Bouza, C.

Cal, R. (Rosa)

Castro, J. (José)

Gómez-Ceruelo, M.C. (María Castora)

Martínez, P.

Piferrer, F.

English

Bouza, C. (Carmen)

Repositorio Institucional Digital del IEO

III  GROWTH AND DEVELOPMENT OF DIPLOID GYNOGENETIC TURBOTExperiment 3Experiment 2INTRODUCTIONThe induction of gynogenesis was investigated in the  turbot (Scophthalmus maximus L). Gynogenesis includes the production ofdiploid individuals with both sets of chromosomes from the female parent. Gynogenesis can be valuable as a rapid method forinbreeding of fish, positional mapping of genes in relation to their centromere, and production of exclusively female populations(Thorgaard and Allen 1987).The production of gynogenetic fish was carried out by fertilizing  eggs with sperm which had its DNA previously inactivated usingUV light radiation so that the genetic material of  spermatozoa would  does not contribute to the genome of the zygote.The radiation dose needs to be adjusted, so that the DNA of the sperm is completely inactivated without losing its mobility andtherefore its capacity to fertilize the eggs and trigger embryonic development (Hertwig effect). The resulting zygotes are usuallyhaploid, and therefore not viable after hatching. Diploidy  must be  restored by applying thermal shock or pressure treatments shortlyafter fertilization (Felip et al, 1999). In this work, we have studied the appropriate conditions for production of diploid gynogenetic  turbot.  Different methods (karyotypeanalysis, nucleoli per nucleus counts, and microsatellite markers) were evaluated for gynogenesis checking. Survival, growth, and sexratio, were analysed in one family of gynogenetics with relation to their corresponding diploid controls.PRODUCTION OF HAPLOID AND DIPLOID GYNOGENETIC TURBOT (Scophthalmus maximus L)R. Cal1, C. Gómez1, J. Castro2, C. Bouza2,  P. Martínez2, and  F. Piferrer3 • 1 Instituto Español de Oceanografía, C.O. de Vigo. Apartado 1552 - 36280 Vigo - Spain • e-mail: rosa.cal@vi.ieo.es • 2 Departamentode Genética. Facultad de Veterinaria. Universidad de Santiago de Compostela. 27002-Lugo - Spain • 3 Institut de Ciènces del Mar, C.S.I.C. Passeig Maritim, 37-49. 08003- Barcelona. Spain.MATERIAL AND METHODSI. Induction to gynogenesisThe induction of meiogynogenesis was carried out by fertilizing the eggs with sperm whose genome had been previously irradiatedwith UV ligth. Diploidy was restored by applying thermal shock treatment to the eggs minutes after fertilization (Piferrer et al., 2000;2002). To achieve optimum induction of gynogenesis, three experiments were designed:Experiment 1: Was performed to determine the effect of UV ligth on sperm mobility. The sperm, diluted in Ringer-200 (Chereguiniet al., 1997), was divided into aliquots which were irradiated with increasing intensities of UV ligth from 5000 to 50000 erg.mm-2except in the case of those used as a control. The mobility of the sperm treated was verified after its reactivation in sea water, accordingto the percentage of mobile cells (Sánchez-Rodríguez, 1975) and  the duration of mobility.Experiment 2:  Was carried out to determine the quantity of UV ligth necessary to inactive the sperm’s DNA  without affecting itscapacity to fertilize the egg and trigger embryos development  (Hertwig effect). Aliquots of eggs, were fertilized with  irradiated sperm with UV ligth at increasing intensities from 300 to 100000 erg.mm-2 , themincubated at 13-14ºC. At  4, 5. 72 and 144 hours, survival rates and the state of the larvae or embryos were determined and theirhaploidy condition was confirmed by counting  chromosomes and nucleoli.Experiment 3:  Was carried out to establish the effect of UV ligth on the sperm in combination with thermal shock treatment appliedto the newly fertilized eggs.The eggs were fertilized with sperm irradiated with UV ligth to 30000-100000 erg.mm-2 , and then a cold shock treatment was applied(Piferrer et al., 2002). At the end of incubation, the fertilization, embryo genesis and hatching rates were  determined.II.  VerificationThe ploidy was examined in larvae collected 1 day after hatching, except for the sperm irradiated group, where the embryos wereused because of the non- viability of haploid larvae. Metaphase plates were obtained according Kligerman and Bloom (1977), andwere stained following the procedure of   Howell and Black (1980). Chromosome counts and the number of nucleoli was examinedin 10 plates and 50 nuclei, respectively in all individuals analysed.The exclusively maternal inheritance of diploid gynogenetic, was determined by applying a set of 11 microsatellite loci, as geneticmarkers ( Bouza et al., 2002). The potential of the microsatellite loci used for monitoring the production of meiogynogenetic  turbot was evaluated by computing the two cumulative exclusion probabilities using the data from an Atlantic turbot population (Bouzaet al., 2002).This population was selected as a reference among those analysed to obtain the potential of exclusion of microsatellites,because the brood stock kept at the IEO were founded with native individuals from this area.III. Growth of gynogenetic turbot One group of gynogenetic diploid turbot and their control, were cultivated separately for two years according to the establishedprotocol for this species. Survival, growth and the percentage of sexes were monitored. The exclusively maternal inheritance wasdetermined in larvae, two days after hatching,  and confirmed at the end of the experiment in each of the fish in the meiogynogeneticgroup.CONCLUSIONSThe production of gynogenetic diploid turbot was achieved by fertilizing eggs with sperm diluted 1:10 and irradiated with UV ligthat a dose of 30000-35000 erg.mm-2. Diploidy was restored by  applying a cold shock shortly after the fertilization, according to (Piferreret al., 2002).The hatching rate of meiogynogenetics diploids larva was 8,6 % compared to the control. with great mortality being observed duringembryogenesis. However, after the larval phase, they exhibited similar survival, resistance to diseases and tolerance to treatment andvaccines as the controls, although their growth was 21% lower at 24 months of age.Gynogenetic diploid turbot had a higher proportion of  females  (25% male and 75% female) than the controls (50 % male and 50%female).Policy determination has been the traditional approach to evaluate gynogenesis in fish,   however  the obtention of good metaphasesplates  in fish  is often hampered by the small size of chromosomes as compared  and the lack of appropriate protocols for obtainingchromosomes of adequate quality. Nevertheless, the estimation of the number of nucleoli is a straightforward method, but theexistence of a NOR-site polymorphism in the case of turbot, disturbs ploidy estimation by this approach. The use of hyper variablemicrosatellite loci described here constitutes a powerful and sound method to confirm the exclusive maternal inheritance in turbotgynogenetics.REFERENCESBouza, C.,  Presa, P.,  Castro, J., Sánchez, L., and  Martínez, P. 2002. Allozime and microsatellite diversity in natural and domestic populations of turbot (Scophthalmus maximus) in comparison with otherPleuronectiformes. Can. J. Fish. Aquatic. Sci. (in press).Chereguini, O., Cal, R.M., Dreanno, C., Ogier de Baulny, B., Suquet, M., and Maisse G., 1997. Short- term storage and cryopreservation of turbot (Scophthalmus maximus ) sperm. Aquat. Living Resources 10: 251-255.Felip A., F. Piferrer, M. Carrillo and S. Zanuy. 1999. The relationship between the effects of UV light and thermal shock on gametes and the viability of early developmental stages in a marine teleost fish, the seabass (Dicentrachus labrax L.). Heredity 83: 387-397Howell, W.H. and Black, D.A., 1980. Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a one-step method. Experientia 36: 1014-1015.Kligerman, A.D. and Bloom, S.E., 1977. Rapid chromosome preparations from solid tissues of fishes. J. Fish Res. Board Can, 34: 266-269.Piferrer  F., R. Cal, B. Alvarez-Blazquez, L. Sánchez, P. Martínez. 2000. Induction of triploidy in the turbot (Scophthalmus maximus). I. Ploidy determination and the effects of cold shock. Aquaculture  188: 79-90.Piferrer, F., R.M. Cal, C. Gómez, C. Bouza, P. Martínez (2002).  Induction of triploidy in the turbot (Scophthalmus maximus). II. Effects of cold shock timing and induction of triploidy in a large volume of eggs.Aquaculture (in press).Thorgaard, G.H., and Allen, S.K.Jr., 1987. Chromosome manipulation  and markers in fishery management. Population Genet. Fish. Manag., pp. 319-331.Fig. 2.  Effect of exposure to different doses of UV light on the duration of the mobilityof turbot spermatozoa  and according to different dilutions with Ringer-200.Sperm from the same male at different dilutions showed that the greater thedilution, the lesser  duration of mobility.Fig. 1. Effect of exposure to different doses of UV light on the duration of the mobility of turbot spermatozoa diluted 1:10 with Ringer-200. A, effecton the mobility score, class 0 to 5, according to Chereguini et al. (1997). B, effect on  the duration of mobility. The ID50 dose was calculated as thedose in which the mobility  score on duration was reduced  to 50% of the control.It was observed that as the radiation dose increased, there was a decrease in sperm mobility, both in the percentage of mobile spermatozoids andin the duration of the mobility.Fig. 3.  Relationship between sperm concentration and dose of UV light for which the score of mobility is reducedto 50%.Sperm from different males with similar concentrations of spermatozoids did not respond in the same way tothe application of UV radiation. This indicates that although the dilution of sperm is a factor to be taken intoaccount there are other factors, like the quality of each sperm, which are more important than the concentrationin determining the response to the effects of UV light.Fig. 4.  Representation of a typical Hertwig effect curve in turbot. Survival is depicted at three different times: 4,5 hafter fertilization (AF), embryogenesis  (72 h AF) and hatching (144 h AF). The Hertwig effect takes place between 3x 102  and 3 x 104 erg.mm-2.In the fertilisations made with sperm irradiated with increasing doses from 300 erg.mm2 onwards an increase ofsurvival of larvae  and embryos at  72 hours was seen between doses of  30 000-35 000 erg.mm-2. These results aretypical of the Hertwig effect,  and for this reason it was considered appropriate the dose of 30 000-35 000 erg.mm-2to inactivate the turbot sperm while maintaining in most cases their capacity to activate embryo development.Fig. 5.  Effect of gamete manipulation, fertilization with irradiated sperm  with UV light , (30000-35000 erg.mm-2 )  applied  in combination with a cold shockon the viability of turbot during early developmental stages: fertilization 4,5 HPF (F); embryogenesis, 72 HPF (E); and 2 days post hatch, 144 HPH (L). (a), controldiploid group, made with  diluted sperm (control of gamete quality, with survival at fertilization set to 100%, to which the other treatments  were  compared).(b), gynogenetic diploid group made with diluted and irradiated sperm (30000-35000 erg.mm-2 ) and thermally shocked (0ºC, applied at 6,5 min. post fertilization,during 25 min.) to restore diploidy (effect of UV light + thermal shock).Fig. 6.  Embryos (Fig. 2a) and hatched larvae (Fig. 2b) from de diploidcontrol group, had a morphologically normal appearance, as expected.In contrast, embryos originating from eggs fertilized with sperm exposedto 30000-35000 erg.mm-2 , of UV light exhibited aberrant development(Fig. 2c). The few larvae that hatched were deformed, exhibing a typical" haploid syndrome" (Fig. 2d) . Group fertilized with UV-irradiated sperm,and thermally shocked  had  a external morphology similar to that ofcontrol diploids. (Fig. 2e, f).Fig. 7.  Ploidy verification in turbotobtained after UV-irradiation ofsperm. Typical karyotype and Ag-stained  nuclei of cells obtainedfrom control diploid (a and b,respectively; 2n= 44), gynogenetichaploid (c and d, respectively,2n=22), and gynogenetic diploid(e and f, respectively, 2n=44)turbot embryos and larvae at 2days after hatching and analysedin this study.GINOGENETICOS (2n)7,16 5,3221,64020406080100120F E LdeformenormalCONTROL (2n)100 80,01 70,64020406080100120F E LdeformenormalFig. 10.  Showing the microscopic appearance of testes (a, b) and ovaries (c, d) from  diploid andmeiogynogenetic diploid  turbot at 24 months of age. ( Stained with toluidine blue. Originalmagnification 200 x.).Note the presence of spermatids and spermatozoa in the testis of both males diploid (a) andmeiogenetic diploid (b), however a lower number of spermatozoa were evident in males meiogeneticdiploid compared with those observed in males control diploid.In contrast to males, clear differences in the gonadal development were observed in females betweenthe control diploid group and the meiogynogenetic diploid. Whereas it was possible to identify matureoocytes in the ovary of females control diploid, in females meiogynogenetic diploid only perinucleolaroocytes were recognized.Since it is not surprising the existence of undeveloped gonads in 24 month old turbot, the abnormalgonadal development in females meiogynogenetic diploid is an intriguing possibility worthy offurther investigation.Table I: Measures of genetic diversity and probabilities of exclusion for the 11 microsatellite loci usedin the present work in an Atlantic natural population of turbot (Scophthalmus maximus; Bouza et al.,2002), where breeders used in the present work putatively proceeded. Calculations were performedwith the computer program CERVUS 2.0 (Marshall et al., 1998). Primer sequences and PCR amplificationconditions for the all the loci are described in Bouza et al. (2002).Fig. 9.  Evolution from 6 to 24 months of age, of body weigth (a) and body length (b) of diploid and meiogynogenetic diploid turbot.Experiment 1Locus na H(E) PIC Excl(1) Excl(2)Smax-01 13 0.742 0.704 0.355 0.532Smax-02 24 0.845 0.829 0.545 0.707Smax-03 9 0.635 0.581 0.227 0.390Smax-04b 17 0.825 0.800 0.483 0.655Sma3-8INRA 12 0.870 0.853 0.579 0.735Sma3-12INRA 10 0.808 0.781 0.452 0.628Sma4-14INRA 14 0.794 0.774 0.451 0.631Sma5-111INRA 14 0.896 0.884 0.643 0.784Sma1-125INRA 9 0.768 0.732 0.378 0.558Sma3-129INRA 20 0.920 0.911 0.713 0.833Sma1-152INRA 9 0.733 0.689 0.330 0.505Mean/Total 13.73 0.803 0.776 0.999368 0.999992A Fig. 8.  Demonstration of the exclusivematernal contribution to gynogeneticoffspring using the diagnosticmicrosatellite loci Smax-02 (a) andSma3-129INRA (b) in each of the twofamilies analysed, respectively. Theelectrophoretic  patterns presentedshow in lane 1 the external standardsize marker including an allelic ladder.Additionally, each lane contains twointernal standard size markers (80-230bp (a); 114-230bp (b)) forgenotyping accuracy. Lanes 2 to 8contain correlatively theelectrophoretic patterns of the twoparents (a: 107/133 _ and 95/95 _;b: 169/169 _ and 179/185 _) and asample of five gynogenetic offsprings(a: 107/107, 133/133, or 107/133; b:all 169/169).BEvolution of body length0510152025303540456 9 12 15 18 21 24Age (months)ControlGynogeneticEvolution of body weigth02004006008001000120014001600180020006 9 12 15 18 21 24 Age (months)ControlGynogenetic3 3I INDUCTION OF GYNOGENESISII VERIFICATIONRESULTSmachos ginogeneticosmachos controlhembra ginogeneticohembra control Weigth (g) Length (cm)

Production of haploid and diploid gynogenetic turbot (Scophthalmus maximus L)

Cal, Rosa

Gómez-Ceruelo, María Castora

Castro, José

Bouza, Carmen

Digital.CSIC

http://www.repositorio.ieo.es/e-ieo/bitstream/10508/9100/1/PRODUCTION%20OF%20HAPLOID%20AND%20DIPLOID%20GYNOGENETIC%20TURBOT.pdf

Production of haploid and diploid gynogenetic turbot (Scophthalmus maximus L)

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