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

Lead Research Organisation: University of St Andrews
Department Name: Sch of Medicine


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.

Technical Summary

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.

Planned Impact

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.


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Kalujnaia S (2016) Myo-inositol phosphate synthase expression in the European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus): effect of seawater acclimation. in American journal of physiology. Regulatory, integrative and comparative physiology

Description The concentration of a variety of organic osmolytes within the skin and gill of juvenile Atlantic salmon was determined in Atlantic salmon during the period of smoltification in freshwater (FW) and for up to 3 weeks following seawater (SW) transfer. Fish were fed a series of diets supplemented with various pyrimidine/purine nucleotide contents to determine any effects of these metabolites on gene expression or tissue osmolyte levels. FW/SW transfer induced characteristic increases in the abundance of some well-known osmolytes (taurine/betaine) as well as glycine and creatine. In contrast both glucose and inositol concentrations fell following SW exposure and there was no significant changes in the levels of a number of other metabolites including glycerophosphocholine, sorbitol, lactate, urea, TMAO or alanine. Hypoxanthine levels increased by 10-20% during smoltification in FW and again by a further 10-20% on SW transfer. The tissue-specific expression of over 80 genes (including 23 directly associated with purine and pyrimidine metabolism) were characterised during the smotification period and following FW/SW transfer. A few genes were identified as potential smoltification and/or salinity biomarkers however the majority of genes including all associated with nucleotide metabolism showed no significant changes in expression with development, salinity or diet. Enzyme assays designed to determine gill xanthine dehydrogenase and xanthine oxidase activities (XOR) were developed. Although there were no changes in dehydrogenase activity in any experimental group of fish, xanthine oxidase activity was significantly increased by up to 4-fold but in a sporadic fashion across all fish groups during the smoltification period. No changes in expression of XOR were found in the gill at either mRNA or protein levels in any fish group. A large number of subsequent experiments using various immunoassays, q-RT-PCR and immunoprecipitation/mass spectrometry suggested that the increased oxidase activity was associated with the transient but large increases in expression of an isoform of salmon peroxiredoxin. Initial experiments indicated that this was associated with Saprolegnia infections in FW smolts, however most recent results suggest that the induction of expression of this gene was due to the repeated hydrogen peroxide/picene treatments used to treat the fungal infections rather than the fungal infection itself. Studies were confined to healthy and Saprolegnia-infected Atlantic salmon as it proved impossible to obtain samples from other fish species or fish with other infections/infestations, as originally promised from our collaborators in Norway and Chile. In conclusion, hypoxanthine levels do increase in the skin and gill of juvenile salmon during smoltification and after transfer to SW although there appears to be no change in expression of the XOR gene at transcriptional or translational levels. Supplementation of diets with purine/pyrimidine nucleotides resulted in no significant changes in skin osmolytes or expression of any gene investigated. Fish exhibit transient and large changes in both mRNA and protein expression of an isoform of peroxiredoxin and increased oxidase activity within the gill, which is likely to be associated with chemicals used for fungal treatments, rather than a direct result of the underlying Saprolegnia infections.
Exploitation Route Future investigations are required to understand the mechanisms responsible for the increases in tissue hypoxanthine concentration during smoltification/salinity transfer. Also the mechanisms controlling the elevated expression and function of peroxiredoxin in salmon gill require investigation. Could this gene be a biomarker of stress?
Sectors Agriculture, Food and Drink,Environment

Description Studies by other groups have demonstrated that supplementation of salmonid diets with purine nucleotides is beneficial to the survival and subsequent growth rates of smolts after their transfer to seawater. The purine supplements are reported to augment the hypo-osmolatory abilities of fish in SW and also reduce the incidence of infections and disease that often appear some 6-10 weeks after SW transfer. The results of this study suggest that these dietary supplements do not augment the concentrations of the osmolyte hypoxanthine in the skin or fin and therefore are likely to induce their actions by some other unknown mechanism. Likewise as changes in dietary nucleotide supplements do not appear to change the expression (at the levels of transcription) of a wide range of genes involved in the general metabolism and transport of pyrimidine and purine nucleotides within the fish, the mechanisms behind the protective actions of dietary nucleotide supplements remain to be determined.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Environment
Impact Types Economic

Description EWOS Innovation 
Organisation University of Oxford
Department Oxford University Innovation
Country United Kingdom 
Sector Private 
PI Contribution Research into the effects of certain nutritional additives on the efficiency of osmoregulation of salmon smolts when moved to seawater.
Collaborator Contribution Provision of animals, materials and in house facilities to mediate our research
Impact None as yet
Start Year 2010
Description Marine Harvest (Scotland) 
Organisation Marine Harvest
Country Norway 
Sector Private 
PI Contribution Conducting research in an area that has potential to discover a new biomearker of smoltification in the salmon aquaculture industry.
Collaborator Contribution Provision of fish and on-site facilities for small scale experimentation.
Impact So far outputs have been limited to presentations at national and international conferences. Full publications are currently in preparation.
Start Year 2008