Genetic Regulation Of Spodoptera Litura Population

Spodoptera litura belonging to Lepidoptera, Noctuidae, is an important agricultural pest worldwide widely distributed abroad and mainly distributed in Africa, sub-tropical region, the Mediterranean region, Southeast Asia and South Asia region, and mainly distributed in the Yangtze River and the Yellow River in China. Currently the prevention and regulation of Spodoptera litura mainly relyed on chemical pesticides, but with the increased resistance of pests and the security issues of pesticides, the demand of developing new control methods for Spodoptera litura becomes urgent, and genetic regulation technique of insect population is an ideal choice for Spodoptera litura management.This study was based on the principles of the genetic regulation technique of insect population, trying to utilize insect transgenic technology and genome editing technique to construct a population genetic regulation system for Spodoptera litura.Insect transgenic technology is the basis for genetic regulation technique. Based on the established transgenic technology for the silkworm in our laboratory, we construct some piggyBac transposon based transgenic plasmids. After comprehensive understanding of the oviposition manners of Spodoptera litura, we established and optimized the embryo microinjection platform in Spodoptera litura. In the present study, four different transgenic plasmids:Oxitec4319, IE1-DsRed, 3XP3-EGFP and IE1-DsRed-ovoP-1.2k-EGFP were injected to validate the feasibility of our transgnic operation. Fourty-eight hours post microinjection, strong transient expression of reporter gene was observed which indicated that these reporter genes can be expressed and translated into corresponding proteins in the Spodoptera litura embryos. These results suggested the possibility of transgenic research in Spodoptera litura.The newly developed genome editing technologies show great potentials to be the new direction of insect genetic regulation, particularly the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system has already been applied in insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, few reports confirmed the utility of CRISPR/Cas9 in agricultural pests due to the shortage of genomic information and embryonic microinjection techniques. Here we proved that the CRISPR/Cas9 system could induce efficient gene mutagenesis in Spodoptera litura. We targeted the S. litura Abdominal-A(Slabd-A) gene which is an essential embryonic development gene and plays a significant role in determining the identity of abdominal segments of insects. Direct injection of Cas9 mRNA and Slabd-A-specific sgRNA into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. The PCR-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in S. litura and can be further applied in the future genetic regulation.