A DNA metabarcoding workflow for the detection of terrestrial gastropods from ingested DNA

DNA metabarcoding is a powerful approach for assessing biodiversity and unravelling trophic interactions, particularly in the context of biological invasions where introduced predators may impact native communities. However, the analysis of predator digestive contents with DNA metabarcoding poses technical challenges including the specificity of primers used to amplify the target DNA, amplification bias and contamination issues, which must be carefully considered when working on new taxa. This study addresses these challenges by developing and validating a methodology for assessing the diversity of terrestrial gastropods predated by the exotic flatworm Obama nungara . Specifically, an optimized primer pair providing taxonomic resolution for at least 40 gastropod species and genetic lineages, including taxonomically complex and diverse slug genera such as Arion and Deroceras is proposed. The primer pair was optimized through in silico predictions, laboratory controls, and field testing. Mock communities, laboratory-fed individuals, and technical replicates from the field were used as metabarcoding controls to calibrate filter thresholds for Operational Taxonomic Units. Preliminary field results revealed significant dietary diversity in O. nungara , suggesting a broad feeding spectrum. Beyond O. nungara , the primers and the assay developed here can also be applied to study the diets of other terrestrial predators or to detect gastropods in environmental DNA samples, providing a useful tool for ecological and conservation research.