LC/MS/MS identification of 20-hydroxyecdysone in a scorpion (Liocheles australasiae) and its binding affinity to in vitro-translated molting hormone receptors

Recent advances in mass spectrometry (MS) technology have facilitated the detection and quantification of minor components in organisms and the environment. In this study, we successfully identified 20-hydroxyecdysone (20E) in first instar nymphs (7 days after hatching) of the scorpion Liocheles australasiae, using tandem mass spectrometry combined with high-performance liquid chromatography (LC/MS/MS). This substance was not found in adults after the fifth stage. Other possible molting hormone candidates such as makisterone A (MaA) and ponasterone A (PoA), both of which are reported to be the molting hormones of a few arthropod species, were not detected in this scorpion. The ligand-receptor binding of 20E and its analogs was quantitatively evaluated against the in vitro-translated molting hormone receptor, the heterodimer of ecdysone receptor (EcR) and the retinoid X receptor (RXR) of L. australasiae (LaEcR/LaRXR). The concentrations of ecdysone (E), MaA, 20E, and PoA that are required to inhibit 50% of (3)H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 μM, respectively. The activity profiles of these 4 ecdysteroids are consistent with those obtained for the molting hormone receptors of several insects. The binding of a non-steroidal E agonist, tebufenozide, to EcR was not observed even at high concentrations, indicating that the structure of the ligand-binding pocket of LaEcR is not favorable for interaction with tebufenozide.