Metabolite Profiling of Drosophila Larvae

Larval metabolic studies shown in Figure 1D were conducted using 25 staged late second instar larvae from at least three independent collections. Metabolite levels were normalized to protein levels and combined to determine average concentration and standard error of the mean, relative to the metabolite levels found in w¹¹¹⁸ controls. Triglyceride (TAG) measurements were performed using a coupled colorimetric assay (Sigma T2449) as described (Palanker et al., 2009 (link); Tennessen et al., 2014 (link)). Glycogen and glucose concentrations were determined using the Hexokinase (HK) and/or Glucose Oxidase (GO) assay kits (Sigma GAHK20, GAGO20) as described (Tennessen et al., 2014 (link)). Total protein levels were determined in parallel by Bradford assay. For adults, samples of five flies per replicate were collected at one week of age and homogenized in 100 µl of 1× PBS. Each assay was repeated at least three times for a combined total of 11–24 replicates per genotype, per sex. Metabolite levels were normalized to protein levels and combined to determine average concentration and standard error of the mean, relative to the metabolite levels found in btl-GAL4; UAS-DHR78 controls. Both control and tracheal-rescued mutant females were maintained with w¹¹¹⁸ males to ensure that mating took place.