Quantification of 20-Hydroxyecdysone From Drosophila Larvae at the Onset of Metamorphosis

Metamorphosis in class Insecta is defined as the drastic transition of an organism from a larval stage to a morphologically very different adult form. Insects such as Drosophila melanogaster undergo metamorphosis, which requires precise temporal control, exerted by ecdysteroids, the primary regulators of these developmental transitions. The active form of ecdysone is 20-hydroxyecdysone (20E), which acts in pulses that trigger the onset of different developmental stages. A massive pulse of 20E occurs between the prepupal and pupal stages, triggering pupariation. Precise quantification of 20E levels is critical for understanding how this hormonal signaling governs the developmental timing of different stages. This protocol is sensitive for detecting low levels of 20E and describes a methanol-based extraction method coupled with a competitive ELISA to quantify whole-body 20E levels in third-instar Drosophila larvae. Further, this protocol enables detection of differences in ecdysone titers among genotypes affecting ecdysone biosynthesis, particularly at the late third-instar larval stage. This protocol can also be utilized for disease-causing insects (Aedes aegypti), as well as economically relevant beneficial insects (Apis mellifera), to study the effects of ecdysteroid analogs during development. Key features • 20E-dependent Ecdysone signaling induces morphological changes in insects known as metamorphosis. • This protocol provides details for 20E quantification, based on the competitive ELISA between unconjugated and HRP-conjugated 20E. • Spectrophotometry-based color intensity is directly proportional to the conjugated 20E and inversely proportional to the amount of 20E present in the sample.