Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth,Plutella xylostella

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.In the current study,we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella,in addition to its known functions in the ovarian development in female adults and egg hatching.Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein(GFP)gene into autosomal Pxfl(2)d for labelling mutants could be achieved.However,this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein(CFP)expression cassettes on a Z-linked region in P.xylostella,which can also be used for early sex detection.By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain,the gene editing efficiency reached 29.73%,confirming the success of expressing a functional Cas9 gene.Taken together,we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P.xylostella,while the establishment of a heritable strain required the positioning of appropriate sites.This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

Identification of chorion genes and RNA interference-mediated functional characterization of chorion-1 in Plutella xylostella

Choriogenesis is the last step of insect oogenesis,a process by which the chorion polypeptides are produced by the follicular cells and deposited on the surface of oocytes in order to provide a highly specialized protective barrier to the embryo.The essential features of chorion genes have yet to be clearly understood in the diamondback moth,Plutella xylostella,a worldwide Lepidoptera pest attacking cruciferous crops and wild plants.In this study,complete sequences for 15 putative chorion genes were identified,and grouped into A and B classes.Phylogenetic analysis revealed that both classes were highly conserved and within each,branches are also species-specific.Chorion genes from each class were located in pairs on scaffolds of the P.xylostella genome,some of which shared the common promoter regulatory region.All chorion genes were highly specifically expressed in the P.xylostella adult females,mostly in the ovary with full yolk,which is a crucial period to build the shells of the eggs.RNAi-based knockdown of chorion-1,which is located on the Px_scaffold 6 alone,although had no effect on yolk deposition,resulted in smaller eggs and sharply reduced hatchability.Additionally,inhibition of PxCho-1 expression caused a less dense arrangement of the columnar layers,reduced exochorion roughness and shorter microvilli.Our study provides the foundation for exploring molecular mechanisms of female reproduction in P.xylostella,and for making use of chorion genes as the potential genetic-based molecular target to better control this economically important pest.