Dissecting structural and functional genomic factors underlying the resistance of Atlantic salmon fry to infectious pancreatic necrosis.
Lead Research Organisation:
University of Stirling
Department Name: Institute of Aquaculture
Abstract
Infectious pancreatic necrosis (IPN) is currently the most serious viral disease affecting the UK salmon farming industry. IPN is caused by infectious pancreatic necrosis virus (IPNV) which results in damage to the pancreas, intestine and liver of infected salmon. The annual economic loss to the UK aquaculture industry from IPN is estimated to be £5-10 million. Surviving salmon can become carriers of infection and then spread the disease to other susceptible fish, perpetuating IPN in both farmed and wild fish populations. Atlantic salmon vary in susceptibility to IPNV infection as they proceed through their life cycle. Newly-hatched salmon, i.e. fry, live in freshwater. They are particularly susceptible to IPN, and epidemics in hatcheries are typified by sudden large-scale mortalities. Subsequently, at the smolt life cycle stage, salmon alter their physiology in readiness for the move from freshwater to seawater. Salmon smolts (more specifically post-smolts) are susceptible to IPN during a period lasting from 2 - 10 weeks after seawater transfer. Certain families show genetic resistance to IPN, and we have previously shown that it is possible to identify resistant and susceptible salmon smolts using genetic markers. However, a number of questions relating to IPN resistance remain unanswered. In particular, whilst there is evidence that these genetic effects are consistent across different stages of the salmon life-cycle, i.e. consistent in smolts and fry, it is not conclusive. Furthermore, it is not known which specific genes and molecular pathways underlie genetic resistance to IPN. In an attempt to answer these questions we have three major objectives. First, we will confirm and describe genetic resistance to IPN in salmon fry and identify specific genomic regions affecting resistance. Second, we will determine which salmon genes work differently between genetically resistant and susceptible fish following infection. This will give insight into which biological mechanisms lead to genetic differences in resistance. Third, we will bring together all of the results to identify specific genes that may be responsible for the genetic resistance. The results of the study will strengthen salmon breeding programmes by providing genetic marker tests to identify IPN resistant fish early in the salmon life-cycle, thus reducing costs and reducing the number of diseased fish. The improved knowledge of the crucial genes defining IPN resistance may also contribute to the rational development of control measures against IPNV infections, including vaccination, and provide sensitive diagnostic tests. This project will be undertaken by researchers based at the Roslin Institute and the Institute of Aquaculture at Stirling University, and will also utilise the facilities and expertise of the Centre for Environment, Fisheries and Aquaculture Science, Weymouth. These UK researchers will collaborate with the Genomic Research on Atlantic Salmon Project (GRASP) in Canada, providing access to world-leading salmon genomics resources. The involvement of the innovative salmon breeding company Landcatch Natural Selection ensures that a clear route exists for the immediate commercial application of the results. This project is relevant to research supported by the BBSRC aimed at the analysis of the mechanisms of immune function and disease, and fits the priority relating to the control of infectious diseases, including the genetics of host resistance to infection.
Technical Summary
Infectious pancreatic necrosis (IPN) is the most serious viral disease affecting the UK salmon farming industry and is responsible for annual losses of £5-10 million. IPN affects two distinct life cycle stages of Atlantic salmon / fry and smolts. We have previously defined strong genetic variation in IPN resistance and mapped resistance QTL within salmon smolts but, beyond clear indications that it exists, little is known about resistance in fry. Detailed knowledge of genetic resistance in salmon fry would be particularly valuable and the comparison with resistance in smolts would be of obvious scientific interest. Therefore we propose to work with Atlantic salmon fry to i.) quantify genetic variation in IPN resistance and resolve it to specific QTL regions, ii.) determine genes and pathways underlying differences in resistance, and iii.) synthesize structural and functional genomics outputs to identify candidate quantitative trait genes. Using established experimental IPN virus challenge models, observed patterns of mortality of fry will allow i.) genetic variation to be quantified and related to resistance in smolts and ii.) QTL to be mapped using a genome scan and related to those in smolts. The differential transcription patterns of i.) fry from susceptible and resistant families, and ii.) full siblings carrying alternative QTL alleles will be determined by profiling infected fish using the TRAITS Atlantic salmon cDNA microarray and qRT-PCR. This will help to establish the molecular mechanisms that underlie genetic differences in resistance and will lead to candidate genes that will be integrated with a fine-mapping and structural genomics approach to bring us closer to the QTG. Information on the genes and pathways underlying genetic resistance to IPN will lead to more robust and effective selection for resistance, facilitate the design of vaccines and immunostimulants and improve our understanding of the basic biology of an unusual viral infection.
Publications
Ross Houston (Author)
(2010)
SNP discovery and mapping using RAD sequencing in Atlantic salmon
Ross Houston (Speaker)
(2009)
The genetic basis of resistance to Infectious Pancreatic Necrosis in Atlantic salmon.
Ross Houston (Speaker)
(2009)
The resistance of Atlantic salmon fry to IPN in freshwater is largely controlled by a major QTL
Scullion Hall LE
(2017)
The influence of facility and home pen design on the welfare of the laboratory-housed dog.
in Journal of pharmacological and toxicological methods
Tsai HY
(2016)
Construction and Annotation of a High Density SNP Linkage Map of the Atlantic Salmon (Salmo salar) Genome.
in G3 (Bethesda, Md.)
Description | A successful fry challenge methodology was developed as part of the current work that has been demonstrated to mimic the differential infection levels experienced by marine phase salmon smolts. The developed protocols will substantially assist further research in this area and can also function to shortcut breeding programmes by a generation by reducing the need for marine challenges as an indicator of disease resistance phenotype. As part of this project a 44K Atlantic salmon Agilent oligo microarray (Salar_1 / Salar_2) was designed and employed for analyses. This microarray is open to use by other researchers in the UK and internationally. This project has generated an archive comprising fry tissue from resistant and susceptible families subject to IPN challenge over a timecourse and including respective uninfected control groups. This archive, stored as tissue and trizol homogenates at -80oC, will provide material for further study of individual gene expression, microRNA and individual host responses. Refinements of techniques for preparation of libraries for RAD sequencing were developed as part of the current project, which are currently being employed by other UK laboratories. The produced libraries and RAD sequences provide a key resource for ongoing work. The success of this project has fostered a number of complementary studies investigating the genetic and transcriptomic basis for disease resistance to a number of other Atlantic salmon pathogens including infectious salmon anaemia (ISA) and salmon lice. The work undertaken in the current project has also provided the basis for a BBSRC career-path fellowship entitled "Towards the Causal Factors Underlying the Resistance of Salmon to Infectious Disease." Amongst other objectives, this fellowship will build upon the current transcriptomics study in order to identify the QTG underlying the observed resistance phenotype. |
Exploitation Route | As noted above under routes to exploitation, the research undertaken in this project is currently being used by industry for improving marker assisted selection for resistance to IPN in commercial Atlantic salmon stocks. The data produced is also being used to help develop a dense SNP chip for Atlantic salmon that will be employed by industry for selection of a broad range of farm salmon traits. Fry tissue from this project is being employed to examine family differences in susceptibility to IPN at the transcriptomic (using Illumina RNA-Seq) and genomic (using RAD -Seq) levels, which will similarly inform industry breeding programmes for resistance in Atlantic salmon. The work carried out within this project involved a direct collaboration with the Atlantic salmon breeding industry (Landcatch Natural Selection (LNS) Ltd.). This project has identified genetic markers that are currently being used to select for IPN resistance by our commercial partner, LNS, who are currently implementing SNP-based selection using these markers. This is a simple and cost-effective form of marker-assisted selection, and one which can be transferred across populations. Unlike traditional marker-assisted selection, there is NOT a prior requirement for phenotypes and genotypes on the same animals, thus the results can be applied more quickly, easily, cheaply and widely. This is a significant positive output from the project, and an excellent example of high quality science moving straight to implementation. This project has provided clear evidence of the transcriptomic differences between resistant and susceptible families, highlighting key pathways involved in resistance to infection and providing a number of gene candidates that may be indicative of the mechanism of resistance. Genes identified in the project are serving as markers for both disease state and for resistance or susceptibility in a family or population and are being employed to assist breeding strategies and for disease diagnostics. Resources generated by this project, including SNPs, archived fry tissue samples, BAC sequences and RAD-Seq sequences are all currently being employed by industry and academia to further understanding of the mechanisms of IPN resistance in Atlantic salmon with outcomes of research being employed directly by industry to improve breeding programmes for resistance providing commercially available resistant Atlantic salmon populations. The work undertaken within this project has fostered a wide diversity of follow-on work, which is similarly industry-oriented. These include: A knowledge transfer project on marker-assisted selection (MAS) for IPN resistance, a successful European Fisheries Fund project to investigate the genetic control of resistance to sea lice in salmon, a TSB Genomes UK initiative for the purposes of developing a dense salmon SNP chip, and funding from Scottish government and from the Scottish Partnership for Animal Science Excellence (SPASE) for work examining transcriptomic responses of Atlantic salmon to key viral pathogens as well as further funding from Scottish Government to look specifically at Pancreas Disease (PD) in Scottish salmon. |
Sectors | Agriculture, Food and Drink |
Description | TSB sustainable protein |
Amount | £24,391 (GBP) |
Funding ID | TP-5771-40229 |
Organisation | Innovate UK |
Sector | Public |
Country | United Kingdom |
Start | 08/2011 |
End | 07/2014 |
Title | 454 sequencing of BACs relevant to the identified QTL |
Description | 454 sequencing of BACs relevant to the identified QTL |
Type Of Material | Cell line |
Provided To Others? | No |
Impact | These sequences obtained are available for use by other researchers seeking to control viral diseases in salmon |
Title | DNA and transcriptomic sequences |
Description | DNA and transcriptomic sequences from IPN challenged / control fry families |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | Genomic and transcriptomic data generated through this project are being used to improve understanding of the mechanisms of resistance of farmed fish and are helping in the development of improved breeding strategies |
Title | Development of IPN challenge model for Atlantic salmon fry |
Description | A successful fry challenge methodology was developed as part of the current work that has been demonstrated to mimic the differential infection levels experienced by marine phase salmon smolts. The developed protocols will substantially assist further research in this area and can also function to shortcut breeding programmes by a generation by reducing the need for marine challenges as an indicator of disease resistance phenotype |
Type Of Material | Technology assay or reagent |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | The fry challenge developed through this project in now used routinely by ourselves and other research groups including government labs e.g. Cefas Weymouth and others |
Title | RAD sequencing refinements |
Description | Refinements of techniques for preparation of libraries for RAD sequencing were developed as part of the current project, which are currently being employed by other UK laboratories. The produced libraries and RAD sequences provide a key resource for ongoing work. |
Type Of Material | Technology assay or reagent |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | RADseq is being widely used by other researchers and industry to examine aspects of disease resistance in fish and many of those using this approach are using the refined techniques developed as a result of this work |
Title | Reduction and replacement of use of fish for IPN research |
Description | While this project has not directly employed techniques to reduce use of animals in science, the results of the transcriptomics analyses conducted in the current project clearly indicate that gene expression patterns observed in in vitro cell culture (see preceding BBSRC project "A transcriptomic analysis of the host-pathogen relationship in infectious pancreatic necrosis of salmon") are reflective of in vivo responses to infection. This information can be employed in future work to replace live fish experiments with alternative cell culture models. The successful challenge model developed in the current work provides a baseline for more accurate power calculations, allowing researchers to better calculate the minimum number of fish needed to resolve particular traits and to produce more parsimonious experimental designs. The identification of a highly predictive QTL in the current study reduces the need to challenge fish in order to assess disease resistance phenotype and thus directly reduces the number of fish that will be employed in experiments. |
Type Of Material | Technology assay or reagent |
Provided To Others? | No |
Impact | Following from the work conducted for IPN, similar methods are being used to look at other viral diseases in fish e.g. SAV and these studies are helping industry to control disease |
Title | Salar_1 and Salar_2 44K Atlantic Salmon oligo microarray platform |
Description | Salar_1 and Salar_2 44K Atlantic Salmon oligo microarray platform |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | These microarray designs are held by Agilent and are free for use by any interested researcher worldwide. They have been used by a number of groups including University of Aberdeen and CSIRO in Australia and a number of research projects examining responses of salmon to disease and nutritional variables. The project researchers have been happy to support and advise on the use of these microarrays as required. Many of the applications of these microarrays are directly associated with industry interests including development of improved feeds, development of parasite vaccines, understanding of the mechanisms of disease and resistance to disease. |
Title | 454 sequencing of BACs relevant to the identified QTL |
Description | 454 sequencing of BACs relevant to the identified QTL |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | These BACs are proving an important resource for homing in on the QTL identified through this project |
Title | Tissue archive of resistant and susceptible IPN challenged and control fry families |
Description | Tissue archive of resistant and susceptible IPN challenged and control fry families |
Type Of Material | Database/Collection of data |
Year Produced | 2008 |
Provided To Others? | Yes |
Impact | This material is available for use by any researchers who are working in this area. Use of this material continues to inform progress towards solving this problem in farmed salmon |
Description | Collaboration with Genepool University of Edinburgh |
Organisation | University of Edinburgh |
Department | The GenePool |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | New collaboration with Genepool, University of Edinburgh |
Start Year | 2008 |
Description | Collaborations with Glasgow Vet School |
Organisation | University of Glasgow |
Department | School of Veterinary Medicine Glasgow |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | A new collaboration with glasgow vet school has arisen through this project |
Start Year | 2008 |
Description | Collaborations with Norwegian and French Researchers |
Organisation | French National Institute of Agricultural Research |
Department | INRA Toulouse |
Country | France |
Sector | Public |
PI Contribution | Project involved new collaborations with Norwegian and French researchers |
Start Year | 2008 |
Description | Collaborations with Norwegian and French Researchers |
Organisation | NOFIMA Ås |
Country | Norway |
Sector | Private |
PI Contribution | Project involved new collaborations with Norwegian and French researchers |
Start Year | 2008 |
Description | Collaborations with Norwegian and French Researchers |
Organisation | Norwegian School of Veterinary Science |
Country | Norway |
Sector | Academic/University |
PI Contribution | Project involved new collaborations with Norwegian and French researchers |
Start Year | 2008 |
Title | SNP licensing |
Description | A licence agreement is currently being drawn up, which will govern the use by LNS of SNPs discovered in this project that are in population-wide linkage disequilibrium with IPN resistance. Governed by the rules of this agreement LNS intend to market salmon that have been selected for IPN resistance using these SNPs. LNS also intend to market the SNPs as a genetic test for IPN resistance to customers of their genetic services. |
IP Reference | |
Protection | Trade Mark |
Year Protection Granted | 2012 |
Licensed | Yes |
Impact | The SNPs developed through this project are part of a larger panel that is being used by industry to examine a range of farm production traits |
Description | 'omics in the Institute |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Article in popular press Aquaculture News Arcticle in Aquaculture News 2009 vol 36 2-3 no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
Description | Genetic Discovery Brings Welfare Benefits |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Popular press article on Genetic Discovery Brings Welfare Benefits Article in popular press Fish Update 2008 no actual impacts realised to date |
Year(s) Of Engagement Activity | 2008 |
Description | Hosting of Nuffield Students from local secondary schools |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Schools |
Results and Impact | Drs T and B have hosted / supervised one or more Nuffield students from local secondary schools per annum, with two students winning a gold award for paired work at the "Big Bang" science festival. Student projects on aspects of fish disease and molecular biology Two students winning a gold award for paired work at the "Big Bang" science festival. Student projects on aspects of fish disease and molecular biology |
Year(s) Of Engagement Activity | 2008,2009,2010 |
Description | IPN resistance isn't futile! |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Article in popular press, Aquaculture News, on IPN resistance isn't futile! Article in popular press Aquaculture News 2009 vol 36 pg 3 no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
Description | Improving disease resistance in farmed salmon |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Popular press publication on Improving disease resistance in farmed salmon Article in popular press Fish Farmer 2010 Sept/Oct 18-19 no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |
Description | Presentation on the subject of "Fish parasites in Scottish Waters" to members of the Scottish Wildlife Trust |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Presentation on the subject of "Fish parasites in Scottish Waters" to members of public belonging to the Scottish Wildlife Trust Talk on parasites in Scottish waters no actual impacts realised to date |
Year(s) Of Engagement Activity | 2009 |
Description | Primary School Visits for science education |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Schools |
Results and Impact | Over the course of this project, considerable effort has been made to enhance public engagement with science. Work with schools has included annual visits to primary schools to talk about science as part of the World of Work Day (Drs B, T and S), including demonstrations centred around viruses, aquaculture and aquatic organisms / environments. Talks on careers in science and on special topics related to aquaculture and fish disease Additional schools have requested visits following on from initial visits and we have visited further primary schools as a result |
Year(s) Of Engagement Activity | 2008,2009,2010 |