A7420

Five flies that were fed or starved from each group were homogenized with 0.5 mL PBS containing 0.2% Triton X-100 and heated at 70 °C for 5 min. The supernatant was collected after centrifugation at 12,000 rpm for 10 min at 4 °C. Ten microliters of supernatant was used for protein quantification using Bradford Reagent (E111-01, Vazyme). Whole-body glycogen levels were measured from 10 μL of supernatant treated with or without 0.4 μL Amyloglucosidase (2 mg/mL, A7420, Sigma) to degrade glycogen into glucose at 37 °C for 30 min using glucose assay reagent (K-GLUC, Megazyme) following the manufacturer’s protocol. We subtracted the amount of free glucose from the measurement and then normalized the subtracted values to protein levels in the supernatant. To measure whole-body triglycerides, we processed 10 μL of supernatant using a Serum Triglyceride Determination kit (TR0100, Sigma). We subtracted the amount of free glycerol in the supernatant from the measurement and then normalized the subtracted values to protein levels in the supernatant.

We assayed nutritional indices in pools of 10 adult males from each line aged 3–6 days after eclosion. We measured free glucose, glycogen stores, total triglycerides, free glycerol, and soluble protein in groups of 10 male flies, with three biological replicates of rearing on each diet. Each group of flies was weighed using a MX5 microbalance (Mettler-Toledo, Columbus, OH) and then homogenized in 200 μL buffer (10 mM Tris, 1 mM EDTA, pH 8.0 with 0.1% v/v Triton-X-100) using lysing matrix D (MP Biomedicals, Santa Ana, CA) on a FastPrep-24 homogenizer (MP Biomedicals). We immediately froze 50 μL of the homogenate to be used for the total protein assay and incubated the remaining 150 μL at 72° for 20 min to denature enzymes naturally present in the homogenate. Each nutritional index was assayed using modifications of commercially available kits (see Unckless et al. unpublished data; Ridley et al. 2012 (link)): glucose with the oxidase kit (GAGO-20; Sigma-Aldrich); glycogen using the glucose kit and amyloglucosidase from Aspergillus niger (A7420; Sigma-Aldrich) in 10 mM acetate buffer at pH 4.6; free glycerol and triglycerides using reagent kits F6428 and T2449, respectively (Sigma-Aldrich); and soluble protein with the DC Protein Assay (BIO-RAD, Hercules, CA).

Squash (Guangmi No. 1) weighing 4.0–5.0 kg was harvested in July 2022 (Guangdong, China). To minimize differences, we selected squash that were the same size and had similar skin and flesh colors for treatment. After the squash was cleaned, the skin and seeds were removed. Next, the flesh of the squash was freeze-dried and crushed (100 mesh). Glucose, galactose, mannose, rhamnose, fucose, xylose, arabinose, glucuronic acid, galacturonic acid, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2-2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) salt (ABTS) were purchased from Solarbio Technology Co., Ltd. (Beijing, China). α-Amylase (A-3176-500KU, 10 U/mg), pepsin (P6887-5 G, 3706 U/mg), pancreatin (P1750-100 G) and amyloglucosidase (A7420, 31.2 U/mg) were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA).