Expanding the genetic toolkit for codling moth: Phenotypic and fluorescent markers for transgenesis

The codling moth, Cydia pomonella (L.), (Lepidoptera:Tortricidae), is a global pest of apple, pear and walnut. CRISPR/Cas9 gene editing serves as an excellent way to study the biological effects of individual genes, in vivo, of non-model organisms, and has previously been demonstrated to function efficiently in codling moth. However, challenges remain in tracking the CRISPR-edited genotypes without utilization of good visual markers, either phenotypic or fluorescent. Producing a phenotypic white eye moth by knocking out the white transporter gene would provide a convenient visual marker to facilitate targeting other genes for CRISPR editing. When a homozygous white eye strain of moths is created, CRISPR could be used to knock in a wild-type copy of the white gene into the exon of any target gene of interest, thereby rescuing the white eye phenotype. This approach would necessarily require promoter constructs coupled to the white rescue gene to effectively drive white gene expression in appropriate tissues to manifest the rescue phenotype. For this study, we have targeted the white gene in codling moth for CRISPR/CAS9 editing. We report on the phenotypic effects of the white mutant strains as well as genomic dynamics and fitness effects of CRISPR editing at this locus. Furthermore, we report on promoter-fluorescent reporter construct activity in codling moth embryos and larvae. In addition to novel characterizations of promoter activity in codling moth, constructs may also serve as transgenic markers in lieu of the white gene to facilitate phenotypic tracking of CRISPR knock-in/knock-out genome editing.