Resistance to pyrethroid and neonicotinoid insecticides has emerged indicating that the use of these broad-spectrum insecticides may be short lived 6, 7. Control of GWSS has focused on quarantine restrictions across California, as well as the application of chemical insecticides. The costs of PD alone are considerable, as GWSS endangers the Californian grape and wine industry that generates $57.6 billion annually 4, 5. It is a xylem-feeder with more than 100 host plants 1 and is an important vector of Xylella fastidiosa, which causes Pierce’s Disease (PD) in grapes and other pathologies its host plants 1, 2, 3. GWSS is a polyphagous hemipteran pest that, while invasive to California, is native to the south-eastern United States and north-eastern Mexico. Here we describe deployment of this technology in Homalodisca vitripennis Germar (glassy-winged sharpshooter, GWSS). All that is required for direct mutagenesis is a high-quality draft genome, a means to introduce the Cas9 protein and single-guide RNAs (sgRNAs) to the germline, and the ability to perform genetic crosses. This will facilitate novel genetic control strategies.ĬRISPR/Cas9 technology provides the means to direct mutagenesis to a specific site in a genome its application to insect pests brings genetic control strategies to the forefront in insect species where genetic manipulations have either proven elusive or not been attempted. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. Other diseases include Alfalfa Dwarf, Phony Peach Disease, and Citrus Variegated Chlorosis.In combination with an efficient vector such as GWSS, Xf is a significant risk to billions of dollars of agricultural production and cultural value.CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. Xf is linked to many other plant diseases, including leaf scorch of almond, oleander, olive, and plum. In 2000, APHIS issued a Declaration of Emergency to control and prevent the spread of GWSS and PD in California, and the California Department of Food and Agriculture (CDFA) organized the Pierce’sĭisease Control Program (PDCP). The biology, feeding habits, and large host range of GWSS make it more effective in transmitting PD than native insects.
PD, present in California for over 100 years, did not pose a severe threat to agriculture until the introduction of GWSS in the 1990’s. GWSS is native to the southeastern US and northeastern Mexico. A subspecies of Xf, also called fastidiosa, causes Pierce’s Disease (PD) in vinifera grape varieties. Vitripennis (Cicadellidae family), vectors the bacterium Xylella fastidiosa ( Xf).
The Glassy-Winged Sharpshooter (GWSS), Homalodisca