3. Results
3.1. Endomitosis Ž
. Because of incomplete time series in preliminary experiments , only 11 out of 17
Ž experiments were considered in the estimation of the optimal shock conditions experi-
. ments 1–11 in Table 1 . We identified various peaks in survival rate resulting from the
Ž .
Ž inhibition of meiosis-II meiogynogenesis; till about 16 m.a.a. and first mitosis endo-
. mitosis by varying the moment after activation at which the shock is applied. At 24 h, a
Ž .
clear drop in survival is observed when the shock is applied about 25 m.a.a. Table 1 . Ž
. Survival at 72 h after activation shows the highest although not significant
p 0.05 peak values for different temperatures at the following moments: 2–4, 11–13, 19–22
and 36–38 m.a.a. Ž
. Shock temperature and duration are tightly linked. Long shocks 2–3 min at 418C or
Ž .
428C are lethal results not shown . A 1-min shock at 408C gives relatively high survival Ž
. rates 12, mean of exp. 6 and 7 when the shock is applied 22 and 38 m.a.a.
Ž .
endomitosis . This high percentage is mainly due to one experiment with a female that Ž
. produced a lot of spontaneous gynogens exp. 6 in Table 1 . When this experiment is
Ž .
omitted, the percentages ca 5 obtained with a 1.5–2 min shock at 398C are not Ž
. significantly lower
p 0.05 . Screening of adult gynogenic progeny identified only Ž
phenotypic females n s 233; all 113 adults in Table 2 and 120 additional adults raised .
Ž from gynogenic larvae , except for four males which were typed as non-gynogenic see
. below .
3.2. Determination of maternal identity and degree of heterozygosity Ž
The genetic material of the progeny in the positive control group normal fertilisa- .
Ž .
tion was always traced to both parents Fig. 3; Table 2 . Ž
. Ž
. In the 17 gynogenesis experiments n s 224 , less than 1 two larvae of paternal
Ž .
contamination was found among the larvae Fig. 3B; Table 2 . This percentage was Ž
. higher 7r233 or 3 among presumed gynogens raised to maturity, probably because
of a higher chance of survival of heterozygote individuals. Hence, we further only Ž
. Ž
. considered larvae unless mentioned otherwise to obtain unbiased by selection het-
erozygosity values. Of these mature fish mentioned above, four males and three females were found to carry paternal DNA. Gynogenic males were not observed. In the negative
control we found one biparental individual out of 23 individuals tested. Offspring Ž
. n s 114 that were typed as gynogenic for one marker were also gynogenic when
Ž .
analysed with up to three additional genetic markers Table 2 . Among the true meiogynogenic offspring induced with a shock applied 1 to 16
m.a.a., high percentages of heterozygotes were found. Heterozygosity percentages of Ž
. Ž
. 86, 71 and 17 were recorded at locus Cga01
n s 115 , Cga08 n s 28 and
Ž .
Ž .
Cga09 n s 12 , respectively Table 2 . Ž
. Mitogynogenic progeny induced with a shock applied 17 to 40 m.a.a. should prove
Ž to be homozygous using a heterozygous female. However, at locus Cga01 across all
. Ž
experiments in Table 2; but see for example Fig. 3A , and at Cga09 in 13 larvae and an
Table 2 Ž
. Genotypes of parents and offspring described in Table 1 a of genotyped offspring between brackets .
Bold s paternal contamination, italic sadult offspring and vertical bars s pooled offspring
Ž .
Fig. 3. Photograph of a 4 agarose gel NuSieve GTG; two rows of wells revealing the PCR-products of a Ž
. Ž
. putative mitogynogenic family male: eight, female: nine, and offspring: 10 to 23 , positive 1–4 and negative
Ž .
Ž . Ž .
Ž . control individuals 5–7 . Microsatellite DNA was amplified at the Cga01 A and Cga02 B loci. A reveals
that only five gynogenic individuals are homozygous: genotype ‘CC’ for the negative control in lane 5 and genotype ‘AA’ for the gynogenic individuals heat-shocked at 29, 30, 32 and 38 m.a.a. in lanes 13, 14, 16 and
Ž . Ž .
22, respectively see exp. 13 in Table 2 for more details .
B shows that all except one individual
Ž .
Ž . Ž
heat-shocked at 38 m.a.a. lane 20 are gynogens, excluding the positive control 1–4 M s Molecular
. marker: 100, 200 and 300 bp fragments are visible as separate bands .
. specially large batch of 99 adults at 37 m.a.a.; see exp. 6 in Table 2 we detected
Ž .
Ž .
respectively 75 n s 20 and 26 n s 112 heterozygotes among the true gynogenic offspring. Because attempts to raise these latter individuals to maturity were complicated
by high mortalities and because of low numbers in general, the percentages presented should be considered with caution.
We also analysed the level of heterozygosity in the negative control group. Not Ž
. Ž
. counting the one contaminant see higher we found that 13 out of 18 72 sponta-
Ž .
neous gynogens were heterozygous at locus Cga01 Fig. 3A; Table 2 .
4. Discussion
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