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

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, 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.

Specific and sensitive primers for the detection of predated olive fruit flies, Bactrocera oleae (Diptera: Tephritidae)

“…barbarus;12,P. globosus;13,PCR negative control. gene in a similar work on sharpshooters (6 pg/µL, León et al, 2006).…”

Identifying the predator complex of Homalodisca vitripennis (Hemiptera: Cicadellidae): a comparative study of the efficacy of an ELISA and PCR gut content assay

“…It is important to remember two main diVerences between the two types of gut assays used. First, the ELISA only detects predation on the GWSS egg and adult (gravid) female life stages, and the PCR assay detects predation on all the GWSS life stages de León et al 2006). Second, when compared with ELISA, the PCR detected prey remains for a longer period in the guts of the two predators (C. carnea and H. axyridis) that we have examined thus far ).…”

“…Contemporary assays of stomach content include monoclonal antibody (mAb)-based, enzyme-linked immunosorbent assays (ELISA), which detect species-speciWc proteins (and sometimes life-stage-speciWc proteins) (Greenstone and Morgan 1989;Hagler et al 1991Hagler et al , 1993Hagler et al , 1994Symondson and Liddell 1996;Greenstone 1996;Fournier et al 2006;Harwood et al 2007a), and polymerase chain reaction (PCR)-based assays, which detect species-speciWc DNA (all life stages) (Agustí et al 1999(Agustí et al , 2003Harper et al 2005;de León et al 2006;Harwood et al 2007b). …”

Molecular Markers Discriminate Closely Related Species <I>Encarsia diaspidicola</I> and <I>Encarsia berlesei</I> (Hymenoptera: Aphelinidae): Biocontrol Candidate Agents for White Peach Scale in Hawaii

“…A rapid crude DNA extraction procedure was used as described in de Leó n et al (2006a) and de Leó n and Morgan (2007). In brief, individual specimens were homogenized on ice in 1.5-ml microfuge tubes in 40 l of lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 1% IGEPAL CA-630, and 100 g/ml proteinase K) with one 30-s burst on ice (Pellet Pestle Motor, Bel-Art Products, Pequannock, NJ).…”