Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae.
Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the "Black Box" wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2,22,25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4,7-dien-3-one (3-oxo-Δ4,7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ4,7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4,6,8(14)-trien-3-one, oxidized from 3-oxo-Δ4,7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ4-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ4-steroids into ecdysteroids via 5β-reduction.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Junki
J
Saito
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Ryota
R
Kimura
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Yuya
Y
Kaieda
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Ritsuo
R
Nishida
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Electronic address: onoono@kais.kyoto-u.ac.jp.
Hajime
H
Ono
D000818:Animals; D053898:Biosynthetic Pathways; D004331:Drosophila melanogaster / Q000254:growth & development / Q000502:physiology*; D004440:Ecdysone / Q000096:biosynthesis*; D026461:Ecdysteroids / Q000378:metabolism*; D007814:Larva / Q000254:growth & development / Q000502:physiology; D008675:Metamorphosis, Biological; D010084:Oxidation-Reduction
D026461:Ecdysteroids; D004440:Ecdysone
7-Dehydrocholesterol; Black Box; Cholesta-4,7-dien-3-one (3-oxo-Δ(4,7)C); Drosophila melanogaster; Ecdysone; Ecdysteroidogenesis