Abstract: [Objective] The fall webworm (Hyphantria cunea) is an invasive species, and has been causing severe damage to forestry and agricultural production. It is urgently needed to effectively control the pest population by an environmentally-benign technique. To reveal the mechanism of its strong migration capability which plays an important for its spreading, we analyzed the function of Wnt-1, an essential gene in wing development, by using the genome editing method.[Method] The full length cDNAs of HcWnt-1 were obtained by using the primers designed based on the genome data and transcriptome data of H. cunea. Online softwares was used to predict the structure characteristics of HcWNT-1 protein. RT-PCR and Immunohistochemistry were used to explore the expression pattern of HcWnt-1. The CRISPR/Cas9 was applied to induce mutations of HcWnt-1, the phenotypic and genotypic characteristics of embryonic mutants were observed, and further direct sequencing were used to detect the mutation events.[Result] The complete coding sequence of HcWnt-1 is 1 221 bp, which encodes a protein of 407 amino acids, characterized by helix-turn-helix DNA binding motif and 24 cysteine residues, and the highly conserved motifs are scattered throughout the sequence. During the embryogenesis stages, HcWnt-1 mRNA was highly expressed at early stage, WNT-1 protein expression showed sequential change during the embryonic development stages. The early embryos formation was completed in 24 h, at which the first peak was detected at transcriptional level and the HcWNT-1 expression was mainly concentrated in acron. The expression of HcWnt-1 declined as development progressed and the expression of HcWNT-1 elongated from head to tail along the anterior-posterior body axis. HcWnt-1 expression presented the second peak at 144 h, which occured in the appendages. CRISPR/Cas9-based mutagenesis of HcWnt-1 led to a high mortality rate (99.8%) and a mutation efficiency of 62.5% at embryonic stages following injection of Cas9 mRNA and sgRNAs with 1 000 eggs. Defective phenotypes as missing segments and appendages were observed in unhatched larvae.[Conclusion] The results have revealed that the segmentation model of H. cunea belongs to short and intermediate germ band. CRISPR/Cas9 system is a powerful genome manipulation tool in the invasive pest, and would be a promising tool for gene functional research in forestry and non-model insects. Loss-of-function of HcWnt-1 inhibited the segmentation and appendage development, demonstrating that HcWnt-1 plays an important role in embryonic development of H. cunea. Furthermore, HcWnt-1 was proved as a lethal gene, and it could be used as an appropriate target for future genetic control of H. cunea.

Key words: Hyphantria cunea, CRISPR/Cas9, Wnt-1, genome editing, immunohistochemistry, segmentation, appendage development