The P450 enzyme Shade mediates the hydroxylation of ecdysone to 20‐hydroxyecdysone in the Colorado potato beetle, Leptinotarsa decemlineata. Issue 5 (2nd July 2014)

• Record Type: Journal Article
• Authors: Kong, Y.; Liu, X‐P.; Wan, P‐J.; Shi, X‐Q.; Guo, W‐C.; Li, G‐Q.
• Is Part Of: Insect molecular biology. Volume 23:Issue 5(2014:Oct.)
• Page Start: 632
• Page End: 643
• Publication Date: 2014-07-02
• Subjects: Insects -- Molecular aspects -- Periodicals; 595.7
• DOI: 10.1111/imb.12115
• Languages: English
• ISSNs: 0962-1075

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <p>Ecdysone 20‐monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20‐hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene <italic>Shade</italic> (<italic>Shd</italic>) encoding E20MO in the Colorado potato beetle, <italic>Leptinotarsa decemlineata</italic>. LdSHD has five conserved motifs typical of insect P450s, ie the Helix‐C, Helix‐I, Helix‐K, PxxFxPE/DRF (PERF) and heme‐binding motifs. <italic>LdShd</italic> was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in <italic>Spodoptera frugiperda</italic> 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double‐stranded RNA (dsRNA) of <italic>LdShd</italic> into the second instar larvae successfully knocked down the <italic>LdShd</italic> expression level, decreased the mRNA level of the <italic>ecdysone receptor</italic> (<italic>LdEcR</italic>) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of <italic>LdShd</italic>‐dsRNA by the fourth instars also down‐regulated <italic>LdShd</italic> and <italic>LdEcR</italic> expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the <italic>LdShd‐</italic>dsRNA‐ingested second instars, and of halofenozide into the <italic>LdShd‐</italic>dsRNA‐ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species <italic>Le. decemlineata</italic>.</p> </abstract>