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Artificial coastal structures associated with coastal defences, energy generation, ports, marinas and other developments, are known to support lower levels of biodiversity than natural coastal environments and tend to be hotspots of invasive non-native species (INNS). In the present study, we attempted to detect INNS through both quantitative (q)PCR and metabarcoding of environmental (e)DNA from seawater samples. A mitochondrial COI based species-specific qPCR assay was developed and deployed to detect Didemnum vexillum, a colonial tunicate that has successfully become established at coastal sites across Europe. Our targeted qPCR assay was able to detect D. vexillum in eDNA seawater samples from all sampled sites where it is currently found in Ireland and Wales. Through metabarcoding of the same eDNA samples, we detected an established INNS at all sites but not D. vexillum even in locations where it is present. We conclude that our qPCR approach is effective for sensitive and targeted screening for specific INNS at coastal sites including those with artificial structures, and while metabarcoding is a less sensitive approach it is a valuable tool to detect a broad taxonomic range of native and non-native species.

Environmental DNA (eDNA) is increasingly being used in aquatic environments for monitoring species, particularly those that are of conservation concern and/or are difficult to visually observe. Quantitative PCR (qPCR) has been employed to detect low abundance species occurring in environmental water samples. However, the qPCR technique has principally been applied to freshwater habitats, with less application to pelagic marine environments. We developed a species-specific eDNA assay for the Chilean devil ray, Mobula tarapacana, to assess the capability of using eDNA to detect transient pelagic marine animals. For this pilot study, seawater samples taken at seamounts around the Azores (NE Atlantic) were tested to determine the suitability of this approach for detecting the target species. Samples were taken at sites where M. tarapacana has been previously observed, in addition to sites where its presence is not known. eDNA detection was compared to observations carried out on the same day as water sampling. The qPCR assay successfully detected M. tarapacana at four of five seamount sampling opportunities where the species was observed, and there is a statistically significant relationship between genetic and visual detection. Target DNA was found at one location between seamounts in the absence of visual observation. Our results highlight the importance of physical environmental factors in relation to sampling eDNA in the ocean, such as currents and eDNA dispersal ability. This method has been shown to be sensitive for detection of M. tarapacana DNA in seawater and, therefore, in the identification of important seamounts requiring conservation.

The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in environmental DNA (eDNA) metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units (OTUs) or/and amplicon sequence variants (ASVs) retrieved from such studies do not get a taxonomic assignment with a reference sequence. To assess and investigate such sequences, we have developed the Dark mAtteR iNvestigator (DARN) software tool. For this purpose, a reference COI-oriented phylogenetic tree was built from 1,593 consensus sequences covering all the three domains of life. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 sequences retrieved from the Midori reference 2 database, which represented 70% of the initial number of reference sequences. Similarly, sequences from 431 bacterial and 15 archaeal taxa at the family level (29% and 1% of the initial number of reference sequences respectively) were retrieved from the BOLD and the PFam databases. DARN makes use of this phylogenetic tree to investigate COI pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of their phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms, such as bacteria and archaea, are also amplified in eDNA samples and we suggest prokaryotic COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and as a Docker image at https://hub.docker.com/r/hariszaf/darn.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans ( P. perurans ) is a disease affecting Atlantic salmon ( Salmo salar ). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha ( tnf- α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.

1. Pink salmon Oncorhynchus gorbuscha was introduced from its native range in the Pacific to Northwest Russia several times since the 1950s. While this species has been regularly observed in rivers in northern Norway since then, there has been an upsurge in the numbers of odd-year O. gorbuscha individuals observed in rivers, particularly in northern Norway in recent years, and particularly in 2017 and 2019. 2. In the present pilot study, an assay was developed to detect O. gorbuscha from eDNA water samples. Positive control water samples were taken at two locations of the River Signaldalselva in northern Norway during the summer of 2019, when adults were spawning in the river. Samples showed positive detection of this species in the river, while negative control samples collected upstream migration barriers in central and southern Norway confirmed the absence of the target species. 3. These findings reveal that eDNA-based methods can be used to track the ongoing O. gorbuscha invasion of northern Europe and other regions where it might be or become invasive. ddPCR, eDNA, invasion, Norway, Oncorhynchus gorbuscha, pink salmon, qPCR

Coastal countries have traditionally relied on the existing marine resources (e.g., fishing, food, transport, recreation, and tourism) as well as tried to support new economic endeavors (ocean energy, desalination for water supply, and seabed mining). Modern societies and lifestyle resulted in an increased demand for dietary diversity, better health and well-being, new biomedicines, natural cosmeceuticals, environmental conservation, and sustainable energy sources. These societal needs stimulated the interest of researchers on the diverse and underexplored marine environments as promising and sustainable sources of biomolecules and biomass, and they are addressed by the emerging field of marine (blue) biotechnology. Blue biotechnology provides opportunities for a wide range of initiatives of commercial interest for the pharmaceutical, biomedical, cosmetic, nutraceutical, food, feed, agricultural, and related industries. This article synthesizes the essence, opportunities, responsibilities, and challenges encountered in marine biotechnology and outlines the attainment and valorization of directly derived or bio-inspired products from marine organisms. First, the concept of bioeconomy is introduced. Then, the diversity of marine bioresources including an overview of the most prominent marine organisms and their potential for biotechnological uses are described. This is followed by introducing methodologies for exploration of these resources and the main use case scenarios in energy, food and feed, agronomy, bioremediation and climate change, cosmeceuticals, bio-inspired materials, healthcare, and well-being sectors. The key aspects in the fields of legislation and funding are provided, with the emphasis on the importance of communication and stakeholder engagement at all levels of biotechnology development. Finally, vital overarching concepts, such as the quadruple helix and Responsible Research and Innovation principle are highlighted as important to follow within the marine biotechnology field. The authors of this review are collaborating under the European Commission-funded Cooperation in Science and Technology (COST) Action Ocean4Biotech – European transdisciplinary networking platform for marine biotechnology and focus the study on the European state of affairs.

POCI-01-0145-FEDER-031045 projects/OSTEOMAR/16.02.01-FMP-0057 ALGASOLE/16.02.01-FMP-0058 Atlantic Area/BLUEHUMAN/EAPA/151/2016 No. PBA/MB/16/01 PROMAC (244244) Platform (294946) 710566 NCN 2016/21/B/NZ9/02304 no. 239 research core funding P1-0245 Marine organisms produce a vast diversity of metabolites with biological activities useful for humans, e.g., cytotoxic, antioxidant, anti-microbial, insecticidal, herbicidal, anticancer, pro-osteogenic and pro-regenerative, analgesic, anti-inflammatory, anti-coagulant, cholesterol-lowering, nutritional, photoprotective, horticultural or other beneficial properties. These metabolites could help satisfy the increasing demand for alternative sources of nutraceuticals, pharmaceuticals, cosmeceuticals, food, feed, and novel bio-based products. In addition, marine biomass itself can serve as the source material for the production of various bulk commodities (e.g., biofuels, bioplastics, biomaterials). The sustainable exploitation of marine bio-resources and the development of biomolecules and polymers are also known as the growing field of marine biotechnology. Up to now, over 35,000 natural products have been characterized from marine organisms, but many more are yet to be uncovered, as the vast diversity of biota in the marine systems remains largely unexplored. Since marine biotechnology is still in its infancy, there is a need to create effective, operational, inclusive, sustainable, transnational and transdisciplinary networks with a serious and ambitious commitment for knowledge transfer, training provision, dissemination of best practices and identification of the emerging technological trends through science communication activities. A collaborative (net)work is today compelling to provide innovative solutions and products that can be commercialized to contribute to the circular bioeconomy. This perspective article highlights the importance of establishing such collaborative frameworks using the example of Ocean4Biotech, an Action within the European Cooperation in Science and Technology (COST) that connects all and any stakeholders with an interest in marine biotechnology in Europe and beyond.

The pink salmon Oncorhynchus gorbuscha was introduced from its native range in the Pacific to Northwest Russia several times since the 1950's. While this species has been regularly observed in rivers in Northern Norway since that time, there has been an upsurge in the numbers of odd-year O. gorbuscha individuals observed in rivers in southern Norway in recent years, and particularly in 2017. Although the wide-scale effects of this species presence are currently uncertain, there are concerns regarding potential competition between O. gorbuscha and native species - most notably the Atlantic salmon Salmo salar. Environmental (e)DNA is becoming a widely used tool to monitor rare and invasive species in aquatic environments. In the present pilot study, primers and a probe were developed to detect O. gorbuscha from eDNA samples taken from a Norwegian river system where the species was observed. Water samples were taken at both upstream and downstream locations of the Lysakerelva river during Autumn 2017 (to coincide with spawning) and during late Spring 2018. Autumn samples were positive for O. gorbuscha at both sampling locations, whereas Spring samples showed positive detection of this species in the upstream region of the river, when smolt should have left, or be in the process of leaving the river. These findings reveal that eDNA-based methods can be used detect the presence of O. gorbuscha during their spawning season. This suggests that odd-year populations have the potential to become established in the studied river system. We recommend that eDNA sampling is repeated to determine whether individuals of this odd-year population have survived at sea and return to spawn. Our assay specificity tests indicate that the tools developed in the present study can be used for detection of O. gorbuscha in both Norwegian and other European river systems where presence/absence data is required. We also suggest some modifications to our methodology that may improve upon the detection capabilities of O. gorbuscha using eDNA. Footnotes * We have included a map of the sampling area in Norway (Figure 2).

Marine organisms produce a vast diversity of metabolites with biological activities useful for humans, e.g., cytotoxic, antioxidant, anti-microbial, insecticidal, herbicidal, anticancer, pro-osteogenic and pro-regenerative, analgesic, anti-inflammatory, anticoagulant, cholesterol-lowering, nutritional, photoprotective, horticultural or other beneficial properties. These metabolites could help satisfy the increasing demand for alternative sources of nutraceuticals, pharmaceuticals, cosmeceuticals, food, feed, and novel bio-based products. In addition, marine biomass itself can serve as the source material for the production of various bulk commodities (e.g., biofuels, bioplastics, biomaterials). The sustainable exploitation of marine bio-resources and the development of biomolecules and polymers are also known as the growing field of marine biotechnology. Up to now, over 35,000 natural products have been characterized from marine organisms, but many more are yet to be uncovered, as the vast diversity of biota in the marine systems remains largely unexplored. Since marine biotechnology is still in its infancy, there is a need to create effective, operational, inclusive, sustainable, transnational and transdisciplinary networks with a serious and ambitious commitment for knowledge transfer, training provision, dissemination of best practices and identification of the emerging technological trends through science communication activities. A collaborative (net)work is today compelling to provide innovative solutions and products that can be commercialized to contribute to the circular bioeconomy. This perspective article highlights the importance of establishing such collaborative frameworks using the example of Ocean4Biotech, an Action within the European Cooperation in Science and Technology (COST) that connects all and any stakeholders with an interest in marine biotechnology in Europe and beyond.

The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in eDNA metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units or/and amplicon sequence variants are retrieved from such studies and referred to as “dark matter”, and do not get a taxonomic assignment with a reference sequence. For a thorough investigation of this dark matter, we have developed the Dark mAtteR iNvestigator (DARN) software tool. A reference COI-oriented phylogenetic tree was built from 1,240 consensus sequences covering all the three domains of life, with more than 80% of those representing eukaryotic taxa. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 retrieved from the Midori reference 2 database. Similarly, sequences from 559 bacterial genera and 41 archaeal were retrieved from the BOLD database. DARN makes use of the phylogenetic tree to investigate and quantify pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms such as bacteria and archaea are also amplified in eDNA samples and we suggest bacterial COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and you may find it as a Docker at https://hub.docker.com/r/hariszaf/darn. DARN is a software approach aiming to provide further insight in the COI amplicon data coming from environmental samples. Building a COI-oriented reference phylogeny tree is a challenging task especially considering the small number of microbial curated COI sequences deposited in reference databases; e.g ~4,000 bacterial and ~150 archaeal in BOLD. Apparently, as more and more such sequences are collated, the DARN approach improves. To provide a more interactive way of communicating both our approach and our results, we strongly suggest the reader to visit this Google Collab notebook where all steps are described step by step and also this GitHub page where our results are demonstrated. Our approach corroborates the known presence of microbial sequences in COI environmental sequencing samples and highlights the need for curated bacterial and archaeal COI sequences and their integration into reference databases (i.e. Midori, BOLD, etc). We argue that DARN will benefit researchers as a quality control tool for their sequenced samples in terms of distinguishing eukaryotic from non-eukaryotic OTUs/ASVs, but also in terms of understanding the unknown unknowns.