Nanophotometer

dsRNA was synthesized in vitro using the T7 RiboMAX^TM Express RNAi System (Promega, cat. no. P1700, USA) according to the manufacturer’s instructions. ACT fragments of different length were PCR amplified using specific primers containing the T7 promoter sequence (see Supplementary Table S2). The PCR products were purified with a gel extraction kit (Omega, China). The reaction mixture consisted of 2 µl T7 Express Enzyme Mix, 10 µl RiboMAX™ Express T7 2 × Buffer, and 1 µg of DNA template. The mixture was incubated at 37 °C for 2–6 h and 70 °C for 10 min, then slowly cooled to room temperature (~20 min) to anneal of the dsRNA. The DNA template and single‐stranded RNA (ssRNA) were removed by DNase (removing DNA template) and RNase A (removing ssRNA) treatments, respectively. The yield of dsRNA was determined by ultraviolet absorbance at a wavelength of 260 nm with a Nano Photometer (Implen, Germany) and the integrity of the full length was verified by RNA gel electrophoresis.
For ssRNA synthesis, the ACT200 fragment was amplified with primer pair act200-F/T7act200Rev (see Supplementary Table S2). In vitro transcription reactions were conducted with T7 RNA polymerase (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. The RNA yield was determined with a Nano Photometer (Implen, Germany) and kept at −80 °C until further use.

Recombinant plasmid including serpin7 gene fragment was extracted by using EZgene™ Plasmid Miniprep Kit (Biomiga, San Diego, CA, USA). Using the recombinant plasmid as template, the serpin7 gene was amplified by primers serpin7‐2F/serpin7‐2R (Table 1). The amplified PCR products were then purified with TIANgel Midi Purification Kit (Tiangen), followed by quantification through NanoPhotometer™ (Implen GmbH, Munchen, Germany). Serpin7 double‐stranded RNA (dsRNA) was synthesized using the T7 RiboMAX™ Express RNAi System Kit (Promega, Madison, WI, USA). The expected size of serpin7’s dsRNA was 602 bp (Fig. 1). dsRNA concentration of serpin7 was detected by a NanoPhotometer™ (Implen, GmbH, München, Germany), and the final concentration was adjusted to 1 μg·μL⁻¹ for further analysis.
Twenty‐five female L. migratoria were selected from each photoperiod within 24 h after adults were injected with 10 μL serpin7 dsRNA (μg·μL⁻¹) between the third and fourth abdominal segments. Double‐distilled water (ddH₂O) as control was injected in a similar manner to the selected females. Dissecting the whole bodies of dsRNA‐injected and control group’s adult locusts after 36 h, we obtained hind leg, ovary and fat body. The efficiency of RNAi‐mediated knockdown was determined with real‐time quantitative PCR (RT‐qPCR).

Ascorbic acid determination was carried out by a colorimetric method (Stevens et al., 2006 (link)) with modifications reported by Rigano et al. (2014) (link). Briefly, 500 mg of frozen powder were extracted with 300 μl of ice cold 6% TCA. The mixture was vortexed, incubated for 15 min on ice and centrifuged at 14000 rpm for 20 min at 4°C. Twenty microliters of supernatant were placed in an eppendorf tube with 20 μl of 0.4 M phosphate buffer (pH 7.4) and 10 μl of double distilled (dd) H₂O. Then, 80 μl of color reagent solution were prepared by mixing solution A [31% H₃PO₄, 4.6% (w/v) TCA and 0.6% (w/v) FeCl₃] with solution B [4% 2,2′-dipyridil (w/v)]. The mixture was incubated at 37°C for 40 min and measured at 525 nm by a NanoPhotometer^TM (Implen). Three separated biological replicates for each sample and three technical assays for each biological repetition were measured. The concentration was expressed in nmol of AsA according to the standard curve, designed over a range of 0–70 nmol; then the values were converted into mg/100 g of fresh weight (FW).