Xs3du microbalance

Larvae were picked up from fruit vials and were transferred into a glass spot plate containing water. The larvae were then heat-killed by microwaving for 10 s. The bodies of the larvae straightened out after microwaving and were arranged and photographed on a slide under an Amscope MU300 microscope (Amscope, Irvine, CA). A ruler was set beside the larvae as a reference. Newly emerged flies were collected and weighed on a Mettler Toledo XS3DU microbalance (Mettler Toledo, Greifensee, Switzerland). Flies that emerged from the sucrose food were weighed in pairs, and the weight of each adult was calculated afterward. Flies from the microbe rescue and nutrition rescue experiments were weighed individually. Quantification of wing length was performed as previously described (86 (link)). In brief, one wing from each emerged fly was removed and wings from the same treatment were positioned under a coverslip on a glass slide. The pictures were taken using ×10 magnification and a Zeiss Axio Observer Z1 microscope and measured with ZEN 2012 (blue edition) (Carl Zeiss, Inc., Oberkochen, Germany). Wing length was measured as the linear distance from the intersection of the anterior cross vein to the wing margin at the distal end of the third longitudinal vein. Both female and male wings were cut and measured.

Each generation, five replicates of five flies per sex were sampled from the CON and from the ETP. These adult flies (10–15 days old) were frozen at −20°C for approximately 1 h, then weighed on a Mettler Toledo XS3DU microbalance. The samples were placed in an incubator for 24 h at 50°C and then reweighed. Dried flies were homogenized using a powered hand pestle in 300 µl of phosphate buffer (25 mmol/L KHPO4, pH 7.4). Following initial homogenization, 700 µl of buffer was added, mixed by vortexing and 950 µl of supernatant was saved for the metabolic pool assays. Glycogen assays (Pointe Scientific G7521-500, Roche 10102857001, Thermo Fisher Scientific J16445-06), triglyceride assays (Pointe Scientific T7532-120, T7531-STD) and soluble protein assays (Sigma-Aldrich B9643-1L) were then carried out in triplicate according to the kit instructions. The means were normalized using the respective sample dry weights.