Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium.
Peptides. 2010 Mar;31(3):456-67. doi: 10.1016/j.peptides.2009.08.002. Epub 2009 Aug 12.
20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. The biosynthesis of ecdysteroid is triggered and under the control of the neuropeptide, prothoracicotropic hormone (PTTH). To date, five cytochrome P450 enzymes, namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. These genes are called Halloween genes and mediate the biosynthesis of 20E from cholesterol. In this study, we extended these works to a major pest insect in agriculture, the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes, and the converted amino acid sequences were compared with those of other insects. The phylogenetic analysis clearly showed separated clusters of each gene and the evolutional conservation in insects with a high similarity in Lepidoptera. Spo, phm, dib and sad were predominantly expressed in prothoracic glands, and shd was expressed in fat body and Malpighian tubules at the last instar larvae. Spo expression was kept high level between day 2 and day 4 after ecdysis. The expression of phm and dib peaked at day 2, and sad and shd expressions peaked at day 2 and day 4 after ecdysis. In addition, the hemolymph ecdysteroid titer showed a small peak at day 2 and a large peak at day 4 after ecdysis. These results suggest the importance of Halloween genes in ecdysone biosynthesis by prothoracic glands and conversion of ecdysone into 20E by fat body in larval-pupal metamorphosis.
20-羟基蜕皮甾酮（20E）是一种蜕皮甾酮的活性形式，是昆虫生长发育的关键激素。蜕皮甾酮的生物合成是由神经肽促前胸腺激素（PTTH）触发和控制的。迄今为止，已经鉴定出与蜕皮甾酮生物合成相关的五种细胞色素 P450 酶，即 Spook（Spo）、Phantom（Phm）、Disembodied（Dib）、Shadow（Sad）和 Shade（Shd），并且后四种酶的特性在果蝇、家蚕和烟青虫中得到了很好的研究。这些基因被称为万圣节基因，介导胆固醇向 20E 的生物合成。在本研究中，我们将这些工作扩展到农业上的一种主要害虫——棉铃虫（鳞翅目：夜蛾科）。我们鉴定了五个万圣节基因的序列，将转化的氨基酸序列与其他昆虫进行了比较。系统发育分析清楚地显示了每个基因的分离簇，并且在昆虫中具有高度相似性的 Halloween 基因在进化上是保守的。Spo、phm、dib 和 sad 在前胸腺中主要表达，shd 在幼虫末期的脂肪体和马氏管中表达。蜕皮后第 2 天到第 4 天，Spo 的表达水平保持较高。phm 和 dib 的表达在第 2 天达到高峰，sad 和 shd 的表达在蜕皮后第 2 天和第 4 天达到高峰。此外，血液中蜕皮甾酮的浓度在蜕皮后第 2 天和第 4 天出现小高峰和大高峰。这些结果表明，万圣节基因在幼虫-蛹变态过程中前胸腺的蜕皮甾酮生物合成和脂肪体中蜕皮甾酮转化为 20E 中起着重要作用。
