Mismanagement and poor transparency in the European processed seafood supply revealed by DNA metabarcoding.

In the global processed seafood industry, disparate actors play different roles along the supply chain, creating multiple opportunities for mistakes, malpractice, and fraud. As a consequence, consumers may be exposed to non-authentic products, which hinder informed purchasing decisions and broader efforts to improve trade transparency and sustainability. Here, we characterised the taxonomic composition of 62 processed seafood products in Italian, British and Albanian retailers, purposefully obtained from different supply routes, using multiple DNA metabarcoding markers. By combining molecular results with metadata reported on labels, we revealed patterns of mislabelling in 24 products (39%) across sampling regions, denoting lack of transparency of processed seafood products based on resources sourced from either Europe or globally. We show that the accuracy of label claims and the mis-represented and underestimated levels of traded biodiversity are largely determined by the management of raw material by global processors. Our study shows that DNA metabarcoding is a powerful and novel authentication tool that is mature for application at different stages of the seafood supply chain to protect consumers and improve the sustainable management of fish stocks.

Decoding Seafood: Multi-Marker Metabarcoding for Authenticating Processed Seafood.

Given the recognized nutritional value of fish and shifting consumer lifestyles, processed seafood has become increasingly prevalent, comprising a significant portion of global food production. Although current European Union labeling regulations do not require species declaration for these products, food business operators often voluntarily provide this information on ingredient lists. Next Generation Sequencing (NGS) approaches are currently the most effective methods for verifying the accuracy of species declarations on processed seafood labels. This study examined the species composition of 20 processed seafood products, each labeled as containing a single species, using two DNA metabarcoding markers targeting the mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes. The combined use of these markers revealed that the majority of the products contained multiple species. Furthermore, two products were found to be mislabeled, as the declared species were not detected. These findings underscore that NGS is a robust technique that could be adopted to support routine food industry activities and official control programs, thereby enhancing the 'From Boat to Plate' strategy and combating fraudulent practices in the complex fisheries supply chain.

Integrative approach to monitoring metazoan diversity and distribution in two Mediterranean coastal sites through morphology and organismal eDNA.

Marine and coastal ecosystems respond to climate change in various ways, such as the type of ecosystem, the species composition, interactions, and distribution, and the effect of local stressors. Metazoan organisms, particularly zooplankton, are important indicators for monitoring the effects climate-driven warming in marine coastal ecosystems over the long term. In this study, the diversity and distribution of zooplankton communities in the Mediterranean Sea (Canyon Dohrn and LTER-MareChiara, Gulf of Naples), a known biodiversity and climate changes hotspot, have been assessed using the integration of morphological-based identification and organismal eDNA. Our findings showed that the multi-locus strategy including the mitochondrial cytochrome c oxidase I (COI) gene and the hypervariable region V9 of the 18S rDNA (18S V9) as targets, improved the taxonomic overview, with the COI gene being more effective than the 18S V9 region for metazoans at the species level. However, appendicularians were detected only with the 18S V9 region. Overall, organismal eDNA is a powerful approach for revealing hidden biodiversity, especially for gelatinous and meroplankton components, and provided new insights into biodiversity patterns. The ecological importance of calanoid copepods in coastal ecosystems has been confirmed. In contrast, the discovery of 13 new metazoan records in the Mediterranean Sea, including two non-indigenous copepod species, suggested that local stressors affect zooplankton community structure and resilience, highlighting the importance of biomonitoring and protecting marine coastal ecosystems.

Inter-comparison of marine microbiome sampling protocols.

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22-3 µm, 3-20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and "whole water" (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume.

Preliminary survey on the occurrence of microplastics in bivalve mollusks marketed in Apulian fish markets.

Microplastics (MPs) are a relevant threat to food safety because they are ingested by humans through various foods. Bivalves are at high risk of microplastic contamination due to their filter-feeding mechanism and pose a risk to consumers as they are ingested whole. In this work, microplastics were detected, quantified, identified, and classified in samples of mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas) marketed in the Apulia region. The total number of plastic debris was 789 particles in the mussel samples and 270 particles in the oyster samples, with size ranging from 10 to 7350 µm. Fragments with size within the category of 5-500 µm were the predominant findings in both species, with blue as the predominant color in mussels and transparent in oysters; most of the debris was polyamide and nylon polymers in the mussels and chlorinated polypropylene in the oysters. These results show that mussel and oyster samples purchased at fish markets are contaminated with microplastics. The sources may be diverse and further studies are needed to assess the impact of the marketing stage on microplastic contamination in bivalves to better define the human risk assessment associated with microplastic exposure from bivalves consumption.

Encapsulated in sediments: eDNA deciphers the ecosystem history of one of the most polluted European marine sites.

The Anthropocene is characterized by dramatic ecosystem changes driven by human activities. The impact of these activities can be assessed by different geochemical and paleontological proxies. However, each of these proxies provides only a fragmentary insight into the effects of anthropogenic impacts. It is highly challenging to reconstruct, with a holistic view, the state of the ecosystems from the preindustrial period to the present day, covering all biological components, from prokaryotes to multicellular eukaryotes. Here, we used sedimentary ancient DNA (sedaDNA) archives encompassing all trophic levels of biodiversity to reconstruct the two century-natural history in Bagnoli-Coroglio (Gulf of Pozzuoli, Tyrrhenian Sea), one of the most polluted marine-coastal sites in Europe. The site was characterized by seagrass meadows and high eukaryotic diversity until the beginning of the 20th century. Then, the ecosystem completely changed, with seagrasses and associated fauna as well as diverse groups of planktonic and benthic protists being replaced by low diversity biota dominated by dinophyceans and infaunal metazoan species. The sedaDNA analysis revealed a five-phase evolution of the area, where changes appear as the result of a multi-level cascade effect of impacts associated with industrial activities, urbanization, water circulation and land-use changes. The sedaDNA allowed to infer reference conditions that must be considered when restoration actions are to be implemented.

Host-associated and Environmental Microbiomes in an Open-Sea Mediterranean Gilthead Sea Bream Fish Farm.

Gilthead seabream is among the most important farmed fish species in the Mediterranean Sea. Several approaches are currently applied to assure a lower impact of diseases and higher productivity, including the exploration of the fish microbiome and its manipulation as a sustainable alternative to improve aquaculture practices. Here, using 16S rRNA gene high-throughput sequencing, we explored the microbiome of farmed seabream to assess similarities and differences among microbial assemblages associated to different tissues and compare them with those in the surrounding environment. Seabream had distinct associated microbiomes according to the tissue and compared to the marine environment. The gut hosted the most diverse microbiome; different sets of dominant ASVs characterized the environmental and fish samples. The similarity between fish and environmental microbiomes was higher in seawater than sediment (up to 7.8 times), and the highest similarity (3.9%) was observed between gill and seawater, suggesting that gills are more closely interacting with the environment. We finally analyzed the potential connections occurring among microbiomes. These connections were relatively low among the host's tissues and, in particular, between the gut and the others fish-related microbiomes; other tissues, including skin and gills, were found to be the most connected microbiomes. Our results suggest that, in mariculture, seabream microbiomes reflect only partially those in their surrounding environment and that the host is the primary driver shaping the seabream microbiome. These data provide a step forward to understand the role of the microbiome in farmed fish and farming environments, useful to enhance disease control, fish health, and environmental sustainability.

DNA metabarcoding for identification of species used in fish burgers.

The absence of morphological identification characters, together with the complexity of the fish supply chain make processed seafood vulnerable to cases of species substitution. Therefore, the authentication and the traceability of such products play a strategic role in ensuring quality and safety. The aim of the present study was to detect species used in the production of multi-species fish burgers and to evaluate mislabelling rates, using a DNA metabarcoding approach by sequencing a fragment of the 16S rRNA mitochondrial gene. The study highlighted the presence of 16 marine and 2 mammalian taxa with an overall mislabelling rate of 80%, including cases of species substitution, the undeclared presence of molluscs and of taxa whose use is not permitted by current Italian legislation. The presence of swine DNA as well as the inclusion of undeclared taxa potentially causing allergies raise concerns regarding consumer safety and protection regarding ethical or religious issues. Overall, the study shows that the application of DNA metabarcoding is a promising approach for successfully enforcing traceability systems targeting multi-species processed food and for supporting control activities, as a guarantee of an innovative food safety management system.

Phenological segregation suggests speciation by time in the planktonic diatom Pseudo-nitzschia allochrona sp. nov.

The processes leading to the emergence of new species are poorly understood in marine plankton, where weak physical barriers and homogeneous environmental conditions limit spatial and ecological segregation. Here, we combine molecular and ecological information from a long-term time series and propose Pseudo-nitzschia allochrona, a new cryptic planktonic diatom, as a possible case of speciation by temporal segregation. The new species differs in several genetic markers (18S, 28S and ITS rDNA fragments and rbcL) from its closest relatives, which are morphologically very similar or identical, and is reproductively isolated from its sibling species P. arenysensis. Data from a long-term plankton time series show P. allochrona invariably occurring in summer-autumn in the Gulf of Naples, where its closely related species P. arenysensis, P. delicatissima, and P. dolorosa are instead found in winter-spring. Temperature and nutrients are the main factors associated with the occurrence of P. allochrona, which could have evolved in sympatry by switching its phenology and occupying a new ecological niche. This case of possible speciation by time shows the relevance of combining ecological time series with molecular information to shed light on the eco-evolutionary dynamics of marine microorganisms.

Occurrence of hepatitis E virus (HEV) in Calabrian wild boars.

Hepatitis E virus (HEV) is an emerging pathogen in industrialized countries. HEV infections in humans are mainly related to the HEV-3 genotype, predominant in Europe and widespread in wild boars' food products. However, there are little relevant data around HEV prevalence in wild boars, although they are considered the main HEV reservoir and used for typical food products such as liver sausages. Our study aimed to assess HEV occurrence and genetic variability in Calabrian wild boars hunted in the central and ionic area of Catanzaro's province. A total of 86 wild boar liver samples were analyzed showing an overall HEV RNA prevalence of 26.7% (23/86). All positive samples were characterized molecularly as genotype 3 and predicted as HEV-3c subtype despite the shortness of fragment employed for the molecular analysis. This data is in line with previous studies conducted in Europe highlighting the public health concern of these results. Biomolecular methods performed in our study detected only the HEV RNA positivity of analyzed samples without information about the virus viability. Consequently, it is not possible to fully estimate the risk related to the consumption of wild boar's liver sausages or wild boar meat products. Our results highlight the need for further studies in order to investigate the virus viability and to link wild boar's meat consumption with HEV human seroprevalence in Italian regions (Abruzzo, Lazio, Campania and Calabria) where typical wild boar's products are consumed. In this way, the Competent Authorities could perform a complete risk assessment, implement risk management and establish proper measures to ensure the public health and prevent relative human disease.

5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species.

Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

gltA typing of Anaplasma strains related to A. platys: Taxonomical and one health implications.

Species belonging to the genus Anaplasma (Rickettsiales) include bacteria of veterinary and public health importance. Beside the zoonotic Anaplasma phagocytophilum, A. platys, the etiological agent of canine cyclic thrombocytopenia, has been sporadically reported in clinically ill human patients. The ongoing emergence of novel strains related to this species in vertebrate hosts emphasises the need for genetic comparisons among strains identified in different regions of the world. In this paper we developed a PCR test suitable for amplification of the still undescribed gltA gene of Anaplasma strains related to A. platys from Mediterranean ruminants and applied on a panel of 248 samples. gltA sequencing allowed phylogenetic comparison with strains related to A. platys recently identified in China, and strains representative of the Anaplasmataceae family. Results suggest the designation of Candidatus A. turritanum, including Mediterranean A. platys - like strains, and Candidatus A. cinensis, including strains isolated in China. Data generated in this study are a solid reference for future epidemiological studies of novel unclassified strains related to A. platys and for their diagnosis and raise concern on their potential veterinary and public health implications encouraging investigating the suspected unexplored diversity within the genus Anaplasma in animals and human.

Emergence of Anaplasma Species Related to A. phagocytophilum and A. platys in Senegal.

The genus Anaplasma (Anaplasmataceae, Rickettsiales) includes tick-transmitted bacterial species of importance to both veterinary and human medicine. Apart from the traditionally recognized six Anaplasma species (A. phagocytophilum, A. platys, A. bovis, A. ovis, A. centrale, A. marginale), novel strains and candidate species, also of relevance to veterinary and human medicine, are emerging worldwide. Although species related to the zoonotic A. platys and A. phagocytophilum have been reported in several African and European Mediterranean countries, data on the presence of these species in sub-Saharan countries are still lacking. This manuscript reports the investigation of Anaplasma strains related to zoonotic species in ruminants in Senegal by combining different molecular tests and phylogenetic approaches. The results demonstrated a recent introduction of Candidatus (Ca) Anaplasma turritanum, a species related to the pathogenic A. platys, possibly originating by founder effect. Further, novel undetected strains related to Candidatus (Ca) Anaplasma cinensis were detected in cattle. Based on groEL and gltA molecular comparisons, we propose including these latter strains into the Candidatus (Ca) Anaplasma africanum species. Finally, we also report the emergence of Candidatus (Ca) A. boleense in Senegal. Collectively, results confirm that Anaplasma species diversity is greater than expected and should be further investigated, and that Anaplasma routine diagnostic procedures and epidemiological surveillance should take into account specificity issues raised by the presence of these novel strains, suggesting the use of a One Health approach for the management of Anaplasmataceae in sub-Saharan Africa.

Resolving cryptic species complexes in marine protists: phylogenetic haplotype networks meet global DNA metabarcoding datasets.

Marine protists have traditionally been assumed to be lowly diverse and cosmopolitan. Yet, several recent studies have shown that many protist species actually consist of cryptic complexes of species whose members are often restricted to particular biogeographic regions. Nonetheless, detection of cryptic species is usually hampered by sampling coverage and application of methods (e.g. phylogenetic trees) that are not well suited to identify relatively recent divergence and ongoing gene flow. In this paper, we show how these issues can be overcome by inferring phylogenetic haplotype networks from global metabarcoding datasets. We use the Chaetoceros curvisetus (Bacillariophyta) species complex as study case. Using two complementary metabarcoding datasets (Ocean Sampling Day and Tara Oceans), we equally resolve the cryptic complex in terms of number of inferred species. We detect new hypothetical species in both datasets. Gene flow between most of species is absent, but no barcoding gap exists. Some species have restricted distribution patterns whereas others are widely distributed. Closely related taxa occupy contrasting biogeographic regions, suggesting that geographic and ecological differentiation drive speciation. In conclusion, we show the potential of the analysis of metabarcoding data with evolutionary approaches for systematic and phylogeographic studies of marine protists.

Empirical evidence for concerted evolution in the 18S rDNA region of the planktonic diatom genus Chaetoceros.

Concerted evolution is a process of homogenisation of repetitive sequences within a genome through unequal crossing over and gene conversion. This homogenisation is never fully achieved because mutations always create new variants. Classically, concerted evolution has been detected as "noise" in electropherograms and these variants have been characterised through cloning and sequencing of subsamples of amplified products. However, this approach limits the number of detectable variants and provides no information about the abundance of each variant. In this study, we investigated concerted evolution by using environmental time-series metabarcoding data, single strain high-throughput sequencing (HTS) and a collection of Sanger reference barcode sequences. We used six species of the marine planktonic diatom genus Chaetoceros as study system. Abundance plots obtained from environmental metabarcoding and single strain HTS showed the presence of a haplotype far more abundant than all the others (the "dominant" haplotype) and identical to the reference sequences of that species obtained with Sanger sequencing. This distribution fitted best with Zipf's law among the rank abundance/ dominance models tested. Furthermore, in each strain 99% of reads showed a similarity of 99% with the dominant haplotype, confirming the efficiency of the homogenisation mechanism of concerted evolution. We also demonstrated that minor haplotypes found in the environmental samples are not only technical artefacts, but mostly intragenomic variation generated by incomplete homogenisation. Finally, we showed that concerted evolution can be visualised inferring phylogenetic networks from environmental data. In conclusion, our study provides an important contribution to the understanding of concerted evolution and to the interpretation of DNA barcoding and metabarcoding data based on multigene family markers.

High-throughput sequencing of 5S-IGS in oaks: Exploring intragenomic variation and algorithms to recognize target species in pure and mixed samples.

Measuring biological diversity is a crucial but difficult undertaking, as exemplified in oaks where complex patterns of morphological, ecological, biogeographical and genetic differentiation collide with traditional taxonomy, which measures biodiversity in number of species (or higher taxa). In this pilot study, we generated high-throughput sequencing amplicon data of the intergenic spacer of the 5S nuclear ribosomal DNA cistron (5S-IGS) in oaks, using six mock samples that differ in geographical origin, species composition and pool complexity. The potential of the marker for automated genotaxonomy applications was assessed using a reference data set of 1,770 5S-IGS cloned sequences, covering the entire taxonomic breadth and distribution range of western Eurasian Quercus, and applying similarity (blast) and evolutionary approaches (maximum-likelihood trees and Evolutionary Placement Algorithm). Both methods performed equally well, allowing correct identification of species in sections Ilex and Cerris in the pure and mixed samples, and main lineages shared by species of sect. Quercus. Application of different cut-off thresholds revealed that medium- to high-abundance (>10 or 25) sequences suffice for a net species identification of samples containing one or a few individuals. Lower thresholds identify phylogenetic correspondence with all target species in highly mixed samples (analogous to environmental bulk samples) and include rare variants pointing towards reticulation, incomplete lineage sorting, pseudogenic 5S units and in situ (natural) contamination. Our pipeline is highly promising for future assessments of intraspecific and interpopulation diversity, and of the genetic resources of natural ecosystems, which are fundamental to empower fast and solid biodiversity conservation programmes worldwide.

Determining the Authenticity of Shark Meat Products by DNA Sequencing.

Given that the global shark meat market is poised to grow in future years, the aim of this study was to use DNA sequencing of the cytochrome c oxidase I (COI) and NADH dehydrogenase subunit 2 (NADH2) mitochondrial genes to examine the market of shark meat products in Italy. This made it possible to analyze patterns of species utilization and commercialization of threatened, endangered and/or prohibited species, focusing on fraudulent activities in the shark food chain in order to propose seafood safety and environmental sustainability solutions. The study shows that the labeling of shark meat products generally lacks comprehensive information, thus making it difficult for consumers to make informed purchasing decisions and fails to comply with European Union (EU) legislation regarding seafood labelling. Molecular investigation reveals a high mislabeling rate (45.4%), highlighting widespread use of cheaper species either in order to replace species that are better known and more popular, or else in order to sell various threatened species. Considering that seafood mislabeling can circumvent the management of sustainable fisheries and facilitate Illegal, Unreported and Unregulated (IUU) fishing, the routine use of genetic analysis should be encouraged among control and enforcement agencies in order to implement effective management measures. This would help to build a species-specific reporting system for all catches, and enhance control measures, in order to prevent illegal activities connected with shark catches and trade around the world.

Species detection and delineation in the marine planktonic diatoms Chaetoceros and Bacteriastrum through metabarcoding: making biological sense of haplotype diversity.

High-throughput sequencing (HTS) metabarcoding is commonly applied to assess phytoplankton diversity. Usually, haplotypes are grouped into operational taxonomic units (OTUs) through clustering, whereby the resulting number of OTUs depends on chosen similarity thresholds. We applied, instead, a phylogenetic approach to infer taxa among 18S rDNA V4-metabarcode haplotypes gathered from 48 time-series samples using the marine planktonic diatoms Chaetoceros and Bacteriastrum as test case. The 73 recovered taxa comprised both solitary haplotypes and polytomies, the latter composed each of a highly abundant, dominant haplotype and one to several minor, peripheral haplotypes. The solitary and dominant haplotypes usually matched reference sequences, enabling species assignation of taxa. We hypothesise that the super-abundance of reads in dominant haplotypes results from the homogenization effect of concerted evolution. Reads of populous peripheral haplotypes and dominant haplotypes show comparable distribution patterns over the sample dates, suggesting that they are part of the same population. Many taxa revealed marked seasonality, with closely related ones generally showing distinct periodicity, whereas others occur year-round. Phylogenies inferred from metabarcode haplotypes enable delineation of biologically meaningful taxa, whereas OTUs resulting from clustering algorithms often deviate markedly from such taxa.

Global distribution and diversity of Chaetoceros (Bacillariophyta, Mediophyceae): integration of classical and novel strategies.

Information on taxa distribution is a prerequisite for many research fields, and biological records are a major source of data contributing to biogeographic studies. The Global Biodiversity Information Facility (GBIF) and the Ocean Biogeographic Information System (OBIS) are important infrastructures facilitating free and open access to classical biological data from several sources in both temporal and spatial scales. Over the last ten years, high throughput sequencing (HTS) metabarcoding data have become available, which constitute a great source of detailed occurrence data. Among the global sampling projects that have contributed to such data are Tara Oceans and the Ocean Sampling Day (OSD). Integration of classical and metabarcoding data may aid a more comprehensive assessment of the geographic range of species, especially of microscopic ones such as protists. Rare, small and cryptic species are often ignored in surveys or mis-assigned with the classical approaches. Here we show how integration of data from various sources can contribute to insight in the biogeography and diversity at the genus- and species-level using Chaetoceros as study system, one of the most diverse and abundant genera among marine planktonic diatoms. Chaetoceros records were extracted from GBIF and OBIS and literature data were collected by means of a Google Scholar search. Chaetoceros references barcodes where mapped against the metabarcode datasets of Tara Oceans (210 sites) and OSD (144 sites). We compared the resolution of different data sources in determining the global distribution of the genus and provided examples, at the species level, of detection of cryptic species, endemism and cosmopolitan or restricted distributions. Our results highlighted at genus level a comparable picture from the different sources but a more complete assessment when data were integrated. Both the importance of the integration but also the challenges related to it were illustrated. Chaetoceros data collected in this study are organised and available in the form of tables and maps, providing a powerful tool and a baseline for further research in e.g., ecology, conservation and evolutionary biology.