Ecdysteroid Quantification in Insect Larvae

Quantification of ecdysteroids in whole larvae was performed as described by [61 (link),113 (link)] with the following modifications. Briefly, animals were homogenized in 0.25 ml 75% methanol, and then the supernatants were collected following centrifugation at 14,000 g for 15 min. The pellets were re-extracted in 0.1 ml methanol. The supernatants were combined, evaporated using a SpeedVac, and then re-dissolved in 0.5 ml ELISA buffer (Cayman Chemical). Ecdysteroids were measured using a commercial ELISA kit (Cayman Chemical) that detects 20E equivalents. Standard curves were generated using 20E (Cayman Chemical), and absorbance was measured at 405 nm on a microplate photometer (Thermo Scientific).