Navel orangeworm (Amyelois transitella) is a primary pest of tree nuts in the state of California. Larvae of the species bore into nut causing direct damage while also contaminating the kernel with frass, webbing, and aflatoxin producing fungi. As tree nut acreage has expanded and concentrated in California’s Central Valley there has been increased interest in area-wide approaches for the management of this pest, including the use of genetic technologies. Here we describe the application of CRISPR/Cas9 genome editing tools to knockdown three pigment genes (White, Scarlet, and Brown-like) in Amyelois transitella. Knockouts of all three genes were viable through the adult stage and were readily identified phenotypically due to distinct alterations in eye pigmentation. White and Scarlet knockouts also produced visible changes in the larval cuticle pigmentation. Adult Scarlet knockouts produced viable pigmentless larvae, demonstrating a successful germline transformation. Survival and transformation rates of White and Brown-like injected eggs did not produce sufficient adult moths to test viability of progeny. Survival rates for injected eggs were low (2.6-11.4% per sgRNA, averaging 6.5%) as was the transformation rate when calculated across all eggs (0-6.2%), though these rates are in-line with similar insect transformation systems. This work represents the first CRISPR/Cas9 mediated genetic modification of Amyelois transitella, a tool which may be utilized in future research of the pest’s functional genomics, as well as a potential tool for pest management.
