Dumont 5 forceps

Late feeding L3 larvae were gently removed from inside the food media using a paintbrush and rinsed in water to remove food particles. Larval fat bodies (FBs) or guts were dissected in PBS using Dumont #5 forceps (Electron Microscopy Sciences). All tissues were fixed in 4% paraformaldehyde for 20 min at room temperature and rinsed briefly in PBS prior to staining with organelle labeling dyes. Lipid droplets in FBs were stained by incubating FBs in 100 µM monodansylpentane (MDH) LD stain (Abgent) for 30 min at room temperature, in the dark. F-actin structures were stained with Phalloidin (1:400) or Cell Mask Green Actin Tracking Stain (1:1000) according to manufacturer’s instructions (Invitrogen). LDs in guts were stained with 1 µM Nile Red for 30 min at room temperature, in the dark. Next, FBs and guts were rinsed in PBS and mounted on slides in SlowFade Gold antifade reagent with DAPI (Invitrogen) for subsequent imaging.

Feeding larvae washed out from culture vials were collected in 630 µm mesh-fitted baskets (Genesee) and rinsed to get rid of adherent food particles. Larvae were dried and divided onto into piles (10–12 larvae each) on a strip of parafilm. Larvae were bled by tearing the cuticle with Dumont 5 forceps (Electron Microscopy Sciences). Two µl of colorless hemolymph was aspirated from each pile and separately transferred to 96-well plates (Thermo-Scientific) containing 0.1% N-Phenylthiourea (Sigma-Aldrich) in 50 µl PBS. 150 µl of Autokit Glucose reagent (Wako) was added to each well and incubated at room temperature for 20 min before measuring absorbance at 505 nm. Glucose concentration was calculated from a standard curve generated with manufacturer’s glucose standards. For trehalose assays, 8 µl of dilute hemolymph was treated with 5 µl of (diluted 8 X) porcine kidney trehalase (Sigma) overnight at 37 °C. Ten µl of treated sample was assayed for trehalose as described for glucose.