Genetic variation in red cell membrane fragility in Atlantic salmon and rainbow trout

Aquaculture, 98 ( 199 1) 349-354 Elsevier Science Publishers B.V., Amsterdam

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Genetic variation in red cell membrane fragility in Atlantic salmon and rainbowtrout T. Gjedrem, ELM. Gjaen and R. Salte AKVAFORSK, Institute ofAquuculture Research, P.0. Box IO.N-1432h-NLH, Norway

(Accepted 14 February 1991)

ABSTRACT Gjedrem, T., Gjoen, H.M.and Salte, R., 1991. Genetic variation in red cell membrane fragility in Atlantic salmon and rainbow trout. Aquacdture, 98: 349-354. The strength and flexibility of cell membranes are of particular importance for fish which routinely experience changes iu the surrounding te&mperature.496 fingerlings of Atlantic salmon from 50 fulland 25 halfsib groups and i 33 fingerlings of rainbow trout from 14 fullsib groups were used to study the total as well as the genetic variation in cell membrane strength, measured as red cell membrane fragility. Fragility is defined as the salinity at which 50% of the red cells are hemolyzed, and gradient as the regression coefficient of the fragility cume at the turning point. Both parameters showed significant differences between Atlantic salmon and rainbow trout. The mean fragility curve for rainbow trout was similar to the fragility curve for the family of Atlantic salmon which had the strongest cell membranes. Heritabii;t;r was high for fragility and medium for gradient. There was no significant genetic correlation between red cell fragility and susceptibility to experimental infection with furunculosis.

INTRODUCTION

Functional cell membranes are crucial for all living cells, and in poikilotherms, which routinely experience changes in ambient temperature, membrane function and structure are highly dependent on the fatty acid composition of the diet (Cossins and Lee, 1985 ) . This relationship is further accentuated in farmed fish since they are neither allowed to choose their own ration, nor the waters of their liking. The genetic component in this complex appears, however, to be more uncertain. Recently Salte et al, ( 1987) showed that degeneration of endothelial and red cell membranes was part of a basic pathological process in ‘Witra disease” in farmed Atlantic salmon. This disease affects any age-class of salmon and is more prevalent in winter; rainbow trout is, on the other hand, rarely affected (Salte et al., 1987). Choosing the erythrocyte as a model cell, it was shown that the physical (and presumably the functional) properties of the cell mem00448486/9 l/$03.50

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brane of affected salmon could be greatly improved by increasing the level of highly unsaturated fatty acids of the n-3 family in the diet (Salte et al., 1988 ). These findings raised the question whether the ability to strengthken the cell membranes by restructuring the membrane lipids is correlated with susceptibility to disease. Tnbreeding programs one purpose is to increase disease resistance; one major problem is, however, $3 measure the trait. The purposes of this investigation were, therefore: ( 1) to study the total as well as the genetic variatiun in cell membrane strength, measured as red cell membrane fragility, in Atlantic salmon and rainbow trout; and (2) to study the relationship between red cell membrane fragility and resistance to disease. Furunculosis was chosen because an experiment studying genetic variation in resistance to experimental Aeromonas salmonicida infection was carried out simultaneously (Gjedrem et al., 1991). MATERIAL AND METHODS

The experiment was carried out at AKVAFORSK’s research station at Sunndalsnrra. Atlantic salmon and rainbow trout fingerlings were used. Samples of ten Atlantic salmon fingerlings were drawn at random from each of the 50 fullsib- and 25 halfsib families; the same families were used by Gjedrem et al, ( 199 1) for testing resistance to furunculosis. Data for rainbow trout were obtained on 133 fingerlings sampled from 14 fullsib families. The families of Atlantic salmon and rainbow trout are part of a national selection program where the environmental conditions for all families within species are standardized. Rainbow trout and Atlantic salmon are reared in different barns, but are fed the same diet, have the same water supply, and are given the same management. Blood was drawn from the caudal vein into heparinized evacuated bloodcollecting tubes ( VenojectR ) . For rainbow trout this was done in the fall when the fish had been fed for about 7 months; Atlantic salmon were about 1 year old. The method uked for measuring cell membrane fragility was previously described by Salte et al. ( 1988): 12 salinities varying from 0.28 to 0.64% NaCl and from 0.20 to 0.56% NaCl were used f& Atlantic salmon and rainbow trout, respectively. The fish were kept in freshwater at 9°C during the bloodsampling period. In order to get comparable characters of fragility for each individual, the regression line through the observed percent hemolysis for each NaCl solution had to be estimated for each fish. These observations fit well to a rem versed logistic curve. The curve could be estimated by the formula: Y=eZ/( 1+e’), where z=b, +b, *:X

GENETIC VARIATION IN RED CELL MEMBRANE FRAGILITY IN SALMON AND TROUT

351

This new variable (z) is obtained by transforming the observations according to Z=lO&(Y/(l-Y)) bOand bl, regression coeffkients, are then estimated for each fish, and in this way a nonlinear regression line is obtained for each fish. To describe this line two measures were chosen (Fig. I): 1. “Fragility”: the MC1 value (%) at the turning point of the estimated fragility curve. This is also the point where 50% of the red cells are hemolyzed. 2. “Gradient”: the gradient of the fragility curve at the turning point. Harvey’s LSMLMW program (Harvey, 1987) was used to estimate the heritability for fragility and gradient based on sire and dam components, and genetic correlation between the mw.surements. The following model was used: Yijkl=p+si+du+wk+eijkl where: Y-IJkl P si dly wk eijki

fragility or gradient of the I-th fish with k-th weight, the j-th dam within the i-th sire = overall mean =random effect on i-th sire, i= 1 to 25 =random effect ofj-th dam within i-th sire?j= 1 to 2 =random effect of k-th weight, k= 1 to 496 = random residual effect, mean = 0, variance = $ and covariances assumed = 0. =

RESULTS AND DISCUSSION

The average fragilities for all families of Atlantic salmon and rainbow trout, measured as the salinity at which 50% hemolysis occurs and as the gradient, and the body weights are given in Table 1. There are large and significant differences between species for both measurements, which is illustrated in Fig. 1. It is interesting to note that th.e family of Atlantic salmon with the TABLE I Average (_f) and standard deviatiord ((T)for measurement of cell membrane fragility and body weight Character

Fragility Gradient Body weight No. of fingerlings

Rainbow trout

Atlantic salmon R

0

x

t7

0.41 - 5.58 57.40 496

0.05 0.96 12.75

0.34 -4.87 70.90 133

0.09 1.26 18.51

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Atlantic Fig. 1. Red cell fragility curves. - - - average for all families of Atlantic salmon, msalmon family with lowest fragility (strongest cell membranes); - - family with highest fragility (weakest cell membranes ); - average for rainbow trout. l

TABLE 2

Heritability and its standard error for estimates of red cell membrane fragility in Atlantic salmon Heritability Sire component

Dam component

Fragility

0.60 f 0.20

0.82 + 0.20

Gradient

0.26kO.13

0.241kO.13

“strongest” cell membranes has a fragility curve very similar to that of rainbow trout. For Atlantic salmon significant differences were found between sires and between dams within sires. For rainbow trout there were significant differences between fullsib families for both measures of cell membrane fragility. Heritability for fragility was very high, h2 = 0.60 -I-0.20, (Table 2) which is quite rare for quantitative traits. The coefficient of variation (CV) was 12, which means a quite high genetic variance. The gradient shows a heritability of 0.26 2 0.13 which together with a CV of 17 gives a lower genetic variation than for fragility. The genetic correlation between fragility and gradient was 0.74 I?0.15, and the phenotypic correlation was 0.67. Based on these results fragility measured as the salinity at which 50% hemolysis occurs is a better expression for the genetic variance in cell membrane fragility than the gradient.

GENETIC VARIATION

IN RED CELL MEMBRANE

FRAGILITY

IN SALMON AND TROUT

353

Results from an observational study on the seasonal variation in red cell membrane fragility (Salte, unpublished) indicate that rainbow trout tolerate changes,particularly drops in the surrounding water temperature, far better than Atlantic salmon; where rainbow trout respond by restructuringtheir cell membranes within days or even within hours, salmon may need weeks on the same feed and in the same environment. The present results strongly indicate that there is a substantial difference in this ability even among families within species. Such differences in physical properties of cell membranes may reflect genetic differences in the ability to respond to changes in ambient temperature, which is primarily a function of the composition of membrane lipids (Hazel, 1979). One likely explana?ion ftir these differences is a genetic variation in fatty acid desaturase activity; 6desaturase is the first and (probably) speed-limiting enzyme in the metabolism of linolenic acid, and 4-desaturase forms the end product of this pathway, docosahexaenoic acid ( DHA), which has been shown to be of prime importance in the adaptation of fish cell membranes to cold (Sellner and Hazel, 1982; Hazel, 1984). A second possible explanation is the existence of genetic difierences in the ability to incorporate fatty acids into the membrane, which involves mobilization of fatty acids from fat depots, from liver or even the intestine, and activation of available fatty acids which precedes any incorporation; little is known about species differences in this respect. Genetic differences may also exist in the rates of incorporation into particular lipid classes (acylation of phospholipids) or in the turnover rates for molecular species of lipids. Thirdly, the inter- and intraspecies differences observed may reflect differences in the susceptibility of red cell membrane lipids and proteins to oxidative damage (Hochstein et al., 1981). Since Gjedrem et al. ( 199 1) submitted the above 50 Atlantic salmon families to experimental A. saZmonicida infection, it was possible to estimate genetic correlations between fragility and mortality due to furunculosis. Correlations between family means for the traits studied would have been genetic correlations if samples from each family were significantly large. In this investigation, the number was ten fish for measurements of fragility and 100 for mortality., The correlations were low both between mortality and fragility, Y=0.14, and between mortality and gradient, r= 0.07; none of these were significant. Since one of two main extracellular enzymes and the lethal toxin produced by A.salmonicidais a hemolysin (Lee and Ellis, 1989) a correlation between fragility and mortality might have been expected. The toxin is a glycerophospholipid: cholesterol acyltransferase which in the native state is complexed with lipopolysaccharide (Lee and Ellis, 1989), and its mechanism of action may be the production of lysophospholipids in the membrane which eventually may lead to cell lysis; our results indicate that this enzyme does not require a weakene’d membrane to exert its effect.

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CONCLUSION

The results show that red cell membrane fragility has a rather large genetic

variance. It is therefore possible to strengthen the cell membrane through sekecticn. On a short term basis it is, however, more easily strengthened through dietary manipulation (Salte et al., 1988). According to the present findings, selection will not give a correlated effect on susceptibility to furunculosis. Salte et al. ( 1987 ) showed a relationship between “Hitra disease” and disorders of enduthelial and red cell membranes. Further, Standal and Gjerde ( 1987 ) estimated a heritability of h ‘=0.08 for survival in connection with “Hitra disease”, which means that there is some genetic variation in resistance to this disease. Thus, a genetic correlation between fragility and susceptibility to “Hitra disease” cannot be excluded. ACKNOWLEDGEMENTS

This work was financed by the Norwegian Council for Fishery Research. The authors wish to thank Mrs. Brit Seljebra,Mr. Gjermund ujeldnes and Mr. Petter Wdahl for technical assistance. REFERENCES Ccassins,A.R. and Lee, J.A.C., 1985. The adaption of membrane structure and lipid composition to cold. In: R. Gilles (Editor), Circulation, Respiration and Metabolism. Springer, Berlin, pp* 543-552. Gjedrem, T., Salte, R. and Gjoen, H.M., 1991. Genetic variation in susceptibility ofAtlantic salmon to furunculosis. Aquaculture, 97: l-6. Harvey, W.R., 1987. User’s guide for LSMLMW PC-l Version, Mixed Model Least-squares and Maximum Likelihood Computer Program. Hazel, J.R., 1979. Influence of thermal acclimation on membrane lipid composition of rainbow trout liver. Am. J. Physiol,, 236: R91-RlOl. Hazel, J.R., 1984. Effects of temperature in the structrire and metabo!ism of cell membranes in fish. Am. J. Physiol., 246: R460-R470. Hochstein, P,, Jain, SK. and Rice-Evans, C., 1981. The physiological significance of oxidative pertubations in erythrocyte membrane lipids and proteins. The Red Cell, Fifth Ann Arbor Conference on Red Cell Metabolism and Function, pp. 449-459. Lee, K.-K. and El!is, A.E,, 1989,The quantitative relationship of lethality extracellular protease and haemolysin of Aeromonas snlmonicida in Atlantic salmon, Saimo suhr. FEMS Microbiol. Lett., 6 1: 127-i 32. Salte, R., Nafstad, P. and Asgird, T., 1987. Disseminated intravascular coagubdtion in “Hitra disease” (Hemorrhagic syndrome) in farmed Atlantic salmon. Vet. Pathol., 24: 378-385. Salte, R., Thomassen, MS. and Wold, K., I988. Do high levels of dietary polyunsaturated fatty acids (EPA/DHA) prevent diseases associated with membrane degeneration in farmed Atlantic salmon at low water temperatures? Bull. Eur. Assoc. Fish Pathol., 83,63-66. Sellner, P.A. and Hazel, J.R., 1982. Desaturation and elongation of unsaturated fatty acids in hepatocytes fromthermally-acclimated rainbow trout. Arch. Biochem. Biophys., 213: 5866. Stand& M. and Gjerde, B., 1987. Genetic variation in survival of Atlantic salmon during the sea-rearing period. Aquaculture, 66: 197-207,