of approximately 700 – 800 mm. At this time, they were transferred to upwelling system and the densities adjusted according to need as spat grew.
2.5. Flow cytometry 2.5.1. LarÕae
Approximately 10,000 48-h-old larvae were sampled from each culture for flow Ž
. cytometric FCM analysis to determine the percentage of tetraploids produced. In
preparation for flow cytometry, larvae were concentrated into a 1 ml suspension, then pelleted by centrifugation at 1500 = g for 10 s in a microcentrifuge. Supernatant
Ž seawater was withdrawn and 0.5 ml of DAPIrdetergentrDMSO solution 146 mM
NaCl, 10 mM Tris, adjust to pH 7.4, 2 mM CaCl , 22 mM MgCl , 0.05g BSA, 0.1
2 2
. Triton X, 10 mgrml DAPI and 10 DMSO was added to the tube. Larvae were
resuspended by vortex. Larval suspensions were frozen at y808C, and, after at least 1 h, larvae were thawed and disaggregated by repeated aspiration with a 1-ml syringe fitted
with a 26G needle. Cell suspensions were passed through a 25-mm screen immediately before the assay. FCM analyses were accomplished on a Partec CA-II flow cytometer.
Analysis of larval samples yielded data from a population of larvae, about 10,000 prepared by disaggregation. The proportion of cells in each ploidy class was calculated
relative to the proportion of observations in all ploidy classes, after curve fitting with
Ž . Ž
. Modfit Verity Software House, Topsham, ME, USA
Allen and Bushek, 1992 . 2.5.2. Spat
When the spat reached about 1000 – 2000 mm, about 30 individual spat from each experiment were examined by flow cytometry to assess ploidy. Spat were placed into
separate tubes and crushed gently. About 0.5 ml of DAPI solution was added to each tube, and the contents vortexed and screened on a 25-mm Nytex screen before FCM
analysis.
3. Results
3.1. Treatment from 5 to 20 min PF Ž
. Treatment according to strict criteria of timing described by Guo and Allen 1994b
Ž .
were relatively unsuccessful Fig. 1A–D according to the ploidy determined 48 h PF. Generally, these profiles show significant numbers of aneuploid cells with indistinct
ploidy peaks. Although not well separated, these peaks seem organized around two Ž
. values relative to a diploid standard peak value s 62 : about 75–76 for the first peak
Ž .
Ž .
about 2.40–2.45n and 104–108 for the second 3.30–3.48 n . These values are shown in Table 1. None of the replicates presented major peaks located at the tetraploid level
Ž .
expected mean relative DNA content s 124 , indicating very few, if any, tetraploids. It Ž
. is interesting to note that for the first replicate Fig. 1A the second peak is dominant
Ž .
whereas in the three other replicates Fig. 1B–D , the first peak is dominant, indicating some variability among females for the same treatment.
Fig. 1. Flow cyometric ploidy profiles of 48-h-old larvae obtained from triploid eggs=diploid sperm treated Ž
. with CB 0.5 mgrl from 5 to 20 min after fertilization. Ploidy levels have been calculated relative to a diploid
standard.
3.2. Short and long treatments The results of our experiments with short and long treatments were highly repro-
ducible. Fig. 2 shows the ploidy profiles obtained from the larvae at 48 h PF in three of
Table 1 Ž
. Mean relative DNA content of 48-h old larvae from unmodified treatments from Guo and Allen, 1994b to
induce tetraploidy in C. gigas. The mean and coefficient of variation are derived from a curve fitting program. Ploidy is calculated relative to the diploid standard
Replicate Peak 1
Peak 2 Mean
CV Ploidy
Mean CV
Ploidy 1
76 15.8
2.45 104
10.4 3.35
2 76
16.7 2.45
107 11.9
3.45 3
75 19.6
2.42 102
14.7 3.30
4 75
11.3 2.40
108 7.8
3.48 2 n std.
62 9.2
2.00 –
– –
Ž .
Fig. 2. Flow cytometric ploidy profiles of 48-h-old larvae resulting from short 15–35 min PF — left column Ž
. Ž
. and long 7–43 min PF — right column CB treatments 0.5 mgrl of triploid eggs fertilized with diploid
sperm. Ploidy levels have been calculated relative to a diploid standard.
the five replicates. For reference, the mean relative DNA content of the diploid standard was 61, placing the theoretical tetraploid peak at 122. Although the histograms of
relative DNA content are well defined, they have high coefficients of variation, reflecting the wide distribution of DNA contents in the cells of the larvae, and so, the
presence of a high proportion of aneuploids.
Table 2 Ž
. Ž
. Percent survival among replicates of short 15–35 min PF and long 7–42 min PF CB treatments of triploid
eggs for the induction of tetraploidy in C. gigas, at 48 h and eight days after treatment. Values are calculated from the number of fertilized eggs
Replicate 48 h
8 days Short
1 5
0.6 2
9 3
3 13
3 4
12 4
5 2
1 Mean
8.2 2.3
Long 1
2 1.6
3 1.5
4 0.6
5 Mean
0.7 0.0
2 n std 68
14
Ž .
Short treatment Fig. 2A,C, and E yielded ploidy profiles with a major tetraploid Ž
. peak at about 120, and a secondary peak at 210, corresponding to heptaploid cells 7n .
Replicate 3 has an additional peak with a mean relative DNA content of 74, correspond- ing to 2.5n larvae.
Ž .
The ploidy profiles obtained from long treatments Fig. 2B,D, and F had their Ž
. principal peak at 210 7n with virtually no tetraploid cells. For all the treatments, the
Ž .
percentage of tetraploids after 48 h e.g., eight days of larval rearing was difficult to estimate accurately because of their high variance due to aneuploids. However, we can
reasonably deduce that after the aneuploids and heptaploids died, larvae from short treatments will yield higher levels of viable tetraploids than will long treatments.
Larval survival at 48 h and eight days PF are presented for all five replicates in Table 2. Larval survival at 48 h in short treatments ranged from 2 to 13 averaging 8.2.
After 8 days, this mean was 2.3. For long treatments, larval survival was very low, ranging from 0 to 1.6 at 48 h PF, with no larvae surviving to day 8. For the diploid
standard, larval survival was 68 at 48 h and 14 at day 8.
3.3. Adapting the treatment to meiosis timing Adapting CB treatments to individual females was highly successful. Fig. 3 shows a
typical ploidy profile obtained from larvae 48 h PF. There are two principal peaks whose positions were determined in relation to a diploid standard peak located to a mean
relative DNA content of 57. The principal peak is the tetraploid with a mean relative DNA content of about 117 in each replicate. The secondary peak has a mean relative
DNA content of 69 – 72, corresponding to a 2.5n peak.
Fig. 3. Flow cyometric ploidy profiles of 48-h-old larvae obtained from eggs of an individual triploid Ž
. female=diploid male treated with CB 0.5 mgrl based on the meiotic landmark, 50 PB one. Ploidy levels
have been calculated relative to a diploid standard.
Compared to treatments based on time alone, those adapted to individual females were consistent for producing tetraploid larvae. Table 3 shows the percentage of
tetraploid larvae and their survival at 48 h and at day 8 PF for all eight replicates. Percentages of tetraploids ranged from 30 to 84 at 48 h PF with a mean of 58 and,
Table 3 Percent survival and percent tetraploidy at 48 h and eight days PF from CB treatments of eggs from triploid C.
Ž .
gigas, using biological criteria observed milestones of meiosis adapted to treatment of individual females. nd s not detected
Experiment 48 h
8 days Survival
Tetraploidy Survival
Tetraploidy IndiÕidual females
X4N14 01 26
84 07
92 02
18 33
10 14
03 10
70 06
90 X4N15 01
04 nd
02 13
02 13
30 02
36 03
22 nd
04 63
04 15
nd 02
77 P4N21
07 73
02 nd
Mean 14.4
58.0 4.4
55.0 2 n std.
65 –
37 –
Pooled eggs, multiple females P4N2301
15 11
0.2 12
after eight days, when most of the aneuploid larvae have died, tetraploids ranged from 13 to 92, with a mean of 55.0. Survival at 48 h ranged from 4 to 26, averaging
14.4. After eight days, survival ranged from 2 to 10, averaging 4.4. In the diploid standard, survival was 65 at 48 h and 37 at day 8.
Treatments of pooled eggs from different females produced larvae with 11 te- Ž
. traploids and 15 survival after 48 h Table 3 . At day 8, survival decreased to 0.2
and the percentage of tetraploids remained 12. Treatment was started at 12 min PF and stopped after 37 min, the maximum allowable time under our default parameters.
According to our observations, 50 PB 1 was not reached in this pooled batch of triploid eggs.
4. Discussion
