title: Quantitation and localization of pospiviroids in aphids.
abstract: In this paper, the potential role of aphids in viroid transmission was explored. Apterous aphids were fed on pospiviroid-infected plants and viroid targets in the aphids were consequently quantified through RT-qPCR and localized within the aphid body using fluorescence in situ hybridization (FISH). Based on the analytical sensitivity test, the limit of detection (LOD) was estimated at 1.69×10(6) viroid copies per individual aphid body. To localize the viroids in the aphids, a pospiviroid-generic Cy5-labelled probe was used and the fluorescent signal was determined by confocal microscopy. Viroids were clearly observed in the aphid's stylet and stomach, but not in the embryos. Viroids were detected in 29% of the aphids after a 24h feeding period, which suggests only a partial and low concentration viroid uptake by the aphid population including viroid concentrations under the LOD. However, these results show that viroids can be ingested by aphids while feeding on infected plants, thus potentially increasing the transmission risk. The combination of FISH and RT-qPCR provides reliable and fast localization and quantitation of viroid targets in individual aphids and thus constitutes a valuable tool in future epidemiological research.
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium; Plant Sciences Unit - Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan 96, 9820 Merelbeke, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Van Bogaert
authors.affiliation: Plant Sciences Unit - Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan 96, 9820 Merelbeke, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: De Jonghe
authors.affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
authors.forename: E J M
authors.initials: EJ
authors.lastname: Van Damme
authors.affiliation: Plant Sciences Unit - Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan 96, 9820 Merelbeke, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Maes
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium. Electronic address: guy.smagghe@ugent.be.
authors.forename: G
authors.initials: G
authors.lastname: Smagghe
mesh_terms: D000825:Animal Structures / Q000821:virology; D000818:Animals; D001042:Aphids / Q000821:virology*; D004772:Entomology / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007303:Insect Vectors / Q000821:virology*; D018613:Microscopy, Confocal; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*; D014773:Virology / Q000379:methods
keywords: Fluorescent in situ hybridization; Myzus persicae; Potato spindle tuber viroid; Tomato apical stunt viroid; Transmission