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Fisheries enforcement relies on visual catch identification and quantification at sea or when landed. Silage (fish dissolved in acid) and fish blocks (block frozen fish) are promising methods for on-board processing and storage of low-value catches. We examined the use of non-destructive sampling and two DNA-based methods, quantitative PCR (qPCR) and metabarcoding, to assess species composition and relative abundance in industrial grade experimental silage and fish blocks. We demonstrate the ability to identify and quantify DNA from fish species in both products. qPCR analysis of small silage samples collected over 21 days detected all target control species. DNA from one species (Atlantic wolffish) was consistently overrepresented while, for three species of gadoids (Atlantic cod, haddock and whiting), the DNA content matched input tissue proportions with high accuracy. qPCR and metabarcoding of fish blocks, sampled as run-off water and exterior swabs, provided consistent species detection, with the highest variance observed in quantification from swab samples. Our analysis shows that DNA-based methods have significant potential as a tool for species identification and quantification of complex on-board-processed seafood products and are readily applicable to taxonomically and morphologically similar fish. There is, however, a need for establishing DNA/weight calibration factors for primary fisheries species.

Environmental DNA (eDNA) is increasingly used for monitoring marine organisms; however, offshore sampling and time lag from sampling to results remain problematic. In order to overcome these challenges a robotic sampler, a 2nd generation Environmental Sample Processor (ESP), was tested for autonomous analysis of eDNA from four commercial fish species in a 4.5 million liter mesocosm. The ESP enabled in situ analysis, consisting of water collection, filtration, DNA extraction and qPCR analysis, which allowed for real-time remote reporting and archival sample collection, consisting of water collection, filtration and chemical preservation followed by post-deployment laboratory analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify target molecules from the most abundant species (Atlantic mackerel) both in real-time and from the archived samples. In contrast, detection of low abundant species was challenged by both biological and technical aspects coupled to the ecology of eDNA and the 2G ESP instrumentation. Comparison of the in situ analysis and archival samples demonstrated variance, which potentially was linked to diel migration patterns of the Atlantic mackerel. The study demonstrates strong potential for remote autonomous in situ monitoring which open new possibilities for the field of eDNA and marine monitoring.

Monitoring the distribution of marine nonindigenous species is a challenging task. To support this monitoring, we developed and validated the specificity of 12 primer-probe assays for detection of environmental DNA (eDNA) from marine species, all nonindigenous to Europe. The species include sturgeons, a Pacific red algae, oyster thief, a freshwater hydroid from the Black Sea, Chinese mitten crab, Pacific oyster, warty comb jelly, sand gaper, round goby, pink salmon, rainbow trout and North American mud crab. We tested all assays in the laboratory, on DNA extracted from both the target and non-target species to ensure that they only amplified DNA from the intended species. Subsequently, all assays were used to analyse water samples collected at 16 different harbours across two different seasons during 2017. We also included six previously published assays targeting eDNA from goldfish, European carp, two species of dinoflagellates of the genera Karenia and Prorocentrum, two species of the heterokont flagellate genus Pseudochattonella. Conventional monitoring was carried out alongside eDNA sampling but with only one sampling event over the one year. Because eDNA was relatively fast and easy to collect compared to conventional sampling, we sampled eDNA twice during 2017, which showed seasonal changes in the distribution of nonindigenous species. Comparing eDNA levels with salinity gradients did not show any correlation. A significant correlation was observed between number of species detected with conventional monitoring methods and number of species found using eDNA at each location. This supports the use of eDNA for surveillance of the distribution of marine nonindigenous species, where the speed and relative easy sampling in the field combined with fast molecular analysis may provide advantages compared to conventional monitoring methods. Prior validation of assays increases taxonomic precision, and laboratorial setup facilitates analysis of multiple samples simultaneously. The specific eDNA assays presented here can be implemented directly in monitoring programmes across Europe and potentially worldwide to infer a more precise picture of the dynamics in the distribution of marine nonindigenous species.

The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targetingL. salmonisDNA in environmental samples. Here, we developed and tested a newL. salmonisspecific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of differentL. salmonisisolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages ofL. salmonisto investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed forL. salmonisdetection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages ofL. salmoniswas established in the laboratory conditions. In the field,L. salmonisDNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that onlyL. salmonistissue or eDNA residues were detected. This novel study opens for a fully automatizedL. salmonisDNA quantification using ESP robotic to monitor the parasitic load, but the challenge remains the adequate sampling of a volume of seawater sufficiently large to be representative of outbreaks and load around fish farms.

Monitoring, understanding, and communicating the impact of offshore oil and gas exploration and production (E&P) activities on marine mammal (MM) biodiversity can be challenging. Here, we share methods, results, and experiences accumulated during seven years of monitoring MM and underwater sound in a mature E&P activity area in the North-East Atlantic. We collected data on the distribution and behaviour of MM, their prey and changes associated with impulsive (e.g., seismic) and continuous (e.g., platform) sound generated by E&P operations. A program was created as an incentive for offshore staff to report systematically incidental MM sightings. Underwater acoustic recorders were placed around producing facilities and across a 3D seismic survey area before, during and after acquisition. A 2nd Generation Environmental Sample Processor (ESP) was deployed to collect and analyse environmental DNA (eDNA) from seawater to identify MM species and their potential prey. Monitoring data were shared with relevant stakeholders through publications in scientific journals, presentations at conferences and meetings, or through social media. Our data provided evidence of MM activity in the E&P activity area year-round. A total of eight species were sighted from platforms and vessels; harbour porpoise being the most common MM. Harbour porpoises were recorded as being within 800m of the platform three times more often than at stations further away. Near installations, acoustic data showed porpoises actively searched for prey whilst eDNA confirmed the presence of prey species - validating a strong reef-effect. Decrease in porpoise echolocation to 8-12 km from an active seismic vessel, is suggestive of a temporary displacement of animals in the corresponding area. There was no large scale or long-term displacement as harbour porpoises were detected again in the area few hours after airgun operations ended. The results indicate that despite elevated sound level in the E&P area, porpoise distribution appears to be linked to higher availability of prey around the artificial reef created by subsea O&G structures. The monitoring efforts generate valuable scientific knowledge, which form a sound basis to support management and regulation of E&P activities in Denmark. Additionally, several simultaneous tangible benefits resulted from the study such as staff awareness of biodiversity and increased stakeholder interactions. Results and methodologies may be used by HSE practitioners and O&G project managers to assess the potential impact of sound generated by O&G industry on marine mammals. Our success illustrates the value of long-term monitoring and should inspire and support HSE practitioners in their future environmental monitoring initiatives.

Biomass assesment of fish stocks is a difficult task that often involves costly fisheries trawl surveys. Trawl surveys rarely return replicate samples, as trawling attempts are expensive and difficult to reproduce. Furthermore, traditional benthic trawling is often detrimental to habitats and the organisms associated with the sea bottom. The rapidly developing field of environmental DNA (eDNA) analysis offers a new approach to non-invasive monitoring of fish. In the present study, we develop and test species-specific primers and probes for qPCR detection of eDNA from Atlantic herring (Clupea harengus ), Atlantic cod ( Gadus morhua ), European flounder ( Platichthys flesus ), European plaice ( Pleuronectes platessa), and Atlantic mackerel ( Scomber scombrus ) in the Baltic Sea. A recently published qPCR system was applied for European eel (Anguilla anguilla ). Filtered water samples were collected during a stratified benthic trawl survey, enabling parallel comparisons of eDNA concentrations with biomass caught by trawling. No significant correlation was found between eDNA concentrations and the biomass of fish caught by the trawl, although an association was observed between the measured concentrations of eDNA and the known distributions and main abundances of cod, herring, plaice and flounder. This indicates that while eDNA concentrations may not be directly comparable to results from existing methods – likely because aquatic eDNA concentrations are not controlled by the same combinations of factors as e.g. trawl biomass catch – eDNA analysis could prove a useful supplement for monitoring fish stocks in the future.

Project manager Jesper H. Andersen. The MONIS 3 project reports the development of 22 species-specific eDNA-based test systems for monitoring and mapping of the occurrence of non-indigenous species in Danish marine waters. The development has been undertaken by the MONIS partnership and the species have been selected based on the outcome of the MONIS 2 project. It should be noted that the test systems are considered operational cf. the MONIS definition (see Chapter 1). It should also be noted that the 22 test systems have been applied in the MONIS 4 project, where monitoring of 16 Danish harbours have been carried out to map occurrence of nonindigenous marine species using both conventional methods and molecular methods (eDNA). Development of additional test systems is being planned and expected to be carried out in 2019 pending funding. The MONIS partnership encourages interested institutions, especially in neighbouring countries (Germany, Norway, Poland and Sweden) to make use of the test systems.

Application of environmental DNA (eDNA) analysis has attracted the attention ofresearchers, advisors and managers of living marine resources and biodiversity. The apparent simplicity and cost-effectiveness of eDNA analysis make it highly attractive as species distributions can be revealed from water samples. Further, species-specific analyses indicate that eDNA concentrations correlate with biomass and abundance, suggesting the possibility for quantitative applications estimating abundance and biomass of specific organisms in marine ecosystems, such as for stock assessment. However, the path from detecting occurrence of an organism to quantitative estimates is long and indirect, not least as eDNA concentration depends on several physical, chemical and biological factors which influence its production, persistence and transport in marine ecosystems. Here, we provide an overview of basic principles in relation to eDNA analysis with potential for marine fisheries application. We describe fundamental processes governing eDNA generation, breakdown and transport andsummarize current uncertainties about these processes. We describe five major challenges in relation to application in fisheries assessment, where there is immediate need for knowledge building in marine systems, and point to apparent weaknesses of eDNA compared to established marine fisheries monitoring methods. We provide an overview of emerging applications of interest to fisheries management and point to recent technological advances, which could improve analysis efficiency. We advise precaution against exaggerating the present scope for application of eDNA analysis in fisheries monitoring, but also argue that with informed insights into strengths andlimitations, eDNA analysis can become an integrated tool in fisheries assessment and management

This report is the outcome of MONIS 2 – or in full, “Monitoring of Non-Indigenous Species in Danish Marine Water, phase 2” – and includes three deliverable: (1) a national Target Species List including 50 species, (2) a draft Technical Guidance Report, and (3) in silico designed and tested primers and probes for 48 of the 50 species on the Target Species List. The list is based on discussions at a workshop and subsequent scoring and ranking of relevant species. The draft Technical Guidance report is anchored in existing Standard Operating Procedures (i.e. protocols for sampling, storage and analysis) and adapted to the requirements of the Danish NOVANA programme. In addition, the report includes suggestions for next steps to take to implement and improve monitoring and assessment activities in regard to non-indigenous species in Danish marine waters. Danish Nature Agency

While genetics has assisted fisheries management for over 50 years, genetic applications aiming to alleviate or eliminate discards have received little attention. In this chapter, we focus on how genetics can be applied under the EU Landing Obligation, to identify and prevent unwanted catches and to estimate the composition of products made from such catches. Three themes are covered: (i) the genetic identification of bycatch; (ii) the genetic analysis of species composition in nutritional products made from unwanted fish; (iii) the potential of using so-called environmental DNA (DNA shedded from aquatic organisms into the water) to reduce bycatch. For all themes, we introduce and explain the relevant genetic techniques, including data formats and analyses. We present the most significant limitations of the methodologies for their implementation in fisheries and provide examples of their use through relevant case studies. Finally, we discuss the potential future perspectives, with emphasis on the rapid progress in portable and automatic DNA devices, which may revolutionize the use of real-time onsite genetic analyses.