Green premix ex taq 2 kit

The head (without antennae), thoraxes (without legs), abdomens were dissected from 15 virgin 1-day-old of females or males, respectively. These tissues were immediately transferred into 1.5 mL RNA-free tube, super-cooled via liquid nitrogen, and then stored at − 80 °C freezer. These tissues were used for RNA extraction with RNAiso Plus (TAKARA, 9109, Dalian, China) and then cDNA synthesis with A Prime Script RT reagent Kit with gDNA Eraser (TAKARA, RR047, Dalian, China). The quantitative PCR reactions were conducted on an ABI QuantStudioTM 6 Flex system (Thermo Fisher Scientific, Massachusetts, USA). The PCR reaction was performed with each reaction was performed with Green Premix Ex Taq II Kit (TAKARA, RR820A, Dalian, China) and prepared as introduced^{42 (link)}. The expression level of target gene was normalized with reference gene TUB1 (α-tubulin) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) via 2^-∆∆CT method according to our previous works^{39 ,42 (link)}. The primers used in this research were listed in the Table S1.

Ten fruit flies in each group were collected into each EP tube after 3 consecutive days of treatment, quick-frozen with liquid nitrogen, and stored at −80 °C for follow-up detection. As the bimodal activity pattern with a morning peak around lights-on and an evening peak before lights-off in fruit fly^{53 (link),67 (link)}, samples were taken at 18:00 in this study. The relative expression levels of major clock genes (period (per), timeless (tim), Clock (Clk), cycle (cyc) and cryptochrome (cry)) and genes related to the synthesis of the neurotransmitters dopamine (DA), serotonin (5-HT) and gamma-aminobutyric (GABA) (dopa decarboxylase (Ddc), pale (ple), tryptophan hydroxylase (Trh), and glutamic acid decarboxylase 1 (Gad1)) were detected. Total RNA was extracted by TRIzol reagent and reverse transcribed by the PrimeScript RT Master Mix Perfect Real Time kit (Takara, Japan), and the Green Premix Ex TaqII kit (Takara, Japan) was used for real-time fluorescence quantitative PCR experiments. All samples were tested three times, and the CT values of the target genes were normalized to the CT values of the reference gene rp49⁸² . The relative quantitative analysis was carried out by the 2^−ΔΔCT method^{83 (link)}. The primers used are shown in Supplementary Table 1. The experiment was repeated three times.