Mar. Biotechnol. 2, 466–475, 2000
DOI: 10.1007/s101260000016
© 2000 Springer-Verlag New York Inc.
Triploid and Tetraploid Zhikong Scallop, Chlamys farreri
Jones et Preston, Produced by Inhibiting Polar Body I
Huiping Yang,1 Fusui Zhang,1 and Ximing Guo2,*
1
Experimental Marine Biology Laboratory, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao,
Shandong 266071, China
2
Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, 6959 Miller Avenue,
Port Norris, NJ 08349, U.S.A.
Abstract: Triploid scallops are valuable for aquaculture because of their enlarged adductor muscle, and tetraploids are important for the commercial production of triploids. We tested tetraploid induction in the zhikong
scallop by inhibiting polar body I in newly fertilized eggs. The ploidy of resultant embryos was determined by
chromosome counting at 2- to 4-cell stage and by flow cytometry thereafter. Embryos from the control groups
were mostly diploids (79%), along with some aneuploids. Embryos from the treated groups were 13% diploids,
18% triploids, 26% tetraploids, 13% pentaploids, and 36% aneuploids. Tetraploids, pentaploids, and most
aneuploids suffered heavy mortality during the first week and became undetectable among the larvae at day 14.
Five tetraploids (2%) were found among a sample of 267 spat from one of the replicates, and none was detected
at day 450. The adductor muscle of triploid scallops was 44% heavier (P < .01) than that of diploids, confirming
the value of the triploid technology in this species.
Key words: triploid, tetraploid, aneuploid, polar body I, cytochalasin B, scallop, aquaculture.
I NTRODUCTION
Triploids are organisms with three sets of chromosomes
instead of the normal two sets in diploids. Triploid shellfish
are useful for aquaculture because of their sterility, superior
growth, and improved meat quality, and sometimes for increased disease resistance (Allen et al., 1989; Hand et al.,
1998; Guo, 1999). Triploid Pacific oyster has been commercializd on the West Coast of the United States and now
accounts for one third to one half of the total production
Received December 8, 1999; accepted March 3, 2000.
*Corresponding author: telephone 856-785-0074; fax 856-785-1544; e-mail
[email protected]
(Chew, 1994). Triploid scallops are particularly valuable in
aquaculture. All studies so far have demonstrated that triploid scallops have greatly enlarged adductor muscles. Compared with those of normal diploids, the adductor muscles
of triploids are 73% larger in the bay scallop Argopecten
irradians (Tabarini, 1984), 67% larger in the noble scallop
Chlamys nobilis (Lin et al., 1995), and 167% larger in the
catarina scallop Argopecten ventricosus (Ruiz-Verdugo et al.,
1998). The adductor muscle is the marketed product for
scallops, and an increase in adductor muscle size will not
only increase the yield, but also attract higher prices per
unit.
Triploid shellfish can be produced by blocking the release of polar body I (PBI) or II (PB2) (Stanley et al., 1981,
Triploid and Tetraploid Scallops 467
1984), or by mating tetraploids and diploids (Guo et al.,
1996). Blocking polar bodies with chemical or physical
treatments is complicated, expensive, and rarely 100% effective. The most effective way to produce triploids is
through diploid × tetraploid crosses. Triploids produced
from diploid × tetraploid crosses are 100% pure and free of
genetic defects from polar body inhibition (Guo et al.,
1996).
Tetraploid production remains a major challenge in
shellfish breeding. Theoretically, tetraploid mollusks can be
produced by several methods, including the inhibition of
mitosis I, PB1, gynogenesis, and cell fusion (Beaumont and
Fairbrother, 1991; Guo, 1991; Guo et al., 1993, 1994). The
difficulty is that tetraploid embryos have very limited viability in mollusks. So far, tetraploid production has succeeded in only one mollusk, the Pacific oyster (Guo and
Allen, 1994). Development of a tetraploid Pacific oyster led
to a patent and rapid commercialization. Tetraploids have
also been reported in the mussel Mytilus galloprovincialis
(Scarpa et al., 1993) and the Manila clam Tapes philippinarum (Allen et al., 1994), from which only a few tetraploids
were produced and later lost.
Zhikong scallop, Chlamys farreri Jones et Preston, is the
most important scallop cultured in China. Zhikong scallop
accounts for about 75% to 80% of the scallop production
from aquaculture in China, which was 1 million tons in
1996 (Guo et al., 1999). In one attempt at tetraploid induction reported in the zhikong scallop, tetraploid embroyos
did not survive beyond metamorphosis (Yang et al., 1997).
In the present study, we tested tetraploid induction in the
zhikong scallop by blocking the release of PB1 and report
for the first time that tetraploid scallops suvived to the
juvenile stage.
were filtered through an 80-µm screen, collected on a 25µm screen, and resuspended for fertilization. Sperm suspension was filtered through a 25-µm screen. For fertilization, the sperm were added to the egg suspension to a
density of about 5 to 10 sperm per egg. Fertilization, treatment, and embryo culture were all conducted at 20° ±
0.5°C.
Cytochalasin B Treatment
Blocking PB1 was selected for this study because it has
produced tetraploids in other species (Stanley et al., 1984;
Stephens, 1989; Guo et al., 1992a, 1992b). Cytochalasin B
(CB) was used to inhibit the release of PB1. Stock solution was prepared by dissolving CB in dimethylsulfoxide
(DMSO) at the concentration of 1 mg/ml. The final concentration used in this study ranged between 0.5 and 0.9
mg/L. The CB was applied to fertilized eggs at 7 to 10
minutes postfertilization (PF). The CB treatment was
stopped as soon as PB2 was observed in the control group
(about 37–42 minutes PF). The CB was removed by rinsing
eggs on a 25-µm screen. The eggs were then returned to
fresh seawater and cultured at 20°C. Eggs in the control
groups were treated with 0.1% DMSO.
The CB treatment and its control were repeated 9 to 12
times using different sets of parents. The first nine replications, each with a diploid control group (2n-1 to 2n-9) and
a treatment group (PB-1 to PB-9), were conducted at small
scales and cultured in 60-L tanks. Three additional replicates of the treated group (PB-A, PB-D, and PB-E) were
made at a commercial hatchery and cultured in 15,000-L
tanks, without diploid controls.
Determination of Ploidy
M ATERIALS
AND
M ETHODS
Gametes
Scallop broodstocks used in this study were from Qingdao
and Rizhao, Shangdong province, China. Scallops were culturd indoors briefly to accelerate gonad maturation. Gametes were obtained from induced spawning of scallops. To
induce spawning, the parental scallops were left dry for 1
hour. Males and females were separated according to the
gonad coloration: orange for females and milky white for
males. Scallops were then put into 20°C seawater, which was
2° to 3°C higher than the conditioning temperature. Eggs
Ploidy of early embryos from both control and treated
groups was determined by chromosome number. Samples
were taken at 2- to 4-cell stage. Embryos were treated with
0.01% colchicine for 20 to 30 minutes and fixed in Carnoy
fixative (3:1, methanol–acetic acid). The fixative was
changed twice. Embryos were dropped on slides and stained
with 0.5% hematoxylin dissolved in 45% acetic acid with
ammonium iron (III) sulfate dodecahydtate (2%) under
coverglass. After staining for 10 to 15 minutes, the slides
were heated on an alcohol burner, squashed, and sealed
with cytoseal or nail polish. Only metaphase plates that
showed no signs of chromosome loss or gain were counted.
Fifty or more embryos were counted in each group. The
468 Huiping Yang et al.
diploid chromosome number for the zhikong scallop is 38
(Wang et al., 1990). The ploidy was classified as follows: 19,
haploid; 37 to 38, diploid; 55 to 57, triploid; 73 to 76,
tetraploid; and 91 to 95, pentaploid. All the others were
considered to be aneuploid.
The ploidy of larvae (D-stage and beyond) and juveniles was determined by flow cytometry (FCM). Several
hundred larvae were taken from each group and pooled for
FCM. FCM analysis was conducted with a 4,6-diamidine2-phenylindole (DAPI) stain (Guo et al., 1993). The ploidy
of individual juvenile scallops (0.2–0.4 cm) was determined
by FCM. The whole body of each scallop was used for
analysis. Before FCM, all samples were frozen and then
thawed to room temperature. They were vortexed and syringed with a 26-gauge needle three times and then filtered
through a 25-µm screen. Sperm from normal diploid scallops was used as the haploid standard.
Fertilization, survival, and larval size were determined
for each group every other day at water change. Fertilization
level was determined as the percentage of eggs that divided
at 2 hours PF. Cumulative survival of fertilized eggs to day
2 (D-stage), day 6, day 14, and spat was calculated. The
length of larvae was measured under a microscope. At juvenile stage, both length and width were measured with
calipers.
All the data in this study were analyzed using the SYSTAT program. Differences between the control and treated
groups were examined by two-sample t test. Percentage data
for fertilization level, survival, and ploidy composition were
arcsine-transformed before the analysis.
R ESULTS
Laboratory Replicates
In control groups, fertilized eggs started to release PB1
around 8 to 12 minutes PF and PB2 around 35 to 40 minutes PF (at 20°C). The timing of PB1 and PB2 releases
varied slightly among replicates, and the timing of CB treatment varied accordingly (Table 1). In the CB-treated
groups, no PB1 was released at 10 to 15 minutes PF during
the CB treatment. The treated eggs started to release polar
bodies 3 to 5 minutes after the removal of CB. Most treated
eggs released only one PB, which was often irregularly
shaped. Some eggs had two PBs, usually positioned side by
side. There was no noticeable difference in the appearance
of mitosis I between control and treated eggs, except that
development was less synchronized in the treated eggs.
Table 1. Cytochalasin B Treatment Parameters Used to Block
Polar Body I in Fertilized Eggs of Zhikong Scallop
Group
CB
(mg/L)
Begin
(min PF)
End
(min PF)
PB-1
PB-2
PB-3
PB-4
PB-5
PB-6
PB-7
PB-8
PB-9
PB-A
PB-D
PB-E
0.75
0.75
0.75
0.75
0.90
0.50
0.75
0.75
0.75
0.75
0.75
0.90
12
10
9
9
5
10
8
7
9
8
8
8
42
40
37
35
36
42
37
38
39
40
40
42
Duration
(min)
30
30
28
26
31
32
29
31
30
32
32
34
*CB indicates cytochalasin B; PF, postfertilization.
Fertilization level was variable among replicates, and
there was no significant difference (P = .231) between the
treated and control groups (Table 2). Overall, fertilization
levels ranged between 24% and 51% in both control and
treated groups—lower than we normally observe with this
species. The low fertilization level is probably caused by
poor egg quality and mechanical damage from the handling
of eggs before fertilization. At day 2, an average of 34% of
fertilized eggs in the control groups developed into D-stage
larvae, and most larvae were normal and active. In the
treated groups, only 2% of the fertilized eggs developed to
D-stage, which was significantly (P < .001) lower than in the
control groups. The majority of larvae in the treated groups
remained at the trochophore stage and appeared abnormal.
The abnormal trochophores could not swim normally and
often rotated in the same spot. Compared with larvae in the
control groups, D-stage larvae in the treated groups appeared to be slow or retarded in movement.
Ploidy composition, as determined by chromosome
counts at 2- to 4-cell stage, is presented in Table 3. In the
control groups, most (79%) of the embryos were diploid,
and the others were aneuploids (17%), haploids (3%), and
triploids (2%). The percentage of aneuploids in the control
groups varied somewhat among replicates, ranging from
5% to 32%. Ploidy composition in the CB-treated groups
varied considerably among replicates. Aneuploids were the
most frequent ploidy, averaging 36% (ranging from 14% to
53%). Tetraploids were produced in all CB-treated groups,
Triploid and Tetraploid Scallops 469
Table 2. Number of Eggs Used, Fertilization Level, and Survival to Day 2, 6, 14, and 60 of Experimental Groups
Group
2n-1
2n-2
2n-3
2n-4
2n-5
2n-6
2n-7
2n-8
2n-9
Average
PB-1
PB-2
PB-3
PB-4
PB-5
PB-6
PB-7
PB-8
PB-9
Average
P value†
Eggs
(×1000)
Fertilization
(%)
Day 2
(%)
Day 6
(%)
Day 14
(%)
12,757
2,952
13,080
472
2,940
2,304
7,260
6,030
2,940
51
48
51
49
24
49
50
38
28
43
43
44
48
24
23
46
51
33
26
38
.231
19.2
45.9
43.1
0.1
—
34.3
48.9
48.3
33.2
34.1
0.3
0.6
6.1
0.0
0.7
2.8
3.7
2.1
2.3
2.1
<.001
5.3
10.7
11.2
—*
—
29.1
27.5
27.4
—
18.5
0.11
0.01
0.57
—
0.09
0.15
0.11
0.09
—
0.16
<.001
3.3
5.9
6.5
—
—
4.2
4.3
5.9
—
5.0
0.01
—
0.16
—
0.05
0.04
0.01
0.04
—
0.05
<.001
36,255
7,248
35,840
1,860
21,216
12,480
12,780
18,330
9,620
Day 60
(No.)
0
3
4
0
0
37
3
0
0
0
0
0
0
20
0
0
0
0
*Numbers were not available or discarded.
†P values are from a two-sample t test of the mean.
although the frequency varied greatly, ranging from 6% to
72%. The average induction efficiency was 26%. Triploid
embryos (18%) and pentaploid embryos (13%) also were
observed in the CB-treated groups. Ploidy composition of
the CB-treated groups was significantly (P < .05) different
from that of the control groups for all ploidy categories
(Table 3). The chromosome number of aneuploids from the
treated groups was mainly distributed into 42–48, 62–69, or
83–89. Some aneuploids had two or more metaphases with
the same chromosome number. In treated groups, some
metaphases (less than 1%) had more than 100 chromosomes. Example metaphases of diploid, triploid, tetraploid,
and aneuploid (65 chromosomes) cells are presented in
Figure 1.
FCM analysis of D-stage larvae at day 2 showed approximately the same ploidy composition for all groups. As
expected, the control groups had only a diploid peak and a
small G2 peak (Figure 2, A). In the CB-treated groups, the
ploidy composition of larvae was complicated and variable,
as the chromosome counts had shown. One of the treated
groups (PB1-3) had a dominant tetraploid peak (Figure 2,
B), suggesting most of the larvae were tetraploid. Some
groups (PB1-8) had distinctive diploid, triploid, tetraploid,
and pentaploid peaks (Figure 2, C). Others had aneuploid
and pentaploid peaks (Figure 2, D). Pentaploids, tetraploids, and the majority of aneuploids died off rapidly during the first week. At day 6, tetraploids and pentaploids
were no longer the dominant peaks (Figure 2, E) At day 14,
the ploidy of larvae was diploid, and sometimes triploid
(Figure 2, F).
Cumulative survival to day 6 was 18% for the control
groups and only 0.2% for the treated groups (Table 2).
Larvae in both control and treated groups suffered heavy
mortality after day 6 for unknown reasons. Cumulative survival to day 14 was 5% for the control groups and 0.05% for
the treated groups. At day 60, five of the nine control
groups produced no spat, and the other four produced only
3 to 37 spat. Eight of the nine treated groups suffered com-
470 Huiping Yang et al.
Table 3. Ploidy Composition of Zhikong Scallop Embryos from the Control and PB1-Inhibited Groups as Determined by Chromosome
Counts
Group
n
1n
2n
3n
4n
5n
2n-1
2n-2
2n-3
2n-4
2n-5
2n-6
2n-7
2n-8
2n-9
Average
PB1-1
PB1-2
PB1-3
PB1-4
PB1-5
PB1-6
PB1-7
PB1-8
PB1-9
Average
P value*
50
50
38
50
54
50
50
50
50
2
2
13
2
0
0
2
0
4
3
0
0
0
0
2
0
0
0
0
0.2
.020
70
88
82
88
80
78
64
86
76
79
8
28
2
13
14
11
14
21
6
13
<.001
0
0
0
2
0
0
2
2
8
2
31
16
6
10
18
16
26
19
14
18
<.001
0
0
0
0
0
0
0
0
0
0
27
6
72
23
30
9
18
19
32
26
<.001
0
0
0
0
0
0
0
0
0
0
4
28
0
2
22
11
14
11
24
13
<.001
48
50
50
60
50
55
50
57
55
An
28
10
5
8
20
22
32
12
12
17
29
22
20
52
14
53
28
30
24
36
.021
*P values are from a two-sample t test of the mean.
plete mortality by day 60, and one produced 20 spat, 65%
of which were triploids. There was no significant difference
in larval size between the control and treated groups at days
2, 6, and 14 (Table 4). But larvae in treated groups were
more variable in size than those in the control groups.
Hatchery Replicates
Three hatchery replicates of the CB treatment, PB-A, PB-D,
and PB-E, were produced without controls, at a commercial
hatchery. Large numbers of eggs, 92 to 180 million per
group, were used and cultured in 15 m3 production tanks
(Table 5). The fertilization level varied among replicates,
ranging from 22% to 51%. FCM analysis of larvae at day 2
showed variable proportions of diploids, triploids, tetraploids, and pentaploids in the three replicates. At day 10,
only diploid and triploid peaks were detected by FCM.
All three hatchery replicates produced large numbers of
spat at day 60 (Table 5). Cumulative survival of fertilized
eggs to spat ranged from 0.4% to 0.7%. The ploidy of these
juvenile scallops was individually determined by FCM
(Table 5). In the PB-A group, scallop spat were either diploid (89.8%) or triploid (10.2%). Similarly in PB-D, only
diploids (77.7%) and triploids (22.3%) were found. In PBE, five tetraploids (or 1.9%) were discovered among the 267
spat sampled, and the others were diploid (76.8%) and
triploid (21.3%). FCM histograms of sperm (standard) and
diploid, triploid, and tetraploid spat are presented in Figure
3. Besides the normal diploids, triploids, and tetraploids,
some spat (less than 1%) had two different peaks and were
probably mosaic for different ploidy levels. PB-D and PB-E
groups were sampled again at day 450. No tetraploids were
found among 137 scallops sampled in each replicate. The
frequency of triploids decreased from 22% to 10% in PB-D,
and from 21% to 15% in PB-E.
The length and height were measured for each scallop
spat that was sampled for FCM analysis at days 60 and 450
(Table 6). In PB-A, triploids were significantly larger (P <
Triploid and Tetraploid Scallops 471
Figure 1. Example metaphase of
diploid (A), triploid (B), tetraploid (C),
and aneuploid (D, 65 chromosomes)
zhikong scallop embryos resulting from
polar body I inhibition.
.05) than diploids in both length and height. But in PB-D
and PB-E, there were no differences in length and height
between diploids and triploids. Tetraploids from the PB-E
group were not statistically different from diploids in length
and height, and from triploids in length and height. At day
450, triploids were measured in two of the three replicates.
The other replicate did not contain enough triploids for
growth comparison. In both replicates, there was no significant difference between diploids and tripliods in shell
and meat measurements; however, the adductor muscle of
triploids was 31% and 57% heavier than that of diploids,
and the difference was highly significant (P < .01).
D ISCUSSION
PB1 inhibition is possible in mollusks because mature eggs
of most mollusks rest at prophase or metaphase of meiosis
I, and PB1 is only released after fertilization (Strathmann,
1987). In the Pacific oyster, segregation analysis has revealed
that PB1 inhibition results in complicated segregation patterns that lead to the formation of triploids, tetraploids,
pentaploids, and a wide range of aneuploids (Guo et al.,
1992a, 1992b). Results of this study show that PB1 inhibition in the zhikong scallop has similar effects on chromosome segregation and ploidy in the resultant embryos. It
produces triploids, tetraploids, pentaploids, and aneuploids
in highly variable proportions. The pentaploids are most
likely produced by accidiental inhibition of both PB1 and
PB2. Tetraploids and triploids are produced through segregation patterns that lead to the release, respectively, of one
set and two sets of chromosomes as “PB2.” Aneuploids are
probably produced through complicated tripolar segregations (Guo et al., 1992b).
The efficiency of tetraploid induction by blocking PB1
varied greatly in this study, from 6% to 72%. The variation
may be partly a reflection of the complicated segregation
patterns caused by PB1 inhibition. The CB treatment parameters were about the same in all replicates, and it is not
clear what factors affected chromosome segregation differently among replicates. One possibility is variation in egg
quality among females, although we could not tell the difference under a microscope. Further studies are needed to
identify factors that affect PB1 inhibition and treatments
that promote tetraploid formation. Withouth significant
improvement, PB1 inhibition cannot be regarded as a reliable method of tetraploid induction in the zhikong scallop.
PB1 inhibition is not an effective method for triploid
production in this species either. The hatchery replicates of
this study produced only about 20% triploids. One of the
laboratory replicates produced 65% triploids, which is still
not good enough for commercial production and lower
than what PB2 inhibition can produce. Another major
problem with PB1 inhibition is the greatly reduced larval
mortality during the first few days after fertilization. The
heavy mortality is a direct consequence of the high levels of
472 Huiping Yang et al.
Table 4. Larval Length of Zhikong Scallop Larvae from Control
and Treated Groups at Day 2, 6, and 14
Mean length ± SD (µm)
Group
Day 2
Day 6
2n-1
2n-2
2n-3
2n-6
2n-7
2n-8
2n-9
Average
PB-1
PB-2
PB-3
PB-5
PB-6
PB-7
PB-8
PB-9
Average
P value†
116 ± 4
115 ± 4
114 ± 4
113 ± 4
113 ± 5
109 ± 4
108 ± 4
113
119 ± 5
115 ± 6
114 ± 5
119 ± 4
114 ± 6
113 ± 8
108 ± 5
106 ± 4
114
.583
136 ± 6
127 ± 5
132 ± 5
135 ± 6
123 ± 9
123 ± 8
—*
129
138 ± 9
—
125 ± 11
139 ± 19
123 ± 8
111 ± 8
113 ± 3
—
125
.411
Day 14
170 ± 10
136 ± 8
166 ± 11
166 ± 15
145 ± 11
137 ± 10
—
154
166 ± 18
—
149 ± 22
150 ± 20
143 ± 11
124 ± 8
124 ± 9
—
146
.273
*Not available or discarded.
†P values are from a two-sample t test of the mean.
Figure 2. Flow cytometry analysis of zhikong scallop larvae from
polar body I inhibition. A: Day 2 larvae from a control group.
B–D: Day 2 larvae from three treated groups. E: Day 6 larvae from
a treated group. F: Day 14 larvae from a treated group.
aneuploids, tetraploids, and pentaploids produced. Nevertheless, PB1 inhibition has been suggested as a possible
method for triploid production (Stanley et al., 1984; Jiang et
al., 1991).
A significant finding of this study is that a few tetraploids can survive to at least the juvenile stage (day 60), or
about 2 to 4 mm. In a previous study, tetraploid embryos
were produced in the zhikong scallop, but no tetraploids
were detected at juvenile stage (Yang et al., 1997). This
study provides the first evidence that tetraploid scallops can
survive beyond early larval stages and metamorphosis.
Metamorphosis is the most important milestone in molluscan development. Survival beyond metamorphosis is a
strong indication that tetraploid scallops can survive to sexual maturation, which is encouraging for the development
of tetraploid scallops.
No tetraploids were detected at day 450 among hun-
dreds of scallops sampled. It is clear that most tetraploid
larve died within the first 2 weeks, as demonstrated by FCM
analysis of larvae. Low survival has also been observed for
tetraploid embryos produced in oysters (Stanley et al., 1984;
Stephens, 1989; Guo, 1991). The exact cause for the poor
survival of tetraploid mollusks remains unknown. One hypothesis has attributed the poor survival to a deficiency in
cytoplasm or cell number due to the cleavage of a normal
egg by a large tetraploid nucleus (Guo, 1991; Guo et al.,
1994; Guo and Allen, 1994). At the end of cleavage, tetraploid embryos may have either fewer cells than normal
diploids, or the same number of cells with an inadequate
cytoplasm-nucleus ratio. Unlike fish and amphibians, in
which the development is regulative, most mollusks follow a
mosaic type of development in which a cell’s fate is programmed by the number of divisions and distribution of
morphogenic determinants (Gilbert, 1988). A deficiency in
cell number or cytoplasm at the end of cleavage may effectively block further development in mollusks. The deficiency can be corrected by an increase in the egg’s volume.
Viable tetraploids in the Pacific oyster are produced using
Triploid and Tetraploid Scallops 473
Table 5. Number of Eggs Used, Fertilization Level, Survival to Day 60, and Ploidy Composition of Surviving Spat as Determined by Flow
Cytometry
Group
Eggs
(×1000)
Fertilization
(%)
Survivors,
N (%)
Diploid,
N (%)
Triploid,
N (%)
Tetraploid,
N (%)
PB-A
PB-D
PB-E
180.0
173.9
92.2
21.9
50.5
32.0
192,000 (0.4)
64,800 (0.7)
112,200 (0.4)
230 (89.8)
157 (77.7)
250 (76.8)
26 (10.2)
45 (22.3)
57 (21.3)
0
0
5 (1.9)
Table 6. Size Measurements of Diploid and Triploid Zhikong
Scallops at Day 60 and 450
Group
Day 60
PB-A
PB-D
PB-E
Day 450
PB-D
PB-E
Figure. 3. Flow cytometry analysis of juvenile zhikong scallops
produced by inhibiting PB1. A: Sperm. B: Diploid. C: Triploid. D:
Tetraploid.
larger eggs from triploids (Guo and Allen, 1994). Tetraploid
oysters produced from normal eggs did not survive beyond
metamorphosis (Guo et al., 1994).
The cytoplasm or cell deficiency hypothesis provides
one explanation for the poor viability of tetraploid embryos
observed in this study, although a few tetraploids can clearly
overcome the deficiency and reach juvenile stage. A few
tetraploids produced from normal eggs even survived to
adult stage in the mussel Mytilus galloprovincialis (Scarpa et
al., 1993) and the Manila clam Tapes philippinarum (Allen
et al., 1994). If the cytoplasm deficiency is the main problem, an increase in cytoplasm volume may greatly improve
Measurement
Diploid
Triploid
Number
Length (cm)
Height (cm)
Number
Length (cm)
Height (cm)
Number
Length (cm)
Height (cm)
230
0.253
0.289
157
0.221
0.249
205
0.168
0.188
26
0.309
0.356
45
0.228
0.257
57
0.161
0.183
Number
Length (cm)
Height (cm)
Meat weight (g)
Adductor (g)
Number
Length (cm)
Height (cm)
Meat weight (g)
Adductor (g)
149
4.265
4.698
12.957
1.379
116
4.499
4.939
14.829
1.644
17
4.252
4.588
14.178
2.169
21
4.464
4.857
14.912
2.158
P value*
.038
.040
.589
.578
.703
.851
.945
.563
.428
.001
.840
.595
.947
.007
*P values are from a two-sample t test of the mean.
the survival of tetraploid scallops. There may be other explanations for the poor survival of tetraploid mollusks such
as the disruption of gene expression by duplication. The
poor survival in laboratory replicates was clearly caused by
problems with larval culture because diploid controls experienced catastrophic mortalities. This is unfortunate because some of the laboratory replicates produced the highest levels of tetraploids.
Another major finding of this study is that triploid
474 Huiping Yang et al.
zhikong scallops have significantly larger adductor muscles
than diploids, confirming previous observations in other
scallop species. This finding is remarkable considering triploid scallops did not differ from diploids in overall growth
in this study. Clearly the triploid advantage in scallops is
manifest in adductor muscle size. The increase in adductor
muscle size observed in this study, 44% on average, is
smaller than the 67% to 167% reported in other scallops
(Tabarini, 1984; Lin et al., 1995; Ruiz-Verdugo et al., 1998).
Even with an average of 44% increase in adductor muscle
size, triploid zhikong scallops will make scallop farming
significantly more profitable. It should be noted that the
triploid from this and some other studies were produced by
blocking PB1, PB1 triploids often grow faster than PB2
triploids (Stanley et al., 1984; Jiang et al., 1991).
In summary, this study clearly demonstrates that triploids are highly valuable for the culture of zhikong scallops.
The finding of a few tetraploids surviving to juvenile stage
should stimulate further development of tetraploids in this
species. The triploid-tetraploid technology, if successfully
developed, should make a significant contribution to scallop farming worldwide.
A CKNOWLEDGMENT
We thank Ms. Tian Deng for assistance with FCM, Professors Yichao He and Jianghu Ma, Dr. Huayong Que, and Mr.
Zhongcheng Bao for advice and help with broodstock and
larval rearing. Dengfeng Fisheries Company provided
hatchery resources for this study. This study was supported
by grant 819-01-07 from China’s National 863 Development Program, the “100 Scholars” program of the Chinese
Academy of Science, and the China’s Natural Science Foundation (No. 39825121). This is publication No. 3680 of the
Institute of Oceanology, Chinese Academy of Sciences, and
No. 2000-05 of IMCS, Rutgers University.
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