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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 lusitanicus (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 ribosomal 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 exhibited 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 specific 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. [ABSTRACT FROM AUTHOR]

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts. [ABSTRACT FROM AUTHOR]