Rapid molecular methods for in-field and laboratory identification of the yellow-legged Asian hornet (Vespa velutina nigrithorax)

The yellow-legged Asian hornet (Vespa velutina nigrithorax) is an invasive species that presents a threat to apiculture in Europe; first introduced into France in 2004, it has subsequently spread into neighbouring European countries. There is a risk of invasion and establishment in the UK, and in 2016, nests were found and destroyed in Alderney in the Channel Islands, and in Tetbury, Gloucestershire, illustrating a need for screening of suspect specimens so that invading hornets can be rapidly identified, and their nests destroyed. In this study, loop-mediated isothermal amplification (LAMP) and real-time PCR assays were developed to enable both in-field and laboratory testing. Species-specific identification assays and generic invertebrate control assays were developed. All the assays were validated according to the European Plant Protection Organisation standard PM 7/98. The assays were tested successfully against V.velutina nigrithorax obtained from France, Asia and the UK. Eight non-target species, that were closely related or morphologically similar to the Asian hornet, gave negative results with the species-specific assays, and positive results with the control assays. The assays could be used to detect target DNA at concentrations as low as 5pg per reaction. LAMP was rapid, and cable of generating positive results within 10min. Using simplified sample homogenization protocols that could be performed in the field, the LAMP assay was successful when tested against all developmental stages and nest samples, assisting with identification of samples that cannot be determined morphologically and allowing detection away from the laboratory. These assays provide a valuable tool for fast and reliable detection of this invasive species, offering the ability to identify damaged/incomplete specimens and immature life-stages.