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

Abstract

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.

Keywords: Adriatic Sea; European hake; Merluccius merluccius; feeding habits; metabarcoding.

Figure 1

European hake picture. The European hake is a nektobenthic predator of communities inhabiting the Mediterranean shelf and upper slope (image courtesy Stefano Guerrieri).

Figure 2

Map of the sampling hauls in the Adriatic Sea. Further details can be found in Supporting Information Table S1. This map was created using ArcViewGIS version 3.2a (https://geonet.esri.com/thread/36365). Image courtesy of Chiara Manfredi. The Adriatic cartography used is freely available at http://www.faoadriamed.org/html/adr_inf_centre.html#cart

Figure 3

Comparison of dietary richness among M. merluccius size classes. (a) Sample‐based species richness curves for each size class for the metabarcoding data (left) and morphological data (right); (b) Main prey of M. merluccius by size classes as identified by metabarcoding approach (left) and morphological data (right). Frequency occurrence data of species are reported. The 10 most recurrent items across all classes are showed

Figure 4

Comparison of mean ORA for each size class. Mean ORA (±SD) per sample for (a) Crustacea, (b) Mollusca and (c) Teleostei. No significant comparison was revealed after Kruskall‐Wallis test (Kruskal & Wallis, 1952) (p‐value = >0.05)

Figure 5

Principal Component Analysis. PCoA of relative occurrence‐based Bray‐Curtis dissimilarity of samples from all size classes (permutest p‐value = 0.017). Triangles depict the centroids of the distributions. Black color: size class 1, red: size class 2, green: size class 3, blue: size class 4, light blue: size class 5

Figure 6

The food web related to the predator M. merluccius. Brown nodes indicate the predator divided into five size classes and green nodes represent the prey. The size of nodes is proportional to number of links connected (degree), and the size of links is proportional to the number of times the link prey–predator was found in the samples. Species are distributed according to their linkage with predator size classes: the prey species common to all size classes are in the middle

Figure 7

Feeding strategy diagram. Prey‐specific abundance (PSA) plotted against frequency of occurrence of prey. Only the species identified with both the metabarcoding and morphological analyses were considered. In bold the species found with the metabarcoding approach

Figure 8

Prey–predator functional relationship. Number of M. merluccius stomachs containing E. encrasicolus in relation to the abundance of E. encrasicolus estimated for the same hauls (data from MEDITS 2014 survey). The bubbles size is proportional to the number of M. merluccius stomach data (specimen, e.g., number of individuals) available per haul