Identification of Agriotes spp. (Coleoptera: Elateridae)by loop-mediated isothermal amplification (LAMP)

The larvae of Agriotes spp. (Coleoptera: Elateridae), also known as wireworms, are abundant soil-dwell-ing insects and can be a serious pest of a wide range of crops, such as potatoes, maize, onions, ... When wireworm numbers rise above the economic threshold of a crop, the grower needs to take control measures. To monitor the number of wireworms in a field, bait traps are being used. As Agriotes look-alikes often occur in these traps, a correct identification of the captured insects is needed. Morpholog-ical identification of wireworms to species level is extremely difficult, even for experts. Identification via multiplex PCR or barcoding is a reliable solution but can only be performed by well-trained staff in laboratories equipped with adequate molecular facilities. Hence, a cheaper, highly specific, sensitive, and faster detection tool, based on a loop-mediated isothermal amplification technology (LAMP) is being developed. In this study, we will discuss the LAMP-primer design, the validation parameters such as specificity, sensitivity, and robustness for the three most important species for Belgium viz. Agriotes lineatus, A. obscurus and A. sputator. All tests were performed using the portable and user-friendly Genie II (OptiGene®). Primer design is based on mitochondrial COI sequences from Genbank combined with the COI sequencing obtained from our own ILVO Agriotes collection. The selectivity was tested on 13 different wireworms species collected in the bait traps during a three-years monitoring campaign. In addition, larvae from Carabidae and Staphylinidae found in these bait traps were also considered to test the species selectivity. To find out how robust the primers are, we run the LAMP-PCR on more than 200 wireworm specimens from different countries (France, Germany, Austria, and Switzerland). The LAMP primers for A. sputator are highly specific and result in a reliable detection and their applicability will be further investigated in the field. The LAMP primers designed for A. obscurus and A. lineatus are not sufficiently species-specific, so adjustment of the primers and further validation is required to dis-tinguish A. lineatus from A. obscurus.