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.

Environmental DNA (eDNA) metagenomics sequences all DNA molecules present in environmental samples and has the potential of identifying virtually any organism from which they are derived. However, due to unacceptable levels of false positives and negatives, this approach is underexplored as a tool for biodiversity monitoring across the tree of life, particularly for non‐microscopic eukaryotes. We present SeqIDist, a framework that combines multilocus BLAST matches against several reference databases followed by an analysis of sequence identity distribution patterns to disentangle false positives while revealing new biodiversity and increasing the accuracy of metagenomic approaches. We tested SeqIDist on an eDNA metagenomic dataset from a riverine site and compared the results to those obtained with an eDNA metabarcoding approach for benchmarking purposes. We start by characterising the biological community (~2000 taxa) across the tree of life at low taxonomic levels and show that eDNA metagenomics has a higher sensitivity than eDNA metabarcoding in discovering new diversity. We show that limited representation of whole genome sequences in reference databases can lead to false positives. For non‐microscopic eukaryotes, eDNA metagenomic data often consist of a few sparse, anonymous sequences scattered across the genome, making metagenome assembly methods unfeasible. Finally, we infer eDNA source and residency time using read length distributions as a measure of decay status. The higher accuracy of SeqIDist opens the discussion of the potential of eDNA metagenomics for archived samples and its implementation in long‐term biodiversity monitoring at a planetary scale.

Leptocephali are the characteristic larvae of the superorder Elopomorpha that are difficult to identify at the species level. In this study, we used DNA barcoding (i.e. short genetic sequences of DNA used as unique species tags) coupled with classical taxonomic methods to identify leptocephali in the southern Adriatic Sea. This information will provide an assessment of the biodiversity of the eel larvae in this region. A total of 2,785 leptocephali were collected, and using external morphology were assigned to seven morphotypes: Ariosoma balearicum, Conger conger, Gnathophis mystax, Facciolella sp., Nettastoma melanurum, Dalophis imberbis and Chlopsis bicolor. Collectively, these seven morphotypes are considered to be a good proxy for the Anguilliformes community (the main order of the Elopomorpha) in the southern Adriatic Sea (to date, seven families and sixteen species have been recorded in this region). Interestingly, the higher number of G. mystax larvae collected suggests an increased abundance of this genus. To validate the morphological identifications, we sequenced 61 leptocephali (at a 655 bp fragment from the cytochrome oxidase subunit 1 mitochondrial region) and developed barcode vouchers for the seven morphotypes. Using genetic information from reference databases, we validated three of these morphotypes. Where reference sequences were unavailable, we generated barcodes for both adult and juvenile forms to provide additional genetic information. Using this integrated approach allowed us to characterize a new species of Facciolella in the Adriatic Sea for the first time. Moreover, we also revealed a lack of differentiation, at the species level, between G. mistax and G. bathytopos, a western Atlantic Ocean species. Our morphological and barcode data have been published in the Barcoding of the Adriatic Leptocephali database. This work represents the first contribution to a wider project that aims to create a barcode database to support the assessment of leptocephali diversity in the Mediterranean Sea.

Environmental DNA (eDNA) metabarcoding has revolutionized ecological and environmental research by describing communities without relying on direct observations, making it a powerful, non-invasive, and cost-effective tool in biodiversity monitoring. However, implementation of eDNA as a standard protocol in long-term monitoring programs, that have traditionally relied on capture-based methods, poses challenges in terms of data comparability. Here, we compared freshwater fish communities assessed through eDNA metabarcoding and electrofishing, across 35 sampling sites in the lower Tagus River basin, Portugal. For most species or species-groups analyzed individually (13 out of 17), there was a significant correspondence between electrofishing and eDNA metabarcoding detections. The correspondence was weaker when comparing the number of specimens captured by electrofishing with the number of eDNA metabarcoding reads, with seven out of 13 taxa showing significant relationships. Species richness estimates based on the two methods were very similar at the basin level. The methods yielded significantly different species compositions, although these differences were driven by samples collected in the Tagus main channel, which is wider and has higher flow rates than tributaries. Benthic and shoreline fish communities showed similar species composition in the two methods, but this was not the case for pelagic communities, probably due to the higher water turnover of the pelagic zone and electrofishing inefficiency. Our results highlight the high potential of eDNA metabarcoding as a complementary method to electrofishing for freshwater fish monitoring, though further validation is needed to assess biases related to site-specific hydrological conditions and the ecology of the target species.

Environmental DNA (eDNA) metagenomics sequences all DNA molecules present in environmental samples and has the potential of identifying virtually any organism from which they are derived. However, due to unacceptable levels of false positives and negatives, this approach is underexplored as a tool for biodiversity monitoring across the tree of life, particularly for non-microscopic eukaryotes. We present SEQIDIST, a framework that combines multilocus BLAST matches against several reference databases followed by analysis of sequence identity distribution patterns to disentangle false positives while revealing new biodiversity and increasing the accuracy of metagenomic approaches. We tested SEQIDIST on an eDNA metagenomic dataset from a riverine site and compare the results to those obtained with an eDNA metabarcoding approach for benchmarking purposes. We start by characterizing the biological community (∼ 2000 taxa) across the tree of life at low taxonomic levels and show that eDNA metagenomics has a higher sensitivity than eDNA metabarcoding in discovering new diversity. We show that limited representation of whole genome sequences in reference databases can lead to false positives. For non-microscopic eukaryotes, eDNA metagenomic data often consist of a few sparse, anonymous sequences scattered across the genome, making metagenome assembly methods unfeasible. Finally, we infer eDNA source and residency time using read length distributions as a measure of decay status. The higher accuracy of SEQIDIST opens the discussion of the archival potential of eDNA metagenomics and its implementation in biodiversity monitoring actions at large planetary and temporal scales.

Lactococcosis is a bacterial warm water disease whose occurrence is enhanced by climate change. It induces chronic stress and immunodepression in trout, thereby enhancing host susceptibility to bacterial pathogens. Lesions are typically systemic, with involvement of hematopoietic organs and of central nervous system. Clinical signs include lethargy and early anorexia, abnormal swimming behaviour, exophthalmia, cutaneous, visceral and ocular haemorrhages, ascites and splenomegaly. As conventional preventive measures are becoming less effective, complementary, long‑term strategies aimed at improving host resistance. In this context, selective breeding of broodstock for disease resistance is regarded as an effective adaptive measure to mitigate climate related health risks in aquaculture. Two complementary approaches were used to identify genetic markers associated with lactococcosis. The first was a candidate gene approach based on the MHC class II gene on 739 rainbow trout reared in three Mediterranean countries: Italy, Greece and Türkiye. Different single nucleotide polymorphisms of the MHC class II gene were found to have a protective role. Nevertheless, the most interesting results came from the haplotype-level analysis of the gene. Haplotype A126T130A131 emerged as a promising genetic marker associated with resistance in populations affected by both Lactococcus garvieae or Lactococcus petauri as the causative agents of the disease. The second approach, applied to a subset of 271 fish, combined genome-wide case-control association (GWAS) with a detection of selection signatures (SS). The functional analysis of genes located in chromosomal regions harbouring significant signals in the association study revealed several GO terms associated with neuronal development, vision, infection and disease response and platelet development. Ten SNPs were identified in common by GWAS and SS. The incorporation of molecular markers into marker‑assisted or genomic selection programmes may contribute to the development of trout stocks better adapted to future environmental conditions, reducing the impact of lactococcosis and improving animal welfare while decreasing reliance on chemotherapeutic treatments. Further studies aimed at validating these associations across different environmental conditions and genetic backgrounds are essential to support their implementation at a commercial level.