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Assessment of homozygosity and fertility in meiotic gynogens of the European sea bass (Dicentrarchus labrax L.)
Aquaculture 243 (2005) 93 – 102 www.elsevier.com/locate/aqua-online
Assessment of homozygosity and fertility in meiotic gynogens of the European sea bass (Dicentrarchus labrax L.) Antonia Francescona,*, Alvise Barbaroa, Daniela Bertottoa, Angelo Libertinia, Fulvio Cepollarob, Jacopo Richardc, Paola Belvedereb, Lorenzo Colombob a
CNR-Ismar, Istituto di Scienze Marine/Biologia del Mare, Castello 1364/A, I-30122 Venezia, Italy b Dipartimento di Biologia, Universita` Padova, Via Colombo 3, I-35121 Padova, Italy c Veneto Agricoltura Centro Ittico Sperimentale, strada Murazzi, 200, I-30100 Pellestrina (Ve), Italy Received 3 March 2004; received in revised form 26 October 2004; accepted 30 October 2004
Abstract Analysis of 5–6 microsatellite loci was used to measure the increment of homozygosity in two meiogynogenetic progenies (A and B) of sea bass with respect to their mother. In progeny A and B, 20% and 12% of the meiogynogens retained heterozygosity for all investigated maternal loci, respectively, while complete homozygosity was observed only in 6% of B and in none of A, indicating the occurrence of significant allelic recombination during meiosis. The overall increment of homozygosity for the investigated loci was 27% for A and 47% for B. Although survival at hatching of meiogynogens was about half that of controls, they subsequently grew as controls and displayed the same onset of puberty and reproductive potential at adulthood during three consecutive years when crossed between themselves (11 crosses) or with control fish (13 crosses). In particular, meiogynogenetic females (n=24) underwent vitellogenesis and yielded eggs of good quality upon stimulation with LH–RH analogue similarly to normal fish. Sperm released by meiogynogenetic males (n=23) was equivalent to that of controls in terms of volume, quality and fertilization capability. Second generation meiogynogens were obtained by chromosome set manipulation from meiogynogenetic females and found to be morphologically normal at 3 years of age. Interestingly, under our culture conditions, the percentage of meiogynogenetic males in the second generation was 7% as opposed to 39% in the first generation. D 2004 Elsevier B.V. All rights reserved. Keywords: Sea bass; Dicentrarchus labrax; Gynogenesis; Homozygosity; Microsatellite DNA; Fertility
1. Introduction * Corresponding author. Tel.: +39 41 2404711; fax: +39 41 5204126. E-mail address: [email protected] (A. Francescon). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.10.023
Owing to high fecundity and easy access to gametes, teleost fish are suitable models for research on gynogenesis and its application to improve fish
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culture. To this end, it is essential to establish for each species whether gynogens are not only viable and growable to sexual maturity, but also if their gametes are normally fertile. Induced gynogenesis obtained by sperm genetic inactivation and diploidy restoration by inhibition of the second meiosis with consequent retention of the second polar body (meiogynogenesis) ensures viable uniparental chromosome transmission and all-maternal genetic inheritance. This reproductive mode enhances homozygosity in the progeny where the retention of the second polar body replaces the male pronucleus in the karyogamic process and maintains the property of a sexual reproductive interaction. In the case of endomitosis or double haploid gynogenesis, the first embryonic cleavage is inhibited (mitogynogenesis) and the manipulation targets the inception of the somatic lineage. Hence, it modifies asexually the course of the embryo formation. Induction of mitogynogenesis produces homozygous offspring, while meiogynogens show residual heterozygosity due to recombination events. Nevertheless, meiogynogenesis can be used to obtain isoallelic lines as a preliminary effort to discharge deleterious alleles. Moreover, meiogynogens exhibit greater survival than mitogynogens and are usually preferred (because progeny have not been made completely homozygous) to produce all-female progeny as well as all-pseudomale progenys by androgen treatment in species with strict XX/XY sex determination (Zanuy et al., 2001). Sex ratio analysis can reveal the heterogametic sex or evidence the occurrence of autosomal or environmental factors in sex determination. Meiogynogens are also invaluable for the estimation of allele recombination frequencies and gene–centromere mapping (Purdom, 1993). The European sea bass (Dicentrarchus labrax L.) is an economically important species whose farming has greatly expanded in recent times along the Mediterranean and Atlantic coasts of Europe, Asia Minor and North Africa. These species have been the subject of several studies on chromosome set manipulation to induce gynogenesis and polyploidy (Carrillo et al., 1993; Colombo et al., 1995; Gorshkova et al., 1995; Barbaro et al., 1998; Knibb et al., 1998; Peruzzi and Chatain, 2000; Felip et al., 2002). It has been conclusively demonstrated that sea bass meiogynogens are viable and grow to adulthood
showing variable sex ratios, a fact that has hampered the production of all-female stocks in this species. Felip et al. (2002) has reported as unpublished results that female meiogynogens display normal vitellogenesis and are capable of producing eggs of excellent quality after hormonal treatment. No information is currently available on the fertility of male meiogynogens. The objective of this work was to assess the homozygosity and the fertility in meiogynogen sea bass. We compared gamete quality, and fertilization and hatching rates in crosses, including female and male meiogynogen lots, meiogynogen and normal fish lots, and normal crosses. We also established that a second generation of meiogynogens might be obtained from meiogynogenetic parents. Moreover, microsatellite analysis was applied to a first generation of meiogynogens to check the absence of paternal genetic contribution and provide a preliminary estimate of levels of homozygosity.
2. Materials and methods 2.1. Founder fish stock Wild specimens of European sea bass (D. labrax L.) were captured by angling in the North Adriatic sea and kept in an outdoor 500-m3 tank at 32–35 ppt salinity under ambient water temperature (14–28 8C) and photoperiod (May–October), as well as in an indoor 20-m3 tank at 14 8C and 34 ppt of salinity under natural photoperiod (November–April). All fish in this study were electronically tagged and reared communally (diploid meiogynogens and controls). 2.2. Spawner selection and hormonal treatment During the reproductive season (January–March), mate pairs were transferred separately into 9-m3 spawning tanks with recirculating water and a net (500-Am mesh) basket at the outlet for egg collection. Preovulatory females were subjected to ovarian biopsy and selected for hormonal treatment on the basis of the diameter of 20 postvitellogenic follicles (N700 Am). Oviposition was induced by a single intramuscular injection of 10 Ag/kg body weight of GnRH analogue (des-Gly10, [d-Ala6] LH–RH ethyl-
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amide; Sigma, USA). Sperm fluency in males was assessed after stripping by measuring the sperm volume and counting spermatozoa in a Bqrker haemocytometer. Sperm motility duration (time in seconds from activation to propulsive motion cessation; Fauvel et al., 1999) was checked in duplicate following dilution (1:10) with extender solution (0.1875 M KCl, pH 7.35; Colombo et al., 1995) and further mixing (1:10) with seawater (35 ppt). Males were not hormonally treated. 2.3. Gamete collection and artificial fertilization When fish were judged as proximate to spawning according to their behaviour, they were rapidly netted and anaesthetized. About 1-g sample of eggs was stripped from each female to establish good quality based on egg buoyancy (35 ppt at 14 8C) and morphology (regular shape, transparency, presence of cortical granules, unsplit lipid droplet and absence of flocculation). If good quality was confirmed, a second full stripping was performed and the total egg mass was volumetrically estimated. Eggs were maintained at 14 8C for V15 min. Absolute fecundity (AF) was inferred from the total number of stripped eggs (calculated as total egg volume/volume of a single egg); relative fecundity (RF) was given by the ratio AF/postspawning female body weight. Collected sperm was stored at 2–4 8C until use. Artificial fertilization was carried out mixing 1 part of eggs, 4 parts of seawater and 1 part of sperm diluted 1:100 with extender. Three minutes later, eggs were washed out of sperm excess. Approximately 100 eggs were withdrawn from the hatching tanks to estimate the percentage of fertilization [number of blastulae at 12 h postfertilization (pf)] and 300–500 eggs were cultured in 2-l flasks to determine the percentage of hatching at 96 hpf (number of larvae100/number of fertilized eggs). All samples were carried out in duplicate. 2.4. Production of meiogynogens Meiogynogenesis was obtained by egg fertilization with UV-irradiated (254 nm; 3300 erg mm 2) sperm, and diploidy was restored by cold shock (0 8C, 5 min after fertilization for 20 min; Barbaro et al., 1998)
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which effectively blocks the extrusion of the second polar body. For each egg batch, three control crosses were produced in addition to gynogens: a normal diploid progeny to check egg quality, a haploid progeny to check effectiveness of sperm inactivation, and a triploid progeny to verify efficacy of the cold shock. Determination of ploidy was carried out by flow cytometry by measuring the nuclear DNA amount in pooled embryos (haploids), single larvae or in individual samples of erythrocytes or caudal finclips from adult fish (Libertini et al., 2002). Two meiogynogenetic progenies, A and B, were genetically typed using microsatellite markers (n=6 in A and n=5 in B; see below) at 43 days after hatching (larvae) and at 1 year of age (fin-clips), respectively. Maternal transmission was determined based on the allelic sets of the respective parents. The overall heterozygosity was obtained as percentage by cumulating the number of heterozygotes at each loci in each progeny. Because both males and females may be obtained by meiogynogenesis in the sea bass, as reviewed by Felip et al. (2001), a stock of 21 female and 24 male meiogynogens were raised to adulthood for 4 years and were utilized during the subsequent 3 years as spawners through 1–3 cycles. Control diploids were similarly cultured and analysed as reference stocks. 2.5. Reproduction of meiogynogens Both gynogenetic and control males with fluent sperm and females with postvitellogenic follicles of at least 700 Am in diameter and hormonally stimulated as above were selected to accomplish a total of 24 meiogynogenetic and 20 control crosses by artificial fertilization according to all four possible crossing combinations. Four gynogenetic females were allowed to spawn spontaneously and separately with four gynogenetic males. Here, AF was calculated according to the formula: number of laid eggs=volume of eggs (2865 1796 egg mean diameter) (Fornie´s et al., 2001). The applicability of chromosome set manipulation to gametes obtained from meiogynogenetic female sea bass was investigated by subjecting eggs fertilized with UV-irradiated sperm to meiotic block by cold shock to produce second-generation meiogynogens as described above.
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2.6. Genetic analysis Fin-clips or whole larvae were stored in ethanol. Genomic DNA was purified according to Laird et al. (1991) and DNA quality was verified by 0.8% agarose gel electrophoresis. Genetic variation was assayed using up to 6 microsatellite loci: Dla-6 (Castilho and McAndrew, 1998), Labrax-6, Labrax8, Labrax-13, Labrax-17 and Labrax-29 (Garcı´a de Leo´n et al., 1995). All loci are on separate linkage groups (Chistiakov et al., 2004; Chris Haley, BASSMAP research panel, Rosling University UK, personal communication). Selection of primer pairs (MWG-Biotech, Ebersberg, Germany; Perkin-Elmer/ Applied Biosystems Division; Milan, Italy) was based on their polymorphism, amplification repeatability and coamplificability in a single PCR reaction without artefact bands. To distinguish products with overlapping size ranges, a total of six primer sets was labelled with three different fluorochromes used in a multiplex DNA PCR reaction. Three primer sets were 5V-labelled with Hex dye, two primer sets with 6-Fam and one set with Ned. The multiplex DNA amplifications were performed in a T-Gradient Thermal Cycler (Whatman Biometra, Gfttingen, Germany), using a touch-
down PCR protocol. It allowed the simultaneous amplification of loci having different optimal annealing temperatures. PCR reactions were carried out in 20-Al total volume containing 40 ng of genomic DNA, 2 Al of 10 polymerase reaction buffer, 0.5–5 AM of each primer set, 1.0 mM dNTPs, 2.0 mM MgCl2 and 2.5 units of AmpliTherm Hot Start DNA polymerase (Societa` Italiana Chimici, Rome, Italy). PCR conditions were: initial denaturation at 95 8C for 3 min, followed by 10 cycles at 95 8C for 1 min, 59 8C for 1 min ( 0.5 8C per cycle) and 72 8C for 1 min; these were followed by 28 additional cycles at 95 8C for 1 min, 55 8C for 1 min, 72 8C for 1 min and a final extension of 72 8C for 10 min. After amplification, samples were diluted 1/20 with water. Two microliters of diluted PCR products were electrophoresed on a PE/ABI 3700 automatic DNA sequencer with 0.5 Al of GeneScan 400-HD-ROX as a size standard internal lane. DNA fragments were analysed with GENESCANVIEW 1.1 software (http://bmr.cribi.unipd.it). 2.7. Statistical analysis Means were expressed with confidence limits ( Pb0.05). Differences between means were checked
Table 1A Upper panel: maternal transmission of diploid sets of 6 microsatellite loci and residual heterozygosity per locus in meiogynogenetic progeny A with lack of paternal transmission of loci not already present in the mother (homozygosity evidenced in grey)
Indicated in the first column is the number of fishes analyzed which have a certain haplotype.
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Table 1B Upper panel: maternal transmission of diploid sets of 5 microsatellite loci and residual heterozygosity per locus in meiogynogenetic progeny B with lack of paternal transmission of loci not already present in the mother (homozygosity evidenced in grey)
with Student’s t test or ANOVA 1. Before analysis, percentages were normalized by arc sin transformation. Statistical significance was fixed at 0.01% ( Pb0.01). In the calculation of mean values of fertilization and hatching rates, data biased by technical failures were not considered.
3. Results The meiogynogenetic nature of the first generation crosses was confirmed by the expected outcomes in the control progenies: haploids manifested the typical haploid syndrome (100% mortality at hatching and DNA values around 0.5 times that of diploid controls). All triploids had DNA relative values around 1.5 times that of diploid controls. Genetic typing by microsatellite analysis of the two female parents and their respective meiogynogenetic offspring (A and B) confirmed only-maternal inheritance of allelic variants (Tables 1A and B). Female A was heterozygous for 5 out of 6 loci, while female B was heterozygous for 3 out of 5. As expected, all maternal homozygous loci were maintained in the offspring. Regarding the heterozygous loci, only 20% and 12% of the meiogynogens in progeny A and progeny B, respectively, retained the maternal heterozygosity, indicating a loss of initial heterozygosity. Gene–centromere recombination rates (Y) were generally high (Table
1A) and ranged from 40% for Labrax-29 to 94% for Labrax-8. Complete homozygosity (for all loci examined) was observed in 6% of the meiogynogens in progeny B, while no full homozygotes occurred in progeny A (Tables 1A and B). The values of 27% and 47% increase of homozygosity in progenies A and B, respectively, indicate that about one quarter and half of the investigated loci were not recombined.
Table 2 Means with confidence limits ( Pb0.05) and minimum and maximum values of body weight, ovarian follicle diameter, relative fecundity (RF) and stripped egg diameter in meiogynogen females as opposed to controls Female group
Meiogynogen Number of fish MeanFc.l. Min Max Control Number of fish MeanFc.l. Min Max Student’s t test
Weight (kg)
Follicle diameter (Am)
Egg RF (103/kg)
Diameter (Am)
24 2.5F0.3 0.8 3.7
24 758F15 710 827
24 285F47 113 573
24 1107F14 1029 1185
20 2.6F0.3 1.4 4.0 ns
20 777F19 720 853 ns
20 266F56 36 493 ns
20 1102F22 1042 1196 ns
ns: Means not significantly different at Pb0.01.
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Table 3 Means with confidence limits ( Pb0.05) and minimum and maximum values of body weight and sperm output (sperm quantity relative to body weight, and spermatozoa concentration and motility) in meiogynogen males as opposed to controls Male group
Meiogynogen Number of fish MeanFc.l. Min Max Control Number of fish MeanFc.l. Min Max Student’s t test
Weight (kg)
Sperm Quantity (ml/kg)
Spermatozoa (109/ml)
Motility (s)
23 1.9F0.2 1.1 2.8
23 2.5F0.8 0.4 9.9
23 40F6 25 83
23 56F5 36 77
20 1.7F0.2 0.9 2.6 ns
20 2.4F0.9 0.6 9.5 ns
20 42F9 14 89 ns
20 63F8 0 85 ns
ns: Means not significantly different at Pb0.01.
During the 3 years of experiments, 16% of gynogenetic females ovulated spontaneously, 56% completed vitellogenesis, 18% achieved only partial vitellogenesis, while 9% underwent atresia. Among normal diploid controls, no females ovulated spontaneously, 52% reached postvitellogenesis, 20% attained partial vitellogenesis and 28% were atretic. No significant differences were noted between gynogenetic and control normal females ovarian follicle and stripped egg diameters and relative fecundity (Table 2). Only 4% of meiotic gynogenetic males were spontaneously fluent (i.e., running milt which could be easily stripped: class 3), 84% were fluent at first stripping (class 2), 6% emitted little sperm after repeated stripping (class 1) and 6% were not fluent
(class 0). Among normal controls, 10% were of class 3, 61% of class 2, 11% of class 1 and 18% of class 0. Means volumes of stripped sperm, spermatozoa concentration and motility of meiotic gynogens did not differ from those of normal controls (Table 3). The gynogenetic male with the best reproductive performance was of class 3 and gave on three stripping sessions a total of 9.9 ml of sperm/kg body weight during 2 months with a concentration of 30–65109 spermatozoa/ml having excellent motility (60–63 s) and fertility (77–98%). The best control male was no better (9.5 ml of sperm/kg; 22–37109 spermatozoa/ml with a motility of 68–72 s; 81–98% of fertility). Eleven crosses were carried out with female and male gynogens, whose mean fertility and hatching rates were statistically equivalent to those of the nine crosses between normal females and males (Table 4). Similarly, fertility and hatching percentages in the six crosses between female gynogens and normal males and in the seven reverse crosses were statistically comparable (Table 4). On the other hand, in four crosses in which gametes from gynogenetic parents were spontaneously spawned and fertilization occurred in the tank, fertility and hatching rates were highly variable (0–79% and 3–70%, respectively). Gynogenetic females maintained their fertility during two or three reproductive seasons in 45% of the cases, while 26% of controls did the same. Gynogenetic males remained fertile in 84% of the cases vs. 25% in the controls (Fig. 1). No repeated occurrence of infertility was observed in males of both groups. Eggs from females of the first gynogenetic generation were subjected to chromosome set manipu-
Table 4 Means with confidence limits ( Pb0.05) and minimum and maximum values of fertilization and hatching rates in the four possible crosses between meiogynogenetic and/or control sea bass Cross
Fertilization rate
Females Meiogynogen Meiogynogen Control Control ANOVA 1
x x x x
Larval hatching
Males
Number
Range (% fertilized eggs)
Mean (% fertilized eggs)
Range (% larvae)
Mean (% larvae)
Meiogynogen Control Meiogynogen Control
11 6 7 9
49–93 57–92 40–98 54–97
74F10 77F16 73F18 78F12 ns
3–87 10–95 5–81 9–96
48F21 58F30 51F24 69F26 ns
ns: Means not significantly different at Pb0.01.
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Fig. 1. Reproductive performance of female and male meiogynogens as opposed to controls during three testing years in terms of full (in all 3 years) and partial (only in some years) fertility and infertility (in all 3 years).
lation to produce meiogynogens of second generation. Of these fish (n= 84), 7% are male (at 3 years old).
4. Discussion Reproduction by meiotic gynogenesis is theoretically expected to yield in the resulting progeny an increase of homozygosity in maternal heterozygous loci ranging from 100% to 0%, depending upon the level of recombination. In the absence of crossovers, all recombined heterozygous loci would become homozygous, as the incorporation of the second polar body chromosome set prevents allelic segregation. In this case, meiogynogens would be equivalent to
mitogynogens because they are completely homozygous. Conversely, in the presence of a single crossover, all recombined heterozygous loci would remain heterozygous. With two crossovers, the percentage of recombined heterozygous loci that remain heterozygous would average 50%. With a greater number of crossovers, the percentage of residual heterozygous loci would tend to 67% for an infinite number of crossovers, according to the formula by Purdom et al. (1976): P (x)=1 1/2 P (x 1) where P (x) is the probability of heterozygosity with x crossovers. The more proximal the site of chromatid exchange to the centromere, the greater the number of loci that would preserve their heterozygosity. In metacentric chromosomes, recombination events occur more
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frequently in the distal regions of the chromatid arms due to centromere interference. Furthermore, the greater the chromosome length, the higher the probability of multiple crossovers. In the case of sea bass, most of the chromosomes are acrocentric (2n=48; karyotype: 46 acrocentric+2 submetacentric/ subtelocentric; Arefye´v, 1989; Vitturi et al., 1990), thus reducing centromere interference. Their length, however, is relatively short (1.5–3 Am; Arefye´v, 1989), since their diploid genome size is small (Cvalue 0.8 pg). This may entail higher chiasma interference and consequently a higher frequency of obligate single chiasmata. This means that, in this species, a high number of heterozygous loci in meiotic gynogens may arise from single crossovers, thus reducing the tendency towards homozygosity due to meiogynogenesis. This is consistent with our findings and the high levels of heterozygosity at certain loci observed in our two meiogynogenetic progenies. A similar phenomenon has been described in other teleost species, as discussed by Galbusera et al. (2000). Usually, increments of homozygosity are expressed as inbreeding coefficients calculated from allelic frequencies in parental vs. filial generations. In our experiments, each genotyped mother (A and B) was regarded as representative of a virtual isoallelic population in which only the heterozygous loci are considered as to their conversion to homozygosity in the offspring. Our calculations show that between about 25% and 50% of the examined maternal heterozygous loci were not recombined and were, thus, inherited in homozygous condition by both progenies A and B, respectively. It should be noted that progeny B, with a fixation index ( F) close to the most frequent value of 0.5 (Taniguchi et al., 1990), was tested at an older age than progeny A (1 year vs. 45 days of age). If the measured homozygosity values are extrapolated to the whole genome from the small number of investigated microsatellite loci, they are still prone to be underestimated because selective death of fish with higher number of homozygous loci may have occurred whenever they harboured deleterious recessive alleles. Although selective death has not been measured, meiogynogenetic offspring constantly exhibited about half the hatching rate of controls (18% vs. 49%), as a consequence of either the manifestation of genetic defects or eventually
damage caused by the thermal shock to block meiosis. Harmful effects of recessive alleles can be exerted not only on survival and development, especially at early stages, but also on reproductive capability at adulthood. Impairment of gonadal development and maturation has been documented in some meiogynogenetic fish belonging to different species, such as common carp, Cyprinus carpio (Nagy et al., 1978; Komen et al., 1992), coho salmon, Oncorhynchus kisutch (Piferrer et al., 1994) and honmoroko, Gnathopogon caerulescens (Fujioka, 1998). On the other hand, apparently normal fertility was exhibited by meiogynogenetic loach, Misgurnus anguillicaudatus (Suzuki et al., 1985) and thai walking catfish, Clarias macrocephalus (Na-Nakorn, 1995). Similarly, meiogynogenetic female sea bass underwent normal vitellogenesis and yielded good eggs upon hormonal treatment (Felip et al., 2002). It is generally assumed that these conflicting results reflect an increased phenotypic variation due to the enhanced homozygosity of recessive alleles negatively affecting reproductive capacity with respect to the normal parental stock. The present work suggests the possibility that the residual heterozygosity observed in meiogynogens might be sufficient to ensure a rather normal reproductive function. Meiogynogenetic sea bass of both sexes were found to be normally fertile when compared with controls in terms of age at puberty, season of reproduction, quantity and quality of gametes, and offspring viability. Their performance was equivalent not only to our controls but also to those of wild and captive fish reported by other authors. For example, in wild female sea bass fished along the English coasts, the RF values, derived from the number of ovarian follicles with a diameter greater than 200 Am, ranged between 273,000 and 538,000 follicles/kg (Mayer et al., 1990). Captive females subjected to an equivalent hormonal treatment to induce oviposition as in our experiments, showed an average RF value of 420,000 eggs/kg with a hatching rate of 22–27% (Fornie´s et al., 2001). These data are comparable to those observed in meiogynogenetic females of the present work (average RF=285,000; hatching rate=48%). A sperm concentration of 60109 spermatozoa/ml with motility up to 44 s and artificial fertilization rate of 70% using 66,400–615,000 spermatozoa/egg has
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been reported for normal captive males (Fauvel et al., 1999). Meiogynogenetic males in the present study had similar values. Meiogynogenetic sea bass males had a reproductive potential equivalent to that of normal males, despite the fact that they bear only maternal chromosomes. This demonstrates that maleness does not require a paternal sex chromosome. Furthermore, 7% of males were found among second-generation meiogynogens as opposed to 39% reported for firstgeneration meiogynogens under the same culture conditions (Colombo et al., 1998), indicating that the sex ratio of the progeny from meiogynogenetic females is more skewed in favour of females (although some few males can still arise). This observation provides the first evidence regarding the sex ratio of second-generation meiogynogens and, although still a preliminary result, it suggests the possibility that environmental factors alone do not drive the masculinization of meiogynogenetic sea bass. Pavlidis et al. (2000) has clearly demonstrated that increasing the rearing temperature from 13 to 20 8C at the stage of half-epiboly until middle metamorphosis increases the proportion of males from 27% to 74%, verifying a previous suggestion by Colombo et al. (1998) based on meiogynogen sex ratios at three hatcheries with different culture conditions. A possible explanation for the very low percentage of males among second-generation meiogynogens is that, in meiogynogens differentiating as females, there are less male-inducing genetic components responsive to environmental factors with respect to meiogynogenetic males. A genetic polymorphism of autosomal factors influencing in both directions the sex ratios in the progenies of YY males of Nile tilapia, Oreochromis niloticus, has been lately documented (Tariq Ezaz et al., 2004), and evidence that they are amenable to selection for less skewed sex differentiation has been reviewed (Beardmore et al., 2001). Hence, repeated gynogenetic matings may be an approach to eliminate masculinizing genetic determinants in sea bass, as males would be excluded from gynogenesis. Even the thermosensitivity of the sex ratio in this species might be quenched by raising gynogenetic offspring at high temperature during the critical period and selecting for gynogenesis those with lower percentages of males. If this is correct, then almost all-female stocks with more uniform
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growth performance may be eventually obtained to improve sea bass culture.
Acknowledgements This investigation was supported by grants 5C53 and 5C117 of the Ministero per le Politiche Agricole e Forestali (Rome, Italy) and by the EU Research Project BASSMAP Q5RS-2001-01701.
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Fornie´s, M.A., Manano´s, E., Carrillo, M., Rocha, A., Laureau, S., Mylonas, C.C., Zohar, Y., Zanuy, S., 2001. Spawning induction of individual European sea bass females Dicentrarchus labrax using different GnRHa-delivery systems. Aquaculture 202, 221 – 234. Fujioka, Y., 1998. Survival, growth and sex ratios of gynogenetic diploid honmoroko. J. Fish Biol. 52, 430 – 442. Galbusera, P., Volckaert, F.A.M., Ollevier, F., 2000. Gynogenesis in the African catfish Clarias gariepinus (Burchell, 1822): III. Induction of endomitosis and the presence of residual genetic variation. Aquaculture 185, 25 – 42. Garcı´a de Leo´n, F.J., Dallas, J.F., Chatain, B., Canonne, M., Versini, J.J., Bonhomme, F., 1995. Development and use of microsatellite markers in sea bass Dicentrarchus labrax Linnaeus, 1758 Perciformes: Serranidae. Mol. Mar. Biol. Biotechnol. 4, 62 – 68. Gorshkova, G., Gorshkov, S., Hadani, A., Gordin, H., Knibb, W., 1995. Chromosome set manipulations in marine fish. Aquaculture 137, 157 – 158. Knibb, W., Gorshkova, G., Gorshkov, S., 1998. Genetic improvement of cultured marine finfish: case studies. Trop. Maric. 4, 111 – 149. Komen, J., Wiegertjes, G.F., van Ginneken, V.J.T., Eding, E.H., Richter, C.J.J., 1992. Gynogenesis in common carp (Cyprinus carpio L.): III. The effects of inbreeding on gonadal development of heterozygous and homozygous gynogenetic offspring. Aquaculture 104, 51 – 66. Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenisch, R., Berns, A., 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19, 4293. Libertini, A., Francescon, A., Bertotto, D., Benedetti, P., Poltronieri, C., Barbaro, A., 2002. Further investigations on tetraploidy in the European seabass, Dicentrarchus labrax (L.). Biol. Mar. Mediterr. 9, 562 – 565. Mayer, I., Shackley, S.E., Witthames, P.R., 1990. Aspects of the reproductive biology of the bass, Dicentrarchus labrax L.: II. Fecundity and pattern of oocyte development. J. Fish Biol. 36, 141 – 148. Nagy, A., Rajki, K., Horvath, L., Csanyi, V., 1978. Investigation on carpo, Cyprinus carpio L., gynogenesis. J. Fish Biol. 13, 215 – 224.
Na-Nakorn, U., 1995. Comparison of cold and heat shocks to induce diploid gynogenesis in thai walking catfish (Clarias macrocephalus) and performances of gynogens. Aquat. Living Resour. 8, 333 – 341. Pavlidis, M., Koumoundouros, G., Sterioti, A., Somarakis, S., Divanach, P., Kentouri, M., 2000. Evidence of temperaturedependent sex determination in the European sea bass (Dicentrarchus labrax L.). J. Exp. Zool. 287, 225 – 232. Peruzzi, S., Chatain, B., 2000. Pressure and cold shock induction of meiotic gynogenesis and triploidy in the European sea bass, Dicentrarchus labrax L.: relative efficiency of methods and parental variability. Aquaculture 189, 23 – 37. Piferrer, F., Benfey, T.J., Donaldson, E.M., 1994. Gonadal morphology of normal and sex-reversed triploid and gynogenetic diploid coho salmon (Oncorhynchus kisutch). J. Fish Biol. 45, 541 – 553. Purdom, C.E., 1993. Genetics and Fish Breeding. Chapman & Hall, London, pp. 277. Purdom, C.E., Thompson, D., Dando, P.R., 1976. Genetic analysis of enzyme polymorphisms in plaice (Pleuronectes platessa). Heredity 37, 193 – 206. Suzuki, R., Oshiro, T., Nakanishi, T., 1985. Survival, growth and fertility of gynogenetic diploids induced in the cyprinids loach, Misgurnus anguillicaudatus. Aquaculture 48, 45 – 55. Taniguchi, N., Hatanaka, H., Seki, S., 1990. Genetic variation in quantitative characters of meiotic- and mitotic-gynogenetic diploid ayu, Plecoglossus altivelis. Aquaculture 85, 223 – 233. Tariq Ezaz, M., Myers, J.M., Powell, S.F., McAndrew, B.J., Penman, D.J., 2004. Sex ratios in the progeny of androgenetic and gynogenetic YY male Nile tilapia, Oreochromis niloticus L. Aquaculture 232, 205 – 214. Vitturi, R., Mazzola, A., Catalano, E., Lo Conte, M.R., 1990. Karyotype analysis, nucleolar organizer regions (NORs), and Cbanding pattern of Dicentrarchus labrax (L.) and D. punctatus (Block, 1792) (Pisces, Perciformes) with evidence of chromosomal structural polymorphism. Cytologia 55, 425 – 430. Zanuy, S., Carrillo, M., Felip, A., Rodrı´guez, L., Bla´zquez, M., Ramos, J., Pifferer, F., 2001. Genetic, hormonal and environmental approaches for the control of reproduction in the European sea bass (Dicentrarchus labrax L.). Aquaculture 202, 187 – 203.
Assessment of homozygosity and fertility in meiotic gynogens of the European sea bass (Dicentrarchus labrax L.) Antonia Francescona,*, Alvise Barbaroa, Daniela Bertottoa, Angelo Libertinia, Fulvio Cepollarob, Jacopo Richardc, Paola Belvedereb, Lorenzo Colombob a
CNR-Ismar, Istituto di Scienze Marine/Biologia del Mare, Castello 1364/A, I-30122 Venezia, Italy b Dipartimento di Biologia, Universita` Padova, Via Colombo 3, I-35121 Padova, Italy c Veneto Agricoltura Centro Ittico Sperimentale, strada Murazzi, 200, I-30100 Pellestrina (Ve), Italy Received 3 March 2004; received in revised form 26 October 2004; accepted 30 October 2004
Abstract Analysis of 5–6 microsatellite loci was used to measure the increment of homozygosity in two meiogynogenetic progenies (A and B) of sea bass with respect to their mother. In progeny A and B, 20% and 12% of the meiogynogens retained heterozygosity for all investigated maternal loci, respectively, while complete homozygosity was observed only in 6% of B and in none of A, indicating the occurrence of significant allelic recombination during meiosis. The overall increment of homozygosity for the investigated loci was 27% for A and 47% for B. Although survival at hatching of meiogynogens was about half that of controls, they subsequently grew as controls and displayed the same onset of puberty and reproductive potential at adulthood during three consecutive years when crossed between themselves (11 crosses) or with control fish (13 crosses). In particular, meiogynogenetic females (n=24) underwent vitellogenesis and yielded eggs of good quality upon stimulation with LH–RH analogue similarly to normal fish. Sperm released by meiogynogenetic males (n=23) was equivalent to that of controls in terms of volume, quality and fertilization capability. Second generation meiogynogens were obtained by chromosome set manipulation from meiogynogenetic females and found to be morphologically normal at 3 years of age. Interestingly, under our culture conditions, the percentage of meiogynogenetic males in the second generation was 7% as opposed to 39% in the first generation. D 2004 Elsevier B.V. All rights reserved. Keywords: Sea bass; Dicentrarchus labrax; Gynogenesis; Homozygosity; Microsatellite DNA; Fertility
1. Introduction * Corresponding author. Tel.: +39 41 2404711; fax: +39 41 5204126. E-mail address: [email protected] (A. Francescon). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.10.023
Owing to high fecundity and easy access to gametes, teleost fish are suitable models for research on gynogenesis and its application to improve fish
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culture. To this end, it is essential to establish for each species whether gynogens are not only viable and growable to sexual maturity, but also if their gametes are normally fertile. Induced gynogenesis obtained by sperm genetic inactivation and diploidy restoration by inhibition of the second meiosis with consequent retention of the second polar body (meiogynogenesis) ensures viable uniparental chromosome transmission and all-maternal genetic inheritance. This reproductive mode enhances homozygosity in the progeny where the retention of the second polar body replaces the male pronucleus in the karyogamic process and maintains the property of a sexual reproductive interaction. In the case of endomitosis or double haploid gynogenesis, the first embryonic cleavage is inhibited (mitogynogenesis) and the manipulation targets the inception of the somatic lineage. Hence, it modifies asexually the course of the embryo formation. Induction of mitogynogenesis produces homozygous offspring, while meiogynogens show residual heterozygosity due to recombination events. Nevertheless, meiogynogenesis can be used to obtain isoallelic lines as a preliminary effort to discharge deleterious alleles. Moreover, meiogynogens exhibit greater survival than mitogynogens and are usually preferred (because progeny have not been made completely homozygous) to produce all-female progeny as well as all-pseudomale progenys by androgen treatment in species with strict XX/XY sex determination (Zanuy et al., 2001). Sex ratio analysis can reveal the heterogametic sex or evidence the occurrence of autosomal or environmental factors in sex determination. Meiogynogens are also invaluable for the estimation of allele recombination frequencies and gene–centromere mapping (Purdom, 1993). The European sea bass (Dicentrarchus labrax L.) is an economically important species whose farming has greatly expanded in recent times along the Mediterranean and Atlantic coasts of Europe, Asia Minor and North Africa. These species have been the subject of several studies on chromosome set manipulation to induce gynogenesis and polyploidy (Carrillo et al., 1993; Colombo et al., 1995; Gorshkova et al., 1995; Barbaro et al., 1998; Knibb et al., 1998; Peruzzi and Chatain, 2000; Felip et al., 2002). It has been conclusively demonstrated that sea bass meiogynogens are viable and grow to adulthood
showing variable sex ratios, a fact that has hampered the production of all-female stocks in this species. Felip et al. (2002) has reported as unpublished results that female meiogynogens display normal vitellogenesis and are capable of producing eggs of excellent quality after hormonal treatment. No information is currently available on the fertility of male meiogynogens. The objective of this work was to assess the homozygosity and the fertility in meiogynogen sea bass. We compared gamete quality, and fertilization and hatching rates in crosses, including female and male meiogynogen lots, meiogynogen and normal fish lots, and normal crosses. We also established that a second generation of meiogynogens might be obtained from meiogynogenetic parents. Moreover, microsatellite analysis was applied to a first generation of meiogynogens to check the absence of paternal genetic contribution and provide a preliminary estimate of levels of homozygosity.
2. Materials and methods 2.1. Founder fish stock Wild specimens of European sea bass (D. labrax L.) were captured by angling in the North Adriatic sea and kept in an outdoor 500-m3 tank at 32–35 ppt salinity under ambient water temperature (14–28 8C) and photoperiod (May–October), as well as in an indoor 20-m3 tank at 14 8C and 34 ppt of salinity under natural photoperiod (November–April). All fish in this study were electronically tagged and reared communally (diploid meiogynogens and controls). 2.2. Spawner selection and hormonal treatment During the reproductive season (January–March), mate pairs were transferred separately into 9-m3 spawning tanks with recirculating water and a net (500-Am mesh) basket at the outlet for egg collection. Preovulatory females were subjected to ovarian biopsy and selected for hormonal treatment on the basis of the diameter of 20 postvitellogenic follicles (N700 Am). Oviposition was induced by a single intramuscular injection of 10 Ag/kg body weight of GnRH analogue (des-Gly10, [d-Ala6] LH–RH ethyl-
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amide; Sigma, USA). Sperm fluency in males was assessed after stripping by measuring the sperm volume and counting spermatozoa in a Bqrker haemocytometer. Sperm motility duration (time in seconds from activation to propulsive motion cessation; Fauvel et al., 1999) was checked in duplicate following dilution (1:10) with extender solution (0.1875 M KCl, pH 7.35; Colombo et al., 1995) and further mixing (1:10) with seawater (35 ppt). Males were not hormonally treated. 2.3. Gamete collection and artificial fertilization When fish were judged as proximate to spawning according to their behaviour, they were rapidly netted and anaesthetized. About 1-g sample of eggs was stripped from each female to establish good quality based on egg buoyancy (35 ppt at 14 8C) and morphology (regular shape, transparency, presence of cortical granules, unsplit lipid droplet and absence of flocculation). If good quality was confirmed, a second full stripping was performed and the total egg mass was volumetrically estimated. Eggs were maintained at 14 8C for V15 min. Absolute fecundity (AF) was inferred from the total number of stripped eggs (calculated as total egg volume/volume of a single egg); relative fecundity (RF) was given by the ratio AF/postspawning female body weight. Collected sperm was stored at 2–4 8C until use. Artificial fertilization was carried out mixing 1 part of eggs, 4 parts of seawater and 1 part of sperm diluted 1:100 with extender. Three minutes later, eggs were washed out of sperm excess. Approximately 100 eggs were withdrawn from the hatching tanks to estimate the percentage of fertilization [number of blastulae at 12 h postfertilization (pf)] and 300–500 eggs were cultured in 2-l flasks to determine the percentage of hatching at 96 hpf (number of larvae100/number of fertilized eggs). All samples were carried out in duplicate. 2.4. Production of meiogynogens Meiogynogenesis was obtained by egg fertilization with UV-irradiated (254 nm; 3300 erg mm 2) sperm, and diploidy was restored by cold shock (0 8C, 5 min after fertilization for 20 min; Barbaro et al., 1998)
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which effectively blocks the extrusion of the second polar body. For each egg batch, three control crosses were produced in addition to gynogens: a normal diploid progeny to check egg quality, a haploid progeny to check effectiveness of sperm inactivation, and a triploid progeny to verify efficacy of the cold shock. Determination of ploidy was carried out by flow cytometry by measuring the nuclear DNA amount in pooled embryos (haploids), single larvae or in individual samples of erythrocytes or caudal finclips from adult fish (Libertini et al., 2002). Two meiogynogenetic progenies, A and B, were genetically typed using microsatellite markers (n=6 in A and n=5 in B; see below) at 43 days after hatching (larvae) and at 1 year of age (fin-clips), respectively. Maternal transmission was determined based on the allelic sets of the respective parents. The overall heterozygosity was obtained as percentage by cumulating the number of heterozygotes at each loci in each progeny. Because both males and females may be obtained by meiogynogenesis in the sea bass, as reviewed by Felip et al. (2001), a stock of 21 female and 24 male meiogynogens were raised to adulthood for 4 years and were utilized during the subsequent 3 years as spawners through 1–3 cycles. Control diploids were similarly cultured and analysed as reference stocks. 2.5. Reproduction of meiogynogens Both gynogenetic and control males with fluent sperm and females with postvitellogenic follicles of at least 700 Am in diameter and hormonally stimulated as above were selected to accomplish a total of 24 meiogynogenetic and 20 control crosses by artificial fertilization according to all four possible crossing combinations. Four gynogenetic females were allowed to spawn spontaneously and separately with four gynogenetic males. Here, AF was calculated according to the formula: number of laid eggs=volume of eggs (2865 1796 egg mean diameter) (Fornie´s et al., 2001). The applicability of chromosome set manipulation to gametes obtained from meiogynogenetic female sea bass was investigated by subjecting eggs fertilized with UV-irradiated sperm to meiotic block by cold shock to produce second-generation meiogynogens as described above.
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2.6. Genetic analysis Fin-clips or whole larvae were stored in ethanol. Genomic DNA was purified according to Laird et al. (1991) and DNA quality was verified by 0.8% agarose gel electrophoresis. Genetic variation was assayed using up to 6 microsatellite loci: Dla-6 (Castilho and McAndrew, 1998), Labrax-6, Labrax8, Labrax-13, Labrax-17 and Labrax-29 (Garcı´a de Leo´n et al., 1995). All loci are on separate linkage groups (Chistiakov et al., 2004; Chris Haley, BASSMAP research panel, Rosling University UK, personal communication). Selection of primer pairs (MWG-Biotech, Ebersberg, Germany; Perkin-Elmer/ Applied Biosystems Division; Milan, Italy) was based on their polymorphism, amplification repeatability and coamplificability in a single PCR reaction without artefact bands. To distinguish products with overlapping size ranges, a total of six primer sets was labelled with three different fluorochromes used in a multiplex DNA PCR reaction. Three primer sets were 5V-labelled with Hex dye, two primer sets with 6-Fam and one set with Ned. The multiplex DNA amplifications were performed in a T-Gradient Thermal Cycler (Whatman Biometra, Gfttingen, Germany), using a touch-
down PCR protocol. It allowed the simultaneous amplification of loci having different optimal annealing temperatures. PCR reactions were carried out in 20-Al total volume containing 40 ng of genomic DNA, 2 Al of 10 polymerase reaction buffer, 0.5–5 AM of each primer set, 1.0 mM dNTPs, 2.0 mM MgCl2 and 2.5 units of AmpliTherm Hot Start DNA polymerase (Societa` Italiana Chimici, Rome, Italy). PCR conditions were: initial denaturation at 95 8C for 3 min, followed by 10 cycles at 95 8C for 1 min, 59 8C for 1 min ( 0.5 8C per cycle) and 72 8C for 1 min; these were followed by 28 additional cycles at 95 8C for 1 min, 55 8C for 1 min, 72 8C for 1 min and a final extension of 72 8C for 10 min. After amplification, samples were diluted 1/20 with water. Two microliters of diluted PCR products were electrophoresed on a PE/ABI 3700 automatic DNA sequencer with 0.5 Al of GeneScan 400-HD-ROX as a size standard internal lane. DNA fragments were analysed with GENESCANVIEW 1.1 software (http://bmr.cribi.unipd.it). 2.7. Statistical analysis Means were expressed with confidence limits ( Pb0.05). Differences between means were checked
Table 1A Upper panel: maternal transmission of diploid sets of 6 microsatellite loci and residual heterozygosity per locus in meiogynogenetic progeny A with lack of paternal transmission of loci not already present in the mother (homozygosity evidenced in grey)
Indicated in the first column is the number of fishes analyzed which have a certain haplotype.
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Table 1B Upper panel: maternal transmission of diploid sets of 5 microsatellite loci and residual heterozygosity per locus in meiogynogenetic progeny B with lack of paternal transmission of loci not already present in the mother (homozygosity evidenced in grey)
with Student’s t test or ANOVA 1. Before analysis, percentages were normalized by arc sin transformation. Statistical significance was fixed at 0.01% ( Pb0.01). In the calculation of mean values of fertilization and hatching rates, data biased by technical failures were not considered.
3. Results The meiogynogenetic nature of the first generation crosses was confirmed by the expected outcomes in the control progenies: haploids manifested the typical haploid syndrome (100% mortality at hatching and DNA values around 0.5 times that of diploid controls). All triploids had DNA relative values around 1.5 times that of diploid controls. Genetic typing by microsatellite analysis of the two female parents and their respective meiogynogenetic offspring (A and B) confirmed only-maternal inheritance of allelic variants (Tables 1A and B). Female A was heterozygous for 5 out of 6 loci, while female B was heterozygous for 3 out of 5. As expected, all maternal homozygous loci were maintained in the offspring. Regarding the heterozygous loci, only 20% and 12% of the meiogynogens in progeny A and progeny B, respectively, retained the maternal heterozygosity, indicating a loss of initial heterozygosity. Gene–centromere recombination rates (Y) were generally high (Table
1A) and ranged from 40% for Labrax-29 to 94% for Labrax-8. Complete homozygosity (for all loci examined) was observed in 6% of the meiogynogens in progeny B, while no full homozygotes occurred in progeny A (Tables 1A and B). The values of 27% and 47% increase of homozygosity in progenies A and B, respectively, indicate that about one quarter and half of the investigated loci were not recombined.
Table 2 Means with confidence limits ( Pb0.05) and minimum and maximum values of body weight, ovarian follicle diameter, relative fecundity (RF) and stripped egg diameter in meiogynogen females as opposed to controls Female group
Meiogynogen Number of fish MeanFc.l. Min Max Control Number of fish MeanFc.l. Min Max Student’s t test
Weight (kg)
Follicle diameter (Am)
Egg RF (103/kg)
Diameter (Am)
24 2.5F0.3 0.8 3.7
24 758F15 710 827
24 285F47 113 573
24 1107F14 1029 1185
20 2.6F0.3 1.4 4.0 ns
20 777F19 720 853 ns
20 266F56 36 493 ns
20 1102F22 1042 1196 ns
ns: Means not significantly different at Pb0.01.
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Table 3 Means with confidence limits ( Pb0.05) and minimum and maximum values of body weight and sperm output (sperm quantity relative to body weight, and spermatozoa concentration and motility) in meiogynogen males as opposed to controls Male group
Meiogynogen Number of fish MeanFc.l. Min Max Control Number of fish MeanFc.l. Min Max Student’s t test
Weight (kg)
Sperm Quantity (ml/kg)
Spermatozoa (109/ml)
Motility (s)
23 1.9F0.2 1.1 2.8
23 2.5F0.8 0.4 9.9
23 40F6 25 83
23 56F5 36 77
20 1.7F0.2 0.9 2.6 ns
20 2.4F0.9 0.6 9.5 ns
20 42F9 14 89 ns
20 63F8 0 85 ns
ns: Means not significantly different at Pb0.01.
During the 3 years of experiments, 16% of gynogenetic females ovulated spontaneously, 56% completed vitellogenesis, 18% achieved only partial vitellogenesis, while 9% underwent atresia. Among normal diploid controls, no females ovulated spontaneously, 52% reached postvitellogenesis, 20% attained partial vitellogenesis and 28% were atretic. No significant differences were noted between gynogenetic and control normal females ovarian follicle and stripped egg diameters and relative fecundity (Table 2). Only 4% of meiotic gynogenetic males were spontaneously fluent (i.e., running milt which could be easily stripped: class 3), 84% were fluent at first stripping (class 2), 6% emitted little sperm after repeated stripping (class 1) and 6% were not fluent
(class 0). Among normal controls, 10% were of class 3, 61% of class 2, 11% of class 1 and 18% of class 0. Means volumes of stripped sperm, spermatozoa concentration and motility of meiotic gynogens did not differ from those of normal controls (Table 3). The gynogenetic male with the best reproductive performance was of class 3 and gave on three stripping sessions a total of 9.9 ml of sperm/kg body weight during 2 months with a concentration of 30–65109 spermatozoa/ml having excellent motility (60–63 s) and fertility (77–98%). The best control male was no better (9.5 ml of sperm/kg; 22–37109 spermatozoa/ml with a motility of 68–72 s; 81–98% of fertility). Eleven crosses were carried out with female and male gynogens, whose mean fertility and hatching rates were statistically equivalent to those of the nine crosses between normal females and males (Table 4). Similarly, fertility and hatching percentages in the six crosses between female gynogens and normal males and in the seven reverse crosses were statistically comparable (Table 4). On the other hand, in four crosses in which gametes from gynogenetic parents were spontaneously spawned and fertilization occurred in the tank, fertility and hatching rates were highly variable (0–79% and 3–70%, respectively). Gynogenetic females maintained their fertility during two or three reproductive seasons in 45% of the cases, while 26% of controls did the same. Gynogenetic males remained fertile in 84% of the cases vs. 25% in the controls (Fig. 1). No repeated occurrence of infertility was observed in males of both groups. Eggs from females of the first gynogenetic generation were subjected to chromosome set manipu-
Table 4 Means with confidence limits ( Pb0.05) and minimum and maximum values of fertilization and hatching rates in the four possible crosses between meiogynogenetic and/or control sea bass Cross
Fertilization rate
Females Meiogynogen Meiogynogen Control Control ANOVA 1
x x x x
Larval hatching
Males
Number
Range (% fertilized eggs)
Mean (% fertilized eggs)
Range (% larvae)
Mean (% larvae)
Meiogynogen Control Meiogynogen Control
11 6 7 9
49–93 57–92 40–98 54–97
74F10 77F16 73F18 78F12 ns
3–87 10–95 5–81 9–96
48F21 58F30 51F24 69F26 ns
ns: Means not significantly different at Pb0.01.
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Fig. 1. Reproductive performance of female and male meiogynogens as opposed to controls during three testing years in terms of full (in all 3 years) and partial (only in some years) fertility and infertility (in all 3 years).
lation to produce meiogynogens of second generation. Of these fish (n= 84), 7% are male (at 3 years old).
4. Discussion Reproduction by meiotic gynogenesis is theoretically expected to yield in the resulting progeny an increase of homozygosity in maternal heterozygous loci ranging from 100% to 0%, depending upon the level of recombination. In the absence of crossovers, all recombined heterozygous loci would become homozygous, as the incorporation of the second polar body chromosome set prevents allelic segregation. In this case, meiogynogens would be equivalent to
mitogynogens because they are completely homozygous. Conversely, in the presence of a single crossover, all recombined heterozygous loci would remain heterozygous. With two crossovers, the percentage of recombined heterozygous loci that remain heterozygous would average 50%. With a greater number of crossovers, the percentage of residual heterozygous loci would tend to 67% for an infinite number of crossovers, according to the formula by Purdom et al. (1976): P (x)=1 1/2 P (x 1) where P (x) is the probability of heterozygosity with x crossovers. The more proximal the site of chromatid exchange to the centromere, the greater the number of loci that would preserve their heterozygosity. In metacentric chromosomes, recombination events occur more
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frequently in the distal regions of the chromatid arms due to centromere interference. Furthermore, the greater the chromosome length, the higher the probability of multiple crossovers. In the case of sea bass, most of the chromosomes are acrocentric (2n=48; karyotype: 46 acrocentric+2 submetacentric/ subtelocentric; Arefye´v, 1989; Vitturi et al., 1990), thus reducing centromere interference. Their length, however, is relatively short (1.5–3 Am; Arefye´v, 1989), since their diploid genome size is small (Cvalue 0.8 pg). This may entail higher chiasma interference and consequently a higher frequency of obligate single chiasmata. This means that, in this species, a high number of heterozygous loci in meiotic gynogens may arise from single crossovers, thus reducing the tendency towards homozygosity due to meiogynogenesis. This is consistent with our findings and the high levels of heterozygosity at certain loci observed in our two meiogynogenetic progenies. A similar phenomenon has been described in other teleost species, as discussed by Galbusera et al. (2000). Usually, increments of homozygosity are expressed as inbreeding coefficients calculated from allelic frequencies in parental vs. filial generations. In our experiments, each genotyped mother (A and B) was regarded as representative of a virtual isoallelic population in which only the heterozygous loci are considered as to their conversion to homozygosity in the offspring. Our calculations show that between about 25% and 50% of the examined maternal heterozygous loci were not recombined and were, thus, inherited in homozygous condition by both progenies A and B, respectively. It should be noted that progeny B, with a fixation index ( F) close to the most frequent value of 0.5 (Taniguchi et al., 1990), was tested at an older age than progeny A (1 year vs. 45 days of age). If the measured homozygosity values are extrapolated to the whole genome from the small number of investigated microsatellite loci, they are still prone to be underestimated because selective death of fish with higher number of homozygous loci may have occurred whenever they harboured deleterious recessive alleles. Although selective death has not been measured, meiogynogenetic offspring constantly exhibited about half the hatching rate of controls (18% vs. 49%), as a consequence of either the manifestation of genetic defects or eventually
damage caused by the thermal shock to block meiosis. Harmful effects of recessive alleles can be exerted not only on survival and development, especially at early stages, but also on reproductive capability at adulthood. Impairment of gonadal development and maturation has been documented in some meiogynogenetic fish belonging to different species, such as common carp, Cyprinus carpio (Nagy et al., 1978; Komen et al., 1992), coho salmon, Oncorhynchus kisutch (Piferrer et al., 1994) and honmoroko, Gnathopogon caerulescens (Fujioka, 1998). On the other hand, apparently normal fertility was exhibited by meiogynogenetic loach, Misgurnus anguillicaudatus (Suzuki et al., 1985) and thai walking catfish, Clarias macrocephalus (Na-Nakorn, 1995). Similarly, meiogynogenetic female sea bass underwent normal vitellogenesis and yielded good eggs upon hormonal treatment (Felip et al., 2002). It is generally assumed that these conflicting results reflect an increased phenotypic variation due to the enhanced homozygosity of recessive alleles negatively affecting reproductive capacity with respect to the normal parental stock. The present work suggests the possibility that the residual heterozygosity observed in meiogynogens might be sufficient to ensure a rather normal reproductive function. Meiogynogenetic sea bass of both sexes were found to be normally fertile when compared with controls in terms of age at puberty, season of reproduction, quantity and quality of gametes, and offspring viability. Their performance was equivalent not only to our controls but also to those of wild and captive fish reported by other authors. For example, in wild female sea bass fished along the English coasts, the RF values, derived from the number of ovarian follicles with a diameter greater than 200 Am, ranged between 273,000 and 538,000 follicles/kg (Mayer et al., 1990). Captive females subjected to an equivalent hormonal treatment to induce oviposition as in our experiments, showed an average RF value of 420,000 eggs/kg with a hatching rate of 22–27% (Fornie´s et al., 2001). These data are comparable to those observed in meiogynogenetic females of the present work (average RF=285,000; hatching rate=48%). A sperm concentration of 60109 spermatozoa/ml with motility up to 44 s and artificial fertilization rate of 70% using 66,400–615,000 spermatozoa/egg has
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been reported for normal captive males (Fauvel et al., 1999). Meiogynogenetic males in the present study had similar values. Meiogynogenetic sea bass males had a reproductive potential equivalent to that of normal males, despite the fact that they bear only maternal chromosomes. This demonstrates that maleness does not require a paternal sex chromosome. Furthermore, 7% of males were found among second-generation meiogynogens as opposed to 39% reported for firstgeneration meiogynogens under the same culture conditions (Colombo et al., 1998), indicating that the sex ratio of the progeny from meiogynogenetic females is more skewed in favour of females (although some few males can still arise). This observation provides the first evidence regarding the sex ratio of second-generation meiogynogens and, although still a preliminary result, it suggests the possibility that environmental factors alone do not drive the masculinization of meiogynogenetic sea bass. Pavlidis et al. (2000) has clearly demonstrated that increasing the rearing temperature from 13 to 20 8C at the stage of half-epiboly until middle metamorphosis increases the proportion of males from 27% to 74%, verifying a previous suggestion by Colombo et al. (1998) based on meiogynogen sex ratios at three hatcheries with different culture conditions. A possible explanation for the very low percentage of males among second-generation meiogynogens is that, in meiogynogens differentiating as females, there are less male-inducing genetic components responsive to environmental factors with respect to meiogynogenetic males. A genetic polymorphism of autosomal factors influencing in both directions the sex ratios in the progenies of YY males of Nile tilapia, Oreochromis niloticus, has been lately documented (Tariq Ezaz et al., 2004), and evidence that they are amenable to selection for less skewed sex differentiation has been reviewed (Beardmore et al., 2001). Hence, repeated gynogenetic matings may be an approach to eliminate masculinizing genetic determinants in sea bass, as males would be excluded from gynogenesis. Even the thermosensitivity of the sex ratio in this species might be quenched by raising gynogenetic offspring at high temperature during the critical period and selecting for gynogenesis those with lower percentages of males. If this is correct, then almost all-female stocks with more uniform
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growth performance may be eventually obtained to improve sea bass culture.
Acknowledgements This investigation was supported by grants 5C53 and 5C117 of the Ministero per le Politiche Agricole e Forestali (Rome, Italy) and by the EU Research Project BASSMAP Q5RS-2001-01701.
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