Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Marine macroalgae are considered an untapped source of healthy natural metabolites and their market demand is rapidly increasing. Intertidal macroalgae present chemical defense mechanisms that enable them to thrive under changing environmental conditions. These intracellular chemicals include compounds that can be used for human benefit. The aim of this study was to test cultivation protocols that direct seaweed metabolic responses to enhance the production of target antioxidant and photoprotective biomaterials. We present an original integrated multi-trophic aquaculture (IMTA) design, based on a two-phase cultivation plan, in which three seaweed species were initially fed by fish effluents, and subsequently exposed to various abiotic stresses, namely, high irradiance, nutrient starvation, and high salinity. The combined effect of the IMTA’s high nutrient concentrations and/or followed by the abiotic stressors enhanced the seaweeds’ content of mycosporine-like amino acids (MAAs) by 2.3-fold, phenolic compounds by 1.4-fold, and their antioxidant capacity by 1.8-fold. The Sun Protection Factor (SPF) rose by 2.7-fold, and the chlorophyll and phycobiliprotein synthesis was stimulated dramatically by an order of magnitude. Our integrated cultivation system design offers a sustainable approach, with the potential to be adopted by emerging industries for food and health applications.

Background: All-inclusive DNA-barcoding libraries in the storage and analysis platform of the BOLD (Barcode of Life Data) system are essential for the study of the marine biodiversity and are pertinent for regulatory purposes, including ecosystem monitoring and assessment, such as in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). Here, we investigate knowledge gaps in the lists of DNA barcoded organisms within two inventories, Cnidaria (Anthozoa and Hydrozoa) and Ascidiacea from the reference libraries of the European Register of Marine Species (ERMS) dataset (402 ascidians and 1200 cnidarian species). ERMS records were checked species by species, against publicly available sequence information and other data stored in BOLD system. As the available COI barcode data adequately cover just a small fraction of the ERMS reference library, it is of importance to employ quality control on existing data, to close the knowledge gaps and purge errors off BOLD. Results: Results revealed that just 22.9% and 29.2% of the listed ascidians and cnidarians species, respectively, are BOLD barcodes of which 58.4% and 52.3% of the seemingly barcoded species, respectively, were noted to have complete BOLD pages. Thus, only 11.44% of the tunicate and 17.07% of the cnidarian data in the ERMS lists are of high quality. Deep analyses revealed seven common types of gaps in the list of the barcoded species in addition to a wide range of discrepancies and misidentifications, discordances, and errors primarily in the GenBank mined data as with the BINs assignments, among others. Conclusions: Gap knowledge in barcoding of important taxonomic marine groups exists, and in addition, quality management elements (quality assurance and quality control) were not employed when using the list for national monitoring projects, for regulatory compliance purposes and other purposes. Even though BOLD is the most trustable DNA-barcoding reference library, worldwide projects of DNA barcoding are needed to close these gaps of mistakes, verifications, missing data, and unreliable sequencing labs. Tight quality control and quality assurance are important to close the knowledge gaps of Barcoding of the European recommended ERMS reference library.