A multiplex PCR assay for detecting slug species common in European arable land in the diet of carabid beetles

DNA-based diet analysis of natural enemies is a valuable tool for unravelling the food choice of predators in agroecosystems. It enables the rapid identification of potential biocontrol agents of invertebrate pests. Here, we present a new multiplex PCR system for the identification of pest slug species in the diet of their natural enemies such as carabid beetles. It comprises three species-specific primers targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene to detect DNA of the common garden slug, Arion distinctus (Stylommatophora: Arionidae), the Iberian slug, Arion lusitani-cus (Stylommatophora: Arionidae) and the grey field slug Deroceras reticulatum (Stylommatophora: Agriolimacidae). We also include (super)family-specific primers for Arionidae and Limacoidea, which amplify parts of the 28S gene for ribo-somal RNA (rRNA) in order to identify a wider range of slugs. The amplicons for Arionidae can be assigned to a total of seven Central European slug species of this family and the amplicons for Limacoidea to ten species. The multiplex assay showed high specificity against DNA extracts of field-collected slugs and co-occurring invertebrates. The assay also exhib-ited high sensitivity, which was confirmed by testing it with 223 dietary samples from field-collected carabids as potential natural enemies of slugs in agroecosystems. This methodology represents a new, cost-effective, highly sensitive and spe-cific approach for the identification of common Central European slug species as well as for analysing trophic interactions to identify natural enemies for further biological control development. It can also be applied in any study where a rapid and reliable identification of slugs is needed.