Efficient CRISPR/Cas9-mediated white gene editing in the global tortricid fruit pest Grapholita molesta

Abstract

Internal fruit borers (Lepidoptera: Tortricidae) are serious fruit pests. Of particular significance is Grapholita molesta given its pest status of stone and pome fruits. So far, successful control of tortricid moths largely relies on repeated insecticide applications. The development of novel technology by genome manipulation through CRISPR/Cas9 is an environmentally friendly pest management alternative. Due to the small size of eggs, CRISPR/Cas9 protocols have not been developed for tortricid moths like G. molesta. The white gene is widely used as genetic marker in Drosophila. Here, the white gene ( Gmwhite) from G. molesta was identified and it was found that Gmwhite was expressed at all developmental stages. The Gmwhite knockout strain (White-KO) was established using CRISPR/Cas9 genome editing system. When compared with wild type, the eye color of the adult moth in the White-KO strain changed to white and the body color of the fifth-instar larvae of the White-KO strain changed from pink/red to white. The females’ fecundity of the White-KO strain was not affected whereas the flight capacity was significantly decreased. The acquired white compound eye of the White-KO strain is autosomally recessive inherited. The feasibility of the CRISPR/Cas9 methodology was validated by dual-target editing of Gmwhite. Our findings provide the first evidence that the white gene can be used as a visual reference gene for genetic manipulation of tortricid moths. The present study developed CRISPR/Cas9-mediated gene editing technology which can be used for developing genome editing control strategy as well as for gene function analyses in G. molesta.