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Gynogenesis in the African catfish Clarias

ž

/

gariepinus Burchell, 1822

III. Induction of endomitosis and the presence of

residual genetic variation

Peter Galbusera

)

, Filip A.M. Volckaert, Frans Ollevier

Laboratory of Aquatic Ecology, Katholieke UniÕersiteit LeuÕen, Ch. de Beriotstraat 32,

B-3000 LeuÕen, Belgium

Accepted 14 October 1999

Abstract

Gynogenesis is thought to be a useful method to generate fully inbred lines in teleost fish. Endomitosis, which refers to the inhibition of first mitosis, should lead to fully homozygous

Ž .

offspring. In this study, the optimal conditions to induce mitogynogenesis endomitosis by heat shocking of the eggs were determined in the African catfish Clarias gariepinus. Comparable survival rates were obtained with a shock of 408C applied for 1 min and a shock of 398C applied for 1.5–2 min. Applying the shock around 20 or 37 min after activation resulted in the highest

Ž . Ž .

survival rates ca. 5% relative to the positive control . The amount of inbreeding homozygosity and paternal contamination were assayed by using polymorphic microsatellite DNA markers. In less than 1% of the offspring paternal alleles were present, indicating a true gynogenic background for most of the progeny. Meiogynogenetic and mitogynogenetic C. gariepinus showed a fair

Ž .

amount of residual heterozygosity respectively 86% and 75% for one of the markers .

Heterozy-Ž .

gous catfish obtained through mitogynogenesis were attributed to the simultaneous presence of meiogynogens. In addition, we prove that the sex determining system of African catfish

Ž .

C. gariepinus is heterogametic in the male XYrXX . q2000 Elsevier Science B.V. All rights reserved.

Keywords: Clarias gariepinus; Gynogenesis; Microsatellite DNA; Residual heterozygosity; Sex determination;

Teleostei

)Corresponding author. Laboratory for Evolutionary Biology, University of Antwerp RUCA , Groenen-Ž .

borgerlaan 171, B-2020 Antwerpen, Belgium. Tel.:q32-3-218-04-69; fax:q32-3-218-04-74.

Ž .

E-mail address: galbus@ruca.ua.ac.be P. Galbusera .

0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.

Ž .

(2)

1. Introduction

Ž

Induced gynogenesis is a unique tool for inbreeding purposes in teleost fish

Chour-.

rout, 1988; Mair, 1993 . It involves an artificial reproduction, using UV-irradiated sperm to activate the eggs and the application of a physical or chemical shock to restore the diploid status of the embryo. The shocks destroy the aster formation or the microtubules

Ž .

of the spindle figure and inhibit nuclear division Diter et al., 1993 . The result is a

Ž .

diploid embryo containing maternal genetic material only Fig. 1 . Meiogynogenesis is achieved by inhibiting the extrusion of the second polar body. The resulting offspring are homozygous at a locus only if no recombination occurred. By determining the percentage of heterozygous offspring, one can calculate the recombination frequency.

Ž .

Inbreeding F is lower for those traits showing much residual heterozygosity. For some

Ž

species, like rainbow trout, this residual heterozygosity is so high up to 100% at some

.

loci that meiogynogenesis cannot be considered as an efficient inbreeding tool

ŽThorgaard et al., 1983; Guyomard, 1984 ..

Ž .

Mitogynogenesis or endomitosis results in fully homozygous offspring Fs1 since

Ž .

it is achieved by inhibiting the first mitotic cleavage after duplication of the haploid

Ž . Ž .

genome Fig. 1 . Homozygous inbred strains of genetically identical fish clonal lines may be obtained after two generations using this reproduction method. It has been

Ž .

achieved in zebrafish Danio rerio Streisinger et al., 1981 , medaka Oryzia latipes

ŽNaruse et al., 1985 , common carp Cyprinus carpio. ŽKomen et al., 1991 , ayu.

Ž . Ž

Plecoglossus altiÕelis Han et al., 1992 , rainbow trout Oncorhynchus mykiss Quillet et

. Ž .

al., 1991; Scheerer et al., 1991 , hirame Yamamoto, 1999 and Nile tilapia

Ore-Ž .

ochromis niloticus Hussain et al., 1993 . The importance of inbred strains in research

Ž .

and aquaculture has been stressed by Bongers 1997 . Inbreeding represents an impor-tant tool to improve production characteristics with increased chances to directly select specific genotypes. The genome, purged of deleterious genes, may be reconstituted by

Ž .

crossing several inbred lines Yamamoto, 1999 . Also, crossing clonal lines may induce heterotic effects. Finally, as a result of reduced genetic variation in inbred lines, less animals are necessary to obtain statistically significant experimental designs.

The African catfish Clarias gariepinus is an economically important fish species which is cultivated on many continents. In Africa and Asia, an extensive as well as

Ž .

intensive culture exist Huisman and Richter, 1987 , whereas in Europe, they are mostly intensively produced. The specific conditions for the production of meiogynogens through the retention of the second polar body have already been optimised in C.

Ž . Ž .

gariepinus Volckaert et al., 1994, 1997 . High survival rates 46% were obtained when

Ž .

a shock of 418C at 3 min after activation m.a.a. was applied for 2 min. Cold and

Ž

pressure shocks applied 4 m.a.a., generated even higher survival rates 80% and 68%,

.

respectively . The optimal conditions to induce endomitosis remained to be determined. Preliminary experiments showed a higher survival in mitogynogenic embryos using heat

Ž .

shocks, as predicted by Hussain et al. 1993 . Furthermore, androgenesis in C.

gariepi-Ž . Ž

nus has been successfully induced up to 10.5% survival by heat shocking Bongers et .

al., 1995 .

(3)

differ-Fig. 1. Induced and spontaneous gynogenesis in fish. When the oogonium of a female heterozygous at loci T and Q is activated by a UV-irradiated sperm cell, it completes meiosis-II and develops into a haploid

Žnon-viable embryo. Viable diploid 2n offspring can be obtained by inhibiting first mitosis A: mitogyno-. Ž . Ž

. Ž

gens 2n; complete homozygous offspring or second meiosis B: meio-II-gynogens 2n; heterozygous offspring

.

depending on the recombination frequency, r, of the locus; 0% heterozygosity, H, if rs0 . Spontaneous

Ž

gynogenesis can occur via the latter mechanism but also by inhibition of the first meiosis C: meio-I-gynogens

. Ž

2n . In this case, recombination leads to 50% heterozygous offspring 100% heterozygous clonal offspring if

(4)

Ž .

ences in the spermatocrit Mair, 1993 , could generate progeny with paternal genetic

Ž . Ž

material ‘false’ gynogens . This may lead to misinterpretation of the results Carter et

.

al., 1991 . Furthermore, one should verify the homozygous nature of endomitotic

Ž .

gynogens Bongers, 1997 and determine the degree of residual heterozygosity, through recombination, in meiogynogenic progeny. It is possible to assess the level of

recom-Ž

bination and hence the inbreeding rate by using heterozygous females Chourrout,

.

1988 .

Genetic markers are required to detect paternal contamination and to determine the degree of heterozygosity in gynogenic offspring. Morphological markers can be used if

Ž .

the trait is based on a recessive allele; such phenotypic markers e.g., albinism were not available in our broodstock. Multilocus DNA fingerprinting has been used for detecting

Ž

paternal contamination but it is not efficient to verify heterozygosity levels Carter et al.,

. Ž

1991; Volckaert et al., 1994; Bongers, 1997 . Allozyme markers are useful Linhart et

.

al., 1987 but they cannot compete with the level of polymorphism and the information

Ž .

content of single locus microsatellite markers Wright, 1992 . Furthermore, these latter markers allow biopsies.

We show the low feasibility of applying heat shocks to induce endomitosis and the presence of a few ‘false’ and relatively many spontaneous gynogens. We provide alternative hypotheses for the high percentage of residual heterozygosity at one of the DNA markers and suggest an alternative estimation of the recombination rate. Finally, we show evidence for male heterogametic sex chromosomes in C. gariepinus.

2. Materials and methods

2.1. Samples

The catfish used in this study originate from a stock held at the Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Leuven, Belgium, which includes a line

Ž . Ž

originating from the Hulah Swamps Israel and a line from the Ubangui River Central

.

African Republic . Fish were kept under standard conditions as detailed in Huisman and

Ž .

Richter 1987 . Ovulation, egg collection, artificial fertilisation and larviculture have

Ž .

been described previously Volckaert et al., 1994 . We used in each experiment genetically screened and electronically tagged parents belonging to two different lines.

Ž . Ž

Adults were sexed externally genital papilla and internally presence of ovaries or

.

testes .

2.2. Shock experiments

The methods for UV-irradiation and heat shocking have been described in Volckaert

Ž . Ž .

et al. 1994 . We varied the shock-temperature 398C, 408C, 418C and 428C , the

Ž .

duration of the shock 1, 1.5, 2 and 3 min and especially the time of application after

Ž .

(5)

Ž 2.

Fig. 2. Optimal UV-irradiation dosage Jrcm to inactivate the sperm genetically in function of the

Ž 9. Ž .

spermatocrit value number of sperm cells per ml=10 ys0.367q0.493log10x .

conditions. The first mitotic division occurred at about 35 m.a.a. at an incubation

Ž .

temperature of 288C Bongers et al., 1995 . Nevertheless, we applied the shock 1 to 40

m.a.a. to compare yields in survival and heterozygosity in meio- and mitogynogens. For each experimental condition, we obtained the percentage of surviving embryos 24 and

Ž

72 h after activation. To allow for comparison between experiments using different

.

females , standardised survival rates were calculated relative to the positive control

Žwhich is a normal fertilisation. ŽVolckaert et al., 1994 . A negative control eggs. Ž .

activated with irradiated sperm but no shock applied was included to assess the effectiveness of the irradiation. We determined the spermatocrit value of each sperm

Ž

sample in order to adjust the irradiation time accordingly extended irradiation for

.

samples with a high spermatocrit value; see Fig. 2 . Not all combinations of shock

Ž .

temperature and duration were analysed see Results but each experiment was carried

Ž .

out in duplicate except 398C at 1 min . In total, 17 successful experiments were

performed. Embryos were kept at 288C before and after shocking. Putative endomitotic

fish were reared to maturity in order to determine their sex. Survival rates were arcsin-transformed and tested for their significance using a one-way ANOVA and

Ž .

Duncan’s multiple range test Sokal and Rohlf, 1995 .

2.3. Genetic analysis using microsatellite DNA markers

Ž .

We used five Cga01, Cga02, Cga05, Cga09 and Cga10 out of seven microsatellite

Ž .

primer sets which have been developed previously Galbusera et al., 1996 and one additional marker, Cga08, similarly developed through the production and screening of a

Ž

library of short DNA fragments of C. gariepinus. Cga08 GenBank accession number

.

U30869 has the primer sequences F-CATGAGCCAGACACCATTCCC and

R-TTTC-Ž .

(6)

Table 1

Ž .

Survival rate percentage relative to the positive control, after 24 and 72 h, rp24 and rp72, respectively of

Ž . Ž .

positive pos and negative neg control, meiogynogenic and mitogynogenic progeny in each experiment

Žm.a.a.sminutes after activation — other shock conditions are: temperature in8C_duration in minutes.

m.a.a. rp24 rp72 rp24 rp72 rp24 rp72 rp24 rp72 rp24 rp72

39_1 exp1 39_1,5 exp2 39_1,5 exp3 39_2 exp4 39_2 exp5

(7)

Table 1 continued

m.a.a. rp24 rp72 rp24 rp72 rp24 rp72 rp24 rp72 rp24 rp72

40_1 exp6 40_1 exp7 40_1,5 exp8 40_1,5 exp9 41_1 exp10

1 44 7 10 8 5 1 10 9

(8)

Ž .

Table 1 continued

m.a.a. rp24 rp72 m.a.a. rp24 rp72 m.a.a. rp24 rp72 m.a.a. rp24 rp72

41_1 exp11 40_1–2 exp12 40_1–2 exp13 41_2 exp14

1 17 2 2 0 0 20 28 10 1 51 50

range of 158–180 bp and optimal PCR conditions are 628C annealing temperature and

(9)

Table 1 continued

m.a.a. rp24 rp72 m.a.a. rp24 rp72 m.a.a. rp24 rp72

40,5_2 exp15 40_1 exp16 39_1,5 exp17

1 113 98 20 34 27 21 11 2

genomic DNA of fin-clips or whole larvae of about 1 week old was prepared using

Ž

phenol–chloroform extraction or by boiling for 2 h in a 10% Chelex solution Resin

.

100, Bio-Rad Laboratories, Hercules, CA . The DNA was amplified according to

Ž .

Galbusera et al. 1996 .

Three methods are used to visualise these PCR products after electrophoresis depending on the resolution needed to distinguish between alleles. Random labelling

35 Ž

with S or end labelling with a fluorescent molecule Fluos-Phosphororamidite from

.

Eurogentec, Seraing, Belgium is used to visualise the PCR-products which are sepa-rated on a 6% PolyAcrylamide-gel. EtBr is applied after separation of the products on a 4% NuSieve GTG agarose-gel.

In order to check whether there is any paternal contamination in the gynogenic progeny caused by incomplete UV-irradiation, broodstock fish were selected with different alleles for at least one microsatellite locus. Heterozygous females were chosen in order to measure recombination rates. Because the strains used have been

domesti-Ž .

cated for many years about 10 to 20 generations , few females were heterozygous at

Ž .

(10)

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.

Žendomitosis . 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 ns233; 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 ns224 , 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

Žns114 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 ns115 , Cga08 ns28 and

Ž . Ž .

Cga09 ns12 , 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

(11)

Table 2

Ž .

(12)

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 MsMolecular

.

(13)

.

specially large batch of 99 adults at 37 m.a.a.; see exp. 6 in Table 2 we detected

Ž . Ž .

respectively 75% ns20 and 26% ns112 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

The observed yields of meiogynogens were lower than the yields obtained by

Ž .

Volckaert et al. 1994 because of the suboptimal conditions. The higher survival of

Ž .

meiogynogenic individuals compared to mitogynogens Table 1 was expected because at the moment of second meiosis most eggs are simultaneously in metaphase whereas at first mitosis variance in development is much more pronounced. The expression of lethal recessive alleles in mitogynogens might be a lesser factor contributing to the lower

Ž .

survival rates Komen et al., 1991 .

Ž .

To induce endomitosis, a shock of 408C applied for 1 min or 398C for 1.5–2 min at

Ž .

36–38 m.a.a. resulted in the highest survival ca 5% . Since some of these surviving

Ž .

embryos came from a female in exp. 6 of Table 1 that produced many spontaneous gynogens, this result might be partially due to a maternal effect as suggested by Quillet

Ž . Ž .

et al. 1991 and Horstgen-Schwark 1993 . However, shock conditions similar to ours

¨

Ž .

were also most successful in inducing endomitosis Varadi et al., 1999 and

androgene-Ž .

sis Bongers et al., 1995 in C. gariepinus, resulting in similar survival rates. The ranges

Ž16–44 min and 0.6–1.8t , respectively at which shocks were applied in these studies. 0

might be too narrow to observe, respectively, the peak of karyokinesis and cytokinesis.

Ž .

The different optimal time for shocking in androgenesis 33 m.a.a. might be related to

Ž .

the higher incubation temperature 308C . Also in Nile tilapia, O. niloticus, androgenesis

Ž

and endomitosis appeared to occur during the same developmental stages Myers et al.,

.

1995 . A cold shock applied 35–45 m.a.a. resulted in 5% mitogynogenic offspring in the

Ž .

European catfish Silurus glanis Krasznai and Marian, 1986 .

´ ´

The verification of exclusively maternal inheritance is conditional in this sort of experiments. The very low levels of paternal contamination prove the high efficiency of the sperm-irradiation, which was probably obtained by adjusting the irradiation time to the spermatocrit value of each sperm sample. Paternal contamination has been observed

Ž . Ž .

in O. aureus Carter et al., 1991 and rainbow trout Thorgaard et al., 1985 but was

Ž . Ž .

absent in C. gariepinus Varadi et al., 1999 , Salmo trutta Estoup et al., 1993 and

Ž .

Barbus barbus gynogens Castelli et al., 1990 .

We encountered 86% heterozygotes at the microsatellite DNA locus Cga01 and 71% at Cga08 after meiogynogenesis. Without recombination, each pair of homologous chromosomes would have been derived of sister-chromatids which are genetically

Ž .

(14)

is the selective death of the homozygotes due to recessive lethal genes or gene

Ž .

complexes purging of the genetic load , leading to an overestimate of the number of

Ž .

heterozygotes Guyomard, 1984; Guo and Gaffney, 1993 . Ot the other hand, high

Ž

recombination levels have been detected in rainbow trout Thorgaard et al., 1983;

. Ž . Ž

Guyomard, 1984 , Pacific oyster Guo and Gaffney, 1993 , channel catfish Goudie et

. Ž . Ž .

al., 1995 , carp Komen et al., 1991 , tilapia Carter et al., 1991; Mair, 1993 and plaice

ŽThompson et al., 1981 . Theoretically, 67% heterozygosity is the maximum expected.

Ž .

from independent recombination events Purdom et al., 1976; Guo and Gaffney, 1993 . Recombination occurs most frequently in the distal parts of chromosomal arms. The short length of the fish chromosomes might explain the high recombination rates since the consequently higher chiasma interference might lead to one single obligate chiasma

ŽThorgaard et al., 1983; Komen et al., 1992 . There is another explanation, however, that.

does not imply complete chiasma interference.

It implies that part of the induced gynogenic offspring are in fact spontaneous

Ž .

gynogens through the inhibition of meiosis-I as proposed by Quillet et al. 1991 .

Ž .

Komen et al. 1991 suggest that the 1–1.5% spontaneous gynogens are obtained by meiosis-II non-disjunction. No differences were found in the segregation of marker genes in the gynogenic progeny resulting from spontaneous diploidisation of maternal

Ž .

chromosomes SDM and from induced diploidisation by inhibition of meiosis-II in

Ž . Ž .

plaice Thompson et al., 1981 and common carp Cherfas et al., 1995 . However, inhibition of meiosis-I after recombination cannot be excluded based on these results.

Ž .

Cherfas et al. 1995 observed 100% heterozygosity and genotypic identity for all

Ž .

gynogenically without shock produced koi carp using five polymorphic protein genes.

Ž .

This suggests that inhibition of meiosis-I without recombination ameiotic gynogenesis

Ž .

or premeiotic endoreduplication duplication of the genome before meiosis is involved. It is not possible to discriminate between both mechanisms on a genetic basis. Ameiotic

Ž

parthenogenesis occurs naturally in some all-female species e.g., the molly Poecilia

. Ž .

formosa and leads also in nature to obligate heterozygosity and clones Purdom, 1993 .

We cannot present direct evidence for the inhibition of meiosis-I as the cause of spontaneous diploidisation in C. gariepinus but there are some leads in the negative

Ž .

control group. Survivors in this group contain only female DNA except a single male

Ž

and hence are spontaneous gynogens since these eggs did not receive any shock Linhart

.

et al., 1995 . Heterozygosity percentages above 67% observed in the negative control can be explained if we assume that these spontaneous gynogens arise by inhibition of

Ž .

meiosis-I which are heterozygous unless recombination occurred . Since also

homozy-Ž .

gotes 28% at Cga01 have been observed in the negative control group, we cannot exclude recombination before inhibition of meiosis-I. Because only 50% of the eggs that

Ž .

undergo recombination are homozygous Fig. 1 , the recombination rate can be obtained

Ž .

by doubling the percentage of homozygotes rs2=% homozygosity in the negative control, assuming that recombination frequencies in normal and spontaneous gynogenic offspring are comparable. For locus Cga01, the recombination rate has thus been

Ž .

estimated at 56% instead of 72% .

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Whatever the cause, either selective mortality, sudden temperature changes or

over-Ž .

maturation of the eggs Cherfas et al., 1995; Linhart et al., 1995 , the coefficient of

Ž .

inbreeding F is smaller than expected after gynogenesis because off the high residual

Ž .

heterozygosity Ihssen et al., 1990 . Meiotic gynogenesis is by far not as effective for rapid inbreeding as endomitosis where after one generation inbreeding should be, in theory, complete.

Contrary to the expectations, we also detected heterozygotes among the putative mitogynogenic offspring. Using two allozyme markers to analyse heat-treated offspring

Ž .

of the same catfish species, Varadi et al. 1999 revealed only homozygous genotypes. It is not clear, however, whether the three females used in this latter study were homozygous for these loci. Mito- and meiogynogens could not be distinguished

unam-Ž . Ž .

biguously in tilapia Carter et al., 1991 , carp Linhart et al., 1987 and rainbow trout

ŽPurdom et al., 1985; Scheerer et al., 1991; Quillet, 1994; Young et al., 1996 . These.

individuals are not mitogynogenic but are probably spontaneous meiogynogenic and are thought to have arisen by spontaneous inhibition of the first or second meiotic division. Alternatively, at a slow developmental rate of certain eggs, a ‘late’ shock might inhibit the second meiotic division instead of the first mitotic division, resulting in induced meiogynogens. The numbers of these spontaneous and induced meiogynogens are probably quite small but since mitogynogens are also rare, the effect on the residual

Ž .

heterozygosity might be important Quillet et al., 1991 . The putative mitogynogens are

Ž

75% heterozygous at locus Cga01. The expected percentage based on the spontaneous

.

gynogens is around 72% for this locus. Consequently, we cannot expect that we induced endomitosis effectively in the mitogynogenesis experiments analysed with this marker. This probably explains the lack of experimental conditions with significant higher survival rates.

The chromosomal sex of C. gariepinus we deduced seems contradictory to the literature. On one hand, karyological analysis and chromosome manipulation indicated

Ž . Ž

the female as heterogametic ZZrZW for the sex chromosomes Ozouf-Costaz et al.,

.

1990; Teugels et al., 1992; Varadi et al., 1999 , on the other hand, in our study only female ‘true’ gynogens were obtained. However, karyotypic information is not necessar-ily a proof of the true genetic sex, while the circumstances of obtaining the mixed sex gynogenetic progeny are not all that clear. We propose that the sex determining system

Ž .

in African catfish is heterogamous in the male XYrXX as confirmed by test crosses of

Ž .

sex reversed femalesrmales Liu et al., 1996; Eding et al., 1997 . The possibility remains that exogenous factors such as water temperature can influence

sex-differentia-Ž .

tion, as shown for example in hirame Yamamoto 1999 . The genetic sex has major implications for the aquaculture of C. gariepinus; phenotypic males of C. gariepinus

Ž .

grow faster than females Henken et al., 1987 . Males of the American catfish Ictalurus

Ž .

punctatus grow also faster after 10 months Simco et al., 1989; Davis et al., 1990 and

are up to 10% larger and 37% heavier after 26 months. However, all-male production of

C. gariepinus is not feasible using gynogenesis. Androgenesis may provide a

perspec-Ž . Ž .

tive through the production of ‘super-males’ YY Bongers et al., 1995 . Furthermore, this technique might be better suited to produce clonal lines. All five rainbow trout

Ž .

(16)

apparently was derived by spontaneous polar body retention that maintained heterozy-gosity at some loci.

In brief, our findings stress the importance of genetic verification when producing homozygous lines. The application of highly variable microsatellite markers is of special interest in this kind of monitoring. These markers have the additional advantage that they allow an estimation of the heterozygosity level for each marker. Without excluding alternative hypotheses, we propose a hypothesis based on SDM to explain the high percentage of residual heterozygosity.

Acknowledgements

Ž

This research has been funded by the Commission of the European Union FAR

. Ž .

AQ.5.376 and the Research Fund of the Katholieke Universiteit Leuven OTr90r16 . PG was a graduate student supported by a Belgian I.W.T.-scholarship. FV is a research

Ž .

fellow of the Fund for Scientific Research FWO-Vlaanderen . We express our gratitude towards R. Huybrechts and A. De Loof for the use of the laboratory facilities. We thank B. Hellemans for his help with the practical work, T. Wenseleers for the stimulating discussions and two anonymous reviewers for reading earlier versions of this manuscript. E. Holsters and G. Janssens are kindly acknowledged for taking care of the experimental facilities and animals.

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Gambar

Fig. 1. Induced and spontaneous gynogenesis in fish. When the oogonium of a female heterozygous at loci Tand Q is activated by a UV-irradiated sperm cell, it completes meiosis-II and develops into a haploidgens 2n; complete homozygous offspring or second m
Fig. 2. Optimal UV-irradiation dosageŽJrcm2.to inactivate the sperm genetically in function of thespermatocrit value number of sperm cells per mlŽ=109
Table 1Survival rate percentage relative to the positive control, after 24 and 72 h, rp24 and rp72, respectively of
Table 1 continuedŽ.
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