Profiles of free and conjugated ecdysteroids and ecdysteroid acids during pupal‐adult development of Manduca sexta

Abstract

The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from ['4Clcholesterol, in five different age groups of male Manduca sexta during pupal-adult development were determined by HPLC. Eight free ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of each ecdysteroid fraction rose sharply by day4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid. This stage demonstrated high degrees of ecdysone biosynthesis, oxidative catabolism, and phosphorylation. As development proceeded to day 16, total ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was accompanieid by increasing titers of both conjugates and acids resulting from the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were 3-epi-20-hydroxyecdysonoic acid and the phosphate conjugates of 3-epi-20-hydroxyecdysone and 3-epi-20,26-di hydroxyecdysone. The ultimate inactivation of the ecdysteroids of M. sexta during pupal-adult development is possibly mediated by two pairs of metabolically-linked processes, one leading to a 3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates.Key words: ['4C]cholesterol, HPLC, ecdysteroid titers, ecdysteroid metabolism, epimerization, hydroxylation, oxidation, epiecdysteroid acids, epiecdysteroid phosphates INTRODUCTIONPhysiological events that occur during pupal-adult metamorphosis of insects can be correlated with the presence of ecdysteroid hormones whose titer and Acknowledgments: We gratefully thank Lynda J. Liska and Kenneth R. Wilzer, Jr., for their technical support, Sherry A. Cohen for her secretarial assistance, and Martin L. Weinrich for his preparation of the manuscript. We also wish to thank John M. Roman of Program Resources, IRC., NCI-FCRS, Frederick, MD, for the FAB-mass spectra.

Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry

“…20E and E have been found in the hemolymph during the pupal‐adult development23 of M. sexta. [ 14 C]20E and [ 14 C]E were also detected in the whole body extract of pupae after injection of [ 14 C]cholesterol into fifth‐instar larvae 24, 27…”

Phylogenomics of the ecdysteroid kinase-like (EcKL) gene family in insects highlights roles in both steroid hormone metabolism and detoxification

“…Ecdysteroid-phosphate conjugates have also been identified in other insects: the lepidopterans Bombyx mori, Manduca sexta and Pieris brassicae, and the orthopterans Locusta migratoria and Schistocerca gregaria. Lepidopteran conjugates include the 22-phosphates produced by BmEc22K in B. mori (Sonobe et al 2006), but also 2-, 3-and 26-phosphates (Beydon et al 1987;Lozano et al 1989;Thompson et al 1988;Sonobe & Yamada 2004). While Lep1 orthologs of BmEc22K might be responsible for these additional conjugates, genes in other subfamily A clades (Lep2 through Lep8) are also good candidates.…”

Ecdysone oxidase and 3‐oxoecdysteroid reductases in Manduca sexta: Developmental changes and tissue distribution

“…[14C]cholesterol in M. sexta pupae [31,32], accumulate in the gut [33], and are present in the meconium at the end of adult development [XI. Perhaps other tissues contribute to the formation of 3a-hydroxyecdysteroids at this stage of development.…”