Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography–tandem mass spectrometry

Ecdysteroids are polyhydroxylated steroids that function as molting hormones in insects. 20-Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophila melanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography–mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. In vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate.