 rpm) at space temperature (225 ). This timepoint corresponded for the T0 sample (ahead of therapy). The Schneider medium was then replaced with a fresh medium containing 20-hydroxyecdysone (Cayman Chemical cat. #16145) inside a final concentration of 5 54 or equivalent volume of vehicle (absolute ethanol) for 3-6 h soon after which the carcass was frozen.