Primescript first strand synthesis system

Based on the genome database of B. dorsalis (https://i5k.nal.usda.gov/Bactrocera_dorsalis), the BdETH and BdETH-R genes were identified by performing a TBLASTN search for the ETH and ETH-R homologs in D. melanogaster (Park et al., 2002 (link), 2003 (link)). RNA was extracted from different developmental stages (eggs, larvae at the early stage of the 1st, 2nd, and 3rd instar; larvae at the late stage of the 1st, 2nd, and 3rd instar) and specific larval tissues including the central nervous system (CNS), fat body (FB), midgut (MG), Malpighian tubules (MT), integument (IN) and trachea (TR) using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Single-strand cDNA was prepared using PrimeScript first-strand synthesis system (Takara, Dalian, China). Using high fidelity DNA polymerase PrimeSTAR (Takara), the full open reading frame (ORF) of BdETH and the transmembrane domains 4-7 of BdETH-R-A and BdETH-R-B were amplified. Primers were designed based on the genome data of B. dorsalis (Table S1) and PCR conditions were as follows: 98°C for 2 min, 95°C for 30 s, 60°C for 30 s and 72°C for 1 min with 35 cycles, and finally 72°C for 10 min. PCR products were purified and cloned into a pGEMT Easy vector (Promega, Beijing, China) and sequenced (BGI, Beijing, China).

We collected virgin female flies on 0, 5, 10, 15, and 20 days after eclosion. At every time point, four biological replicates were performed and each replicate consisted of four adults. Subsequently, the flies were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, United States) and then RNA was extracted and first-strand cDNA was prepared with PrimeScript first-strand synthesis system (Takara, Dalian, China).
For the RT-qPCR analysis, we selected the ETH signaling genes (ETH, ETHR-A, and ETHR-B) and pathway marker genes (Shade, Spook, JHAMT, Vg1, Vg2, and Vg3). The reactions were performed in duplicate in 384- or 96-well plates on a StepOne system (ABI, Applied Biosystems, Foster City, CA, United States). The 10 μl of each reaction contained 5 μl of SYBR Green Supermix (Novoprotein, Shanghai, China), 3.5 μl of nuclease free water, 0.5 μl of each primer, and 0.5 μl of cDNA sample (∼200 ng/μL). The PCR conditions were 95°C for 2 min, and 40 cycles of 95°C for 15 s and 60°C for 30 s, 95°C for 1 min. This was followed by melting curve analysis (60–95°C). Based on our previous evaluations (Shen et al., 2010 (link)), α-tubulin (GU269902) was used as a stable reference gene. All data were analyzed with qBase software (Jan et al., 2007 (link)).