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Knowing the number of individuals in a population is fundamental for the sustainable management of exploited marine resources but estimating this parameter is often extremely challenging, especially in large, highly mobile and dispersed populations. Abundance estimation traditionally relies on multiple data types that include the relationship between fishery catches and effort (Catch Per Unit Effort or CPUE), scientific research surveys and demographic models that are developed to estimate past and current stock dynamics, but uncertainty is often high. Close-kin mark-recapture (CKMR) is an alternative method for estimating abundance and other demographic parameters (e.g. population trend, survival rates, connectivity), using kinship relationships determined from genetic samples. This methodology is based on a simple concept - the larger the population the less likely to find relatives and vice versa - and was proposed two decades ago although regained considerable attention recently. Refinements in the statistical methodology and advances in high throughput sequencing methods have boosted the efficiency of genomic analysis, promising to revolutionize the field of fisheries stock assessments. In theory, it can be applied to almost any species, provided that there is sufficient information about the life-history/biology of the organism and that the populations are not so small as to be almost extinct or so large that finding relatives becomes extremely difficult. Thus, it has the potential to provide baseline data for the many exploited fish stocks that remain largely unassessed and to reduce uncertainty in those that are regularly evaluated. Here, we provide an overview of the method in the context of fisheries assessments, analyze the advances and synthetize the field studies published in the last five years. Moreover, we evaluate the readiness, viability and maturity of the method to infer demographic parameters in species spanning diverse life histories. We evalua

Stock assessments serve to monitor the condition of fish stocks and exploit them sustainably but require accurate data such as growth and mortality rates as input parameters. Most species fished worldwide lack the data needed to assess their status and even those closely assessed are often based on parameters that are known to contain uncertainty. This has resulted in an increased share of overfished stocks over the last half century, demanding urgently innovative methodologies that can provide novel means to reduce uncertainty of fish stocks assessments and expand the range of assessed species. CKMR has emerged recently attracting a great interest due to its potential to provide accurate demographic parameters of interest in stock assessments. The method is at the crossroads between fisheries science and genomics, requiring specialized knowledge that is usually outside of the experience of fisheries scientist and modellers, complicating the application of the method and its uptake in regular fisheries assessments. In this review, we provide useful information to perform the genomics and bioinformatics steps required to complete successfully a CKMR study. We discuss the most suitable genomics assays, considering the amount of information they provide, their easiness of use and cost of genotyping accurately the large number of individuals needed to assess most fish stocks. We provide an overview of methods of analysis and statistical methodologies that can be used to infer kinship with the accuracy required in a large population setting with sparse sampling, where most individuals are unrelated, determining a low probability of finding closely related individuals. We analyse potential sources of biases and errors and provide recommendations to facilitate the application of CKMR to a wider range of fish stocks

In Europe, research surveys carried out by state governments provide the great majority of fishery-independent data. Member States (MS) in the European Union (EU) regularly conduct research surveys to provide the necessary data to assess the status of exploited fish stocks and to monitor the general condition of the marine ecosystem. In the surveys, samplings are carried out throughout the distribution range of the targeted fish species using standardized gears (e.g., trawls and seines) and other methods (e.g., hydroacoustics and underwater cameras). In the context of fish stock assessment, survey data are important because they provide indices that help tuning the stock assessment models (e.g., the index of fish abundance) and key information about the size and age distributions of the stock, the size-age relationships, the proportion of fish mature at each age, and information on reproductive performance of the stocks. However, research surveys have a number of shortcomings that include, for example, a high economic cost coupled with complex logistics and a long time required for processing the collected data. In addition, some of the parameters that are needed in stock assessment cannot be estimated from survey data for certain commercially important species. For instance, age is usually determined using hard structures (such as otoliths) in fish target species. However, for European hake, age cannot be determined accurately because there are many difficulties in interpreting the ring patterns of the otoliths. This highlights the need to look for alternative methodologies such as genomics, that have the potential of improving the data obtained from research surveys and hence, improve fish stock assessments. Considering this, we carried out a review of the bottom trawl research surveys in the EU with the purpose of: 1) identifying the current approaches for monitoring fishery resources and the ecosystem and 2) determining how genomic techniques can be used to impr

An increasing number of studies using marine environmental DNA (eDNA) approaches are showing its potential application in marine fisheries management by helping and simplifying some of the labor-intensive traditional surveys required to assess exploited populations and ecosystem status. eDNA approaches (i.e. metabarcoding and targeted) can support to ecosystem-based fisheries management by providing information on species composition; surveillance of invasive, rare and/or endangered species; and providing estimates of species abundance. Due to these potential uses in fisheries and conservation sciences, the number of studies applying eDNA approaches in marine habitats has expanded in the very last few years. However, a lack of consistency across studies when applying pipelines for data analyses, makes results difficult to compare among them. Such lack of consistency is partially caused by poor knowledge in the management of raw sequences data, and analytical methods allowing comparative results. Hence, we review here the essential steps of eDNA data processing and analyses to get sound, reproducible, and comparable results, providing a set of bioinformatics tools useful for each step. Altogether this review presents the state of the art of eDNA data analyses towards a comprehensive application in fisheries management promoting sustainability

The Tender contract “FishGenome: Improving cost-efficiency of fisheries research surveys and fish stocks assessments using next-generation genetic sequencing methods” was developed to assess the ability and readiness of several new emerging genomic technologies to enhance fisheries assessments. These assessments are needed to monitor the status of fish stocks and ensure fishing practices that exploit them at sustainable levels. They rely on the collection of data from multiple sources, that include fishery- dependent data -catches, landings, biological information - as well fishery-independent data obtained from research surveys. These surveys provide valuable and systematised information regarding exploited fish populations, marine biodiversity and their environment, and are essential piece in stock assessment and scientific advice. The overall purpose of FishGenome was to evaluate the suitability of three genomic techniques - Close Kin Mark Recapture (CKMR), epigenetic Age Determination by DNA methylation (DNAm) and environmental DNA (eDNA) to estimate various essential parameters for fisheries stock assessments, including absolute abundance, survival, age and biomass. Additionally, it was assessed the potential and ripeness of a fourth genomic technique - restriction site Associated DNA Sequencing (RAD-Seq) - to estimate a series of parameters that are also important to evaluate stocks but have been mostly neglected in fisheries assessments, i.e. fine-scale stock substructure, connectivity and molecular sexing. The results indicated the need and convenience of implementing the genomic sampling into EU-MAP research surveys, as well the high potential of these techniques to contribute to an improved stock assessment of many stocks. On one hand, EU Data collection Framework (EU-DCF) has contributed to improve fisheries management and the implementation of different policies, by harmonising and coordinating the collection of data from the fishery, as well, and more i

An increase in initial growth and decrease in maximum size have been linked to warmer temperatures in fish due to the increase in metabolism. It is essential to understand the dynamics between environmental conditions and growth in order to forecast temperature impacts on fish growth and inform management decisions. This study aimed to determine growth responses to variation of sea bottom temperature in both sexes of seven demersal fish species of commercial interest commonly found in the Flemish Cap, Norwest Atlantic, and to compare the magnitude of growth changes among them. The species were Gadus morhua, Hippoglossoides platessoides, Reinhardtius hippoglossoides, Macrourus berglax, and three species of the genus Sebastes. The study used size-at-age data collected from bottom trawl EU surveys conducting in summer, typically July from 1993 to 2018 and bottom temperature (BT) data from the Copernicus Marine Service (CMEMS). The effect of BT and age on size was analysed using Generalised Additive Models (GAM). The best-fit models were selected using the Akaike Information Criterion (AIC). The best-fit GAMs for both sexes of all species included the variable BT and all explained over 80% of deviance. The interaction between effects of BT and age was significant for both sexes of G. morhua and M. berglax, male R. hippoglossoides, and male S. norvegicus. Based on the models, size-at-age predictions were made for 3, 4, and 5°C BT scenarios, which are within the typical temperature range in the area. The predictions were fitted with von Bertalanffy Growth Functions (VBGF). The GAMs predictions for all species, sexes and scenarios fit the VBGF well (R2 ≥ 0.95) and growth parameters were delivered. The most common response to increased BT was a decrease in L∞, with some species also showing increased k. The estimated maximum sizes at maximum ages generally declined at higher temperatures, with the varying magnitudes among species and sexes. The greatest reductions in maximu

Healthy demographic structures are critical to preserve fish stocks. Yet, understanding the demography of many fish stocks depends on resolving existing uncertainties around the stock-recruitment relationship. One of the reasons for these uncertainties is the use as stock index of the spawning stock biomass, which does not consider variations in the stock reproductive potential and patterns of variation in individual reproductive success. Fecundity and quality of the offspring has been recognized to increase hyperallometrically with the size and age of female spawners (the so called “maternal effects”). Consequently, ignoring the characteristics of the parental stock and the maternal effects prevents the understanding on the drivers of recruitment variability. Here, we used 28 years of data on maturity rates, population abundances and fecundity of Acadian redfish (Sebastes fasciatus Storer, 1854) at Flemish Cap bank to estimate 17 indices of stock reproductive potential (SRP) used to describe interannual changes in reproductive parameters. We compared the performance of these indices to the metrics currently used in stock assessment (Spawning stock biomass: SSB) using a modified Ricker model. We found that 12 of the newly estimated indices were better predictors of recruitment that SSB, including total egg production (TEP) indices. We observed the improvement of recruitment estimation when the TEP data is considered, showing a stronger stock-recruitment relationship compared to SSB (i.e., Cross-correlation TEP > Cross-correlation SSB). Our findings highlight that the incorporation of indices related to maternal effects and parental stock into management can greatly improve our understanding of stock dynamics

Bottom trawling activities can alter significantly deep-water ecosystems, by degrading the seabed integrity and causing the depauperation of benthic biodiversity. Deep-sea environments are often characterized by fragile benthic biodiversity hotspots, known as vulnerable marine ecosystems (VMEs). These ecosystems show structural and functional fragility and include organisms that are unique or rare, with slow growth, large longevity and/or limited dispersion, which makes their recovery difficult when their habitats are altered. VMEs may be negatively affected by fishing, pollution and climate change, including rising water temperatures and ocean acidification. Many aggregations of deep-water corals and sponges are classified by the FAO as VMEs, since they host in their habitat a great biodiversity of epibenthic fauna that is fundamental for the general maintenance at the ecosystem level. The conservation and protection of these habitats requires detailed information on the distribution of VMEs but these are generally poorly described. The general objective of this study is the development of a non-invasive tool to detect and delineate the spatial distribution of VMEs, to ultimately protect them against fishing and develop strategies to prevent their damage. Our case study is focused in the Flemish Cap, Northwest Atlantic, where an annual research survey provides information on the ecosystem and the vulnerable benthic species living in the area. A total of 418 invertebrate samples, collected during the survey in 2022, were characterized using both, classical and molecular taxonomy based on two mitochondrial markers (COI and 16S rDNA). The resulting molecular barcodes are unique to each species inhabiting the ecosystem and served to create a complete and curated reference database to characterize VMEs in this region. The newly created database will help in the immediate future to recognize VME sites through non-invasive tools based on environmental DNA coupled with met

Vulnerable marine ecosystems (VMEs) are characterized by being highly vulnerable to disturbances and an impaired ability to recover that takes a long time and is not always possible. Some of these vulnerable marine habitats include seamounts, hydrothermal vents or cold-water coral reefs. Cold water corals are of significant ecological value and provide a variety of ecosystem services, such as providing habitat for a wide variety of organisms, many of which are of commercial interest. In addition, they are long-lived and have low growth rates, long reproductive cycles and low recruitment rates. Because of their nature and the importance of their ecosystems as biodiversity hotspots, there is an international call to protect these vulnerable marine ecosystems from trawling, the most destructive form of deep-sea fishing, with dramatic consequences for benthic communities. It is therefore of vital importance to develop non-destructive methods to describe and map the seabed in these areas. One of the most promising methodologies is the detection of environmental DNA, a non-invasive tool that only requires the collection of water. This method relies entirely on DNA databases that are neither complete nor accurate, especially for invertebrate species living in poorly described habitats. To improve existing public databases and the biodiversity characterization of the Flemish Cap seabed (NAFO), marine invertebrates were sampled during the 2022 research surveys. These invertebrates were identified both taxonomically and by barcoding using two mitochondrial markers (COI and 16S rRNA). Our preliminary results show difficulties in the genetic identification to species level of the Octocorallia subclass using these widely used barcoding markers and furthermore, that COI amplification is often challenging in echinoderms. These could result in an underestimate of the biodiversity due to the lack of sequence divergence that implies a lack of discrimination power within these groups

This report derives from the implementation of Task 1.5 as described in FishGenome Approved Inception report. The objective of the task was to preliminarily identify any technical, legal, environmental and economic barriers and risks for the practical implementation and roll out of the HTS techniques. Most of this information has been gathered and considered for the elaboration of the review reports about the State of the Art for the different HTS techniques and also for the Fisheries Surveys. The report D.1.5 is made as an extract of identified risks and barriers regarding the suitability and feasibility of the use of the DNA-HTS techniques in fisheries assessments. Apart from technical barriers some others have been considered: policy and regulation related; cost-efficiency and economics; cultural and institutional barriers (aversion to change); etc. The information derives from the revision of literature but mostly from the evidence provided by the experts consulted in and beyond the project partnership, through progress meetings, exchanges with the members of the External Experts Panel and the FishGenome Workshop accomplished remotely on the 28th May 2020. Therefore, the purpose of this deliverable is not to present de novo information, but to extract and organise information about relevant risks and barriers for implementation which can also be found scattered through the different deliverables and reports presented so far.

The status of marine fish stocks needs to be regularly assessed to ensure fishing practices exploit these stocks at sustainable levels. This assessment is based on multiple data types and sources that include catch data, monitoring of fishery landings, biological observers and research surveys, the latest of which provide critical fishery-independent information. Traditional methods to assess the state of fish stocks through scientific surveys have experienced a very slow progress and present recognized shortcomings (Maunder and Piner, 2015). These include a high economic cost coupled with complex logistics, very sparse data in space and time as a direct consequence and a long-time required for treating and analyzing the collected data (Stomatopoulos, 2002). Moreover, some important parameters in fish stock assessment cannot be estimated using traditional methodologies at present. For example, the sex of the younger juveniles cannot be assigned using traditional methods —mainly histology— in those species that lack sexual dimorphism (e.g., Mellon-Duval et al., 2010), despite the importance of sex ratios to evaluate the status of exploited stocks. Similarly, difficulties have been faced when determining the age of individuals of some species, an essential parameter for growth estimates, population dynamic studies and for optimizing the harvesting time (Gursoy et al., 2005). Otoliths of some commercially important fish species such as hake or cod, for instance, have proved unreliable due to the presence of false rings, lack of definition of rings or deposition at irregular intervals (Morales-Nin et al., 1998; Ligas et al., 2011; Hüssy et al., 2010). In this context, High-throughput sequencing (HTS) genomic methodologies can offer the possibility to resolve some of these hurdles and supplement traditional methods used in fisheries assessment. Three HTS methods are considered in FishGenome: Close-kin Mark-Recapture (CKMR), environmental DNA (eDNA) and epigenetics for ag

This report is based on information from two sources; 1) a comprehensive review of the latest genomic literature, and 2) analysis and evaluation of the knowledge gathered during FishGenome. The latest includes the development of Pilot studies to test the use of novel genomic techniques, expert´s advice and opinions gathered during two workshops organized within the framework of FishGenome. These workshops involved European stakeholders, including geneticists, fisheries scientists and fisheries managers (management and policy). The aim of the two workshops was to put FishGenome in the spotlight and to canvas perceptions about how genomics currently informs fisheries management issues as well as future prospects. The two workshops were focused on 1) genomic technologies with potential application to fisheries, specially focused on three novel techniques (CKMR, epigenetics and environmental DNA); their prospects, technical challenges, advantages and barriers; 2) implementation of genomic techniques into the evaluation process, perceptions about their usefulness by fisheries scientist, knowledge gaps and barriers. The key findings were that 1) most geneticists highlighted that the rapid advances, transformative technological developments and plummeting costs within the field of genomics in the last two decades, has expanded its sphere of action tremendously. Despite this, there is a critical knowledge gap between most geneticists and the needs of fisheries scientists and managers that is preventing the integration of genomic information into fisheries management schemes; 2) most fisheries scientist were generally positive about adopting genomic techniques to improve fisheries management, although they were also cautious about their implementation. The importance of avoiding disruption of long time-series and accordingly, the need of coexistence of classical and new methods for an extended period of time, was repeatedly highlighted. Genomic techniques have the potential

The main purpose of this document is to provide guidance on how to integrate the genomic-based approaches tested in FishGenome into data collection carried out in bottom trawl surveys. Firstly, the data types and data collection process in bottom trawl surveys (as is usually carried out) is described. Then, a brief description of the genomic approaches that were tested in the pilot studies in FishGenome is presented. These approaches are: Close-kin mark-recapture (CKMR), Restriction site-associated DNA sequencing (RAD-Seq) for population connectivity and substructure, epigenetic age determination (DNAm) and environmental DNA (eDNA). The next section explains the main aspects to consider for the integration of genomic sampling into bottom trawl surveys, such as the requirements of the genomic method being considered and the survey characteristics (vessel capacity, available staff, etc.). Finally, the sampling experience from FishGenome is presented, including the sampling design and protocols followed for each genomic method. General recommendations are provided at the end of the document.

This report describes the results obtained by the application of several novel HTS genomic techniques - Close Kin Mark Recapture (CKMR), connectivity, stock substructure, epigenetic age determination by DNA methylation (DNAm), environmental DNA (eDNA), and sex marker search - in the Pilot studies performed by the FishGenome Consortium. To discuss the suitability, potentiality pros and constraints of these novel genomic techniques, to complement and improve current fisheries assessments.

An increasing number of studies using marine environmental DNA (eDNA) approaches are showing its potential application in marine fisheries management by helping and simplifying some of the labor-intensive traditional surveys required to assess exploited populations and ecosystem status. eDNA approaches can help to ecosystem-based fisheries management by providing information of species composition; surveillance of invasive, rare and/or endangered species; and providing estimates of species abundance. Because these potential uses in fisheries and conservation sciences, the number of studies applying eDNA approaches in marine realms have expanded in the very last years. However, it is observed a lack of consistency across studies when applying pipelines for data analyses making results difficult to compare among them. Such lack of consistency is partially caused by poor knowledge in the management of raw sequences data, and analytical methods allowing comparative results. Hence, we review here the essential steps of eDNA data processing and analyses to get sound, reproducible, and comparable results, providing a set of bioinformatics tools useful for each step. Altogether this review presents the state of the art of eDNA data analyses towards a comprehensive application in fisheries management promoting sustainability.

A report to describe the protocols used by the FishGenome Consortium to: i) collect biological and environmental samples – Sampling Protocols ii) isolate DNA from biological and environmental samples – DNA isolation Protocols iii) perform the laboratory work required for the application of each technique iv) analyse bioinformatically the sequences derived from the laboratory procedures for the purpose of assessing fish and fish communities using Close Kin Mark Recapture (CKMR), environmental DNA (eDNA) and epigenetic age determination (DNAm). Sampling protocols detailed in this document have been designed tailoring the specific needs of the MEDITS and IBTS research surveys to allow implementation without disturbing the standard procedures carried out on board during the regular Fisheries research surveys. Application to other surveys may require small adjustments of the sampling protocols.

Flemish Cap cod collapsed in the early 90’s after facing multiple threats due to climate variability and anthropogenic pressures. A master 35-year cod growth chronology was reconstructed in the present study by using sclerochronology techniques. Three additional chronologies were developed to account for the age-dependent effect of maturity and fishing intensity on growth. All chronologies showed that cod growth has decreased over the years and it is currently at the minimum level in the historic of data series. Hierarchical mixed-effects models were applied to the master chronology to identify extrinsic drivers of growth. The master chronology was divided into 3 periods: Premoratorium (1988-1998), Moratorium (1999-2009), and Postmoratorium (2010-2019). Our results showed that drivers of cod growth in the Flemish Cap varied between periods (Premoratorium: bottom temperature and fishing mortality, Moratorium: cod and pandalus abundance, and Postmoratorium: cod abundance and thickness of the cold intermediate layer). Our results suggest that density-dependence processes, food availability, and global warming may be behind the lack of recovery of the Flemish Cap cod stock. The Flemish Cap cod growth chronology shows variations on growth over time and identifies its drivers, having the potential to be included in the NAFO stock assessments or to be used to set dynamic reference points that guarantees the stock sustainability