Farmed Atlantic salmon is one of the world’s most popular marine based foods, with a global production of 2.86 million tonnes in 2022. This is a rapidly growing sector of the aquaculture industry, and market demand is expected to increase in the short- and long-term future. A new project led by CIGENE’s Matthew Kent aims to develop a more ethical and sustainable approach to tackle the problem of disease outbreaks in aquaculture.
The majority of salmon product that reaches our dinner plates has been raised from eggs in salmon farms (aquaculture). Today, these farms are located around the world, but for Norway this represents a particularly important industry, with more than half of the global production coming from our fjords and ocean areas.
One major challenge for aquaculture industry today involves keeping farmed fish healthy and disease-free. With large numbers of fish living in close quarters, contagious infections can rapidly spread and have devastating effects. Viral disease outbreaks such as that caused by Infectious Salmon Anemia Virus (ISAV), are major current concerns for industry, and improving disease resistance would be of enormous benefit to the health and well-being of farmed fish.
Challenge testing – a suboptimal approach to fighting fish disease in aquaculture
The most common current practice for reducing disease threats in aquaculture involves an approach called ‘challenge testing’. This relies on the hypothesis that genetic variation exists among fish that will make some individuals more (or less) resistant to a virus like ISAV, and by exposing large numbers of fish to the virus it will be possible to obtain biological samples from a range of survivors and non-survivors. Exploring and contrasting the genetic make-up of these samples can then identify genetic variants associated with resistant fish. Such variations can then be used to guide targeted breeding of more resistant offspring and/or better reveal new knowledge about the biological mechanisms underlying resistance, thereby creating opportunities for therapies (e.g. vaccines).
Matthew Peter Kent is coordinating the PETRI-fish project
Whilst challenge testing has helped to improve disease resistance in aquaculture, there are several problems that make it an unsuitable long-term solution. These include:
- Since farmers purchase their salmon from different egg producers, and cultivate them in diverse environments, a single challenge test experiment will not solve the problem for a particular disease – instead, the procedure must be repeated for many different groups of fish over time.
- The repeated mass infection of thousands of fish, many of which are killed or suffer from the resulting disease, clearly has substantial animal welfare issues
- The tests are expensive and, since there can be many variables, sometimes inconclusive
- Success is entirely dependent upon the existence of resistant fish within the test population; if all fish are sensitive then the test achieves nothing. Since fish have been intensively bred over many generations for traits such as growth, some of the natural variants (especially those related to emerging disease threats) can be absent and a challenge test will be unhelpful.
Collectively, these issues necessitate an alternative solution to identifying the genetic markers that promote resistance to different diseases.
CRISPR-Cas9 screening of salmon cells ‘in the lab’ could replace the use of live fish in challenge testing
The PETRI-fish project was recently funded by the Norwegian Seafood Research Fund (FHF), and aims to transition from repeated testing of live fish to a more sustainable approach for identifying new disease resistance markers.
Instead of using live fish swimming in a tank, the project will use salmon cells grown in petri dishes in the lab to represent fish, and a powerful genetic tool called CRISPR-Cas9 screening to generate novel genetic variation. This cutting-edge approach allows researchers to use a type of molecular scissors (CRISPR-Cas9) to precisely make changes to DNA inside many thousands of salmon cells (micro-fish) to create a vast diversity of variation.
Since ISAV can normally infect and kill these cells, the resulting genetically changed salmon cells represent an ideal substitute for the mass infection of live salmon used in traditional challenge testing (Fig 1). The cells – not salmon – can be treated en mass with an infectious agent. Those that survive infection are collected and the researchers can determine which specific genetic changes enabled them to survive a viral attack. Identifying these genes gives new insights into the biological mechanisms underlying disease resistance, and this information can be used to refine and focus fish breeding programs.
Figure 1: CRISPR screening offers opportunity to replace challenge tests on live fish with lab-based approach using fish cells
A range of benefits to lab-based screening
Applying cell culture and CRISPR-Cas9 screening to aquaculture offers a range of benefits compared to current strategies to improve disease resistance, namely:
- It will help reduce the use of live fish used in experimental testing
- It does not rely on pre-existing variation in resistant fish
- Once the tools are in place, it is cheaper and easier to implement cell-based experiments compared to live fish in a pen.
- It can more precisely identify potential targets for genome editing in breeding programs, if this becomes a feasible practice in the future. Regulations for genome editing in farmed animals are currently very strict, but conversations are ongoing as to how this can be performed in an ethical and responsible manner, especially if it removes the need for approaches such as challenge testing where so many animals are killed.
Early stages for CRISPR-Cas9 screening in salmon
The development of CRISPR-screens is challenging. Although tools exist in model organisms (e.g. mouse) and humans, the technology has not been applied in salmon or other farmed fish. Our research group has recently successfully applied CRISPR-screening in cattle and pigs and will build on our experience to develop this approach in farmed fish species. The Petri-fish project therefore represents an exciting potential development for the field.
Initial challenges include developing a panel of salmon cell cultures that can be used for CRISPR-Cas9 screening, identifying which pathogens/toxins/chemical treatments are compatible with testing in cells (some pathogens require whole fish), constructing and optimizing the delivery of CRISPRs molecular scissors, amongst other goals.
Beyond these early challenges, it will also be crucial to bridge the gap between results obtained using cells in the lab and using live salmon. Clearly, a cell is not a fish, and we need to understand how screening results in cells relate to the complex biology underpinning disease resistance in whole animals. Discovering a gene’s crucial role in a disease response is one thing, but turning this discovery into an effective treatment for fish is an even bigger challenge.
Broad long-term applications
The project will initially focus on ISA virus infection of salmon, but once the key tools are developed, the screening approach could be adapted and applied to a range of fish species and different diseases. Further, CRISPR-Cas9 screening would be a powerful tool to answer a range of important biological questions in farmed fish species, pinpointing genes that regulate processes such as metabolism, cell growth, fertility etc.
For more information on the PETRI-fish project, visit the CIGENE website project pages, or contact Project Coordinator Matthew Kent.