Identification of potential insect ecological interactions using a metabarcoding approach.

Species interactions are challenging to quantify, particularly when they happen cryptically. Molecular methods have become a key tool to uncover these interactions when they leave behind a DNA trace from the interacting organism (., pollen on a bee) or when the taxa are still present but morphologically challenging to identify (., microbial or fungal interactions). The decreasing costs of sequencing makes the mass analysis of thousands of target species possible. However, the challenge has shifted to selecting molecular markers which maximize information recovery while analyzing these data at broad biological scales. In this manuscript we use model arthropod groups to compare molecular markers and their analysis across life stages. We develop protocols for two ecologically and economically devastating pests, the spongy moth () and the emerald ash borer (), and a group of pollinators including bees and wasps which regularly deposit eggs in "bee hotels" where the larvae develop. Using Illumina MiSeq and Oxford Nanopore MinION platforms we evaluate seven primer pairs for five molecular markers which target plants, fungi, microbes, insects, and parasitic phyla (, nematodes). Our data reveals hundreds of potential ecological interactions and establishes generalized methods which can be applied across arthropod host taxa with recommendations on the appropriate markers in different systems. However, we also discuss the challenge of differentiating co-occurring DNA signals and true ecological interactions, a problem only starting to be recognized as eDNA from the environment accumulates on living organisms.