AUTHOR=Peng Lu , Wang Lei , Zou Ming-Min , Vasseur Liette , Chu Li-Na , Qin Yu-Dong , Zhai Yi-Long , You Min-Sheng 

TITLE=Identification of Halloween Genes and RNA Interference-Mediated Functional Characterization of a Halloween Gene shadow in Plutella xylostella

JOURNAL=Frontiers in Physiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2019.01120

DOI=10.3389/fphys.2019.01120

ISSN=1664-042X

ABSTRACT=<p>Ecdysteroids play an essential role in controlling insect development and reproduction. Their pathway is regulated by a group of enzymes called Halloween gene proteins. The relationship between the Halloween genes and ecdysteroid synthesis has yet to be clearly understood in diamondback moth, <italic>Plutella xylostella</italic> (L.), a worldwide Lepidoptera pest attacking cruciferous crops and wild plants. In this study, complete sequences for six Halloween genes, neverland (<italic>nvd</italic>), shroud (<italic>sro</italic>), spook (<italic>spo</italic>), phantom (<italic>phm</italic>), disembodied (<italic>dib</italic>), shadow (<italic>sad</italic>), and shade (<italic>shd</italic>), were identified. Phylogenetic analysis revealed a strong conservation in insects, including Halloween genes of <italic>P. xylostella</italic> that was clustered with all other Lepidoptera species. Three Halloween genes, <italic>dib</italic>, <italic>sad</italic>, and <italic>shd</italic> were highly expressed in the adult stage, while <italic>nvd</italic> and <italic>spo</italic> were highly expressed in the egg and pupal stages, respectively. Five Halloween genes were highly expressed specifically in the prothorax, which is the major site of ecdysone production. However, <italic>shd</italic> was expressed predominantly in the fat body to convert ecdysone into 20-hydroxyecdysone. RNAi-based knockdown of <italic>sad</italic>, which is involved in the last step of ecdysone biosynthesis, significantly reduced the 20E titer and resulted in a longer developmental duration and lower pupation of fourth-instar larvae, as well as caused shorter ovarioles and fewer fully developed eggs of <italic>P. xylostella</italic>. Furthermore, after the knockdown of <italic>sad</italic>, the expression levels of <italic>Vg</italic> and <italic>VgR</italic> genes were significantly decreased by 77.1 and 53.0%. Meanwhile, the number of eggs laid after 3 days was significantly reduced in <italic>sad</italic> knockdown females. These results suggest that Halloween genes may play a critical role in the biosynthesis of ecdysteroids and be involved in the development and reproduction of <italic>P. xylostella</italic>. Our work provides a solid basis for understanding the functional importance of these genes, which will help to screening potential genes for pest management of <italic>P. xylostella.</italic></p>