Aquaculture 176 Ž1999.

209–226

Growth and genetic variation in hatchery-reared

larval and juvenile Dover sole, Solea solea žL. /
Athanasios Exadactylos 1, Audrey J. Geffen ) , John P. Thorpe
Port Erin Marine Laboratory, School of Biological Sciences, The UniÕersity of LiÕerpool, Port Erin, Isle of
Man IM9 6JA, UK
Accepted 3 March 1999

Abstract

The variation in multiple-locus and single-locus heterozygosity, and its correlation with growth
rate, were examined in laboratory-reared juvenile Dover sole from two populations. The genetic
structure of the populations was examined to test for genotype differences among individuals
surviving past metamorphosis Ž70 days after hatching.. Using enzyme electrophoresis, specimens
were examined for 14 scorable loci, seven of them polymorphic. Larval growth rate was
significantly affected by rearing treatment, but differences between treatments declined with age.
Larvae from broodstock originating from the Irish Sea sole were larger at hatching, grew faster,
and initiated metamorphosis earlier than larvae from broodstock originating from the Skagerrak–
Kattegat Norwegian sole. The groups were indistinguishable on the basis of multilocus or
single-locus heterozygosity and allele or genotype frequencies, after metamorphosis. Three
measures of genetic diversity Žpercentage of loci polymorphic, number of alleles per locus and
heterozygosity. were considerably lower than those of wild populations; batches from these sole
hatcheries clearly demonstrated loss of genetic diversity, and marked changes in gene frequencies
of cultured batches relative to the wild populations from which the parents were derived. Only two
loci in the Norwegian sample were deficient in heterozygotes, whereas others exhibited heterozy-
gote excess. These heterozygote excesses did not seem to be related to a particular allele or alleles.
There was little evidence in this data set that the degree of multilocus or single-locus heterozygos-
ity correlates with growth rate in S. solea. This conclusion agrees with the general observation
that the correlation is not expected in populations where there is no heterozygote deficiency.
q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Allozyme electrophoresis; Growth rate; Heterozygosity; Heterozygote deficiency

)
Corresponding author. Fax: q44-1624-831001; E-mail: geffen@liverpool.ac.uk
1
Fax: q30-31-473310.

0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 1 1 2 - X
210 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

1. Introduction

In many species, quantitative traits related to fitness are correlated with the number of
allozyme loci for which an individual is heterozygous Žreviews by Mitton and Grant,
1984; Allendorf and Leary, 1986; Zouros and Foltz, 1987; Zouros and Pogson, 1994..
Growth rate is a fitness-related trait in many groups of animals and plants. Positive
associations between growth rate and heterozygosity have been reported in, for example,
several bivalve species Že.g., Zouros, 1987; Gentili and Beaumont, 1988; Koehn et al.,
1988; Alvarez et al., 1989; Gaffney et al., 1990; Pogson and Zouros, 1994.. Various
factors may affect the ability to detect this correlation ŽGosling, 1989; Volckaert and
Zouros, 1989; Booth et al., 1990; Pecon Slattery et al., 1993; Wright and Guttman,
1995.. A positive correlation between heterozygosity and growth rate could be easier to
detect in a large spawning population ŽGaffney and Scott, 1984; Koehn and Gaffney,
1984; Zouros, 1987., or in young animals during their period of maximum growth rate
ŽDiehl and Koehn, 1985.. The number and class of genes analysed may also have an
influence on this result ŽKoehn et al., 1988., as also may environmental stress and
reproductive condition ŽRodhouse et al., 1986; Gentili and Beaumont, 1988; Scott and
Koehn, 1990.. Failures to show heterozygosityrgrowth relationships in hatchery-reared
animals can be explained by the limited genetic background of animals produced by
hatchery spawnings ŽGaffney and Scott, 1984; Mallet et al., 1986.. The environmental
homogeneity of hatchery conditions may reduce any advantages that accrue to heterozy-
gous individuals in the wild ŽMitton and Grant, 1984..
Two main hypotheses have been put forward to explain why faster growth is often
observed among more heterozygous individuals. Zouros and Foltz Ž1987. suggested that
although the alleles studied may not themselves have differential effects on growth rate,
they may serve as markers that are nonrandomly associated with deleterious genes.
Thus, faster growth in more heterozygous individuals may simply result from reduced
homozygosity for deleterious recessive genes. Alternatively, the faster growth of more
heterozygous individuals may be due to greater metabolic efficiency of heterozygotes
relative to homozygotes. Danzmann et al. Ž1988. found a significant negative correlation
between mean heterozygosity and oxygen consumption in the rainbow trout, Salmo
gairdneri, measured under both normal and stressful conditions. They proposed that
heterozygotes may enjoy lower costs of routine metabolism because of decreased
energetic costs of biosynthesis, resulting in more energy for growth. It has also been
suggested that while the relationship between heterozygosity and growth rate may hold
true at an early stage in the life cycle of an organism, it may later become obscured
through differential mortality of particular enzyme genotypes ŽKoehn and Gaffney,
1984; Diehl and Koehn, 1985..
The effects of heterozygosity may have important implications in the aquaculture
industry. Conflicting results from natural populations in the field argue for more
laboratory-based studies where factors such as stocking density, temperature, salinity,
food ration, can be controlled. In this paper, the variations in multiple-locus and
single-locus heterozygosity and their effects on growth rate are examined at early life
stages of two samples of hatchery-reared Dover sole batches. The sizes of homozygote
 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226 211

and heterozygote fish were compared to test the hypothesis that larger Ži.e., faster
growing. individuals would be more heterozygous than smaller individuals.

2. Materials and methods

Fertilised eggs of S. solea were obtained from sole broodstocks held by the CEFAS
laboratory in Conwy, North Wales, UK, in April 1994 and from the Flødevigen Marine
Research Station, Arendal, Norway, in June 1995. Spawning occurred naturally in both
broodstocks. The fertilised eggs were incubated for 3 days at 128C and 138C, respec-
tively, before being shipped to Port Erin Marine Laboratory, Isle of Man and were at
stage IIIA ŽSimpson, 1959. on arrival. The broodstock in Conwy consisted of adult
Dover sole trawled from the Irish Sea, ICES division VIIa. The Arendal broodstock
consisted of fish trawled from the Kattegat–Skagerrak, ICES division IIIa. Hatching
commenced 8 days after fertilization for the Conwy batch and 4 days after fertilization
for the Arendal batch. The day of hatching was denoted as day zero.

2.1. Rearing conditions

One day after hatching, approximately 1000 larvae Žestimated volumetrically. were
stocked into each of eight experimental tanks. Metamorphosis commenced at day 28 for
the Conwy batch and was complete by day 38. For the Arendal batch, metamorphosis
commenced at day 21 and was complete by day 31.
All tanks were supplied with flowing sea water at about 0.2 l miny1 and constant
gentle aeration was provided by air stones. Illumination at 8.0–10.0 mmol my2 sy1 was
provided by fluorescent ‘cool white’ lamps suspended above the tanks. The photoperiod
was 16L:8D which approximated the natural cycle at this latitude. Temperature was
12.4 " 1.38C.
The newly hatched larvae from Norway were small Ž3.22 " 0.16 mm, Fig. 1a.; and
the alga Isocrysis galbana ŽParke, 1949. was added to all experimental tanks to improve
first feeding conditions. Rotifers Ž Branchionus plicatilis . raised in a continuous culture
of I. galbana, were used as prey until complete yolk sac absorption. Feeding on newly
hatched Artemia nauplii ŽArtemia Systems, Ghent, Belgium, AF grade. started at days
six and five in the Conwy and Arendal batches, respectively. From day 20 onward, the
larvae were fed with Artemia nauplii for 24 h with Super Selco ŽArtemia Systems..
Two stocking densities and four feeding rations were tested, each combination
replicated in two tanks. During the larval period Ž1–29 days after hatching., the larvae
were stocked at 18 or 40 larvae ly1 , and fed at 1 or 3 Artemia nauplii mly1 . After
metamorphosis Ž30–70 days after hatching., the Irish Sea sole were grown at stocking
densities of 4 or 8 fish ly1 , and fed at 1 or 3 Artemia nauplii mly1 . After metamorpho-
sis, the Norwegian sole were grown at stocking densities of 8 or 11 fish ly1 , and fed at 1
or 3 Artemia nauplii mly1 . The positions of the replicate tanks were randomised across
all treatments.
212 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

Fig. 1. Changes in Ža. mean lengths and Žb. yolk sac volume for the two groups of sole larvae during the yolk
sac stage, 0 to 7 days after hatching ŽIrish Sea: solid squares; Norwegian: open diamonds.. Each point
represents the mean"1 s.e. of at least 20 larvae, all treatments combined.

2.2. Sampling procedure

Every 5 or 6 days, at least five larvae were sampled at random from each tank. The
larvae were anaesthetised in chilled MS-222 Žtricaine methanesulfonate, Sandoz. Ž25 mg
ly1 . and viewed with a stereomicroscope equipped with a video camera. For each larva,
the standard length Žmm., yolk sac length Žmajor axis, mm. and yolk sac depth Žminor
axis, mm. were measured to the nearest 0.01 mm using the NIH Image Analysis Ž1.58
VDM. software package for Macintosh. After the measurements were made, the larvae
were returned to the rearing tanks.
Yolk sac measurements beyond day seven after hatching were impossible because of
high pigmentation. The yolk sac volume Žmm3 . was calculated using the formula:
2
Vys s 4r3p Ž L ysr2 .Ž Dysr2 . ,
 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226 213

where Vys s yolk sac volume; L ys s yolk sac length; Dys s yolk sac depth ŽBlaxter and
Hempel, 1966..
Larval wet weight Žmg. was measured by weighing ŽMettler A30 balance. individual
sole on pre-weighed aluminium pads, after blotting for 10–15 s on a paper towel. Before
weighing, larvae were starved for 6 h to minimise food residue in the gut ŽIrvin, 1973..
Larval growth was estimated as change in wet weight or length Žboth log 10 -trans-
formed. over time. Mean growth rates for each group were estimated using the formula:
G s Ž log 10 W2 y log 10 W1 . rt ,
where, G is the instantaneous rate of weight increase, or growth coefficient, W1 is the
average weight of fish larvae at the beginning of the growth interval, W2 is the average
weight of fish larvae at the end of the growth interval, and t is the number of days in the
interval ŽHoude, 1989.. Growth was expressed as the percentage increase in wet weight
per day by multiplying G = 100. The mean condition factor K ŽHeap and Thorpe, 1987.
for each group at the end of the growth interval was estimated as:
3
K s Ž Mean weight. r Ž Mean length. .

2.3. Statistical methods

Analysis of variance ŽANOVA. was used to test for fitness of multiple regressions
between treatments. Analysis of covariance ŽANCOVA. was introduced to test for
differences between the slopes of the regression lines. The general linear model ŽGLM.
option was applied since sample sizes between treatments were not equal. A Tukey test
ŽZar, 1984. was used to make multiple comparisons among slopes. Analysis of variance
ŽANOVA. was used to compare yolk sac volumes, standard lengths and wet weights
between the two batches and within the treatments. Student’s t-test was used to test for
differences between means. Regression, ANOVA, and ANCOVA analyses were carried
out on Minitab version 10.1 ŽMinitab, 1994. and SYSTAT 7.0 ŽSPSS, 1997.. The
significance level used for the growth analysis was P - 0.05.

2.4. Analysis of electrophoretic data

By day 70, the sole larvae had grown large enough to provide adequate tissue for
electrophoresis. From each batch, 15 individuals from each tank, in total 240 individu-
als, were chosen randomly for screening. Standard lengths were measured to the nearest
millimeter and wet weights to 0.01 g. Electrophoresis was carried out on skeletal muscle
with standard horizontal starch gel techniques.
Specimens were examined for 14 scorable loci, seven of them polymorphic Žfor
details, see Exadactylos, 1997.. The mean number of alleles per locus, the mean
observed heterozygosity Ž Ho . and mean expected heterozygosity under random mating
Ž He . per locus and per group ŽNei, 1978., plus the proportion of polymorphic loci
ŽHarris and Hopkinson, 1976., were calculated to assess intra-population variation. The
observed heterozygosity values were analysed in order to assess any correlation between
growth rate and heterozygosity. Individuals were classed according to the number of loci
214 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

at which they were heterozygous. The effects of heterozygosity class, source and rearing
treatment on fish lengths were tested by ANOVA. Differences in mean size of
heterozygotes and homozygotes at each locus were tested using a t-test. Positive values
of t indicate that heterozygotes are larger than homozygotes and vice versa.
Departures of genotype frequencies from Hardy–Weinberg expectations were tested
using exact tests ŽLessios, 1992.. Heterozygote deficiencies per locus in each group
were estimated for all polymorphic loci using the inbreeding index FIS and their
significance was tested using the method of Li and Horvitz Ž1953.. For each locus, the
statistical significance of inter-population variation in allele frequencies was calculated
by contingency x 2 analysis. Genetic differentiation between populations was assessed
using unbiased FST values of Weir and Cockerham Ž1984. and significance tested Ž Ho :
FST s 0. using the equation of Workman and Niswander Ž1970.. Modified Rogers
distance Ž D T . ŽWright, 1978. and Rogers Ž1972. genetic similarity were used to

Fig. 2. Length at age of Irish Sea Žsolid squares. and Norwegian Žopen diamonds. groups of sole larvae Ža.
during the larval stage and Žb. after metamorphosis. Each point represents the mean"1 s.e. of at least 20
larvae, all treatments combined.
 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226 215

estimate genetic distances. The statistical significance level for the genetic analyses was
P - 0.001.

3. Results

3.1. LarÕal growth

During the yolk sac stage, larvae from the Irish Sea and Norwegian broodstocks grew
faster at lower densities than at higher densities ŽANCOVA: F1,7 s 4.46, P - 0.01;
F1,7 s 4.72, P - 0.01.. The daily mean yolk sac utilisation rate, estimated from the
change in log 10 -transformed volume over time, was significantly affected by densities
only, in both groups of larvae ŽANCOVA: F1,7 s 2.42, P s 0.02; F1,7 s 2.27, P s 0.02..
Yolk sac volumes in Irish Sea larvae were significantly larger ŽANOVA: F1,478 s 20.58,
P - 0.01. and perhaps were utilised more conservatively over a longer period of time,
than in Norwegian larvae ŽFig. 1b.. Larval growth in terms of daily increase in length
during the yolk sac stage, was significantly faster in the Irish Sea larvae ŽANOVA:
F1,478 s 375.62, P - 0.01, Fig. 1a..
After yolk sac absorption, growth in length was also significantly faster at lower
stocking densities in both groups of sole larvae ŽANCOVA: F1,7 s 2.42, P s 0.02;
F1,7 s 2.13, P s 0.04.. Larvae from the Irish Sea broodstock continued to grow faster
than larvae from the Norwegian broodstock between yolk sac absorption and metamor-
phosis Ž F1,788 s 13.72, P - 0.01; Fig. 2a.. The increase in wet weight was not affected
by stocking density in either group. The weight-specific growth coefficient for the Irish

Table 1
Mean length Žmm., wet weight Žmg., weight growth coefficients Ž G ., and condition factors Ž K . for sole
spawned from Norwegian and Irish Sea broodstocks before and after metamorphosis
Source of broodstock Length Weight Weight-specific Growth
Žmean "1 s.d.. Žmean "1 s.d.. growth coefficient efficiency Ž K .
Ž G%.
Norway
yolk sac stage 3.22"0.16 – – –
Ž0–7 DAH.
pre-metamorphosis 5.55"1.99 5.41"2.83 0.349 0.010
Ž7–29 DAH.
post-metamorphosis 12.77"1.90 20.05"9.05 0.349 0.020
Ž33–76 DAH.

Irish Sea
yolk sac stage 3.62"0.28 – – –
Ž0–7 DAH.
pre-metamorphosis 6.01"1.47 8.86"1.16 3.020 0.013
Ž7–29 DAH.
post-metamorphosis 17.51"1.92 109.17"25.31 4.697 0.020
Ž33–76 DAH.
216 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

Sea sole larvae was about 10 times higher than that of the Norwegian larvae. The mean
condition factor for each group was similar ŽTable 1.. This result suggests that the
growth efficiency of both groups was similar, and that the faster wet weight increase of
Irish Sea larvae was a consequence of the higher initial wet weight values.
After metamorphosis, the increases in larval length ŽANCOVA:F1,7 s 2.39, P s 0.02;
F1,7 s 2.84, P s 0.01. and weight ŽANCOVA:F1,7 s 1.8, P - 0.05; F1,7 s 3.9, P -
0.01. were significantly higher at low stocking densities in both groups of sole.
Metamorphosed sole from the Irish Sea broodstock continued to grow faster in both
length ŽFig. 2b. and weight ŽFig. 3. than those from the Norwegian broodstock.
Significant differences were detected by pairwise comparison of slope coefficients and
one-way ANOVA of standard length data Ž F1,863 s 205.48, P - 0.01. and wet weight

Fig. 3. Weight at age of Ža. Irish Sea Žsolid squares. and Žb. Norwegian Žopen diamonds. groups throughout
the larval stage and after metamorphosis. Each point represents the mean"s.e. of at least 20 individuals, all
treatments combined.
 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226 217

Table 2
Allele frequencies for sole juveniles from Norwegian and Irish Sea broodstocks
Locus Population
Conwy Arendal
AAT-2
100 1.000 1.000

AAT-4
100 1.000 1.000

G6PDH-3
100 1.000 1.000

GPI-1
100 0.967 0.996
120 0.033 0.004

GPI-3
90 0.004 0.050
100 0.996 0.950

G3PDH-1
100 1.000 1.000

LDH-1
100 1.000 1.000

LDH-2
100 0.971 0.988
110 0.029 0.013

LDH4
100 1.000 1.000

MDH-2
100 1.000 1.000

MDHP-1
90 0.000 0.038
100 1.000 0.962

MDHP-2
90 0.000 0.033
100 1.000 0.967

PGM
90 0.017 0.000
100 0.950 0.992
115 0.033 0.008

PGDH
80 0.017 0.000
100 0.983 1.000

N s120 individuals for each group.

218 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

data Ž F1,649 s 220.5, P - 0.01.. The estimated weight-specific growth coefficient Ž G%.
was 13 times higher in the Irish Sea sole than in the Norwegian sole. However, the
estimated growth efficiency Ž K . of the metamorphosed larvae was similar in both
groups, indicating again that the initially smaller size at hatching was the underlying
reason for these growth rate differences.
Larval growth expressed in either length or wet weight at age was not significantly
affected by any of the feeding rations.

3.2. Genetic Õariation

In S. solea juveniles, three alleles were observed at the locus Pgm and two for six
other loci; Gpi-1, Gpi-3, Ldh-2, Mdhp-1, Mdhp-2 and Pgdh. The remaining seven loci
Mdh-2, Aat-2, Aat-4, Ldh-1, Ldh-4, G3pdh-1 and G6pdh-3 were monomorphic, but
were also included in the statistical analysis of genetic diversity ŽTable 2.. The
proportion of loci polymorphic Ž0.99 criterion. in the sole hatched from the Norwegian
broodstock was 42.9% and in the sole hatched from the Irish Sea broodstock was 35.7%.
The mean observed heterozygosities per locus Ž Ho . were 0.017 and 0.019, respectively
ŽTable 3..
Eleven overall exact tests revealed no significant deviation Ž P s 0.05. from Hardy–
Weinberg expectations. The x 2 contingency analysis ŽBonferroni correction for multiple
comparisons. did not detect any significant differences in allele frequencies at any locus,

Table 3
Percentage of polymorphic loci, observed and expected heterozygosity at 14 loci for two groups of
hatchery-reared juvenile sole compared with data from 33 loci in seven populations of adult sole Žsee
Exadactylos et al., 1998.
Population Mean sample Mean no. of Percentage Mean heterozygosity
size per alleles per of loci Observed ws.e.x Expectedb under
locus ws.e.x locus ws.e.x polymorphic a Hardy–Weinberg
equilibrium ws.e.x
JuÕeniles
Irish Sea 120 1.4 w0.2x 35.7 0.019 w0.009x 0.019 w0.008x
Norway 120 1.4 w0.1x 42.9 0.017 w0.007x 0.020 w0.009x
Total 0.018 w0.001x

Adults
1. CUM 50 1.8 w0.1x 63.6 0.035 w0.008x 0.043 w0.011x
2. IOM 72 2.2 w0.2x 72.7 0.071 w0.013x 0.083 w0.015x
3. GER 17.9 w0.1x 1.8 w0.1x 60.6 0.083 w0.017x 0.107 w0.021x
4. IRL 18 1.7 w0.1x 48.5 0.034 w0.008x 0.064 w0.015x
5. EAN 43 2.2 w0.1x 75.8 0.085 w0.012x 0.101 w0.014x
6. FRA 48 2.3 w0.2x 72.7 0.088 w0.013x 0.108 w0.017x
7. GRE 54 2.4 w0.2x 78.8 0.114 w0.016x 0.141 w0.019x
Total 0.073 w0.012x
a
A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.99.
b
Unbiased estimate ŽNei, 1978..
Table 4

A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

Contingency x 2 analysis for heterogeneity of allele frequencies at polymorphic loci, with pooling of rare alleles between the two groups of juvenile sole. Summary of
Weir and Cockerham’s F-statistics and Wright’s nonhierarchical Ž FDT . F-statistics for the seven polymorphic loci. Observed Ž Ho . and expected Ž He . heterozygosity
and x 2 analysis for heterozygote deficiency or excess Ž D . in each group of sole juveniles
Locus All populations Irish Sea Norway
Žno. of alleles. Contingency Weir and Cockerham’s F-statistics and Heterozygosity and x 2 analysis Heterozygosity and x 2 analysis
x2 Wright’s nonhierarchical F-statistics for heterozygote deficiency for heterozygote deficiency
Ž df . FIS FI T FST FDT x2 df Ho He D x2 Ho He D x2
GPI-1 Ž2. 5.55 Ž1. y0.027 y0.0070 0.019 0.0070 9.12 1 0.067 0.065 0.034 0.140 0.008 0.008 0.004 0.00
GPI-3 Ž2. 9.57 Ž1. 0.278 0.3020 0.034 0.0160 16.32 1 0.008 0.008 0.004 0.001 0.067 0.095 y0.298 10.67
LDH-2 Ž2. 1.63 Ž1. y0.021 y0.0180 0.003 0.0001 1.44 1 0.058 0.057 0.030 0.110 0.025 0.025 0.013 0.020
MDHP-1 Ž2. 9.17 Ž1. 0.196 0.2220 0.033 0.0150 15.84 1 0.000 0.000 – – 0.058 0.072 y0.192 4.420
MDHP-2 Ž2. 8.14 Ž1. y0.030 y0.0001 0.029 0.0130 13.92 1 0.000 0.000 – – 0.067 0.065 0.034 0.140
PGM Ž2. 7.82 Ž2. y0.032 y0.0120 0.019 0.0070 18.24 2 0.100 0.097 0.040 0.030 0.017 0.017 0.008 0.010
PGDH Ž2. 4.03 Ž1. y0.013 y0.0001 0.013 0.0040 6.24 1 0.033 0.033 0.017 0.030 0.000 0.000 – –
Mean total 45.91a Ž8. 0.062 0.0830 0.022 0.0090 12.04 1.14
a
P - 0.001.

219
220 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

indicating little heterogeneity in allele frequencies within and between groups. Unbiased
FST values ŽWeir and Cockerham, 1984. and the nonhierarchical FDT values of Wright
Ž1978. were not statistically significant ŽWorkman and Niswander, 1970.; hence, there

Table 5
Locus-specific comparison of mean standard lengths, mm Žs.e.., of homozygous and heterozygous juvenile
soles from two broodstocks
Locus Irish Sea Norway
GPI-1 Heterozygotes 28.10 Ž5.488. nd
Homozygotes 28.59 Ž4.019. nd
T y0.25 nd
df 7 nd
P 0.81 nd

GPI-3 Heterozygotes nd 18.18 Ž0.848.

Homozygotes nd 16.33 Ž2.005.
T nd 5.20
df nd 13
P nd 0.0002

LDH-2 Heterozygotes 25.53 Ž3.596. 13.63 Ž1.629.

Homozygotes 28.48 Ž3.001. 14.18 Ž1.595.
T y2.12 y0.57
df 6 2
P 0.078 0.62

MDHP-1 Heterozygotes nd 13.74 Ž2.279.

Homozygotes nd 13.93 Ž1.151.
T nd y0.22
df nd 6
P nd 0.83

MDHP-2 Heterozygotes nd 16.16 Ž1.003.

Homozygotes nd 17.26 Ž1.717.
T nd y2.81
df nd 10
P nd 0.058

PGM Heterozygotes 29.58 Ž3.328. 14.65 Ž1.626.

Homozygotes 26.96 Ž3.747. 16.06 Ž2.371.
T 2.55 y1.20
df 14 1
P 0.023 0.44

PGDH Heterozygotes 26.65 Ž2.130. nd

Homozygotes 27.44 Ž2.556. nd
T y0.72 nd
df 3 nd
P 0.52 nd

Positive t values: ŽLength het yLength hom . ) 0; negative t values: ŽLength het yLength hom . - 0; nd: no data.
Significant differences are indicated in bold.
 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226 221

was little evidence of inter-population genetic differentiation between the groups ŽTable
4.. A matrix of Modified Rogers Distance Ž D T . complemented the above observations,
giving a low value of genetic distance Ž0.023. between the two groups.

3.3. Heterozygosity and growth

Heterozygote deficiencies occurred only in the Norwegian sole at loci Gpi-3 and
Mdhp-1. The mean observed heterozygosity Ž Ho . was greater than the mean expected
heterozygosity Ž He . in most cases. Therefore, the rest of the loci in both batches
exhibited excesses of heterozygotes, but these values were not statistically significant ŽLi
and Horvitz, 1953. ŽTable 4.. However, there was no indication of an overall heterozy-
gote deficiency or excess Žrelative to Hardy–Weinberg expectations..
The lengths of the 70-day-old sole with different degrees of heterozygosity were
compared separately for each group to evaluate whether multiple-locus or single-locus
heterozygosity had any effect on growth rate. The degree of heterozygosity varied
between 0 and 2 Žhomozygous, heterozygous at one locus, or heterozygous at two loci.
in both groups ŽTable 3.. There was no significant difference in the lengths of
Norwegian sole heterozygous at one or more loci Ž F2,57 s 2.012, P s 0.14.. On the
other hand, homozygous juvenile sole from the Irish Sea broodstock were significantly
longer, and therefore had grown faster, than those heterozygous at one locus only
Ž F2,57 s 4.223, P s 0.02.. However, the homozygous fish were not larger than those
heterozygous at two or more loci. The analysis was extended to individual loci to assess
whether specific loci had any consistently larger or smaller effect on the
heterozygosity–growth rate correlation, which may have been obscured by the other
loci. No locus-specific trends were discerned ŽTable 5.. The mean juvenile standard
length of heterozygotes was larger than that of the homozygotes at only two loci; Gpi-3
in the Arendal batch and Pgm in the Conwy batch. Therefore, in the S. solea juvenile
populations studied, heterozygous individuals did not achieve higher or more uniform
growth rates than homozygous individuals.

4. Discussion

Within each population, the difference in larval standard lengths between the
different experimental treatments was highly significant, although the effects were
stronger early in the developmental period and weaker thereafter. This suggests that
larvae initially at one or both extremes of length may have been eliminated from the
population over the course of the study, resulting in greater uniformity in size and
weight among older larvae. Differential mortality may have resulted in increased
similarity in growth rates between the different stocking densities, but this mortality, if it
occurred, did not appear to be genotype-dependent, since genotype frequencies did not
vary from those expected from Hardy–Weinberg equilibrium for any locus examined.
Sole larvae from the Irish Sea broodstock grew faster and metamorphosed earlier than
those from the Norwegian broodstock, and if newly metamorphosed individuals were
sampled from a wild population, these may exhibit genetic differentiation. Both groups
222 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

were indistinguishable on the basis of multilocus or single-locus heterozygosity and

allelic or genotyping frequencies, after metamorphosis and thus, differential metamor-
phosis of individuals ŽPierce and Mitton, 1982; Mitton and Grant, 1984. does not appear
to explain the lack of a heterozygosity–growth rate correlation among sole juveniles.
Allele frequencies did not differ significantly between groups, although the eggs were
derived from parents from different wild populations. The allele frequencies were in
broad agreement with previous electrophoretic surveys of Dover sole Že.g., Quignard et
al., 1984; Pasteur et al., 1985; Kotoulas et al., 1995.. FST analyses failed to demonstrate
significant genetic differentiation between the two hatchery batches. However, the
genetic distance between the cultured sole was approximately three times lower than that
of wild adult populations from these same regions ŽExadactylos et al., 1998.. The
juveniles from these two different sole hatcheries clearly demonstrated loss of genetic
diversity, and marked changes in gene frequencies relative to the wild populations from
which the parents were derived. All three measures of genetic diversity Žpercentage of
loci polymorphic, number of alleles per locus and heterozygosity. were considerably
lower than those of the wild populations ŽTable 3.. Similar results have been observed
by Stahl Ž1987., Allendorf and Ryman Ž1987. and Verspoor Ž1988. in cultivated
salmonids. Unfortunately, direct comparison of gene frequencies between parent and
first generation populations was not feasible for the Dover sole used. The loss of genetic
variation in cultured populations gives rise to concern over the initial selection of
individuals for mating programs and indicates the care needed in broodstock selection to
avoid loss of alleles. However, procedures designed to reduce the loss of observed
heterozygosity, such as spawning several adults together Žas an alternative to pairwise
matings., do not always improve levels of genetic diversity ŽGharrett and Shirley, 1985;
Whithler, 1988; Gile and Ferguson, 1990; Gaffney et al., 1992.. The reduction in
heterozygosity in hatchery stocks is likely to have severe consequences for aquaculture
since even small amounts of inbreeding appear to have undesirable effects on, for
example, growth rate, fecundity and survival Že.g., Fujino, 1978; Ryman and Stahl,
1980; Stiles, 1981; Gjerde et al., 1983; Kincaid, 1983..
S. solea is the subject of a small, but growing aquaculture industry. Although
considerable gene exchange occurs between wild adult populations within local regions,
there is a significant stock separation over larger areas Že.g., Irish Sea, Mediterranean
Sea. ŽExadactylos et al., 1998.. Local regional populations should be used for aquacul-
ture operations or restocking, in order to protect this biodiversity; similar policies have
been suggested previously for other commercially important species Že.g., Allendorf and
Ryman, 1987; Munro, 1993.. Close attention to the practical logistics of monitoring
broodstocks effectively and carefully planned mating procedures for pairwise batch
production will be required to ensure that the genetic diversity of distant sole popula-
tions and the genetic integrity of local ones are maintained.

4.1. The relationship between growth rate and multiple and single locus heterozygosity

The correlation between the individual degree of heterozygosity and growth rate has
been observed in natural populations of many species Žreview by Zouros and Mallet,
1989.. In populations of marine bivalves, almost all cases of correlations between
 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226 223

heterozygosity and growth occurred in populations with marked heterozygote deficien-

cies ŽZouros, 1987.. However, the converse is not necessarily true; not all populations
with heterozygote deficiencies exhibit a heterozygosityrgrowth correlation. When such
a correlation occurs, its strength often varies with the degree of heterozygote deficiency:
when the deficiency declines due to selective mortality of homozygotes, the heterozy-
gosityrgrowth correlation disappears ŽSingh, 1982; Diehl and Koehn, 1985.; if the
decline of the heterozygote deficiency is for some reason halted, the hetero-
zygosityrgrowth correlation persists ŽZouros et al., 1988..
These observations suggest that the heterozygosityrgrowth correlation should be
studied by examining heterozygote deficiencies in populations over time. In the juvenile
sole examined in this study, heterozygote deficiency was not evenly distributed among
loci. Only two loci in the Arendal batch were deficient, whereas the rest exhibited slight,
nonsignificant, heterozygote excess. Ho was considerably lower than the average
heterozygosity value of adult sole populations ŽExadactylos et al., 1998., but in
agreement with the observation of Kotoulas et al. Ž1995. that in Dover sole, such
heterozygote deficiency is related to age. The heterozygote excesses did not seem to be
related to a particular allele or alleles. Heterozygote excesses suggest that heterozygosity
per se may afford some growth advantage, as been suggested for a number of cultured
mollusc species ŽZouros et al., 1988; Vrijenhoek et al., 1990; Gaffney et al., 1992.. In
the present study, both groups of sole eggs were obtained from a free-spawning
broodstock, and allozymes that act as markers of very large portions of the chromo-
somes could mask heterozygote expression ŽGaffney and Scott, 1984.. Null alleles
Žalleles present, but not expressed. could also cause heterozygous individuals to be
misidentified as homozygous and, consequently, affect estimates of heterozygote defi-
ciency or excess.
The precise relationship between heterozygosity and growth or fitness is unclear Že.g.,
Beaumont and Zouros, 1991.. Nevertheless, there is little evidence that the degree of
multilocus or single-locus heterozygosity correlates well with growth rate in S. solea. As
discussed above, this conclusion agrees with the general observation that the correlation
is not expected in populations where there is no heterozygote deficiency Že.g., Pierce and
Mitton, 1982; Zouros and Foltz, 1984; Whitehurst and Pierce, 1991; McAlpine, 1993..
The absence of the heterozygosityrgrowth correlation in populations showing no
heterozygote deficiency is a prediction of the associative overdominance hypothesis Žsee
Zouros and Foltz, 1987; Zouros and Mallet, 1989.. The observed low level of heterozy-
gote deficiency is assumed to indicate that there is no strong association between
allozyme homozygosity and homozygosity for deleterious recessive genes, and thus
there is only a weak correlation between allozyme heterozygosity and growth rate. Much
larger samples may be needed to establish a statistical correlation of this level ŽBenzie
and Williams, 1996..
It should be noted also that heterozygote superiority may result from the effect of
multiple- or single-locus heterozygosity on fitness-related traits other than growth rate
ŽDiehl and Koehn, 1985; Rodhouse et al., 1986; Zouros and Foltz, 1987.. For example,
juveniles kept in the absence of predators may show differential spending and rebuilding
of phosphogen reserves correlated with heterozygosity ŽBerger, 1976.. Gonad size and,
consequently, enhanced reproductive rates, may also be related to heterozygosity, and
224 A. Exadactylos et al.r Aquaculture 176 (1999) 209–226

there may be a positive correlation between age-specific mean heterozygosity and age
class. Therefore, further studies of the effect of heterozygosity on individual perfor-
mance need to take into account the life history characteristics of the species.

Acknowledgements

We thank B.R. Howell from the CEFAS laboratory at Conwy, Wales, and D.S.
Danielssen from the Flødevigen Marine Research Station, Arendal, Norway for provid-
ing sole eggs and R.D.M. Nash for advice and comments. A. Exadactylos acknowledges
the financial support of the Greek Scholarship Foundation.

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