Aquaculture 204 Ž2002. 337–348
www.elsevier.comrlocateraqua-online
Heterozygosity and body size in triploid Pacific
oysters, Crassostrea gigas Thunberg, produced
from meiosis II inhibition and tetraploids
Zhaoping Wang a , Ximing Guo b,) , Standish K. Allen b,1,
Rucai Wang a
a
Department of Aquaculture, College of Fisheries, Ocean UniÕersity of Qingdao, Qingdao,
Shandong 266003, China
b
Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers UniÕersity,
6959 Miller AÕenue, Port Norris, NJ 08349, USA
Abstract
Triploid molluscs grow significantly faster than diploids in most species studied so far, a
phenomenon that has been referred to as triploid gigantism. Three hypotheses have been proposed,
attributing triploid gigantism to sterility, increased heterozygosity, or cell size. Testing the
heterozygosity hypothesis, the authors measured the body size and allozyme heterozygosity in
three replicates of a normal diploid group Ž2 n., an induced triploid group Ž3nCB. and a mated
triploid group produced from diploid= tetraploid mating Ž3nDT..
Body size measurements at 1 year of age showed that both 3nDT and 3nCB triploids were
significantly bigger than normal diploids, by 26% and 14%, respectively, in meat weight. The
3nDT triploids were 10% bigger than the 3nCB triploids, although the difference was not
statistically significant. Heterozygosity at five polymorphic loci averaged 0.48 for 3nCB and 0.57
for 3nDT triploids, which were 37% and 63% higher, respectively, than that for normal diploids
Ž0.35.. Differences in heterozygosity were highly significant among all three groups. Among the
three groups, there was a strong and positive correlation between meat weight and heterozygosity.
The correlation was weak or undetectable within groups or at the individual level. These results
support the heterozygosity hypothesis, but do not negate the cell size and sterility hypotheses. It is
possible that the cell size is the fundamental cause for triploid gigantism, which is better expressed
in 3nDT Žthan 3nCB. triploids because of further increases in heterozygosity or other genetic
factors. q 2002 Elsevier Science B.V. All rights reserved.
Keywords: Triploidy; Growth; Heterozygosity; Aquaculture; Crassostrea gigas
)
Corresponding author. Tel.: q1-856-785-0074; fax: q1-856-785-1544.
E-mail address: [email protected] ŽX. Guo..
1
Present address: Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine
Sciences, College of William and Mary, Gloucester Point, VA 23062, USA.
0044-8486r02r$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 8 4 5 - 6
338
Z. Wang et al.r Aquaculture 204 (2002) 337–348
1. Introduction
Triploid shellfish are important in aquaculture because of their sterility, improved
meat quality, and superior growth. Superior growth or increased body size has been
recognized as a general feature of triploid molluscs and referred to as triploid gigantism
ŽGuo and Allen, 1994a; Guo, 1999.. Triploid molluscs are significantly bigger than
diploids in almost all species studied so far, including major aquaculture species such as
the American oyster Ž Crassostrea Õirginica. ŽBarber and Mann, 1991; Stanley et al.,
1984., bay scallop Ž Argopecten irradians . ŽTabarini, 1984., the Pacific oyster Ž Crassostrea gigas . ŽAllen and Downing, 1986; Guo et al., 1996., the nobilis scallop
Ž Chlamys nobilis . ŽKomaru and Wada, 1989., the pearl oyster Ž Pinctada martensii .
ŽJiang et al., 1991., and the zhikong scallop Ž Chlamys farreri . ŽYang et al., 2000.. The
magnitude of increases in the body size ranges between 30% and 40% in most species,
and up to 50–70% in some species ŽGuo, 1999; Guo and Allen, 1994a..
Three hypotheses have been proposed, attributing triploid gigantism to sterility,
increased heterozygosity, and cell size. While all three hypotheses are supported by
some data, all of them have some limitations. The heterozygosity hypothesis, by Stanley
et al. Ž1984., proposes that the increased body size in triploids is caused by increased
heterozygosity, not by triploidy. The heterozygosity hypothesis is supported by the
observation that meiosis I triploids are bigger and more heterozygous than meiosis II
triploids and diploids in Ostrea edulis ŽHawkins et al., 1994.. However, no positive
correlation between heterozygosity and body size is observed in other studies on meiosis
I and meiosis II triploids ŽAllen et al., 1982; Beaumont et al., 1995; Li et al., 1992.. It is
important to determine if the heterozygosity plays a role in triploid gigantism. If
heterozygosity is important, triploids can be potentially improved by breeding strategies
that enhance heterozygosity.
With the introduction of tetraploids, the authors can now produce triploids by
diploid= tetraploid mating in the Pacific oyster ŽGuo and Allen, 1994b; Guo et al.,
1996.. Triploids produced from tetraploids are expected to be more heterozygous than
meiosis I and meiosis II triploids. In this study, the authors tested the heterozygosity
hypothesis in the Pacific oyster by analyzing the allozyme markers in three replicates of
a normal diploid group Ž2 n., a triploid group produced by blocking polar body II with
cytochalasin B Ž3nCB., and a triploid group produced from diploid= tetraploid mating
Ž3nDT..
2. Methods and materials
Oysters used in this study were 1-year-old Pacific oysters, Crassostrea gigas Thunberg, from three replicates of a normal diploid control group Ž2 n., a triploid group
produced by blocking polar body II with cytochalasin B Ž3nCB., and a triploid group
produced by diploid= tetraploid mating Ž3nDT.. The oysters were produced in 1995 at
Rutgers University ŽGuo et al., unpublished.. For each replicate, eggs from three to five
females were pooled and then divided into three groups. The first group was fertilized
with a diploid male and used as the 2 n control. The second group was fertilized with the
Z. Wang et al.r Aquaculture 204 (2002) 337–348
339
same diploid male and treated with CB for the polar body II inhibition to produce the
3nCB group. The third group was fertilized with a tetraploid male to produce the 3nDT
group. Different sets of parents were used for different replicates. The tetraploids used in
the crosses were produced from triploid eggs using the Guo and Allen method ŽGuo and
Allen, 1994b..
Gametes were collected by stripping gonads. All fertilization and treatments were
conducted at 23–25 8C, in filtered seawater Žto 1 mm. with a salinity of 20–24 ppt.
Fertilized eggs in 3nCB groups were treated with CB Ž0.5 mgrl. for 20 min starting
when about 50% of the eggs released the first polar body. CB was removed by rinsing
the eggs on a 20-mm-nytex screen. Larvae were reared in 210-l tanks and fed with a
mixture of Isochrysis galbana and Chaetoceros calcitrans, according to protocols
recommended by Breese and Malouf Ž1975.. During the larval culture period, seawater
temperature ranged from 23 to 278C. Larval tanks were drained for complete water
change every 48 h. All groups were set as cultchless oysters using epinephrine treatment
ŽCoon et al., 1986.. Newly set spats were held in indoor downwellers for 7–10 days
before moving to an upweller nursery system. Around Day 40 post-fertilization ŽPF.,
spats from each group were transferred to the upwellers Ž12-in. diameter and 18-in. tall.,
each receiving a 4-l per minute of Delaware Bay seawater, filtered to 100 mm. At 4
months PF, about 2000 oysters from each group were put into bags and suspended at the
over-wintering site. The winter water temperature ranged between y1 and 78C, and
salinity about 30 ppt. At 8 months PF, oysters were moved back from the over-wintering
site and transferred to 2350-gal tanks with daily changes of natural Delaware Bay
seawater. Culture density was gradually reduced to 500 per bag during the first year.
At 1-year PF, 50 oysters were randomly sampled from each replicate of each group.
The shell height, whole body weight Žwith shells., and wet meat weight were measured
individually. Triploid gigantism was calculated as the percent increase in body size of
triploids over their diploid controls ŽGuo et al., 1996.. A piece of about 0.5 g of
digestive gland and adductor muscle was taken from each oyster and stored at y80 8C
for allozyme analysis.
Seven allozymes were analyzed with starch gel electrophoresis ŽAebersold et al.,
1987.. Allozymes examined included aspartate aminotransferase ŽAAT, EC 2.6.1.1..,
Leucine aminopeptidase ŽLAP, EC 3.4.11., glucose phosphate isomerase ŽGPI, EC
5.3.1.9., isocitrate dehydrogenase ŽIDH, EC 1.1.1.42., aconitate hydratase ŽAH, EC
4.2.1.3., malate dehydrogenase ŽMDH, EC 1.1.1.37., and superoxide dismutase ŽSOD,
EC 1.15.1.1..
Two-sample t-tests or ANOVA were used to compare differences in body size and
heterozygosity of the various groups. Significant levels were set at p - 0.05 unless
otherwise noted.
3. Results
The ploidy of sampled oysters from the triploid groups was individually determined
by flow cytometry. Replicates 1–3 of the 3nCB group contained 6%, 16%, and 18%
Z. Wang et al.r Aquaculture 204 (2002) 337–348
340
diploids, respectively. These diploids were removed from the triploid groups for all
analyses. As expected, the 3nDT groups contained 100% triploids.
3.1. Body size
On the average, the 3nDT triploids were the largest among the three groups,
measuring 6.82 g in whole body weight and 1.51 g in wet meat weight ŽTable 1.. The
3nCB triploids, 6.60 g in whole and 1.37 g in meat weight, were the second largest. The
normal diploids, 6.15 g in whole and 1.20 g in meat weight, were the smallest. Overall,
the triploid gigantism was 7% for the 3nCB triploids and 11% for the 3nDT triploids in
whole body weight. Triploid gigantism was higher in meat weight, averaging 14% for
the 3nCB triploids and 26% for the 3nDT triploids.
Differences among the three groups were more complicated when analyzed in detail.
The ANOVA of the whole weight revealed significant difference among replicates
Ž p s 0.018. and groups Ž p s 0.023., and significant interaction between replicates and
groups Ž p - 0.001.. Subsequently, each replicate was analyzed separately for comparison among groups. In Replicate 1, the 3nCB triploids were significantly bigger
Ž p - 0.001. than the normal diploids and 3nDT triploids ŽTable 1.. The latter two were
not different from each other. In Replicate 2, the 3nDT triploids were significantly
bigger than 3nCB and normal diploids, and there was no significant difference between
the latter two. In Replicate 3, there was no significant difference among the three groups
in whole and meat weights.
The replicates have no significant effects on meat weight as determined by ANOVA,
but effects from group and group-replicate interaction were highly significant Ž p 0.001.. The interaction was primarily from the fact that the largest 3nCB group was
Table 1
Body size Ž"s.e.. in three replicates of diploid and triploid Pacific oyster
Group
Sample size
Shell height Žmm.
Whole weight Žg.
Meat weight Žg.
2 n1
2 n2
2 n3
Mean
50
50
50
150
39.6"0.8
39.9"0.7
35.9"0.7
38.1"0.5
6.13"0.24
5.58"0.25
6.73"0.23
6.15"0.14
1.14"0.05
1.12"0.06
1.37"0.05
1.20"0.03
3nCB1
3nCB2
3nCB3
Mean
47
42
41
130
43.8"0.8
33.1"0.3
35.9"0.7
37.9"0.5
8.60"0.33
5.01"0.21
5.95"0.25
6.60"0.19
1.62"0.07
1.17"0.07
1.31"0.07
1.37"0.04
3nDT1
3nDT2
3nDT3
Mean
50
50
50
150
36.1"0.7
39.2"0.8
36.1"0.9
37.1"0.5
6.03"0.29
7.98"0.37
6.44"0.40
6.82"0.22
1.27"0.07
1.89"0.10
1.41"0.09
1.51"0.06
2 n, normal diploids; 3nCB, triploids produced by blocking polar body II with CB; and 3nDT, triploids
produced from diploid=tetraploid mating.
2 n deleted from 3nCB group.
Z. Wang et al.r Aquaculture 204 (2002) 337–348
341
Table 2
Heterozygosity at five allozyme loci in three replicates of diploid and triploid Pacific oyster
Group
Heterozygosity
AAT2
AH2
AH1
IDH1
LAP
Mean
2 n1
2 n2
2 n3
Mean
0.48
0.56
0.46
0.50
0.54
0.72
0.48
0.58
0.10
0
0.54
0.21
0.08
0
0
0.03
0.40
0.52
0.36
0.43
0.32
0.36
0.37
0.35
3nCB1
3nCB2
3nCB3
Mean
0.87
0.83
0.73
0.81
0.74
0.81
0.60
0.72
0.09
0.10
0.44
0.21
0.08
0.00
0.02
0.03
0.60
0.69
0.66
0.65
0.48
0.49
0.49
0.48
3nDT1
3nDT2
3nDT3
Mean
0.86
0.82
0.78
0.82
0.96
0.96
0.74
0.89
0.14
0
0.06
0.07
0.10
0.66
0
0.25
0.76
0.88
0.86
0.83
0.56
0.66
0.49
0.57
2 n, normal diploids; 3nCB, triploids produced by blocking polar body II with CB; and 3nDT, triploids
produced from diploid=tetraploid mating.
Sample size is the same as in Table 1.
found in Replicate 1, while the largest 3nDT group was in Replicate 2. When data from
the three replicates were pooled, analysis showed that 3nDT triploid was significantly
bigger than 3nCB and 2 n diploids. The 3nDT triploids were also bigger than 3nCB
Fig. 1. Heterozygosity and wet meat weight at 1 year of normal diploid Ž2 n., triploids produced by inhibiting
meiosis II with CB Ž3nCB. and triploids produced from diploid=tetraploid mating Ž3nDT.: combined data
from three replicates.
Z. Wang et al.r Aquaculture 204 (2002) 337–348
342
Table 3
Body size Ž"s.e.. of oysters with the same number of heterozygous loci ŽNHL. in diploid and triploid Pacific
oysters
Group
NHL
Sample size
Whole weight Žg.
Meat weight Žg.
2n
0
1
2
3
4
1.7
9
51
61
27
2
5.82"0.53
6.09"0.230
6.39"0.237
5.78"0.331
6.56"2.351
6.15"0.143
1.09"0.13
1.24"0.05
1.23"0.06
1.14"0.07
1.27"0.50
1.20"0.03
0
1
2
3
4
2.3
1
22
42
56
9
3.55
7.12"0.47
6.21"0.41
6.70"0.36
6.90"1.22
6.51"0.21
0.71
1.41"0.10
1.33"0.11
1.39"0.07
1.44"0.26
1.34"0.14
1
2
3
4
5
2.9
9
36
69
34
2
6.25"1.42
6.25"0.71
6.57"0.30
7.86"0.46
7.23"0.70
6.82"0.22
1.31"0.32
1.31"0.32
1.46"0.07
1.80"0.13
1.68"0.04
1.51"0.05
Mean
3nCB
Mean
3nDT
Mean
2 n, normal diploids; 3nCB, triploids produced by blocking polar body II with CB; and 3nDT, triploids
produced from diploid=tetraploid mating.
Diploids were removed from 3nCB groups.
triploids, although the difference was not statistically different. Within replicates, meat
weight followed the same pattern as the whole body weights. The 3nCB triploids were
significantly bigger than normal diploids and 3nDT triploids in Replicate 1. The 3nDT
triploids were significantly bigger than normal diploids and 3nCB triploids in Replicate
2; and there was no difference among the three groups in Replicate 3.
Shell height measurements were not analyzed because the oysters were of irregular
shape.
3.2. Heterozygosity
Eleven loci were scored at seven allozymes for all sampled oysters. Five loci, AAT2,
AH1, AH2, LAP, and IDH1, were polymorphic, and the other six were monomorphic.
The heterozygosity of the five polymorphic loci in all groups is presented in Table 2.
Heterozygosity varied greatly among loci and among replicates Žas different sets of
parents were used.. When all loci were combined, triploids showed much higher levels
Notes to Table 4:
2 n, normal diploids; 3nCB, triploids produced by blocking polar body II with CB; and 3nDT, triploids
produced from diploid=tetraploid mating.
Diploids were removed from 3nCB groups.
Z. Wang et al.r Aquaculture 204 (2002) 337–348
343
Table 4
Meat weight Ž"s.e.. of heterozygotes and homozygotes at five loci in three replicates of diploid and triploid
Pacific oyster
Group
2 n1
2 n2
2 n3
3nCB1
3nCB2
3nCB3
3nDT1
3nDT2
3nDT3
Locus
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
AAT2
AH1
AH2
IDH1
LAP
Heterozygotes
Homozygotes
Sample
size
Meat Žg.
Sample
size
Meat Žg.
P-value
24
5
27
4
20
28
0
36
0
26
23
28
24
0
18
41
4
35
4
24
35
4
34
0
29
30
18
24
1
27
43
7
48
5
43
41
0
48
33
44
39
3
37
0
43
1.14"0.07
0.89"0.10
1.13"0.07
1.13"0.28
1.22"0.07
1.01"0.07
–
1.16"0.07
–
1.08"0.08
1.40"0.08
1.39"0.08
1.44"0.08
–
1.21"0.09
1.64"0.09
1.97"0.27
1.69"0.10
1.53"0.50
1.71"0.12
1.16"0.12
0.82"0.09
1.12"0.12
–
1.14"0.08
1.31"0.10
1.43"0.11
1.29"0.09
0.48
1.35"0.11
1.31"0.08
1.02"0.13
1.28"0.07
1.57"0.13
1.32"0.08
1.92"0.11
–
1.92"0.10
1.92"0.12
1.90"0.101
1.37"0.09
1.48"0.27
1.41"0.10
–
1.38"0.10
26
45
23
46
30
22
50
14
50
24
27
22
26
50
32
6
43
12
43
23
7
38
8
42
13
11
23
17
40
14
7
43
2
45
7
9
50
2
17
6
11
47
13
50
7
1.14"0.07
1.17"0.05
1.15"0.07
1.14"0.05
1.09"0.07
1.26"0.10
1.12"0.06
1.01"0.10
1.12"0.06
1.16"0.08
1.34"0.07
1.34"0.08
1.30"0.07
1.37"0.05
1.45"0.07
1.49"0.19
1.59"0.08
1.43"0.12
1.63"0.08
1.53"0.11
1.20"0.09
1.20"0.11
1.35"0.18
1.17"0.10
1.23"0.28
1.31"0.15
1.21"0.12
1.35"0.15
1.33"0.07
1.22"0.13
1.00"0.09
1.30"0.08
1.02"0.02
1.24"0.08
0.97"0.11
1.75"0.18
1.89"0.10
1.22"0.10
1.83"0.15
1.78"0.17
1.54"0.29
1.40"0.10
1.38"0.22
1.41"0.09
1.57"0.22
0.969
0.090
0.967
0.966
0.219
0.032
–
0.250
–
0.508
0.573
0.701
0.205
–
0.029
0.542
0.186
0.167
0.738
0.265
0.887
0.266
0.382
–
0.696
0.992
0.184
0.800
–
0.437
0.114
0.307
0.471
0.104
0.088
0.500
–
0.154
0.471
0.678
0.458
0.823
0.882
–
0.476
344
Z. Wang et al.r Aquaculture 204 (2002) 337–348
of heterozygosity than normal diploids. The average level of heterozygosity was 0.35 in
normal diploids, 0.48 in 3nCB triploids and 0.57 in 3nDT triploids. Compared with
normal diploids, multilocus heterozygosity was 63% higher in the 3nDT and 37% higher
in the 3nCB groups. Furthermore, the 3nDT triploids were 19% higher than the 3nCB
triploids. All differences were statistically significant.
At highly polymorphic loci such as AAT2, AH2, and LAP, the increase in heterozygosity in triploids is systematic and occurred on all replicates ŽTable 2.. Both 3nDT and
3nCB triploids have consistently higher heterozygosity than normal diploids. The 3nDT
triploids either have the same or considerably higher level of heterozygosity than 3nCB
triploids. At less variable loci, AH1 and IDH1, 3nCB triploids have the same levels of
heterozygosity as normal diploids. The 3nDT triploid was more heterozygous than
diploids and 3nCB triploids at IDH1 because of the heterozygous father in Replicate 2.
3.3. Body size and heterozygosity
The mean body size and heterozygosity of the three groups followed the same
pattern, i.e., 3nDT ) 3nCB ) 2 n. There was a clear and positive correlation between
body size and heterozygosity when the group means were considered ŽFig. 1.. Regression of the group meat weight on heterozygosity is significant Ž r 2 s 0.995; p s 0.031..
The correlation is weak when replicates of the groups were included as separate datum
points Ž n s 9. in the regression Ž r 2 s 0.423, p s 0.034.. The correlation between
heterozygosity and whole body weight is not significant.
Within the diploid and 3nCB, however, there was no correlation between body size
and heterozygosity. The 3nCB1 was the 3nCB group that was significantly bigger than
its diploid control Žby 42% in meat weight.. The heterozygosity of 3nCB1, was not
different from the other two 3nCB replicates, where no body-size increases were
observed. Within the 3nDT group, 3nDT2 was significantly bigger than its diploid
control Žby 59% in meat weight., and it did show a higher heterozygosity than 3nDT1
and 3nDT3 Ž0.66 vs. 0.56 and 0.49.. The increased heterozygosity in 3nDT2 was
entirely due to one locus, IDH1.
To test if the multilocus heterozygosity at individual level affects body size, the
number of heterozygous loci ŽNHL. was calculated for each oyster. Oysters with the
same NHL were grouped for the comparison of body size. In the diploid and 3nCB
groups, there was no significant difference among any of the NHL groups ŽTable 3.. In
the 3nDT group, oysters with four heterozygous loci were significantly bigger than
Table 5
Body size Ž"s.e.. of diallelic and triallelic heterozygotes at the AH2 locus in triploid Pacific oysters produced
by diploid=tetraploid mating Ž3nDT.
Parameter
Diallelic
Triallelic
Sample size
Shell height Žmm.
Whole weight Žg.
Meat weight Žg.
93
37.4"6.2
6.96"2.70
1.57"0.69
40
37.2"5.4
6.85"2.23
1.48"0.60
Z. Wang et al.r Aquaculture 204 (2002) 337–348
345
oysters with three and two heterozygous loci in both meat and whole weight. Oysters
with four NHL were primarily from 3nDT2, which are bigger than the other two
replicates.
Meat weight of heterozygotes and homozygotes at each of the five loci was presented
in Table 4. Analysis showed that two of the 12 comparisons, at AAT2 in 2 n2 and LAP
in 2 n3, were significant in normal diploids. However, the homozygotes were actually
bigger than the heterozygotes. In triploids, none of the 23 comparisons was statistically
significant.
Three alleles ŽA, B, and C. were observed at the AH2 locus in the 3nDT group. Body
size of triallelic ŽABC. and diallelic Že.g., ABB, AAC. heterozygotes are presented in
Table 5. There was no significant difference between the two types of heterozygotes.
4. Discussion
Results of this study show that triploid Pacific oysters are, overall, bigger than normal
diploids at 1 year of age, although, there are considerable variation among replicates.
This finding agrees with early observations in Pacific oysters ŽAllen and Downing,
1986; Guo et al., 1996.. It should be pointed out that the body size data from this study
might not reflect the full potential of triploid Pacific oysters. All oysters are maintained
at land-based systems throughout this study. One data point at 1 year of age may not be
a representative of later performance. In another experiment, superior growth of triploids
becomes more pronounced at 2 and 3 years of age ŽGuo et al., unpublished..
Superior growth or increased body size has been recognized as a general feature of
triploid molluscs and referred to as triploid gigantism ŽGuo, 1999; Guo and Allen,
1994a.. In most molluscs studied so far, triploid molluscs grow faster than diploids.
Superior larval growth has been reported in triploid zhe oyster, Saccostrea cucullata
ŽZeng et al., 1994., Dalianwan oyster, Crassostrea talienwanensis ŽLiang et al., 1994.
and zhikong scallop Chlamys farreri ŽLyu and Wang, 1992.. Juvenile and adult triploids
are significantly bigger than diploids in many species including oysters ŽAllen and
Downing, 1986; Guo et al., 1996; Nell et al., 1994; Stanley et al., 1984., clams
ŽEversole et al., 1996; Guo and Allen, 1994a., and scallops ŽKomaru and Wada, 1989;
Tabarini, 1984; Yang et al., 2000..
Three hypotheses have been proposed to explain triploid gigantism in molluscs. First,
the sterility hypothesis suggests that triploids are bigger because of the sterility of
triploids and energy relocation from reproduction to somatic growth. The sterility
hypothesis is supported by studies where triploids are bigger than diploid only after
sexual reproduction ŽAllen and Downing, 1986; Eversole et al., 1996.. It cannot explain
the observations that triploids are bigger than diploids at D-stage and before sexual
reproduction ŽGuo and Allen, 1994a; Guo et al., 1996..
Secondly, increased heterozygosity has been proposed as a cause for triploid gigantism ŽStanley et al., 1984.. The heterozygosity hypothesis is supported by findings that
meiosis I triploids are bigger and more heterozygous than meiosis II triploids and
diploids ŽHawkins et al., 1994; Stanley et al., 1984.. On the other hand, results from
other studies do not show any correlation between body size and heterozygosity ŽAllen
346
Z. Wang et al.r Aquaculture 204 (2002) 337–348
et al., 1982; Beaumont et al., 1995; Li et al., 1992.. Results of this study support the
heterozygosity hypothesis. Levels of heterozygosity in diploids, 3nCB, and 3nDT
triploids have a strong and positive correlation with overall body sizes in the three
groups ŽFig. 1.. However, it is possible that the increase in body size is caused by other
factors rather than increased heterozygosity. Because two different males are used in
3nCB and 3nDT groups Žone diploid and one tetraploid., other genetic or nongenetic
factors may be confined in the difference in heterozygosity. The diploid and tetraploid
parents used in this study are all from the same random mated base population. The
chance that they carry very different quantitative loci is small. It is possible that
heterozygosity is the major genetic difference among the three groups.
Interestingly, there is no detectable or real correlation between heterozygosity and
body size within diploids, 3nCB, and 3nDT triploids. Triploids from 3nCB1, which are
significantly bigger than other 3nCB groups, did not show increased heterozygosity at
the five loci analyzed. For 3nDT triploids, the replicate Ža2. with considerably higher
levels of heterozygosity is significantly bigger than the other replicates. The increased
heterozygosity in 3nDT2 is entirely due to IDH1, and there is no correlation between
heterozygosity and body size at IDH1 in the 3nDT group. Therefore, the observed
increases in heterozygosity and body size in 3nDT2 are probably coincidental. The
finding of positive correlation between body size and heterozygosity at the group level,
but not within groups or at the individual level, suggests that the difference in body size
is not caused directly by heterozygosity at the five loci analyzed. It supports the
Aassociative overdominanceB hypothesis, that other loci, rather than the five measured,
are responsible for the differences in body size ŽBeaumont et al., 1995.. Heterozygosity,
at the five loci used, may accurately measure the true differences among the three
groups, because the differences are significant and easy to detect, and therefore is
positively correlated with the body size. Within the diploid and triploid groups,
differences in heterozygosity among replicates or individuals are too small to be
accurately measured by the five loci, and the homozygosity at particular recessive
deleterious genes becomes important and dominates the phenotypic variation. Positive
correlation between the body size and heterozygosity has been demonstrated in normal
diploids ŽBeaumont et al., 1995; Koehn and Gaffney, 1984; Zouros et al., 1988..
Finally, the cell size hypothesis suggests that the increased in cell size is the
fundamental cause for triploid gigantism in molluscs ŽGuo and Allen, 1994a; Guo,
1999.. Because triploid cells have 50% more DNA, they may require more cytoplasm to
maintain a given cytoplasmrnucleus ratio. Thus, triploid cells are expected to be bigger
than normal diploids, which have been confirmed in most organisms studied so far
ŽFankhauser, 1945.. In higher animals such as fish and amphibians, the development is
regulatiÕe where the increase in cell size is compensated with a reduction in cell number
ŽGilbert, 1988.. In molluscs and other low invertebrates, the development is mosaic,
where the development is programmed by the number of cell divisions and segregation
of morphogenic determinants. Under the mosaic development, the increases in cell size
may not be compensated by a reduction in cell number and therefore results in triploid
gigantism. Results of this study do not support the argument that the cell size is the sole
cause for triploid gigantism. While both 3nDT and 3nCB triploids are triploids with
presumably the same DNA content and cell size, 3nDT triploids are significantly bigger
Z. Wang et al.r Aquaculture 204 (2002) 337–348
347
than 3nCB triploids. This observation argues that increased heterozygosity or genetics
plays a role in the expression of triploid gigantism. Results of this study, however, do
not eliminate increased cell size as an important or even a fundamental cause for triploid
gigantism. A possible and unifying theory could be that increased cell size is the
fundamental cause for triploid gigantism. It gives triploid molluscs the potential to be
bigger than normal diploids. The expression of triploid gigantism is negatively affected
in 3nCB triploids because of the homozygosity or genetic imbalances caused by the
retention of polar body II. Triploid gigantism is better expressed in 3nDT triploids,
possibly because of the increased heterozygosity. Triploid gigantism may not be
expressed in nutrient-limiting environments. Sterility and energy relocation from reproduction to somatic growth may also contribute to the overall expression of triploid
gigantism, particularly at later ages post-maturation.
Acknowledgements
This study is conducted at Rutgers University and supported by grants from National
Sea Grant ŽBrT-4 and RrTAQ-9969. and New Jersey Commission on Science and
Technology Ž00-2042-007-20.. Dr. Zhaoping Wang’s participation is partly supported by
Ocean University of Qingdao. This is publication 01-24 of IMCS, Rutgers University
and 01-471 of NJMSC.
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