• CRISPR/Cas9-Mediated Vitellogenin Receptor Knockout Leads to Functional Deficiency in the Reproductive Development of Plutella xylostella

      Peng, Lu; Wang, Qing; Zou, Ming-Min; Qin, Yu-Dong; Vasseur, Liette (Frontiers Media, 2020)
      The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) gene superfamily and plays an indispensable role in Vg transport, yolk deposition, and oocyte development. For this reason, it has become a promising target for pest control. The involvement of VgR in Vg transport and reproductive functions remains unclear in diamondback moths, Plutella xylostella (L.), a destructive pest of cruciferous crops. Here, we cloned and identified the complete cDNA sequence of P. xylostella VgR, which encoded 1805 amino acid residues and contained four conserved domains of LDLR superfamily. PxVgR was mainly expressed in female adults, more specifically in the ovary. PxVgR protein also showed the similar expression profile with the PxVgR transcript. CRISPR/Cas9-mediated PxVgR knockout created a homozygous mutant of P. xylostella with 5-bp-nucleotide deletion in the PxVgR. The expression deficiency of PxVgR protein was detected in the ovaries and eggs of mutant individuals. Vg protein was still detected in the eggs of the mutant individuals, but with a decreased expression level. However, PxVg transcripts were not significantly affected by the PxVgR knockout. Knockout of PxVgR resulted in shorter ovarioles of newly emerged females. No significant difference was detected between wild and mutant individuals in terms of the number of eggs laid in the first 3 days after mating. The loss of PxVgR gene resulted in smaller and whiter eggs and lower egg hatching rate. This study represents the first report on the functions of VgR in Vg transport, ovary development, oviposition, and embryonic development of P. xylostella using CRISPR/Cas9 technology. This study lays the foundation for understanding molecular mechanisms of P. xylostella reproduction, and for making use of VgR as a potential genetic-based molecular target for better control of the P. xylostella.
    • Identification of Halloween Genes and RNA Interference-Mediated Functional Characterization of a Halloween Gene shadow in Plutella xylostella

      Peng, Lu; Wang, Lei; Zou, Ming-Min; Vasseur, Liette; Chu, Li-Na; Qin, Yu-Dong; Zhai, Yi-Long (Frontiers Media, 2019)
      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, Plutella xylostella (L.), a worldwide Lepidoptera pest attacking cruciferous crops and wild plants. In this study, complete sequences for six Halloween genes, neverland ( nvd ), shroud ( sro ), spook ( spo ), phantom ( phm ), disembodied ( dib ), shadow ( sad ), and shade ( shd ), were identified. Phylogenetic analysis revealed a strong conservation in insects, including Halloween genes of P. xylostella that was clustered with all other Lepidoptera species. Three Halloween genes, dib , sad , and shd were highly expressed in the adult stage, while nvd and spo 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, shd was expressed predominantly in the fat body to convert ecdysone into 20-hydroxyecdysone. RNAi-based knockdown of sad , 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 P. xylostella . Furthermore, after the knockdown of sad , the expression levels of Vg and VgR genes were significantly decreased by 77.1 and 53.0%. Meanwhile, the number of eggs laid after 3 days was significantly reduced in sad 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 P. xylostella . 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 P. xylostella.