Metabarcoding analysis of European hake diet in the Mediterranean Sea.

European hake (EH), Merluccius merluccius, is a demersal fish distributed from the North Sea and Atlantic to the Levantine Sea in the Mediterranean. EH is an important predator of deep Mediterranean upper shelf slope communities and it is currently characterised by growth overexploitation. EH adults feed mainly on fish and squids whereas the young (<16 cm) feed on crustaceans. All current EH diet studies relied on the morphological identification of prey remains in stomach content, however this method is labour intensive and it precludes the identification of strongly digested food. The development of High-Throughput Sequencing (HTS) approaches provide more accurate methods for dietary studies revealing many consumed species simultaneously (DNA metabarcoding). The aim of this study is to use a HTS approach based on COI amplification, contextually to classic microscopic morphological identification, to analyse EH stomach content and to evaluate the efficiency of the molecular method. HTS sequencing has been carried out on the amplicons obtained by PCR amplification (Leray et al. 2013) of stomach remains and all the Miseq Illumina paired-end reads have been analysed by using bioinformatic tools (Boyer et al. 2015) for taxonomic assignment. The selected sequences clustered in OCTUs (Operational Clustered Taxonomic Units) and taxonomically assigned, will be used in diversity analyses to compute distance matrices among samples, to compare taxa summaries from different samples, to create networks and perform PCA and PcoA analysis. Classic microscopic morphological analyses on stomach content remains have been carried out contextually to compare the results of the two methods. The molecular approach has proven a promising method to study marine fish dietary habits. All the data will be summarized to reconstruct EH trophic dynamics in the Mediterranean Sea.