Targeted metagenomics enables species-level detection of arthropod contaminants in food

Regulatory oversight of arthropod contamination in food still relies largely on microscopy, a labor-intensive approach that often cannot identify fragmented material to the species level. We developed a targeted metagenomic next-generation sequencing workflow that combines mitochondrial DNA hybridization capture with the Mitok-mer pipeline, a k-mer–based taxonomic assignment approach for eukaryotes. Two arthropod bait panels were designed: SnackAttack-BugTrakr v1, targeting 1,988 species, and v2, targeting 5,522 species. Panel v1 was evaluated in mock genomic DNA (gDNA) communities containing insect gDNA alone or mixed with wheat flour or cornmeal gDNA backgrounds. In 30-taxon wheat mixtures, all target species were detected in both no-bait and bait-capture libraries, but bait capture increased the mean on-target arthropod signal from 80.9% to 98.4%, while reducing the wheat-derived signal by 1,400–2,000-fold. In cornmeal spike-in experiments, bait capture shifted libraries from food-dominated profiles to insect-dominated profiles, increasing target relative abundance from 4.7–53.2% to 87.9–99.6%. In five-species cornmeal mixtures, target taxa reached 99% relative abundance after bait capture, non-target taxa decreased 39- to 53-fold, and the closely related beetles Tribolium castaneum and T. confusum were resolved to species level. A direct v1–v2 panel comparison across eight wheat-flour mixtures containing 23 insect taxa demonstrated similar bait-capture performance with v2 mainly expanding reference breadth. Two naturally contaminated food samples were also analyzed as a preliminary assessment of real-world applicability. Overall, this proof-of-concept study shows that targeted metagenomics can improve species-level detection of arthropods in food and may complement microscopy, supporting the modernization of the FDA’s filth program. • Hybrid capture boosts on-target arthropod signal and depletes food-matrix DNA • Bait panel expansion increases breadth and intraspecific diversity for key pest taxa • Dedicated k-mer workflow delivers species- or genus-level arthropod identification • Microscopy complements sequencing when life-stage or fragment metrics are needed • Metagenomics + Mitok-mer shows potential to modernize food filth analysis workflows