Appendix A. Quantification of storage molecules.
To quantify storage molecules, we immediately homogenized a subset of animals at the same four points we measured wet mass. This was done in 150–300 µL of 50 mM imidazole buffer, depending on larval wet mass, while on ice. To prevent protein degradation, the imidazole buffer contained a broad spectrum protease inhibitor cocktail at the manufacturer’s recommended concentration (Complete Protease Inhibitor Cocktail, Roche Diagnostics, Penzberg, Germany). This sample was then centrifuged at 1310 g for 10 min at 4° C. Quantification was done colorometrically using a Molecular Devices SpectraMax 190 96-well plate spectrophotometer at a temperature of 25° C. Together with the samples we also ran a concentration range of standards of glucose and triglyceride to construct calibration curves. Each assay was performed in duplicate and the mean of the two measurements was taken as the value for that individual.
To quantify the total amount of free glucose plus glycogen in each sample, we used the Glucose oxidase method ( Trinder 1969) closely following the protocol provided by Thermo Electron ( Louisville, Colorado, USA). We added 75 µL of H2O containing 1 U of amyloglucosidase to 25 µL of the supernatant. This mixture was incubated for 1 hr at 37° C to hydrolyze the glycogen into glucose. Total glucose in the sample was then determined using the Glucose Oxidase Method Kit (Thermo Electron ®) by adding 25 µL of the resulting sample to 200 µL of the enzymatic glucose reagent, and reading the absorbance at 500 nm after 20 min of incubation at room temperature in the dark. To quantify the amount of only free glucose, we followed the same procedure except that the initial 25-µL supernatant sample was added to 75 µL of H2O without amyloglucosidase. The difference in glucose content between these two samples equalled the amount of glucose stored in glycogen. Triglyceride content of a sample was determined using the triglycerides lipase/ peroxidase method ( Fossati and Prencipe 1982, McGowan et al. 1983) closely following the protocol provided by the Infinity Triglyceride kit of Thermo Electron ( Louisville, Colorado, USA). 2.5 µL of supernatant was added to 125 µL of triglyceride reagent, and the absorbance measured at 520 nm after 10 min of incubation at room temperature in the dark.
LITERATURE CITEDFossati, P., and L. Prencipe. 1982. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clinical Chemistry 28:2077–2080.
McGowan, M. W., J. D. Artiss, D. R. Strandbergh, and B. Zak. 1983. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clinical Chemistry 29:538–542.
Trinder, P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals Clinical Biochemistry 6:24–27.