Removal of an Aminopeptidase N From Midgut Brush Border Does Not Affect Susceptibility of Spodoptera litura (Lepidoptera: Noctuidae) Larvae to Four Insecticidal Proteins of Bacillus thuringiensis (Bacillales: Bacillaceae)

Spodoptera litura is one of the most destructive lepidopteran insects of cabbages and cauliflowers in the world. Cry1 and Vip3 toxins from Bacillus thuringiensis have been reported to show toxicity in multiple lepidopteran insects. Binding of toxic molecules to specific receptors on the midgut epithelial cells is known to be a key step in the action mode of Bt toxins. Aminopeptidase N (APN) -like proteins have been reported to be binding sites of multiple Cry toxins in the midgut of Cry susceptible insects. In the present study, we identified six midgut APNs by analysis of the genome and midgut transcriptome of S. litura. CRISPR/Cas9 mediated gene-knockout system was utilized to mutate the GPI-anchor signal peptide at the C terminus of SlAPN1. SlAPN1 was verified to be removed from the midgut brush border membrane vesicles of a homozygous knockout strain of S. litura (SlAPN1-KO). Bioassay results indicated that susceptibility of the SlAPN1-KO strain to Cry1Aa, Cry1Ac, Cry1Ca, and Vip3Aa toxins was close to that of the wild-type strain of S. litura. RT-qPCR results showed that the transcriptional level of SlAPN2-6 was not up-regulated after knockout of the SlAPN1. Results in this study indicated that the SlAPN1 did not play a critical role in the pathway of toxicity of Cry1Aa, Cry1Ac, Cry1Ca, and Vip3Aa toxins in S. litura.