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HAVBRUK2-Stort program for havbruksforskning

Development of an in vitro model to study environmental factors influencing the gill epithelial function

Alternative title: Karakterisering av en cellemodell for studier av miljøfaktorer på gjellefunksjonen til Atlantisk laks

Awarded: NOK 4.0 mill.

Project Number:

294876

Application Type:

Project Period:

2019 - 2022

Location:

Partner countries:

Characterization of a cellular model to study environmental factors influencing the gill of Atlantic salmon (GILLMODEL). Gill disease in Atlantic salmon is a significant problem. Poor gill health, often caused by infectious agents (viruses, bacteria) or poor water quality, leads to both poor animal welfare and sometimes reduced growth. With the increasing use of water recycling in fish farms (RAS facilities), as well as more intensive production, new challenges related to gill health are expected. We therefore need more tools to gain a better understanding of the gills' biology. The gills' surface cells (epithelium) have an important barrier function and their integrity is very important for the salmon to be able to maintain homeostasis. So far, the lack of a cell line from the gill epithelium of Atlantic salmon has limited such research. Now members of this project group have managed to establish a promising epithelial cell line (ASG10) from the gills of Atlantic salmon. GILLMODEL, will through characterization of this cell line, develop a cell culture model to study the biology of the gill epithelium. Results: To compare the ASG10 cell line with gill tissue (obtained from salmon in the hatchery phase), we performed transcriptomic and proteomic analysis. These analyzes revealed high similarity between the gill tissue and the ASG10 cells. Furthermore, it seems that ASG10 cells produces mucus, because the growth-medium, gradually becomes quite viscous and the cells express several mucus genes. This suggests that the cell line, in addition to epithelial cells consists of mucus producing goblet cells. We have also investigated whether there are chloride cells in the cell culture, which is another type of specialized epithelial cells that are important for ion regulation in gills, but these results were negative. When grown on transwell inserts, where the ASG10 cells grow on a thin porous membrane in a separate upper compartment, the cells create a tight barrier, measured by the generation of transepithelial electrical resistance (TER) and tight junctions. Furthermore, even higher TER were obtained when the fibroblast gill cell line (ASG13) were grown together with the ASG10 cells, on the other side of the membrane, facing down to the lower compartment of the transwell. Interestingly, when carrying out direct water exposure of the ASG10 cells in the upper compartment, with ordinary growth-medium in the lower compartment, the cells withstand direct freshwater exposure (tap water), but not seawater (35%o). Water from RAS facilities (freshwater; fiskelaboratoriet, Ås) had similar effects as ordinary tap water. Exposure of ammonia dissolved in culture media or freshwater had similar effects; vacuolization due to a process called lysosome trapping, as also observed in the trout cell line, RTgill-W1. The cells did not tolerate any water exposure when grown on ordinary plastic dishes. Furthermore, we have found that ASG10 has enzymes that are important for detoxification (phase I / phase II enzymes) of contaminants / environmental toxins. In order to change gene expression in the cells, we have in collaboration with Bio-Direct (internal project at NVI) tested several transfection methods for the introduction of foreign DNA and RNA into the cells. Plasmid electroporation (Amaxa) and mRNA transfection (JET messenger) gave the best results with a minimum of 50% transfection efficiency. In conclusion: So far, we can conclude that ASG10 is a very promising model system with regard to research on how different environmental factors affect the salmon's gills.

The multifunctional nature of the fish gill combined with the large surface area exposed to the external environment makes poor gill health in Atlantic salmon is a major challenge. The cause of gill problems is commonly multifactorial, often linked to infectious agents, bad water quality or a combination of several factors. With the increasing use of recirculation aquaculture systems new challenges with the local environment occur, linked to in-house microorganisms and water quality. These factors will affect fish health, including the epithelial function of the gill. Fundamental in vitro tools for research on the tolerance and barrier function of the gill epithelium are therefore needed. The lack of a gill epithelial cell line from Atlantic salmon has hampered in vitro experiments, but members of this project group have now managed to establish a promising Atlantic salmon gill epithelial cell line (ASG10). GILLMODEL will, through a thorough characterization of the ASG10 cell line, develop a model for studying epithelial gill biology as a replacement for experimental animals. This model can be used to test environmental factors influencing the gill toxicity and integrity in Atlantic salmon. To perform this task, the Norwegian Veterinary Institute has gathered a multidisciplinary team with expertise on fish pathology and physiology, cell biology, molecular biology, infection medicine and toxicology. In addition, several national and international external partners, possessing high key competence are involved. Overall, this project will provide an important tool that can replace parts of the experimental animals used for gill research. Furthermore, the model can be useful as a monitoring system to assess water-borne challenges related to gill disease.

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Funding scheme:

HAVBRUK2-Stort program for havbruksforskning