Quantitative prey species detection in predator guts across multiple trophic levels by DNA shotgun sequencing

O_LIMetabarcoding has revolutionized the study of ecological communities, but PCR bias hampers quantitative analyses, as required in studies of trophic interactions. Direct DNA shotgun sequencing can avoid the amplification step when unassembled reads are mapped to a reference database, enabling identification and quantification of prey items. C_LIO_LITwo feeding bioassays tested the precision and accuracy of quantitative assessments with the coccinellids Harmonia axyridis and Hippodamia convergens, the chrysopid Chrysoperla externa, and the aphid Myzus persicae. Guts were dissected and the total DNA extracted was directly sent to sequence by Illumina HiSeq2500 (insert 350 bp, PE 250). Predator gut content reads were blasted against an arthropod mitochondrial DNA reference database for taxonomic assignment and the matches curated for false positive prey identification using a series of bioinformatics pipelines, mostly in R. Taxonomic assignment through KrakenUnique, which is based on the counts of unique k-mers, was compared. C_LIO_LIIn a Prey Quantity bioassay, the number of prey reads was correlated to the amount of prey consumed and the elapsed time since consumption. In a Direct and Indirect Predation bioassay, prey was detectable in the predator 6 h after feeding (direct predation) and the preys prior prey was detectable 3 h after feeding (indirect predation). Detection of indirect predation was related to the species-specific decay rates of the two predators, but not to the order of predation. C_LIO_LIWe demonstrate that degraded prey DNA was quantifiable across trophic levels with high accuracy (98.4% positive predictive value) and taxonomic resolution. C_LI