Molecular analysis of trophic interactions for ecological network construction: EcoNet 2025 workshop v1

Constructing ecological networks is vital for understanding how interactions dynamically change over space and time, and how they structure wider ecosystems. This has, however, traditionally involved laborious observations and datasets that are poorly taxonomically resolved, biased by observational interference or otherwise poorly suited to their purpose. The construction of ecological networks using molecular data has become increasingly commonplace over the last decade. Molecular dietary analysis has generated interaction data for ecologically cryptic taxa and interactions that are otherwise difficult, if not impossible, to observe. The taxonomic resolution of molecular methods unlocks not only the potential for inclusion and delineation of the interactions of morphologically cryptic taxa in networks, but also study of phylogenetic structuring of those interactions. Methods like DNA metabarcoding are therefore an increasingly important component of the network ecologist’s toolbox. This protocol represents a workshop run for the VII Symposium on Ecological Networks, EcoNet 2025, at Newcastle University, UK. The protocol covers the process of analysing the gut contents of invertebrate predators using dietary DNA metabarcoding. From considerations for field sampling through DNA extraction and PCR, to DNA library preparation and nanopore sequencing, this protocol covers the full process. The protocol uses high-throughput robotics equipment hosted at the Newcastle University Molecular Diagnostics Facility to expedite various steps, but the equivalent manual processes are described in a separate step case. The main methods presented offer a cost effective and high-throughput method for molecular dietary analyses of individual invertebrate predators using this specialised lab equipment, but the standard alternatives signposted throughout are accessible using standard molecular lab equipment.