A metabarcoding approach for the feeding habits of European hake in the Adriatic Sea

European hake (Merluccius merluccius) is one of the most economically important fish for the Mediterranean Sea. It is an important predator of deep upper shelf slope communities currently characterized by growth overexploitation: the understanding of hake's diet might support next generation management tools. However, all current European hake diet studies depend on the morphological identification of prey remains in stomach content, with consequent limitations. In this study, we set up a metabarcoding approach based on cytochrome oxidase I PCR amplification and Miseq Illumina paired-end sequencing of M. merluccius stomach content remains and compared the results to classic morphological analyses. A total of 95 stomach contents of M. merluccius sampled in the North-Central Adriatic Sea were analyzed with both the metabarcoding and morphological approaches. Metabarcoding clearly outperformed the morphological method in the taxonomic identification of prey describing more complex trophic relationships even when considering the morphological identification of 200 stomach contents. Statistical analysis of diet composition revealed a weak differentiation among the hake's size classes, confirming an opportunistic feeding behavior. All the analyses performed showed the presence of a core of shared prey among the size classes and a cloud of size-specific prey. Our study highlights the exceptional potential of metabarcoding as an approach to provide unprecedented taxonomic resolution in the diet of M. merluccius and potentially of other marine predators, due to the broad-spectrum of detection of the primers used. A thorough description of these complex trophic relationships is fundamental for the implementation of an ecosystem approach to fisheries.