Hypoxanthine metabolism in salmon: roles in osmoregulation and the innate immune response.

Atlantic salmon accumulate various metabolites in the skin and fin when fish are in seawater (SW) environments. One metabolite, called guanine, is known to be responsible for the skin "silvering" that accompanies developmental changes associated with the migration of young salmon (called smolts) to SW. The elevated concentrations in the skin result in the formation of guanine crystals that give the skin its classic iridescent/pearlescent metallic lustre, characteristic of all salmonids. Recently we have identified that a structurally related metabolite called hypoxanthine, is also found at high levels within the skin and fin of Atlantic salmon. The functional role of this metabolite however, appears to be completely different from guanine. Although guanine is essentially insoluble in the aqueous environment of the skin cells (and hence explains its precipitation as translucent crystals), hypoxanthine is eminently soluble and in SW salmon, accumulates to very high concentrations suggesting that it may function as an organic osmolyte. Organic osmolytes increase the concentration inside cells to near-equivalent concentrations to that of the sodium chloride and other ions in the surrounding SW. This equilibration of solute concentrations inside and outside the cell limits the osmotic loss of cell water to the extracellular SW environment and therefore prevents cell dehydration, shrinkage and inevitable cell death. Another potential function of hypoxanthine, is that of an anti-microbial compound. In addition to being a precursor of guanine, hypoxanthine can be enzymatically converted to uric acid, but at the same time this processes releases hydrogen peroxide. Essentially three times as much hydrogen peroxide can be generated as hypoxanthine present within the cell. It is proposed that this highly reactive metabolite acts as a cellular disinfectant and the first line of the host's defense against microorganisms or parasites that invade the skin or fin. In higher vertebrates, hydrogen peroxide and uric acid are known to function as so-called chemo-attractant molecules, stimulating the recruitment of blood lymphocytes and macrophages to sites of injury and initiating an immune response against any invading organism. The same functions are proposed to take place in the salmon. Preliminary results suggest that this hypothesis may indeed be the case as skin regions chronically infected with sea lice exhibit higher levels of expression of a key enzyme involved in this hydrogen peroxide generating pathway. Salmon are an important species in the aquaculture industry, especially in Scotland, and a critical period in the farming of this fish is during and just after SW transfer. This is a very stressful period of the salmon's life cycle and if fish are not transferred at the optimal time the physiological adaptations required for life in SW can be compromised, resulting in increased fish mortalities from osmotic disturbances or increased infection rates. Virtually nothing is known about the roles of hypoxanthine during this critical period of the salmon's life cycle. This project will characterize the role of hypoxanthine as an osmolyte in cell volume regulation and its potential function as a hydrogen peroxide generator in skin and fin cells during sea lice infestation. The project will also determine whether various nutritional supplements added to the diet of pre-SW transfer smolts will i) affect the expression of enzymes and transporters associated with the general metabolism of hypoxanthine, ii) influence hypoxanthine levels in skin and fin, iii) alter the osmoregulatory capacity of fish following acute SW transfer and determine if iv) selective nutritional supplements can reduce the incidence of stress-induced skin lesions/infections and mortalities that often arise within 2-3 months of SW transfer.

Seawater (SW)-acclimated Atlantic and Coho salmon accumulate hypoxanthine in the skin and fin. The main function of this nucleotide is thought to be associated with the skin "silvering" process during smoltification. Recent investigations however, suggest that hypoxanthine also acts as an essential osmolyte that may also be a component of the innate immune response within the skin. Hypoxanthine is present at concentrations of 10-50 mM in tissue extracts indicating it is a major functional organic osmolyte in the skin. Consequently hypoxanthine is essential for the regulation of epithelial cell volume and maintenance of cell viability when salmon enter SW. Hypoxanthine can also be metabolised by xanthine dehydrogenase/oxidase to uric acid. Depending if the enzyme functions as a dehydrogenase or an oxidase, this pathway can generate two moles of hydrogen peroxide for every mole of hypoxanthine oxidized. In addition, the enzyme uricase oxidises uric acid and generates yet another molecule of hydrogen peroxide. Both hydrogen peroxide and uric acid can act as anti-microbial agents and also as chemo-attractants signaling the recruitment of various immune cells. Does hypoxanthine have a role in the immune response to microbial and/or parasite infections of the skin? Preliminary results indicate that this may indeed be true as skin regions chronically infected with sea lice exhibit higher levels of expression of mRNA for xanthine dehydrogenase/oxidase. This project will determine if purine-based nutritional supplements added to the diet of pre-SW transfer smolts will i) affect the expression of enzymes/transporters associated with hypoxanthine metabolism, ii) influence hypoxanthine levels in skin and fin, iii) alter the osmoregulatory capacity of fish following acute SW transfer and determine if iv) these nutritional supplements reduce the incidence of stress-induced skin lesions/infections and mortalities that often arise after SW transfer.

The main beneficiaries of this study will be the aquaculture feed companies and the aquaculture industry itself. It is well recognised that there are major cost implications in salmon production are in operations associated with increases in mortality and disease followed closely by low growth rates in salmon smolts and post-smolts. The research programme will investigate the hither-to unknown role of hypoxanthine as a novel osmolyte and component of the innate immune system within the skin and fin of SW-acclimated salmonids. The possibility that tissue hypoxanthine levels can be boosted with dietary supplements will be a major scientific finding and one which could have substantial impacts to the aquaculture industry both in terms of improvements in fish production and animal welfare. The study will determine if purine nucleotide-containing dietary supplements can increase the salinity tolerance and reduce skin lesions/infections in SW-acclimating smolts and therefore increase growth rates and reduce mortalities and disease post SW transfer. In addition to obvious benefits for the feed companies and aquaculture industry, any reduction in stress and disease incidence will be reflected in the increased flesh quality and together with lowered mortalities will reflect increased on-farm production and reduced fish costs to the consumer. The aquaculture industry is approaching a point where the production of fish is at the limits of capacity of existing farm sites, with any increases in production dependent upon new initiatives that enhance fish survival and growth. Increasing the number of farmed sites does not come without substantial expenditure particularly if this involves developing offshore aquaculture production sites. If successful the project will make a major impact on increased fish production through lowered mortalities and increased growth rates following the stressful FW/SW transfer period. Enhanced output of Scottish salmon will be of benefit for the increasing demand for this product on the global markets, has the potential to increase the number of people directly employed in the salmon farming industry in Scotland (estimated at over 2200 in 2012) and also increase the number of jobs reliant on the aquaculture industry in the UK (estimated at over 6000 in 2012).

In addition, the potential development of robust metabolic biomarkers related to hypoxanthine metabolism raises the potential for future development of improved diagnostic tools for use in the aquaculture industry in particularly smolt testing in salmon farming. By determining new biomarkers for smoltification it will be possible to develop novel and improved tests for determining the optimal time for smolts to be transferred to sea cages. This will reduce the number of incidents of 'failed smolts' in the culture process, which has lead to significant financial losses. It is also possible that certain dietary regimes may increase fish resistance to sea lice, a major parasite and recurring problem for the salmon farming industry and the well documented effects on wild stock welfare and the environment. The research outputs are directly related to key research objectives prioritised by the Ministerial Group for Sustainable Aquaculture (MGSA) Aquaculture Science and Research Strategy Nutrition Task Group including, 1. Exploration of the metabolic interactions of dietary amino acids, soluble carbohydrates, fatty acids and lipids and 2. immunological effects of dietary ingredients. The project also addresses the NERC and BBSRC's strategic research priorities to promote research on sustainable economies and the impact of land management on biodiversity and, end users will include CEFAS, Marine Scotland, The Marine Directorate of the Scottish Parliament, SEPA and SSPA.