Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and Homalodisca liturata for use in predator gut content examinations

To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy-winged sharpshooter, Homalodisca coagulata (Say), and smoke-tree sharpshooter, Homalodisca liturata (Ball) (Homoptera: Cicadellidae). Two different types of markers were compared, those targeting single-copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multicopy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed, two SCAR and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata specific and HcF6/HcR9 was H. coagulata / H. liturata specific. COI (HcCOI-F/R) and COII (HcCOII-F4/R4) markers were H. coagulata specific, COII (G/S-COII-F/R) marker was H. coagulata / H. liturata specific, and lastly, COII marker (Hl-COII-F/R) was H. liturata specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66-fold more sensitive than marker Hl-COII-F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2-fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we describe the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing the polymerase chain reaction (PCR). This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.