Quantitation and localization of pospiviroids in aphids

• Detection and quantification of pospiviroids in Myzus persicae aphids were achieved by means of RT-qPCR with a detection limit of 1.69 × 106 viroid copies per aphid.
• Localization of Tomato apical stunt viroid (TASVd) in the stylet and foregut of feeding M. persicae aphids was accomplished by fluorescence in situ hybridization and confocal laser scanning microscopy.
• The incidence of viroids in M. persicae aphids after 24 h of feeding on infected plants was 29%.

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 × 106 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 24 h 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.