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The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.

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The blue shark (Prionace glauca) is an oceanic species that occurs in temperate and tropical waters around the globe (Robins and Ray, 1986). This species is a major bycatch of pelagic longline fleets that operate to supply the world’s growing demand for tunas and swordfish (Xiphias gladius) (Stevens, 1992; Bailey et al., 1996; Francis, 1998; Francis et al., 2001; Macias and de la Serna, 2002); numerically, the blue shark is the top nontarget species captured by the U.S. longline pelagic Atlantic fleet (Beerkircher et al.