|De Leon, Jesus|
Submitted to: Entomologia Experimentalis et Applicata
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2005
Publication Date: 4/6/2006
Citation: De Leon, J.H., Fournier, V., Hagler, J.R., Daane, K. 2006. Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and H. liturata (Homoptera: Cicadellidae: Proconiini) for use in predator gut content examinations. Entomologia Experimentalis et Applicata. 119:109-119. Interpretive Summary: Two types of diagnostic markers were developed, SCAR and COI, COII mitochondrial markers. The mitochondrial markers were significantly more sensitive than the SCAR markers; furthermore, the COI markers were slightly more sensitive than the COII markers. All markers were shown to be highly specific toward their targets and were able to detect prey [glassy-winged sharpshooter (GWSS) eggs or adults] in predator (green lacewings, earwigs, and ground beetles) gut contents. This study represents an important step in identifying key predators of the GWSS (H. coagulata) and allows us to begin to understand the ecology of GWSS-predator interactions in natural environments. This information will be included in an area-wide pest management approach to aid in controlling this devasting pest, the vector of Pierce’s disease of grapevines.
Technical Abstract: 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 (GWSS) Homalodisca coagulata (Say) and smoke-tree sharpshooter (STSS) Homalodisca liturata (Ball) (Homoptera: Cicadellidae: Proconiini). Two different types of markers were compared, those targeting single-copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multi-copy 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 GWSS-specific and HcF6/HcR9 was GWSS and STSS (GWSS/STSS)-specific. COI (HcCOI-F/R) and COII (HcCOII-F4/R4) markers were GWSS-specific, COII (G/S-COII-F/R) marker was GWSS/STSS-specific, and lastly, COII marker (Hl-COII-F/R) was STSS-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 mitochondrial 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 PCR. This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.