Genetica 111: 175–195, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Induction of triploidy and gynogenesis in teleost fish with emphasis onmarine species

A. Felip1, S. Zanuy1, M. Carrillo1 & F. Piferrer2,∗1Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Cientıficas (CSIC), 12595 Riberade Cabanes, Castellon, Spain (Phone: +34-964-319500; Fax: +34-964-319509; E-mails: alicia@iats.csic.es,zanuy@iats.csic.es, carrillo@iats.csic.es); 2Institut de Ciències del Mar, CSIC, Passeig Maritim, 37-49, 08003Barcelona, Spain (Phone: +34-93-2309500; Fax: +34-93-2309555; E-mail: piferrer@icm.csic.es); ∗Author forcorrespondence

Key words: chromosome set manipulation, Dicentrarchus labrax, gonadal development, growth, gynogenesis,marine teleosts, sex determination, sex ratio, sterility, triploidy

Abstract

The induction of triploidy and gynogenesis by chromosome set manipulation has traditionally been studied moreintensively in freshwater than in marine fish. In the last years, however, several studies have applied these manipula-tions in about a dozen marine species, including mainly sparids, moronids and flatfishes. This paper focuses on themethodologies used to induce, verify, and assess performance of both triploids and gynogenetics of these marinespecies. Since many of them are batch spawners and have small and fragile eggs and larvae, peculiarities relatingto broodstock management, gamete quality and mortality assessment during early larval stages are also taken intoaccount. However, data show that if handling is correct and the treatments are optimized, triploid and gynogeneticrates of 100% can be easily achieved. Survival of triploids with respect to the controls is about 70–80%, whereasin gynogenetics it is generally low and more variable, depending on the species considered. In the marine fishinvestigated so far, triploidy has not resulted in significantly higher growth rates. On the other hand, the inductionof gynogenesis has resulted in the production of both all-female and mix-sex stocks. Throughout the paper, specialreference is made to the European sea bass (Dicentrarchus labrax L.), a species of both basic and applied interest,for which a comprehensive study has been carried out on the induction, verification and performance of triploidsand gynogenetics.

Introduction

Sexual maturation in fish is often accompanied bya reduction in growth, survival and flesh quality. Inaddition, sex related growth differences, usually infavour of females, occur in many cultured species. To-gether, this results in lower profitability (Carrillo et al.,1995a; Piferrer, Felip & Blázquez, 1995; Donaldson,1996). The application of chromosome set manipula-tion techniques (e.g., the induction of triploidy andgynogenesis) have been studied as a means to im-prove the production of many cultured fish species,especially freshwater, including mainly salmonids,cyprinids, cichlids, and ictalurids (Thorgaard, 1983;

Benfey, 1989; Ihssen et al., 1990; Solar, Donald-son & Douville, 1991). Induced triploidy resultingin genetic sterility and gynogenesis can be used toobtain all-female stocks. To date, the only speciesin which triploids are routinely cultured is the rain-bow trout, Oncorhynchus mykiss (Dillon, Schill &Teuscher, 2000). Sterilization can be useful to neg-ate precocious sexual maturation usually in males(Donaldson, 1997; Felip et al., 2001a, b). Moreover,triploidy induction can control unwanted reproductionin culture (Mair, 1993) and can be an advantage inthose species with high mortality during or after thespawning season (Ihssen et al., 1990; Benfey, 1999),as well as in those with a high gonadosomatic index

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(Knibb, 2000). The production of sterile triploids hasother applications, for example, to prevent the interac-tion between farmed and wild fish (Donaldson, 1996,1997). On the other hand, all-female stocks producedthrough gynogenesis may be exploited for roe pro-duction in sturgeon and salmons (Ihssen et al., 1990;Shelton et al., 1997) and can be used in basic research(see § 6).

In comparison, fewer attempts on chromosome setmanipulation have been made in marine fish, eventhough pioneering works were carried out to inducetriploidy in flatfishes at the beginning of the 1970’s(see refer. in Lincoln 1981a). Since the late 1980’s,however, research focusing on assessing the condi-tions for ploidy manipulation has been carried out inabout a dozen different marine species, particularlyof the families Pleuronectidae, Sparidae, Paralich-thyidae, Moronidae, Soleidae, Oplegnathidae, andScophthalmidae. Similar to the situation observed infreshwater species, the induction of triploidy and gyn-ogenesis have been the most widely used types ofchromosome set manipulation (Table 1). There areno reports available to date on the induction of tet-raploidy and androgenesis in marine fish. However,tetraploidy has been induced in several freshwater spe-cies (Solar, Hajen & Donaldson, 1992), although theiractual reduced viability and reproductive performancehas limited their use for the production of triploids(Chourrout et al., 1986). The interest for rearing all-male stocks includes those cases where males growfaster than females (Mair et al., 1997) or have a highermarket value than females due to the presence of male-specific secondary sex characters (Kavumpurath &Pandian, 1992; Piferrer & Lim, 1997).

The purpose of this review is to provide a sum-mary of the studies carried out so far dealing withploidy manipulation, focusing on the methodologiesused for the production and subsequent identificationof triploid and gynogenetic fish. Results concerninggrowth performance of triploid and gynogenetic dip-loids, their reproductive activity and the proportion ofsexes are also summarized. Where appropriate or pos-sible, emphasis is given to marine species, in particularthe European sea bass (Dicentrarchus labrax). Thisspecies has great commercial value and is a modelorganism that has been used as a tool in research ongenetics and reproductive physiology (Carrillo et al.,1999), where a considerable amount of research hasalready been carried out concerning various aspectsof chromosome set manipulation (Zanuy et al., 1994;Carrillo et al., 1995b, 1999; Colombo et al., 1995,

1996, 1997; Gorshkova et al., 1995, 1996; Barbaroet al., 1996; Curatolo et al., 1996; Felip et al., 1997,1999a; Peruzzi & Chatain, 2000).

Induction of tripoidy and gynogenesis in fish

Broodstock and gamete quality for ploidymanipulation

A major requirement for ploidy manipulation in sea-water fish is a sound broodstock management programin order to obtain gametes of the best possible qual-ity (Wohlfarth, 1990; Bartley, 1997). Gametes frommarine species can be treated to yield triploid andgynogenetic fish (Table 2). However, hormonal induc-tion, maturation, ovulation and spawning of brood-stock as well as gamete collection, oocyte qualityassessment and sperm storage can be more demand-ing than for freshwater fish (Bromage, 1988; Planas& Cunha, 1999). This is the case because the eggsand newly hatched larvae from marine pelagic spawn-ers are usually smaller and more fragile than thoseof freshwater species (Bromage, 1988; Zohar, 1989;Planas & Cunha, 1999). If eggs are handled correctlyand collected during the optimal period of spawningfrom post-puberal broodfish, the toll on larval sur-vival can be disregarded (Colombo et al., 1995; Felipet al., 1997; Carrillo et al., 2000). However, thetriploidy yield can change among different femalesor experiments depending on the stage of maturationof the eggs (Díaz et al., 1993; Colihueque et al.,1996; Várkonyi et al., 1998), suggesting an importantgenetic component involved in the outcome.

In general, males spermiate throughout the en-tire reproductive season thus making sperm readilyavailable for collection. In the sea bass, they produceabundant sperm of excellent quality (9.4 ml/kg bodyweight, 5.5–60× 109 spermatozoa/ml and motility of70–100%) (Suquet et al., 1994; Sorbera et al., 1996;Fauvel et al., 1999; Felip et al., 1999a). Furthermore,artificial fertilization rates are usually high (Colomboet al., 1995; Felip et al., 1997, 1999a). However,in flatfish, males mature in captivity but the gam-etes sometimes cannot be easily removed by strippingwhich may represent an inconvenience for chromo-some set manipulation (Howell, Baynes & Thompson,1995).

In sea bass, differences in oocyte final matur-ation among females can difficult the productionof abundant good quality gametes (Alvariño et al.,

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Table 2. Chromosome set manipulation in marine teleost fish

TypeaSpecies (common name) Triploidy Gynogenesis

bPleuronectes platessa x Platichthys flesus (plaice x flounder) Purdom, 1972bHippoglossus hippoglossus (Atlantic halibut) Holmefjord and Refstie, 1997cAcanthopagrus schlegeli (Black Sea bream) Arakawa et al., 1987cPagrus major (Red Sea bream) Arakawa et al., 1987 Sugama et al., 1990

Kitamura, Teong and Arakawa, 1991cSparus aurata (gilthead sea bream) Garrido-Ramos et al., 1996; Gorshkov et al., 1998

Gorshkov et al., 1998dParalichthys olivaceus (hirame) Tabata, 1991 Tabata, 1991eDicentrarchus labrax (European sea bass) Seei Seej

eMorone chrysops x Morone saxatilis (hybrid striped bass) Leclerc et al., 1996eMorone chrysops (white bass) Gomelsky et al., 1998fSolea solea (common sole) Howell, Baynes and

Thompson, 1995gOplegnathus fasciatus (Japanese parrot fish) Murata, 1998hScophthalmus maximus (turbot) Piferrer et al., 2000 F. Piferrer et al. (unpublished

observations)

aFamily: bPleuronectidae, cSparidae, dParalichthyidae, eMoronidae, fSoleidae, gOplegnathidae, hScophthalmidae. iCarrillo et al.,1993,1999; Zanuy et al., 1994; Colombo et al., 1995; Gorshkova et al.,1995; Felip et al.,1997. jCarrillo et al.,1993, 1999; Zanuy et al., 1994;Colombo et al., 1995; Gorshkova et al.,1996; Felip et al., 1999a.

1992). Hence, stocked females need to be periodic-ally checked by ovarian biopsy to select those withoocytes in a similar developmental stage for spawninginduction by hormones. Different useful criteria andprocedures for the selection of eggs have been reported(Carrillo et al., 2000). These include batch size, eggdiameter and buoyancy, and development to the firstdivisions (Howell, Baynes & Thompson, 1995; Felipet al., 1997; Gorshkov et al., 1998).

Induction of triploidy

Because fish exhibit external fertilization, their gam-etes may be easily handled for chromosome set manip-ulation. Table 1 shows treatments that interrupt earlycellular divisions of fertilized eggs for the inductionof triploidy and tetraploidy and the production of indi-viduals with uniparental inheritance (gynogenesis andandrogenesis).

Suppressing meiosis II to retain the second po-lar body in fertilized eggs produces triploids. This isachieved by applying a shock, which can be a cold,heat or pressure shock, always taking into accountthree important variables. These are: (a) the time afterfertilization when the shock is initiated, which canbe expressed in minutes or in mitotic intervals (Thor-gaard, 1983; Ihssen et al., 1990); (b) shock intensity;

and (c) shock duration. The degree of oocyte develop-ment and egg size is also important (Díaz et al., 1993;Colihueque et al., 1996; Várkonyi et al., 1998). Cold,heat and pressure shocks have been successfully usedin marine species (Table 3), while chemical shocksare used less often in fish. The use of cytochalasinB in Atlantic salmon, Salmo salar (Allen & Stan-ley, 1979) and colchicine in brook trout, Salvelinusfontinalis (Smith & Lemoine, 1979) resulted in thepresence of mosaic individuals, although mosaicismalso occurred albeit at a lower frequency when usingphysical treatments (Miller et al., 1994; Teplitz et al.,1994).

Cold shocks are usually applied in the range from−1◦C to 3◦C, yielding 84–100% triploids (Table 3),and have been the most commonly used to inducetriploidy in marine fish, although heat and pressureshocks also work. However, since high numbers oftriploids are obtained using optimized protocols, it isdifficult to say in general that there is an approach thatworks best for marine fish, although there has been apreference for cold shocks, as mentioned above. Theineffectiveness of pressure shock in the common sole,Solea solea (Howell, Baynes & Thompson, 1995), orcold shock in Atlantic salmon (Lincoln, Aulstad &Grammeltvedt, 1974) may have been due to the lack ofprotocol optimization. The advantage of cold shocks

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Table 3. Methods for the induction of triploidy in marine fish

Species Method Results References

Hippoglossus hippoglossus Cold −1◦C, TAF = 15 min, D = 2–3 h 92–95% triploids, 50–82% Sv to 8 Holmefjord and Refstie, 1997

days post-fertilization (dpf)

Heat 24◦C, TAF = 15 min, D = 15–30 min 84% triploids, 20 Sv to 8 dpf Holmefjord and Refstie, 1997

Acanthopagrus schlegeli Cold 2–3◦C, TAF = 1.5–5 min, D = 5–25 min 90% triploids, > 50% Sv Arakawa et al., 1987

Cold 2–3◦C, TAF = 3 min, D = 5–15 min 100% triploids, > 70% Sv Arakawa et al., 1987

Pagrus major Cold 2–3◦C, TAF = 3 min, D = 15 min Triploids, 30% Sv at 2 month-old Kitamura, Teong and Arakawa, 1991

Cold 0◦C, TAF = 3 min, D = 12 min 100% triploids > 61% Sv up to Sugama et al., 1992

first feeding

Sparus aurata Heat 34◦C, TAF = immediately, D = 10 min 100% triploids, 40% Sv Garrido-Ramos et al., 1996

Heat 37◦C, TAF = 3 min, D = 2,5 min 100% triploids, 40% Sv Gorshkov et al., 1998

Heat 29◦C, TAF = 15 min, D = 25 min, Triploids, 0.8% Sv Carrillo et al., 1993

Pressure 8,000 psi, TAF = 5 min, D = 2–3 min Triploids, 13% Sv Zanuy et al., 1994

Cold 0◦C, TAF = 5 min, D = 5 min Not described Gorshkova et al., 1995

Dicentrarchus labrax Cold 0–2◦C, TAF = 5 min, D = 20 min 89–90% triploids, 40–50% Sv Colombo et al., 1995

Cold 0◦C, TAF = 5 min, D = 10 min 95–100% triploids, 80% Sv Felip et al., 1997

Cold 0–1◦C, TAF = 5 min, D = 15–20 min 100% triploids, 56% Sv Peruzzi and Chatain, 2000

Pressure 8,500 psi, TAF = 6 min, D = 2 min 100% triploids, 71% Sv Peruzzi and Chatain, 2000

Scophthalmus maximus Cold 0◦C, TAF = 5 min, D = 20 min 90% triploids, 80% Sv Piferrer et al., 2000

Abbreviations: TAF – time after fertilization when shock is initiated; D – duration of shock. Sv–survival was to hatching unless otherwise indicated.

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over pressure shocks is that no special equipment isneeded and that large volumes of eggs can be treatedat once.

In general, the timing of the shock is the most crit-ical variable (Ihssen et al., 1990; Levanduski, Beck &Seeb, 1990; Felip et al., 1997, 1999a) since even smallvariations result in significant changes in the numberof triploids obtained, as demonstrated in the sea bass(Felip et al., 1997, 1999a). When using temperatureshocks, not only the temperature of the shock but alsothe difference between the pre-shock temperature andthe shock temperature is important and may affect thefinal yields (Chourrout, 1987; Ihssen et al., 1990).Changes in shock temperature are less important thanin shock timing while changes in duration appear tobe the least relevant. Exceptions to this have beendemonstrated in chinook (Levanduski, Beck & Seeb,1990) and pink salmon (Smoker, Crandell & Mat-suoka, 1995) where the duration of the thermal shockwas crucial for the retention of the second polar body.Finally, the outcome of shocks may also be affectedby oocyte quality and the degree of maturation whenthey are obtained from the females (Levanduski, Beck& Seeb, 1990; Díaz et al., 1993; Smoker, Crandell &Matsuoka, 1995; Várkonyi et al., 1998).

Induction of gynogenesis

Gynogenetic diploid fish are obtained by first usingirradiated sperm to fertilize normal eggs and then sup-pressing the second meiotic division of these eggs(meiogynogenetic diploids) or the first mitotic divisionof the zygotes (mitogynogenetic diploids) (Table 1).The suppression of these cell divisions is carried outwith shocks similar to those used for the inductionof triploidy (compare Tables 3 and 4). Meiogyno-genetic fish retain significant levels of heterozygositydepending on the degree of crossing-over betweenhomologous chromosomes during meiosis I, whereasmitogynogenetic fish are completely homozygous atevery locus (Thorgaard, 1983; Ihssen et al., 1990).As a result, meiogynogenetic diploids have higherviability than mitogynogenetic diploids, which carrydeleterious alleles that negatively affect their survival(Leary et al., 1985; Don & Avtalion, 1988; Ihssenet al., 1990). Among species, yields are variable butgenerally low (Table 4). In marine teleosts, inductionof mitogynogenetic diploids has only been reportedin the sea bass with results showing a low percentageof viable embryos. However, survival in juvenile and

adult fish still needs to be determined (Colombo et al.,1995).

Several types of radiation, for example, ioniz-ing (X-rays, gamma rays) and ultraviolet (UV), andalso chemical treatments have been used to inactivatespermatozoan DNA (Thorgaard, 1983; Ihssen et al.,1990). Unlike ionizing radiation, UV light does notinduce small DNA paternal fragments (minichromo-somes) which may reduce the viability of gynogenet-ics (Thorgaard & Allen, 1987; Ihssen et al., 1990).The effects of UV light can be reversed by expos-ure to visible light, thus, UV- irradiated sperm mustbe protected in darkness until just before fertilizationto avoid a putative photoreactivation process (Ijiri &Egami, 1980). An alternative to sperm irradiation isthe use of sperm from an unrelated species (hetero-logous sperm) (Table 5) which, for added security,can also be UV-irradiated. It facilitates the identifica-tion of non-gynogenetic fish if hybrids are non-viable(Goudie et al., 1995; Smoker, Crandell & Matsuoka,1995; Fujioka, 1998; Gorshkov et al., 1998; Váradiet al., 1999). The appropriate dose of UV light isanother important aspect to effectively inactivate thesperm (Table 5). Thus, a range of doses must be as-sayed. Batches of eggs are fertilized by diluted spermirradiated with increasing doses of UV light inducinga paradoxical phenomenon called the Hertwig effect.This has been elicited in both freshwater (Pongthanaet al., 1995; Lin & Dabrowski, 1996; Palti, Li &Thorgaard, 1997) and marine species (Gomelsky et al.,1998; Felip et al., 1999a). Embryos resulting fromthe fertilization of normal eggs with completely in-activated sperm exhibit a typical ‘haploid syndrome’,for example, inviable embryos or newly-hatched lar-vae with severe body malformations (Pongthana et al.,1995; Lin & Dabrowski, 1996; Felip et al., 1999a).

In many fish species, the irradiation of UV lightdecreases the motility of spermatozoa (Goudie et al.,1995; Felip et al., 1999a). Thus, lower viability at-tributed to gynogenetics could simply be due to lowerfertilization rates as a consequence of reduced num-bers of motile spermatozoa. In sea bass, the exposureof sperm to UV light (2,500–55,000 erg mm−2) re-duced the amount of motile spermatozoa but did notsignificantly affect the duration of motility in the re-maining motile spermatozoa (Felip et al., 1999a). Thisstudy also demonstrated that the thermal shock negat-ively influenced the survival at fertilization, althoughits effects decreased during embryogenesis and disap-peared by hatching, suggesting that the lower viabilityof some triploids is a direct consequence of the shock,

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Table 4. Methods for the induction of gynogenesis in marine fish

Species Method Results References

Paralichthys olivaceus UV-irradiated sperm 7,200 erg mm−2 plus Not described Tabata, 1991

Cold 0◦C, TAF = 3 min, D = 45 min

Pagrus major UV-irradiated sperma 3,000 erg mm−2 plus 100% gynogens, > 70% Sv Sugama et al., 1990

Heat 35◦CC, TAF = 3 min, D = 2.5 min beyond yolk sac resorption

Sparus aurata UV-irradiated sperma 3,000 erg mm−2 plus Gynogens, 46.5% Sv Gorshkov et al., 1998

Heat 36.5–37.3◦CC, TAF = 3 min, D = 2.5 min

Dicentrarchus labrax UV-irradiated sperm 40,000 erg mm−2 plus Gynogens, Sv not described Carrillo et al., 1993

Heat 29◦C, TAF = 15 min, D = 25 min; Zanuy et al., 1994

Pressure 8,000 psi, TAF = 5 min, D = 2–3 min

UV-irradiated sperm 3,300–6,600 erg m m−2 plus 83–100% gynogens, 17% Sv Colombo et al., 1995

Cold 0–2◦C, TAF = 5 min, D = 20 min

UV-irradiated sperm plus Not described Gorshkova et al., 1996

Heat 35◦C, TAF = 3–5 min, D = not shown

UV-irradiated sperm 35,000–40,000 erg mm−2 plus 95% gynogens, 30–35% Sv Felip et al., 1999a

Cold 0◦C, TAF = 5 min, D = 10 min

UV-irradiated sperma 32,000 erg mm−2 plus

Cold 0–1◦C, TAF = 5 min, D = 15–20 min 100% gynogens, 76% Sv Peruzzi and Chatain, 2000

Pressure 8,500 psi, TAF = 6 min, D = 2 min 100% gynogens, 76% Sv

Morone chrysops x M. UV-irradiated sperma 140 erg mm−2 plus >75% gynogens, 26% Sv Leclerc et al., 1996

saxatilis Pressure 8,000 psi, TAF = 4 min, D = 90 sec fertilization

Morone chrysops UV-irradiated sperma 8,000 erg mm−2 plus Gynogens, 24–39% Sv Gomelsky et al., 1998

Heat 36◦C, TAF = 3 min, D = 2 min

Solea solea Non-irradiated sperma plus

Cold 2◦C; TAF = 5–10 min; D = 1–2 h 80% gynogens, 81% Sv Howell, Baynes and Thompson, 1995

Pressure 630 kg cm−2, TAF = 10–40 min, D = 10 min No survival of eggs to hatching

Scophthalmus maximus UV-irradiated sperm plus cold shock 100% gynogens F. Piferrer et al. (unpublished

observations)

aHeterologous sperm (see Table 5). Other abbreviations as in Table 3.

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Table 5. Determination of the optimum UV-dose to genetically inactivate the sperm and elicitation of the Hertwiga effect in experiments aiming at gynogenesis inductionin marine teleost fish

Species UV-doses Dilution of sperm Origin of sperm References

(erg mm−2)

Paralichthys olivaceus 4,560–4,800 1 : 50–1 : 100 Homologous See Yamamoto, 1999

Pagrus major 3,000 Not described Heterologous Sugama et al., 1990

(Black Sea bream)

Sparus aurata 3,000 1 : 30 Heterologus Gorshkov et al., 1998

(Red Sea bream)

3,300–6,600 1 : 100 Homologous Colombo et al., 1995

Dicentrarchus labrax 35,000–40,000a 1 : 10 Homologous Felip et al., 1999a

32,000 1 : 20 Homologous/heterologous Peruzzi and Chatain, 2000

(sea bream)

Morone chrysops x M. saxatilis 140 1 : 5 Heterologous Leclerc et al., 1996

(perch Morone americana)

Morone chrysops 8,000a 1 : 30 Heterologous Gomelsky et al., 1998

(stripped bass)

Solea solea Non-irradiated sperm Non-diluted sperm Heterologous (halibut) Howell, Baynes and Thompson, 1995

Scophthalmus maximus 20,000–40,000a 1 : 10 Homologous F. Piferrer et al. (unpublished

observations)

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Table 6. Methods used to identify triploidy in marine fish

Species Method References

Hippoglossus hippoglossus Flow cytometry Holmefjord and Refstie, 1997

Acanthopagrus schlegeli Not described Arakawa et al., 1987

Pagrus major Karyotyping Kitamura, Teong and Arakawa, 1991

Erythrocyte size Kitamura, Teong and Arakawa, 1991; Sugama et al., 1992

Allozyme markers Sugama et al., 1992

Sparus aurata Karyotyping Garrido-Ramos et al., 1996; Gorshkov et al., 1998

Dicentrarchus labrax Karyotyping Carrillo et al., 1993; Zanuy et al., 1994

Gorshkova et al., 1995; Colombo et al., 1995; Felip et al., 1997

Silver staining Felip et al., 1997

Erythrocyte size Felip et al., 1997

Flow cytometry Carrillo et al., 1993; Zanuy et al., 1994

Colombo et al., 1995; Peruzzi and Chatain, 2000

Scophthalmus maximus Karyotyping

Silver staining Piferrer et al., 2000

not of the triploid condition per se. In contrast, UVlight had an effect more pronounced as developmentprogressed from fertilization onwards, reaching itsmaximum at hatching. Therefore, mortality is higherin gynogenetic fish than in triploid fish. This reflectsthe manipulation of both male and female gametes aswell as the effects of the higher homozigosity com-pared to triploids (Leary et al., 1985; Don & Avtalion,1988). Thus, sperm irradiation rather than the thermalshock has been shown to be the major cause of in-creased mortality in gynogenetics (Lin & Dabrowski,1996; Felip et al., 1999a). Deleterious effects of solarUV radiation affecting survival and development havebeen documented in aquatic ecosystems (Häder, 1986,1993), and in amphibians (Ankley et al., 2000) and fish(Bass & Sistrun, 1997; Béland et al., 1999). In the seabass, the yield of triploids and gynogenetics is similarto that observed in freshwater fish rather than to whathad typically been observed in marine (see Tables 3and 4).

Identification of triploids

Methods to identify triploid fish are presented inTable 6. They include chromosome count or karyotyp-ing (Thorgaard & Disney, 1990), selective stainingof the nucleolar organizer regions (NORs) (Flajshans,Ráb & Dobosz, 1992; Kucharczyk et al., 1999), cel-lular size (Felip et al., 1997, 1999b; Benfey, 1999)

and direct measurement of the nuclear DNA content(Allen, 1983; Ewing & Scalet, 1991; Lecommandeur,Haffray & Philippe, 1994). Comparison of the differ-ent methods can be found in Harrell, Van Heukelemand Kerby (1998). In marine species, karyotyping hasbeen the most commonly used method so far to dis-tinguish between diploids and triploids, for example,48 chromosomes versus 72 chromosomes in sea bass,gilthead sea bream and Red Sea bream, whereas turbothad 44 chromosomes in diploids and 66 in triploids(see refs. in Table 6). Karyotyping, however, is time-consuming and silver staining of NORs can be used asan alternative (Harrell, Van Heukelem & Kerby, 1998).Analysis of NORs is only useful in those species withone nucleolar organizer region per haploid genome,with one or two NORs detected per cell in diploidsand up to three NORs in triploids. Thus, one potentialproblem with this method can be the presence of apolymorphism in the number of NORs, resulting ina lack of correlation between the number of NORsand the ploidy level. In such cases, as for examplein the turbot, it needs to be demonstrated that such apolymorphism affects only a low number of fish andthat it does not interfere with the correct assessment ofploidy (Piferrer et al., 2000).

As cells in triploid fish have 50% more DNA dueto an extra set of chromosomes, cell volume increasesabout 1.5 times in triploids. Thus, cells of triploids arelarger than those of diploids (Benfey, 1999). Hence,

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Table 7. Methods used to identify gynogenesis in marine fish

Species Methoda References

Pagrus major Allozyme markers Sugama et al., 1990

Sparus aurata Karyotyping Gorshkov et al., 1998

Dicentrarchus labrax External morphology Carrillo et al., 1993; Zanuy et al., 1994;

Colombo et al., 1995; Felip et al., 1999a

Karyotyping Colombo et al., 1995; Gorshkova et al., 1995;

Felip et al., 1997

Silver staining Felip et al., 1997

AFLP markers Felip et al., 2000

Microsatellites Peruzzi and Chatain, 2000

Morone chrysops x M. saxatilis DNA amplification Leclerc et al., 1996

techniques

Morone chrysops External morphology

Morphological characters Gomelsky et al., 1998

Karyotyping

Solea solea External morphology Howell, Baynes and Thompson, 1995

Scophthalmus maximus External morphology

Karyotyping F. Piferrer et al. (unpublished observations)

Silver staining

Microsatellites

aExternal morphology based on haploid syndrome; Karyotyping based on numbers of chromosomes withthe exception of Sparus aurata showing ‘marker’ chromosomes, that is, number of biarmed chromosomes;Morphological characters in Morone chrysops based on lack of melanophores.

the length of the major axis of erythrocytes has beenused to rapidly confirm triploidy in several species,especially if a particle size analyzer is available (Wat-tendorf, 1986). Cytofluorometric analysis is anotheralternative that allows processing large amounts ofsamples in a relatively short period of time but, as inthe previous case, its major drawback is the high costof equipment.

Identification of gynogenetics

Methods to identify gynogenetic fish are summarizedin Table 7. The restoration of diploidy in gynogen-etic fish can easily be confirmed by karyotyping andNOR analysis. However, the true assessment of fishwith exclusively maternal inheritance can only bedemonstrated by using morphological, biochemical ormolecular genetic markers (Johnstone & Stet, 1995;Leclerc et al., 1996; Van Eenennaam et al., 1996; Felipet al., 2000; Peruzzi & Chatain, 2000).

In fish, the number of studies using heritable mor-phological traits for paternity testing is low (Stanley &

Jones, 1976; Nagy et al., 1978; Suzuki, Oshiro & Na-kanishi 1985; Don & Avtalion, 1988). Moreover, evenin the case of species exhibiting these morphologicalvariants, other methods of verification are simultan-eously carried out to determine parental assignment(Don & Avtalion, 1988; Gomelsky et al., 1998). Al-though the ‘haploid syndrome’ is not a morphologicalmarker, it is an external feature in embryos or develop-ing larvae with only one set of chromosomes. Duringgynogenesis induction, the presence of embryos withthis syndrome indicates genetic inactivation of the ir-radiated sperm and thus serves for the identification ofputative gynogenetic fish (see Table 7).

Studies based on protein and/or allozyme variantshave had few applications for paternity testing in fish.The reason may be associated with two main limita-tions of this methodology: the low proportion of poly-morphic loci and the reduced allelic diversity of theseloci (2–3 alleles/locus) making the heterozygosity rateand the genetic variability low (Magoulas, 1997). Incontrast, molecular markers are increasingly being ap-plied because, firstly, DNA genomic sequence variants

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Table 8. Growth in body weight (BW) of triploid (3n) versus diploid (2n) fish

Environment aSpecies Life stage Growth References

Marine species bPleuronectes platessa x Juveniles 3n = 2n Purdom, 1972

Platichthys flesus

Adults 3n > 2n Lincoln, 1981cPagrus major Juveniles 3n < 2n See Arai, 2001dParalichthys olivaceus Juveniles 3n < 2n See Arai, 2001eDicentrarchus labrax Juveniles 3n = 2n Felip et al., 1999b

Adults 3n < 2n Felip et al., 2001a

Freshwater species fTilapia aurea Juveniles 3n > 2n Valenti, 1975gCyprinus carpio Juveniles 3n = 2n Gervai et al., 1980

Adults 3n < 2n Cherfas et al., 1994gCtenopharyngodon idella Juveniles 3n = 2n Cassani and Caton, 1985

Oliva-Teles and Kaushik, 1990

3n < 2n Friars et al., 2001hIctalurus punctatus Juveniles 3n = 2n Wolters, Libey and Chrisman, 1982

Adults >8 months 3n > 2niOncorhynchus mykiss Juveniles 3n < 2n Thorgaard et al., 1982

Juveniles, 118 days 3n = 2n Myers and Hershberger, 1991

Adults 3n > 2n Chourrout et al., 1986

Guo, Hershberger and Myers, 1990iOncorhynchus kisutch Juveniles, 17 months 3n < 2n Utter et al., 1983

Juveniles, 30 months 3n = 2n Johnson, Dickhoff and Utter, 1986iSalmo salar Juveniles 3n = 2n Benfey and Sutterlin, 1984

McGeachy, Benfey and Friars, 1995iOncorhynchus mykiss x Juveniles 3n < 2n Parsons et al., 1986

O. kisutchiOreochromis mossambicus 21–79 weeks 3n ≤ 2n Penman, Skibinski and Beardmore, 1987iOreochromis aureus 21–79 weeks 3n ≤ 2n Penman, Skibinski and Beardmore, 1987iOreochromis niloticus 21–79 weeks 3n ≤ 2n Penman, Skibinski and Beardmore, 1987

10–40 weeks 3n = 2n Hussain et al., 1995

Adults 3n > 2n Brämick et al., 1995iOncorhynchus gorbuscha Adults 3n = 2n Benfey et al., 1989iOncorhynchus masou 10–13 months 3n = 2n Nakamura et al., 1993iSalmo salar x Salmo trutta Variable results Galbreath and Thorgaard, 1994iHybrids of Oncorhynchus mykiss Adults 3n ≤ 2n Blanc, Vallee and Dorson, 2000jClarias gariepinus 149–205 days 3n = 2n Henken, Brunink and Richter, 1987jClarias macrocephalus 8 months 3n > 2n Fast et al., 1995kPerca flavescens Juveniles 3n < 2n Malison et al., 1993kStizostedium vitreum Variable results Malison and Garcıa-Abiado, 1996lMisgurnus mizolepis 2 months 3n = 2n Kim, Jo and Lee, 1994

3–9 months 3n > 2n Kim, Jo and Lee, 1994mPomoxis annularis x Variable results Parsons and Meals, 1997

P. nigromaculatusnOplegnathus fasciatus Juveniles 3n < 2n See Arai, 2001

aFamily: bPleuronectidae, cSparidae, dParalichthyidae, eMoronidae, fCichlidae, gCyprinidae, hIctaluridae, iSalmonidae, jClariidae,kPercidae, lCobitidae, mCentrarchidae, nOplegnathidae.Data includes both sexes. Diploids and triploids were reared in separate tanks.

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result in a high polymorphism rate and, secondly,small amounts of tissue are required since DNA can beamplified using the polymerase chain reaction (PCR)(Zhang, Tiersch & Cooper, 1994; Takagi & Tanigu-chi, 1995; Partis & Wells, 1996; Williams, Kazianis& Walter, 1998). Thus, molecular approaches provideunambiguous identification of gynogenetics and arepreferred nowadays.

The technique of DNA fingerprinting, based onvariable numbers of tandem repeats, has been the mostwidely used method to identify gynogenetic fish ina variety of freshwater species (Carter et al., 1991;Volckaert et al., 1994; Johnstone & Stet, 1995; VanEenennaam et al., 1996). To date, the only marinespecies for which true gynogenesis has been proven bymolecular markers (DNA amplification or microsatel-lite analysis) are the hybrid stripped bass (Leclercet al., 1996), European sea bass (Felip et al., 2000;Peruzzi & Chatain, 2000) and turbot (F. Piferrer et al.,unpublished observations) (see Table 7). In the case ofthe European sea bass, the number of true gynogeneticfish produced was 96.5% based on amplified fragmentlength polymorphism (AFLP) markers (Felip et al.,2000).

Performance of triploid fish

Survival and growth

The increased cell size in polyploid fish is balancedwith decreased cell numbers, resulting in fish hav-ing larger cells but not necessarily larger size (Ihssenet al., 1990; Benfey, 1999). Furthermore, triploidymay affect different tissues in a different manner, pro-ducing mosaic individuals. However, no informationis available on the effect of mosaicism on the viabilityand growth performance of these fish (Teplitz et al.,1994).

Most studies on chromosome set manipulation inmarine species have focused on finding the right com-bination of treatment variables to induce triploidy orgynogenesis. Hence, very little research has beenconducted in studying the growth and the reproduc-tion of these fish. This contrasts with the situationin freshwater species where, in addition, a varietyof physiological and behavioral aspects have beenstudied including hematological parameters, oxygenconsumption and aerobic capacity, immunocompet-ence, stress response, osmoregulation, diet utilizationand energetics, sensory perception, and behavior (see

Ihssen et al., 1990; Benfey, 1999, for reviews). Theavailable data published so far revealed that the growthof triploids was dependent on the species concernedand, within the same species, on the age (Thorgaard,1983; Ihssen et al., 1990), rearing conditions (Cherfaset al., 1994; Carter et al., 1994; O’Keefe & Benfey,1997), methodologies used (Thorgaard, 1983; Ihssenet al., 1990; Malison et al., 1993), and reproductivestrategy (Benfey, 1999; Knibb, 2000).

With regard to marine species, the accumulateddata on growth in induced triploids are scarce andsummarized in Table 8, which also includes data onfreshwater species for comparison. Female plaice xmale flounder (Pleuronectes platessa x Platichthysflesus) triploid hybrids grew more than the correspond-ing non-hybrid diploids during the spawning season(Lincoln, 1981). However, diploids reached growthrates similar to those of triploids after sexual matura-tion, thus negating any previous advantage in somaticgrowth. In the sea bass, triploids grew in a patternsimilar to diploids during the juvenile stage. Never-theless, in spite of the gametic sterility, adult triploidsgrew slower than adult diploids even when diploidsmature during the spawning season in winter. Thissuggests that the low growth rate of this species duringwinter depends more on temperature than on the ener-getic cost associated with reproduction (Felip et al.,2001a).

In many species where females grow faster thanmales such as the olive flounder, sea bass and tur-bot, triploid females also show enhanced growth rates,indicating that there is a strong genetic, sex-relatedcomponent in this differential growth. However, sincetriploid females are completely sterile while triploidmales are not, a non genetic component in the formsex-related differences in the energy available forgrowth may also be present. Hence, the establishmentof all-female triploid stocks could benefit the rearingof these species. In freshwater species, this approachhas been considered (Benfey et al., 1989; Piferrer,Benfey & Donaldson, 1994; Galbreath & Thorgaard,1995; Sheehan et al., 1999). Research in rainbow trouthas demonstrated better growth in all-female triploidstocks as compared to those with mixed sex stocks(Sheehan et al., 1999). In contrast, all-female triploidAtlantic salmon appear to have no greater advant-age over mixed-sex cultures (Galbreath et al., 1994;Galbreath & Thorgaard, 1995).

The presence of jaw, operculum and vertebralcolumn deformities has been reported in geneticallymanipulated fish but these deformities have not been

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related to chromosome set manipulation practices(Sugama et al., 1992; Felip et al., 1999b). Instead, theyare considered to be due to handling during artificialfertilization, inbreeding or chromosomal aberrations,since captive broodstock are typically used. In fact,these deformities are encountered in a similar pro-portions in wild fish as well as in diploid controlsmaintained under similar rearing conditions (Cerdáet al., 1994; Colihueque et al., 1996; Felip et al.,1999b; Tudor & Katavic, 1999). Together, these factssuggest that development and the external morphologyof triploid fish is essentially similar to that of diploids.

Gonadal development

Induced triploidy disrupts gametogenesis with morepronounced effects in females than in males (Ben-fey, 1999). Rudimentary ovaries in triploid femaleshave been observed in salmonids (Benfey & Sutterlin,1984; Johnson, Dickhoff & Utter, 1986; Nakamuraet al., 1987), channel catfish (Wolters, Libey & Chris-man, 1982), yellow perch (Perca flavescens) (Malisonet al., 1993), and mud loach (Misgurnus mizolepis)(Kim, Jo & Lee, 1994). However, in a few other spe-cies, triploid females have managed to develop ovariessimilar to those of diploids, as it is the case of Tilapiasp. (Freund, Hörstgen-Schwark & Holtz, 1995), whitesturgeon (Acipenser transmontanus) (Van Eenennaamet al., 1990), carp (Cyprinus carpio) (Wu et al., 1993;Cherfas et al., 1994), and brook trout (Benfey, 1995).An inappropriate follicular structure in the ovary anda greatly reduced production of steroid hormones areoften blamed for the absence of gonadal developmentin triploid females (Mol et al., 1994; Hussain et al.,1995; Benfey, 1999; Felip et al., 2001b). Althoughno endocrine signs of sexual maturation occur in fe-male triploids, some studies have reported their re-sponse to stimulation with sex steroids (Benfey et al.,1989; Breton & Sambroni, 1996). In contrast, trip-loid males display testes similar to those of diploidsand reach sexual maturation but produce dilute, ab-normal and non-viable aneuploid sperm (Kawamuraet al., 1999; Koedprang & Na-Nakorn, 2000; Tiwary,Kirubbagaran & Ray, 2000; Wills et al., 2000). Mo-reover, since testes in triploid males contain ster-oidogenic cells, they exhibit hormonal profiles andsecondary sexual characters similar to those of dip-loids (Nakamura et al., 1993; Mol et al., 1994; Hussainet al., 1995).

Impaired gametogenesis as a consequence of trip-loidy has been observed in marine fish species such

as plaice x flounder triploid hybrids (Lincoln, 1981),Red Sea bream (Kitamura, Teong & Arakawa, 1991;Sugama et al., 1992) and sea bass (Felip et al., 2001b).The induction of triploidy in the latter species con-ferred functional sterility in males and females, thusproving a good model to describe the effects of trip-loidy on the gonadal development in the two sexes.It was found that triploidy blocked the initial phasesof meiosis in females and the late phases of meiosisin males, resulting in the absence of or a reduction ingonadal development, respectively. Specifically, in fe-males, triploidy blocked meiosis during the zygotene,preventing the pairing of homologous chromosomes.In contrast, in males, triploidy blocked meiosis dur-ing the transformation of secondary spermatocytesinto spermatids, thus preventing spermiogenesis (Felipet al., 2001b). To our knowledge, this is the first casereported in fish where triploidy completely interruptedspermatogenesis and, since males are the predominantsex of this species in captivity, this may have economicbenefits for its aquaculture.

However, the ultimate reason as to why triploidydifferentially affects gonadal development betweensexes is still unknown. Differences associated with mi-totic rates between spermatogonia and oogonia beforethe start of meiosis have been considered (Thorgaard,1983; Krisfalusi & Cloud, 1997). Recent studies deal-ing with meiotic chromosome behavior can be usefulto determine the causes of aberrant meiosis in poly-ploid fish (Gui et al., 1992; Zhang & Arai, 1999).These studies indicate that abnormalities occur amonghomologous chromosomes, affecting the synaptic pro-cesses during the zygotene stage, although the syn-aptonemal complex is still formed (Gui et al., 1992;Carrasco et al., 1998; Zhang & Arai, 1999). Further re-search concerning meiotic chromosomal associationsin Nile tilapia revealed a drastic aberration in chromo-some pairing in oocytes but not in spermatocytes as acause of gonadal sterility in females (Carrasco et al.,1998). In humans, male sterility has been attributedto an abnormal pairing between sex chromosomes andunpaired autosomal chromosomes (Rosenmann et al.,1985).

Proportion of sexes

Changes in genome size or allelic proportions as aconsequence of triploidy induction apparently shouldnot result in deviations in the proportion of sexes infish (Ihssen et al., 1990; Hussain et al., 1995; Felipet al., 2001a). However, altered sex ratios in favor

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of males have been observed in triploids of Rhodeusocellatus ocellatus (Kawamura et al., 1999), zebrafish,Brachydanio rerio (Kavumpurath & Pandian, 1990),Red Sea bream (Kitamura, Teong & Arakawa, 1991),carp (Cherfas et al., 1994) and Puntius gonionotus(Koedprang & Na-Nakorn, 2000). In contrast, trip-loidy increased the number of females in Oreochromisaureus (Mol et al., 1994), catfish (Goudie et al., 1995)and hybrids of Lepomis spp. (Wills, Sheehan & Allen,2000). In the sea bass, the number of females was 10%higher in triploids than in diploids. The sex ratio intriploids was 2 : 1 (male:female) in comparison to theproportion of sexes found in captive diploid sea bass(3 : 1) in our experiment (Felip et al., 2001a). Accord-ing to Benfey (1999), the abnormalities observed insex proportions in triploids can be due to a differen-tial mortality or the effects of environmental factors.Nevertheless, it remains to be determined if a doublematernal genomic contribution or an extra copy of oneof the sex chromosomes in triploids may influence theexpression of sex determining genes, thus possibly ex-plaining the alterations observed in the sex ratios ofthese fish.

The sex ratios of triploids (and particularly ofgynogenetics) can contribute to elucidate the sex de-termination mechanism of a species (Thorgaard, 1983;Ihssen et al., 1990). Thus, in species with femalehomogamety, the crosses involving XX females withXY males followed by triploidy induction result in asimilar percentage of both sexes, that is, 50% XXXtriploid females and 50% XXY triploid males. Thiswas demonstrated in catfish (Goudie et al., 1995)and Nile tilapia (Hussain et al., 1995). Gynogenesisinduction in these species corroborated a XX-XY sys-tem (Mair et al., 1991a; Goudie et al., 1995). Inthe sea bass, results obtained so far discard an XX-XY system. Firstly, because the sex ratio in triploidsdeviates from a proportion of 1 : 1 and, secondly, be-cause offspring of gynogenetics are of both sexes(Felip, 2000). Meanwhile, in species with male ho-mogamety (ZZ-ZW system), sex ratios in offspringcan be variable depending on the crossing-over, whichcan result in an equal number of males and femalesor an increased number of females (Thorgaard, 1983).In those species with polygenic or environmentalsex determination, the sex ratio will be substantiallyaltered in offspring originating from the same breedersdepending on the balance of sex-determining genesand the influence of environmental factors, respect-ively (Thorgaard, 1983; Yamazaki, 1983; Piferrer,2001).

Performance of gynogenetic diploid fish

Survival and growth

Most studies on gynogenetic diploids stop when thesefish are still quite young probably due to their lowersurvival (Table 4). Thus, information concerningtheir growth performance is available for only a fewspecies. In mud loach (Suzuki, Oshiro & Kakan-ishi, 1985) and Atlantic salmon (Johnstone & Stet,1995), gynogenetics grew less than their correspond-ing controls. In contrast, in Clarias macrocephalus(Na-Nakorn, 1995) and Gnathopogon caerulescens(Fujioka, 1998) gynogenetics had growth rates sim-ilar to those of controls. Comparable growth betweengynogenetics and diploids was found in the two mar-ine species examined so far: Red Sea bream (Sugamaet al., 1990) and sea bass (Felip, 2000). In these spe-cies gynogenetic fish had a survival of 30–35% withrespect to that of the controls one day post-hatching(Felip et al., 1999a, 2000a).

Gonadal development

Gynogenetic females of many species exhibit ovariescontaining undeveloped oocytes or areas filled withconnective tissue. This is likely due to the expressionof deleterious recessive alleles affecting the normalcourse of key reproductive processes (Piferrer, Benfey& Donaldson, 1994; Fujioka, 1998). These alterationshave been demonstrated in carp (Nagy et al., 1978;Komen et al., 1992), coho salmon (Piferrer, Benfey& Donaldson, 1994) and Gnathopogon caerulescens(Fujioka, 1998). In contrast, gynogenetic femalesin mud loach (Suzuki, Oshiro & Nakanishi, 1985)and Clarias macrocephalus (Na-Nakorn, 1995) haveshown normal ovaries and fertility. In the Europeansea bass, histological analysis revealed normal devel-opment of ovaries and testes during the first 2 yearsof age. Furthermore, the fertility and quality of thegametes of these fish was not affected by gynogenesis(Felip, 2000).

The induction of gynogenesis has also been car-ried out for monosex female production (Thorgaard,1983; Price, 1984; Ihssen et al., 1990). To overcometheir low survival, gynogenetic sex-reversed XX fe-males (i.e., phenotypic males) may be produced andcrossed with normal females to produce 100% femaleswith a higher viability (Ihssen et al., 1990; Pandian &Varadaraj, 1990). Crosses between these sex-reversedgynogenetic fish and normal females have been per-formed in several species including Oreochromis

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Table 9. Gynogenesis induction: percentage of females in offspring and sex determining system

Environment Speciesa References Females (%) Sex determination

Marine species bParalichthys olivaceus Tabata (1991) 97.1 XX-XYm

See Yamamoto, 1999 48–93 XX-XYm

cDicentrarchus labrax Felip (2001b) 50 UnknowndSolea solea Howell, Baynes and Thompson (1995) 40 Unknown

Freshwater species eOreochromis mossambicus Pandian and Varadaraj (1990) 100 XX-XYeOreochromis niloticus Mair et al. (1991a) 100 XX-XYfCtenopharyngodon idella Stanley (1976) 100 XX-XYfCyprinus carpio Nagy et al. (1978) 100 XX-XYfCarassius auratus Oshiro (1987) 0–100 XX-XYm

fPuntius gonionotus Pongthana et al. (1995) 100 XX-XYfGnathopogon caerulescens Fujioka (1998) 87.2 XX-XYm

gIctalurus punctatus Goudie et al. (1995) 100 XX-XYhOncorhynchus kisutch Refstie, Stoss and Donaldson (1982) 49.3–100 XX-XYm

hSalmo salar Johnstone and Stet (1995) 100 XX-XYiClarias macrocephalus Na-Nakorn (1995) 100 XX-XYiClarias gariepinus Galbusera, Volckaert and Ollevier (2000) 100 XX-XYjMisgurnus anguillicaudatus Suzuki, Oshiro and Nakanishi (1985) 94.9 XX-XYm

kTakifugu rubripes See Arai (2001) 77.3–100 XX-XYm

lLimanda yokohamae See Arai (2001) 100 XX-XY

aFamily: bParalichthyidae, cMoronidae, dSoleidae, eCichlidae, fCyprinidae, gIctaluridae, hSalmonidae, iClariidae, jCobitidae,kTetraodontidae, lPleuronectidae. mUnexpected presence of males in species with female homogamety.

mossambicus (Pandian & Varadaraj, 1990), O. niloti-cus (Mair et al., 1991a), O. aureus (Mair et al., 1991b)and Puntius gonionotus (Pongthana et al., 1999)giving all-female progenies.

Proportion of sexes

In species with female homogamety (system XX-XY),the induction of gynogenesis should result in mono-sex female production (Table 9). This has been usedto assess female homogamety in fish, although it canalso be determined by integrating gynogenesis induc-tion with endocrine sex reversal (Hunter & Donaldson,1983; Ihssen et al., 1990; Piferrer, 2001). How-ever, one aspect that needs to be mentioned is that insome species in which the XX-XY scheme operates,a variable proportion of gynogenetic males has beenobserved in the progeny (Table 9). In the sea bass,experiments performed to determine sex ratios in off-spring of sex-reversed females (neomales) (Blázquezet al., 1999) as well as a study to identify the sex ofartificially-induced gynogens (Felip, 2000) have beenunable to conclusively assign any particular type ofsex determining system for this species. Furthermore,Blázquez et al. (1998) and Pavlidis et al. (2000) haveshown that the incubation temperature affects the sex

ratio. In species with male homogamety (system ZZ-ZW), gynogenesis induction renders progenies of bothsexes where ZZ is expected to give males and ZW orWW to give females. In this regard, the proportionof females depends on the viability of WW females(super-females) (Bull, 1983; Yamazaki, 1983). Theproduction of a second generation of gynogenetics us-ing potential viable WW females would result in anall-female progeny.

Prospects on triploid and gynogenetic fish

Besides their potential application in aquaculture, trip-loid and gynogenetic fish possess many traits thatrender them attractive for basic research. Triploid fishcan be used in studies dealing with the allocationof energy between growth and reproduction (i.e., therelationship between somatotropic and gonadotropicaxes) or the physiology and mechanism of fat storageand mobilization (McLean et al., 1991, 1993; Sump-ter et al., 1991; Mol et al., 1994). By virtue of theirsterility, triploids can be used as a negative control instudies examining the steroid activation of the brain-pituitary-gonad axis (Benfey et al., 1989; Breton &Sambroni, 1996, Benfey, 1999). In addition, triploids

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can be used as models for cytogenetic studies focusingon meiotic chromosome behaviour (Gui et al., 1992;Carrasco et al., 1998) or in studying the relationshipbetween disruption of gonadal function and the genesinvolved in gametogenesis, as it occurs in transgenicanimals (Reventós & Munell, 1997).

On the other hand, gynogenetic fish can be anadvantage for genetic analysis and chromosome map-ping. Gynogenesis results in a very efficient methodfor rapid production of isogenic lines as well as inbredlines (Thorgaard, 1983; Yamazaki 1983; Ihssen et al.,1990). Thus, the production of homozygotes facilit-ates the determination of the genetic parameters of anindividual or population for development of geneticmaps and markers (Nagy & Csányi, 1982; Gervai &Csányi, 1984; Streisinger et al., 1986; Liu, Zhanzhou& Wang, 1996) and the estimation of heterozigosity(Allendorf & Leary, 1984). Recently, a detailed link-age map was reported in three teleost fish (Kocheret al., 1998; Young et al., 1998; Liu et al., 1999a,b; Agresti et al., 2000). Linkages between markerscan be associated with genes in order to carry outcomparative studies of genomic evolution and gen-ome conservation among fish as well as quantitativetrait locus (QTL) analysis (Danzmann, Jackson & Fer-guson, 1999; Sakamoto et al., 1999) or marker-basedselection in the commercial fish species (Ihssen et al.,1990; Young et al., 1998). Another application ofmeiotic gynogenetic diploids is their use to discrim-inate genetic and environmental effects on traits andphysiological aspects while mitotic gynogenetic dip-loids are useful to study single-locus effects (Ihssenet al., 1990). Finally, integration of chromosome setmanipulation can be useful in transgenic technologiesfor gene transfer (Peek et al., 1997).

Acknowledgements

We thank Drs. S. Abe and K. Arai for giving usthe opportunity to write this review, and Drs. M.Blázquez, L.A. Sorbera and two anonymous reviewersfor helpful comments of the manuscript.

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