Viruses that cause disease and death in farmed salmon harm the UK's economy, increase the carbon and environmental footprint of production, and reduce fish welfare. Two viruses, called piscine myocarditis virus (PMCV) and infectious salmon anaemia virus (ISAV) are particularly damaging. For every eight salmon that die of known causes on fish farms, one death is caused by PMCV. ISAV is less commonly detected in the UK, and the UK is currently considered free of the disease. However, introductions of ISAV have been shown to nearly destroy entire salmon industries in other countries and therefore protecting against new outbreaks remains critical as the UK expands salmon production to meet growing global demand. Despite the severe problems caused by these viruses, we do not fully understand how they transmit between farms. Our lack of understanding of how viruses enter and persist on farms has prevented action that can improve disease control. We propose to use virus genomic sequencing and genomic analyses to determine the routes of ISAV and PMCV transmission, and how and where pathogenic lineages emerge. Many viruses evolve very rapidly, and acquire genetic changes as they transmit between hosts. Using the evolved patterns of virus genetic changes to reconstruct ancestral relationships between viruses can be used to track how they transmit. Generated virus genomes additionally provide important information on the genetic basis for disease severity, and how pathogenic strains emerge. This approach has been widely used in public health to understand disease in humans, but opportunities to study disease transmission in farmed fish are being missed. We will answer several key questions that will improve our understanding of how to eliminate transmission. Fish are moved between farms as they grow, from egg and progeny in tanks, to inland freshwater sites as juveniles, and finally to the sea at adulthood. Firstly, we will reconstruct transmission to determine whether each virus tends to transmit between farms at the same production stage, or between different stages. This would indicate where biosecurity improvements are best focused. Secondly, we will determine what characteristics of a farm make it more likely to become infected or transmit these viruses with other farms. Identified characteristics could be used to improve surveillance for viruses at certain points in the fish supply chain, leading to more rapid detection and control if viruses are present. Thirdly, we will determine whether wild fish are a source of infection of PMCV, or whether they only rarely become infected when they come into contact with infected farms. The distinction is important to determine whether wild fish can easily infect fish on farms, or whether in contrast wild fish must be better protected to prevent establishment of virus transmission within wild populations. Finally, we seek to understand how genetic changes in the virus lead to more or less severe disease. This could be used to improve genetic 'early warning systems' for risk of disease emergence in fish or improve vaccinations, and are more broadly useful to understand how related viruses evolve and cause disease in other species. The approaches that we use in this research will also be broadly applicable to viral diseases, improving our ability to rapidly respond to new viral outbreaks in humans and other animals Together, our results will contribute to reducing disease amongst farmed salmon. This will lead to improved fish welfare, a more sustainable industry, cheaper cost of fish to the consumer because of reduction in loss, and greater economic value of the industry. Our results will be valuable to the UK, where salmon is the second most valuable food export, but also to producers and consumers in other markets worldwide.

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, disease outbreaks have a major negative effect on salmon production and animal welfare. Infectious salmon anaemia (ISA) is one such disease, and is sometimes dubbed 'salmon flu' because it is caused by a virus (ISAV) that is similar in to influenza. At present, ISA is a notifiable disease in the UK, meaning farmers are obliged to cull their stock in the event of an outbreak. Vaccination and biosecurity cannot fully prevent outbreaks, and developing disease resistant strains of salmon is high priority. Selective breeding can result in moderate improvements in disease resistance of salmon stocks and may take many generations. However, a revolutionary approach known as genome editing has potential to rapidly increase the rate at which disease resistant salmon can be produced. Genome editing involves the use of "gene scissors" to precisely cut the genome at a specific location, leading to small-scale targeted changes in the DNA sequence. In this proposal, genome editing technology will be used to investigate genes underlying resistance to ISAV, and potentially to produce a disease-resistant salmon. The first stage of the project is to identify target genes that will be edited. This will be achieved by measuring the ISAV resistance in a selective breeding program. Genetic markers dispersed throughout the salmon genome will then be used to map individual genes that contribute to variation in resistance in the population. Salmon from resistant and susceptible families will also be sequenced and to identify candidate genes and mutations causing this genetic effect on resistance to ISAV. In parallel to the 'forward genetic' approach described above, a 'reverse genetic' approach to identifying ISAV resistance candidates will be employed using cell culture models. A genome editing method known as CRISPR-Cas9 will be applied to destroy the function of key candidate ISAV resistance genes in the cell lines. Two methods of choosing candidate genes will be used. The first is based on prior knowledge of the biology of the interaction between the virus and the host cell, partly harnessing extensive research which has been performed on ISAV's close relative influenza. The second is to use the genes affecting natural resistance identified in the forward genetic screen described above. These edited cell lines will be infected with ISAV, and the impact of the edited gene on ISAV resistance and cellular response to infection will be assessed. This will build on an ongoing project to develop genome editing for salmon cell lines. Finally, genome editing will be used in Atlantic salmon embryos to test the highest priority ISAV resistance genes, especially where knockout of the gene has an impact on resistance in cell culture. Targeted editing of the genes will be performed by microinjecting newly fertilised embryos, which will be reared until the freshwater fry stage. These edited embryos, and unedited controls from the same family, will be challenged with ISAV. The nature and frequency of the edited genes in the resistant and susceptible salmon will be measured. This proposal has potential to create Atlantic salmon with resistance to a problematic viral disease (ISA) using a novel breeding technology. As such, it could have major animal welfare and economic impacts via prevention of outbreaks and subsequent culling of stocks. The approaches will be directly relevant to other viral disease in fish aquaculture. While the regulatory landscape for application of edited animals in food production is uncertain, a successful outcome of this proposal will provide a high profile example of the power of this technology to understand biology and to improve food security and animal health.

Southern ocean processes are intimately linked to some of the most pressing challenges faced by society today: climate change, ocean acidification and the sustainable management of marine resources. To address these challenges we need to improve our understanding of the natural causes and consequences of Southern Ocean change. Sustained observations, which can only be large enough and maintained through international collaboration, will enable us to measure the baseline and future trends in the distribution and function of the ecosystem. The Southern Ocean Network of Acoustics (SONA) represents a group of scientific institutes and industrial partners who have united to measure an under-sampled component of the ecosystem - the mid-trophic level - , to agree common standards and protocols for data collection and processing and with a view to provide that data on an open access basis. The Southern Ocean comprises more than 10% of the world's oceans and plays a critical role in the Earth's climate system. Changes in the Southern Ocean have global ramifications. The Southern Ocean has warmed, freshened, become more acidic and ocean circulation patterns have changed. Climate models suggest that it will continue to warm and freshen with less sea ice and changes in ocean currents. Changes in marine ecosystems in the Southern Ocean have been linked to these changes. The structure and function of Southern Ocean ecosystems are dictated by the unique habitat that exists in the Southern Ocean defined by seasonal light, low temperatures, water chemistry, depth, currents and sea ice. Potential impacts of climate change on the structure and function of the marine ecosystem will depend upon the sensitivity of the organisms to change in the physical environment. Detecting that change will depend on our ability to monitor the environment. Mid-trophic level organisms range in size from small plankton (<2 cm), which drift with currents, to larger nekton (>10cm), which have the ability to swim freely. They are a diverse group that include squid, salps, krill and fish and play a critical role in Southern Ocean ecosystems. They regulate primary production involved in biogeochemical cycles and are prey for top predators (e.g. penguins, seals and whales). In the Southern Ocean alone they have a biomass equal to the human population, and globally they represent the largest unharvested biomass on the planet. Despite their pivotal role they remain one of the least known components of the ecosystem. Making scientific measurements in the Antarctic oceans is not a simple task and bio-acoustic methods (using sound to measure organisms in the water column) present a cost-effective, widely used (frequently found on research and fishing vessels), large scale method for collecting information on the mid-trophic level organisms. However, in order for data to be comparable between vessels, standards and protocols are required in addition to bounding measurements with validation procedures. SONA will set these standards. SONA will use bio-acoustics to monitor mid-trophic organisms at large spatial scales annually along transits to Antarctic research bases and fisheries sites. It will unite multi-national calibrated acoustic data from both research and fisheries vessels into a common accessible database. This data will inform on ecosystem based fisheries management, marine planning and monitoring impacts of climate change. Ultimately the project will input data and knowledge to international bodies such as the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR) and international programmes such as Integrating Climate and Ecosystem Dynamics (ICED) through the SENTINEL programme and the Southern Ocean Observing System (SOOS) and provide a road map for a global acoustic database of the mid-trophic level.

The Arctic Ocean is exhibiting exceptional levels of warming and ice loss, which are expected to profoundly change the types of animal communities that coexist and the nature of interactions between animals and between animals and humans. In particular, the extent of ice cover in regions that are not permanently ice covered has been declining. The loss of summer sea ice has led to an increase in plankton in open ice free waters, while under-ice algae which are also important food-sources to Arctic species have declined with the retreating ice cover. The underlying marine warming is also making it possible for species from warmer waters to move northward. While these incomers have been flourishing, some native Arctic species have declined in abundance. Several of these species including resident Arctic cod and the incoming Atlantic cod are important in fisheries, which constitute the most direct benefit that society derives from these high-latitude waters. The Arctic species have adaptations to cold that may help protect them from the incoming species to some extent, but to date we have very few ideas of what the long-term ecological outcomes of these recent changes will be. The Coldfish project will focus on the fish of these waters, exploring how their behaviour, specifically the types of food they eat, changes across a wide range of sites which vary in ice cover, the extent to which the incoming species are present and in other environmental respects. We will track fish diets by measuring the ratio of different stable isotopes of carbon, nitrogen and sulfur in their tissues. By comparing the range of different isotopic compositions found in populations of fishes living in different communities and under different physical conditions, we can answer a series of important questions about the current and future states of Arctic ecosystems. For instance, the sensitivity of an ecosystem to change depends on how many different species perform similar ecological roles and are therefore able to compensate if some species are lost. We will determine the extent of this so-called 'redundancy' in terms of fish diets by measuring the degree of overlap in isotopic compositions between populations. We will measure how effectively carbon is transferred from surface waters to the seabed, and how this varies in regions of contrasting ice cover and differing fish communities. We will also study how the incoming species are responding as they move northwards into colder waters, whether their feeding habits and metabolism are changing as a result, and whether the incoming species are likely to compete directly with those native to the Arctic. Coldfish investigators bring a mix of expertise in arctic biogeochemistry, polar fish biology, marine ecology and stable isotope ecology, and this blend of methods and approaches will help deliver new insights. Our project builds on ecological study in the Barents Sea sector of the Arctic Ocean, and benefits from close integration with extensive ecological surveys co-ordinated by our project partners in Norway.

Controversy surrounds the actual impacts of Atlantic salmon farming on wild salmonid stocks, fed by the lack of direct evidence for or against many potential impacts, with uncertainty an increasing impediment to sustainable industry development and effective management of wild stocks. This applies to the potential impact of the introgression of farm genomes into locally adapted wild populations from breeding of farm escapes. Escapes do occur and are recognized as inevitable, but are a very small fraction of farm stocks and vary in numbers both locally and temporally. The majority of escapees are expected to die without breeding but some do remain in or ascend rivers and spawn. However, a detailed understanding of actual levels of interbreeding and introgression in most rivers is lacking which, along with an understanding of the adaptive differentiation of farm and wild salmon, is required to establish the actual impact of this potential interaction on the productivity and viability of wild populations. Detection and quantification of interbreeding and introgression requires diagnostic markers for farm and wild genomes. Genetic differentiation of farm and wild genomes can evolve through founder effects, selective breeding and domestication selection and is observed in respect of a variety of molecular markers. However, existing molecular markers are not fully diagnostic and regionally constrained in their usefulness. Unfortunately, marker panels screened for useful variation have been small and arbitrary such that they are unlikely to include the most informative loci and to be context specific, limiting their power and transferability. To properly address the issue of introgression molecular markers are required that are highly diagnostic across all farm and wild populations. These markers will be in genomic regions involved in domestication and controlling the expression of selected economic traits. What is known of the genomic architecture of domestication and most economic traits indicates their control is polygenic, making the targeting of specific gene regions in the search for markers difficult. In contrast, recent advances in genomics make possible genome scanning and genome-wide association studies (GWAS) which can provide a high resolution assessment of molecular differentiation between different individuals or populations across the genome. Different GWAS strategies can be employed but two are deemed optimal in the current context. Firstly, a GWAS will be carried out using a new Atlantic salmon SNP (single nucleotide polymorphism) containing 930k nuclear SNPs, recently developed in collaboration with the salmon farming industry. This will be carried out on a broad base of representative farm and wild stocks. Secondly, GWAS will be carried out to identify temporally stable epigenetic DNA-methylation base changes induced by rearing fish in culture by comparing groups of single source wild fish reared in the wild and in culture. The study will deliver the first general understanding of domestication related molecular genetic differentiation between farmed and wild salmon and identify the best markers for identifying farm salmon in the wild and assessing genetic introgression of farm genes into wild populations. The work will deliver a more robust and generally applicable tool for determining the actual levels of escapes and introgression in wild salmon populations. Following field calibration and independent validation, the diagnostic methodology defined in the study is expected to provide the basis for generating the evidence needed to clarify the debate on levels of escapes and introgression and the long term impacts of introgression on population viability. This will help to define more clearly the path forward for the sustainable development of the salmon farming industry in the UK and elsewhere in the North Atlantic region and help to inform management priorities for wild Atlantic salmon stocks.