Analyzing Nutrient Profiles in Fruit Flies

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 unpublished data; Ridley et al. 2012 (link)): glucose with the oxidase kit (GAGO-20; Sigma-Aldrich); glycogen using the glucose kit and amyloglucosidase from Aspergillus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).