Genetic effects of hatchery rearing in Atlantic salmon

Aquaculture, 33 (1983) 3340 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

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GENETIC EFFECTS OF HATCHERY REARING IN ATLANTIC SALMON

T.F. CROSS and J. KING* Salmon Research Trust of Ireland, Farran Laboratory, *Present address: Dublin 9, Ireland (Accepted

Central

Fisheries

Board,

Newport, Co. Mayo (Ireland)

Balnagowan,

Mobhi

Boreen,

Glasnevin,

1 December 1982)

ABSTRACT Cross, T.F. and King, J., 1983. Aquaculture, 33: 33-40.

Genetic effects of hatchery rearing in Atlantic salmon.

Six polymorphic enzyme loci were examined electrophoretically in a sample of wild Atlantic salmon smolts from the Burrishoole river in western Ireland and in samples of artificially-reared fry hatched in 1981 and parr hatched in 1979. These hatchery reared fish were the progeny of five generations of artificially reared sea ranched salmon which had originally come from the Burrishoole river. Selection for growth and disease resistance was practised and between ten and 30 females and similar numbers of males were used as parents in each generation. Gene frequencies differed significantly at a number of loci between the wild and the artificially reared samples. Erosion of genetic variability, as measured by mean heterozygosity and mean number of alleles over the six loci, was evident in both hatchery reared samples. It is argued that the observed genetic changes are caused by founder effects and genetic drift rather than selection by some aspects of the artificial rearing regime. The importance of using adequate numbers of parents in hatchery rearing is stressed, since it is shown that differences between wild and reared populations are as great as between natural populations from Irish rivers.

INTRODUCTION

Hatchery production can cause loss of genetic variability by inbreeding, because of the use of very few individuals as broodstock. This is particularly so in the case of species with high fecundities like salmonid fishes (Ryman and Stahl, 1980). In Atlantic salmon (Salmo salur), hatchery production can result in 50% survival from egg to smolt compared with 0.5% in the wild (Piggins, 1981). This factor, coupled with the finite size of smolt producing units can lead to the use of small numbers of parents as broodstock. High genetic variability is a prime requirement of any programme of artificial selection (Gosling, 1982). In contrast, inbreeding can result from any form of selection, either purposeful or inadvertent (Newkirk, 1978).

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Particular gene frequencies present in wild populations may have adaptive value. Hatchery production, using small numbers of parents, can alter these frequencies by genetic drift (Allendorf and Phelps, 1980) and may reduce the viability of hatchery reared individuals. Selection pressures in the hatchery may also be different from those in the wild, thus further reducing the fitness of reared individuals to survive in the wild. Specific electrophoreticallydetectable proteins determined by structural genes are a part of the genome which can easily be visualised (Wilkins, 1972). While representing a very small part of the genome, changes observed in structural protein genes can be assumed to be indicative of changes in the entire genome (Allendorf and Phelps, 1980). Inbreeding should lead to a reduction in variability at such loci and to changes in gene frequencies. Here, we compare genetic variability and allele frequencies for six variable enzyme loci in two groups of hatchery reared Atlantic salmon with wild fish from the same river system. MATERIALS

AND METHODS

Artificially-reared Atlantic salmon (S&no salar) fry or parr were obtained from the hatchery operated by the Salmon Research Trust of Ireland (S.R.T.I.) on the Burrishoole river system in Co. Mayo, on the west coast of Ireland. This hatchery has been in operation for more than 20 years and produces salmon smolts for ocean ranching on an experimental basis. In years when there have been large numbers of broodstock, some subjective selection for large maternal size and resistence to ulcerative dermal necrosis has been applied. Ova from females stripped late in the season have been disposed of, thus selecting for early maturation among females and further reducing the numbers of parents of the hatchery population. Equal numbers of progeny were sampled from each parent for the fish hatched in 1979 and 1981. These parents were the fifth generation progeny of hatchery rearing, using grilse (one sea-winter fish). This stock originated from wild grilse from the Burrishoole system. In general, two males have been used to fertilise the ova from each female and males were often used more than once. The generation interval is either 3 or 4 years because approximately l/3 of the fish from each family become smolts 1 year after hatching, whilst the rest become 2-year-old smolts. Thus, there is an overlap of generations. The progeny of ten to 30 females and similar numbers of males have been reared each year. Smaller numbers of parents were used in hatchery operations in earlier years. Wild salmon smolts were collected in 1981 from the traps, operated by S.R.T.I. on the Burrishoole system. These wild smolts do not represent an entirely virgin stock. There has been some natural spawning by hatcheryreared individuals and a small number of eyed ova have been planted in the spawning streams (Piggins, 1981). However, this is estimated to have rarely contributed more than 10% to the annual smolt run.

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Fish were either used fresh or stored at -25°C for up to 3 months. Sections of skeletal muscle or liver were soaked overnight at 4°C in an equal volume of 2% 2-phenoxyethanol in 0.1 M Tris HCl buffer, pH 7.5, to extract enzymes. Extracts were centrifuged at 3000 rpm for 3 min prior to electrophoresis on 13-14s “Sigma” horizontal starch gels (230 X 190 X 6 mm). Details of buffers, run times and staining methods are given in Cross and Ward (1980). The enzyme loci studied are given in Table I. These six loci were found to be the most polymorphic in a study of 59 protein loci in a sample of salmon parr from the River Blackwater in the south of Ireland and amounted to 94.6% of the observed heterozygosity (Cross and Ward, 1980). TABLE I Enzymes and loci examined in Atlantic salmon Enzyme

Sorbitol dehydrogenase Malate dehydrogenase Malic enzyme Isocitrate dehydrogenase Asp&ate aminotransferase s: m:

Enzyme council number

Abbreviation

Locus

1.1.1.14

SDH

Sdh-1 Sdh-2

1.1.1.37

MDH

Mdh,-3

1.1.1.40

ME

Me,-2

1.1.1.42

IDH

Idh-3

2.6.1.1

AAT

Ao t,-2

supematant or cytoplasmic enzyme. mitochondrial enzyme.

RESULTS

Genotype frequencies for these six loci in wild and reared samples from the Burrishoole system are given in Table II. Hardy-Weinberg expectations, derived from the gene frequencies listed in Table III, are given in parentheses in Table II. No significant deviations from Hardy-Weinberg expectations were observed in the wild sample. Deviations occured at S&-l in the reared sample hatched in 1979 (G = 6.16; df = 1;P < 0.05) and at Sdh-1 (G = 7.22; df = 1; P < 0.01) and Me,-2 (G = 12.53; df = 1; P < 0.001) in the reared sample hatched in 1981 (Sokal and Rohlf, 1969). Since one of the assumptions of the Hardy-Weinberg equilibrium is an infinitely large population size, the deviations observed here are due probably to the small number of parents being used to produce each generation. In the last 12 years, the wild run has varied from 300 to 750 pairs (Piggins, 1981).

36 TABLE II Genotype frequencies parentheses

for wild and reared salmon. Hardy-Weinberg

expectations

Reared salmon Locus

Genotype

Wild salmon

Sdh-1

loo/loo 1 OO/- 72 -72/-72 100/100 100/28 28/28 100/l 00 100/8 7 87/87 115/115 115/100 100/100 116/116 116/100 100/100 100/l 00 100/74 74/74

62(63.4) 32( 29.2) 2( 3.4) 94( 94.0) 2(1.9) - (0.1) 124(124.0) l(1.0) -

Sdh-2

Mdh,-3

Me,-2

Idh-3

Aat,-

15(X5) 56(49.5) 29( 32.5) l(1.1) 20( 19.8) fjS(88.1) 79(80.5) 41(38.0) 3(4.5)

Hatched 1979 75(71.4) lg(26.2) 6(2.4) 98(98.0) 2(2.0) 100 47(45.6) 41(43.9) 12(10.5) l( 2.9) 31(27.3) 62(63.9) 83(82.8) 16(16.4) l(O.8)

Hatched 1981 67( 69.9) 35(29.1) - (3.0) 102 103 6( 14.1) 63(46.9) 31( 39.1) - (0.4) 12(11.2) 83(83.4) 84( 84.9) 19(17.2) -_(0.9)

TABLE III Allele frequencies and sample numbers for wild and reared salmon Reared salmon Locus

Alleles

Wild salmon

Hatched 1979

Hatched 1981

Sdh-1

100 - 72 n 100 28

0.813 0.187 96 0.990 0.010 96 0.996 0.004 125 0.430 0.570 100 0.101 0.899 109 0.809 0.191 123

0.845 0.155 100 0.990 0.010 100 1 .ooo

0.828 0.172 102 1.000

100 0.675 0.325 100 0.176 0.824 94 0.910 0.090 100

103 0.375 0.625 100 0.063 0.937 95 0.908 0.092 1.03

Sdh-2

Mdh,-3

I”00 87

Me,-2

115 100

Zdh-3

1;s 100

Aat,-

1:0 74 n

102 1.000

are in

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Significant differences in allele frequency (Table III) occur between the wild sample and the reared sample, hatched in 1979, at Me,-2 (G = 24.54; df = 1; P < O.OOl), Zdh-3 (G = 4.76; df = 1;P < 0.05) and A&,-2 (G = 9.33; df = 1; P < 0.01)and the reared sample, hatched in 1981, at A&,-2 (G = 9.12; df = 1; P < 0.01). There are also significant differences between the reared samples, hatched in 1979 and 1981, at Me,-2 (G = 36.66; df = 1; P < 0.001) and Zdh-3 (G = 11.73; df = 1;P < 0.001). In Table IV, allele frequencies from the wild Burrishoole fish at these loci, are compared with frequencies in samples from the R. Blackwater (Co. Cork) and R. Moy (Co. Mayo) (Cross and Healy, in press). Mean genetic distances (Nei, 1972) between these samples at these six loci (Burrishoole vs Blackwater, b = 0.019, Burrishoole vs Moy, D = 0.020 and Blackwater vs Moy, b = 0.007) are comparable to distances between wild and hatchery reared samples from the Burrishoole system (Wild vs Reared, hatched 1979, Zj = 0.022; Wild vs Reared, hatched 1981, a = 0.003 and Reared, hatched 1979 vs Reared, hatched 1981, Z? = 0.032). TABLE IV Allele frequences and sample numbers for salmon samples from three Irish rivers Locus

Alleles

Burrishoole R.

R. Blackwater’

R. Moy’

Sdh-1

100 -72

Sdh-2

lE0 28 n 100 87

0.813 0.187 96 0.990 0.010 96 0.996 0.004 125 0.430 0.570 100 0.101 0.899 109 0.809 0.191 123

0.582 0.418 73 0.952 0.048 73 0.982 0.014 111 0.453 0.547 118 0.168 0.832 119 0.672 0.328 119

0.564 0.436 70 0.925 0.075 47 0.993 0.007 72 0.521 0.479 96 0.165 0.835 76 0.835 0.165 88

Mdhs-3

Me*-2

1;s 100

Idh-3

116 100 n 100 74 n

Aat,-

‘From Cross and Healy (in press).

Mean heterozygosity, both observed and expected, together with mean number of alleles over the six variable loci in the Burrishoole wild and reared samples, are given in Table V. Observed heterozygosity is the average proportion of heterozygotes per locus, whereas expected heterozygosity is the average proportion of heterozygotes per locus predicted by the Hardy-Wein-

38 TABLE V Details of observed and expected average heterozygosity (together and average numbers of alleles per locus in wild and reared salmon

Wild salmon

with standard errors)

0.240 f 0.086

0.218 + 0.076

2.000

Reared salmon -hatched

1979

0.185

0.195

+ 0.069

1.833

Reared salmon -hatched

1981

0.214 + 0.098

0.173 + 0.074

1.667

t 0.067

go: mean observed heteroaygosity. He : mean expected heterozygosity derived from Hardy-Weinberg N, : mean number of alleles per locus.

expectations.

berg equilibrium. The former value may be more meaningful in the present context, because of the small numbers of parents used in hatchery practice, as discussed above. Mean heterozygosity and average number of alleles per locus measure different things. For the two alleles per locus situation, expected heterozygosity is greatest when both frequencies are 0.5 and decreases as one allele approaches fixation. Thus heterozygosity measures overall genetic variability independent of gene frequencies at individual loci. With reference to the number of alleles per locus, alleles with low initial frequencies are much more likely to be lost due to inbreeding than alleles with higher frequencies. Thus, mean number of alleles per locus varies depending on the initial array of gene frequencies. It can be seen than mean heterozygosity is lower in the reared samples, with a parallel reduction in mean number of alleles per locus. While these differences are not statistically significant (Wilcoxon test), there is an apparent trend of reduced values in the reared samples. DISCUSSION

A decrease in genetic variability with artificial rearing was observed in this study, as in investigations with S&no trutta (Ryman and Stahl, 1980) and Sdmo clurkii (Allendorf and Phelps, 1980). In all of these cases, small numbers of parents were being used as broodstock. The inbreeding coefficient (AF), expressed as 1/2iV,, where N, (effective number of parents ) = T$$ d

, Q

increases most rapidly when the sex ratio varies greatly from equality. Ryman and Stahl (1980) show diagrammatically how inbreeding per generation increases as the effective number of parents decreases. It is not possible to apply this model directly to the present data because of the overlap of generations, but it is clear that the actual numbers of parents being used

39

would partly explain the observed decreases in genetic variability. This model also assumes that selection has not been practised. Any degree of selection results in a greater degree of inbreeding and the selection for female size, early maturation and disease resistance, applied in the present case, might explain the remaining decrease in genetic variability. At the University of Washington, strong selection for growth has been applied for a number of generations to a strain of rainbow trout (Salmo guirdneri). Heterozygosity in the selected strain, as measured electrophoretically, was found to be three times lower than in wild populations (Allendorf and Utter, 1979). It is of interest to note that this is not the case in many other domestic strains of rainbow trout (Busack et al., 1979). Strong selection in the University of Washington strain also appears to have caused inbreeding depression, as evidenced by low survival at hatching (Allendorf and Utter, 1979). In addition, a detrimental effect of inbreeding on growth rate has been documented in rainbow trout (Kincaid, 1976). There is no evidence of inbreeding causing detrimental effects to the freshwater stage of the S.R.T.I. stocks. However, sea survival of reared smolts, as measured by adult returns, is on average, only % as good as wild smolts, although growth of reared fish in the sea is comparable to that of wild (Piggins, 1981). Sea survival of reared Atlantic salmon smolts in Sweden has been shown to be decreased by inbreeding (Ryman, 1970). Decrease in genetic variability at loci which confer disease resistance, may lead to an increase in susceptibility to disease (Allendorf and Phelps, 1980). Changes in allele frequencies and loss of rare alleles were also observed in the present study. The rearing regime has therefore not succeeded in producing fish identical with the wild stock. It has been argued that selection pressures in artificial rearing may differ from those in the wild. The significant differences observed at S.R.T.I. between the two hatchery populations suggests that small numbers of broodstock have had a much more profound effect on genetic variability than any aspect of rearing practice. If selection alone were involved, it could be expected that different year classes, as represented by the two reared samples, would be similar, since conditions during their rearing differed very little. An alternative explanation for the significant allele frequency differences between the wild and reared samples might be that more than one reproductive unit occurs in the Burrishoole system and that each sample represented a different mixture of populations (M@ller, 1970; Stahl, 1981). This seems unlikely since the wild sample, taken throughout the smolt run of 1981, was in Hardy-Weinberg equilibrium, implying that only one breeding unit occurred in the wild salmon of that year. Genetic distances between hatchery-reared and wild salmon from the Burrishoole system, were as great as genetic distances, measured electrophoretically using the same loci, between samples of wild salmon from other Irish rivers (Cross and HeaIy, in press). This suggests that artificial rearing, using small numbers of parents, can lead to significant genetic variation over

40

a few generations. The magnitude of this variation is similar to that between a number of wild populations from geographically diverse rivers in Ireland. ACKNOWLEDGEMENTS

We thank D.J. Piggins and C.P.R. Quigley for technical assistance.

Mills for helpful

discussion

and D.

REFERENCES Allendorf, F.W. and Phelps, S.R., 1980. Loss of genetic variation in a hatchery stock of cutthroat trout. Trans. Am. Fish. Sot., 109: 537-543. Allendorf, F.W. and Utter, F.M., 1979. Population genetics. In: W.S. Hoar, D.J. Randall and J.R. Brett (Editors), Fish Physiology, Vol. 8. Academic Press, New York, pp. 407-454. Busack, C.A., Hallibarton, R. and Gall, G.A.E., 1979. Electrophoretic variation and differentiation in four strains of domesticated rainbow trout (Salmo gairdneri). Can. J. Genet. Cytol., 21: 81-94. Cross, T.F. and Healy, J.A. The use of biochemical genetics to distinguish populations of Atlantic salmon, Salmo salar. Ir. Fish. Invest. Ser., in press. Cross, T.F. and Ward, R.D., 1980. Protein variation and duplicate loci in Atlantic salmon, Salmo salar L. Genet. Res. Camb., 36: 147-165. Gosling, E.M., 1982. Genetic variability in hatchery-produced Pacific oysters (Crassostrea gigas Thunberg). Aquaculture, 26: 273-287. Kincaid, H.L., 1976. Inbreeding depression in rainbow trout. Trans. Am. Fish. Sot., 105: 273-280. MQller, D., 1970. Transferrin polymorphism in Atlantic salmon (Salmo salar). J. Fish. Res. Board Can., 27 : 1617-1625. Nei, M., 1972. Genetic distance between populations. Am. Nat., 106: 283-292. Newkirk, G.F., 1978. A discussion of possible sources of inbreeding in hatchery stock and associated problems. In: J.W. Avault (Editor), Proceedings of the Ninth Annual Meeting, World Mariculture Society, pp. 93-100. Piggins, D.J., 1981. Annu. Rep. Salmon Research Trust, Ireland, No. XXV. Westport, Ireland, 43 pp. Ryman, N., 1970. A gentic analysis of recapture frequencies of released young of Atlantic salmon (Salmo salar L.). Hereditas, 65: 159-160. Ryman, N. and Stahl, G., 1980. Genetic changes in hatchery stocks of brown trout (Salmo trutta). Can. J. Fish. Aquat. Sci., 37: 82-87. Sokal, R.R. and Rohlf, F.J., 1969. Biometry. W.H. Freeman and Co., San Francisco, CA, 573 pp. Stahl, G., 1981. Genetic differentiation among natural populations of Atlantic salmon (Salmo salar) in northern Sweden. In: N. Ryman (Editor), Fish Gene Pools. Ecol. Bull. (Stockholm), 34: 95-105. Wilkins, N.P., 1972. Biochemical genetics of the Atlantic salmon, Salmo salar L. II. The significance of recent studies and their application in population identification. J. Fish Biol., 4: 505617.