Endocrine sex control strategies for the feminization of teleost fish

Transcription

1 Ž. Aquaculture Endocrine sex control strategies for the feminization of teleost fish Francesc Piferrer ) Institut de Ciencies ` del Mar, Consejo Superior de InÕestigaciones Cientıficas CSIC, Passeig Joan de Borbo, srn, Barcelona, Spain Received 10 January 000; accepted 31 December 000 Abstract In many species of cultured finfish, females exhibit higher growth rates than males and attain larger sizes. In addition, in some species, males mature before reaching marketable size. Together, this results in a larger dispersion of sizes and an overall reduction in production. Therefore, there is great interest from the private sector to produce all-female stocks. This review concentrates on the use of oestrogens for sex control, discussing the advantages of producing monosex female stocks for finfish aquaculture, and pointing out those cases in which hormonal sex reversal technology is worth applying. The biological basis on which hormonal sex manipulation rests, the process of sex differentiation which, compared to that of other vertebrates, is quite labile in teleost fish is described in order to understand the effects of treatments. Sex control is typically achieved by exposing sexually undifferentiated fish to exogenous steroids in order to direct the process of sex differentiation towards the desired sex. These treatments finish months or years before marketing and steroid residues disappear in less than a month after the end of treatment. The currently available methods to produce monosex female stocks, the direct and the indirect methods, are explained, comparing their respective advantages and disadvantages. Feminizing treatments are also used to produce all-male stocks in some species. Thus, this review concentrates on the use of oestrogens for sex control, either in the direct method of feminization or in the indirect method of masculinization. So far, oestrogens have been applied to at least 56 different species, using 1 different oestrogenic substances Ž three natural and nine synthetic.. Special attention is given to the method of administration, including immersion and dietary treatment, and to the variables of the hormonal treatment itself: steroids used, dose, timing and duration of treatments. The importance of correct treatment timing in relation to the degree of gonadal development is emphasized and the outcome of the treatment evaluated in terms of survival, ) Tel.: q ext. 35; fax: q Ž. address: piferrer@icm.csic.es F. Piferrer r01r$ - see front matter q 001 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 30 F. PiferrerrAquaculture gonadal morphology and sex ratios, growth performance and deformities. Next, the current methods to produce all-female or essentially all-female stocks are presented for 35 different species, including eels, salmonids, cyprinids, poecilids, cichlids, gouramies and flatfishes. A section on regulatory issues discusses the advantages of using the indirect method of feminization, when feasible, and emphasizes the convenience of using the natural oestrogen estradiol-17b rather than synthetic oestrogens. A guideline for the development of monosex technology in new species is presented. The overall goal is to emphasize the use of the indirect method, which means that fish that reach the marketplace have never been exposed to steroids. If this method is not feasible, as it happens in many species, an alternative is the use of the direct method, applied in an optimized protocol, to achieve maximum treatment efficiency with minimum exposure to steroids. q 001 Elsevier Science B.V. All rights reserved. Keywords: Sex control; Feminization; All-female; Oestrogens; Oestradiol-17b; 17a-ethynyloestradiol; Endocrine disruptors; Sex differentiation; Fish; Teleosts 1. Introduction With the demonstration that the gonadal sex of fish could be influenced by the administration of hormones in the late 1930s and early 1940s, and with the enormous expansion of fish culture in the past 0 years, it soon became apparent that it would be desirable to enhance the expression of the sex with associated morphological, physiological or etiological characteristics that could be advantageous under certain culture strategies Ž Schreck, 1974; Hunter and Donaldson, 1983; Yamazaki, Globally, the objectives that can be achieved with hormonal sex control in fish are: Ž. a to reduce the number of broodstock necessary to obtain a given egg take or, conversely, to increase egg take by rearing mainly females, Ž. b to favor the sex that shows the greatest growth Ž for example, females in salmonids and cyprinids, and males in cichlids., Ž c. to prevent precocious sexual maturation in males and Ž. d to prevent sexual maturation in both sexes, i.e., by sterilization. Sterilization will in turn Ž. 1 permit the marketing of larger fish as it eliminates carcass weight loss associated with gonadal maturation, Ž. allow marketing fish during the entire year, beyond the maturation season, and Ž. 3 maintain optimal quality of flesh. Sex control techniques can also be used in fisheries management, i.e., sea ranching, for example in the production of female and sterile salmon for release into the natural environment Ž Donaldson and Hunter, 198. or in ornamental fish culture Ž Piferrer and Lim, The ultimate goal of sex control in fish is to increase the profitability of the aquaculture operations. Sex control strategies can be coupled to other recently developed biotechnologies aimed at enhancing production ŽDonaldson, 1996; Donaldson and Devlin, 1996; Penman, The actual economic advantage of producing monosex stocks in aquaculture has been evaluated in the case of Pacific salmon Ž Solar and Donaldson, This review focuses on the endocrine Ž hormonal. feminization of teleost fish. It also includes cases where oestrogens are used for the indirect masculinization of fish. Nevertheless, most of the cited literature relates to the direct feminization of teleosts through the use of oestrogens. The use of oestrogens for the production of monosex female stocks in finfish aquaculture has increased during the last years. Table 1

3 F. PiferrerrAquaculture summarizes the species of teleosts that have been treated with natural or synthetic oestrogens for purposes of controlled sex differentiation, citing the published literature to date Ž Most of the published research relates to the deliberate control of sex ratios in fish, but some of it deals with the effects of exogenous steroids on sex differentiation regardless of the relevance for aquaculture. Research that involves the effects of oestrogens on other aspects such as growth, smoltification or gonadal steroidogenesis has not been included. Other possible forms of sex control in fish such as interspecific crosses, to produce all-male stocks Ž Beardmore et al., 001., or chromosome set manipulation Ž Arai, 001. are covered in other reviews in this volume. Likewise, the use of oestrogens for purposes of controlled sex differentiation in fish other than teleosts is very limited. However, some examples of oestrogen treatments in agnathans, e.g., the river lamprey Ž Lampetra fluõiatilis., and chondrostei, e.g., the sterlet sturgeon Ž Acipenser ruthenus., can be found in Olesen-Larsen Ž and Akhundov and Fedorov Ž 1994., respectively. In all, oestrogens have been applied for purposes of controlled sex differentiation to at least 56 different species of teleosts belonging to 4 different families. These families, in turn, belong to 1 out of the 38 orders of extant teleosts Ž Nelson, Considering that there are more than 5,000 identified species of teleosts, these figures should be kept in mind when attempting generalizations. Classified according to their habitat andror life cycle, the species of teleosts treated with oestrogens are 64% freshwater, 1% marine, 11% anadromous and 4% catadromous. According to their type of sex, most treated species are gonochoristic Ž 91%. and the rest hermaphrodites, usually protandrous in order to promote sex change towards the female sex. There is no point in feminizing unisexual Ž all-female. species, so there are no reports on the use of oestrogen in such species. Previous reviews on hormonal sex control in fish Ž masculinization, feminization and sterilization. include those of Schreck Ž 1974., Hunter and Donaldson Ž 1983., Yamazaki Ž 1983., Billard Ž 1989., Dunham Ž 1990., Pandian and Sheela Ž and Patino Ž Sex in fish A detailed discussion of sex in fish is beyond the scope of this review. Therefore, only a brief summary is given to provide the essential background needed to understand the biological basis on which endocrine sex control rests. The types of reproduction that fish exhibit are classified as: Ž. 1 gonochorism or the existence of separate sexes, Ž. hermaphroditism Ž both sexes present in the same individual., and Ž 3. unisexuality Ž all-female species. Ž Chourrout, 1988; Price, Mechanisms for the expression of sex The expression or manifestation of sex depends on two processes: sex determination and sex differentiation. Sex determination is responsible for the genetic Žalso called genotypic. sex, whereas sex differentiation is responsible of the development of distinct types of gonads, testis or ovary Ž the gonadal or phenotypic sex.. Together, these

4 Table 1 Species of teleost fish that have been treated with natural or synthetic oestrogens for purposes of controlled sex differentiation Ž b c d Family Species Common name Oestrogens Route Refs. Anguillidae Anguilla anguilla European eel EE Diet Colombo and Grandi Ž 1990, E Diet Degani and Kushnirov Ž 199., Andersen et al. Ž A. japonica Japanese eel DES Diet Satoh et al. Ž 199. E Diet Chiba et al. Ž Cyprinidae Cyprinus carpio Common carp E Injection Castelnuovo Ž E Diet Sathyanarayana Rao and Satyanarayana Rao Ž 1983., Komen et al. Ž DES Diet Basavaraja et al. Ž 1989, E, E Diet Sehgal and Saxena Ž 1997a,b. 1 Carassius auratus Goldfish E, E Diet Yamamoto and Kajishima Ž E Diet Yamamoto Ž 1975b. 1 Pimephales promelas Fathead minnow DES Diet Schreck Ž E Immersion Miles Ž 1997., Kramer et al. Ž 1998., Miles-Richardson et al. Ž Cobitidae Misgurnus anguillicaudatus Mud loach E? Kubota and Hatakeyama Ž , Kubota et al. Ž M. mizolepis Mud loach E Immersion Kim et al. Ž Ictaluridae Ictalurus punctatus Catfish E Diet Goudie et al. Ž 1983., Gannam and Lovell Ž Clariidae Clarias lazera Airbreathing catfish E Diet Liu et al. Ž Salmonidae Oncorhynchus mykiss Rainbow trout E Immersion Padoa Ž 1937, 1939a. 1 E, E Diet Okada Ž 1973, E Diet Jalabert et al. Ž E Immersion Simpson Ž 1976., andror diet Johnstone et al. Ž 1978., Bye and Lincoln Ž 1986., Goryczko et al. Ž DES, E Diet Sower et al. Ž E, E, EE, Hexestrol Diet Chevassus et al. Ž a 3 F. PiferrerrAquaculture 197 ( 001 ) 9 81

5 Salmonidae Salmo trutta Brown trout E Immersion Ashby Ž Estrogenic compound Immersion Poston Ž SalÕelinus namaycush Lake trout E Diet Wenstrom Ž 1975., Herman and Kincaid Ž S. fontinalis Brook trout E Diet andror immersion Johnstone et al. Ž 1979b., Parks and Parks Ž Salmo salar Atlantic salmon E Immersion andror diet Simpson Ž 1976., Johnstone et al. Ž 1978., Herman and Kincaid Ž Oncorhynchus kisutch Coho salmon DES, E Diet Sower et al. Ž E Immersionq diet Goetz et al. Ž 1979., Donaldson and Hunter Ž 198., Hunter et al. Ž 198. E Immersion Hunter et al. Ž 1986., Piferrer and Donaldson Ž 1989, 1994., Son Ž 1991., Foyle Ž 1993., Piferrer et al. Ž 1994b. E Diet Redding et al. Ž O. tshawytscha Chinook salmon DES Diet Schreck and Fowler Ž E Immersionqdiet Donaldson and Hunter Ž 198., Wertheimer and Barnum Ž E Immersion Hunter et al. Ž E, EE Immersion Piferrer and Donaldson Ž 199. E, EE, ME Immersion Solar et al. Ž O. masou Masu salmon E Immersion Nakamura Ž 1978, 1981, 1984., Nakamura and Takahashi Ž O. gorbuscha Pink salmon E Immersionq diet Donaldson and Hunter Ž 198. O. keta Chum salmon E Immersion Nakamura Ž E Diet Redding et al. Ž Mugilidae Mugil cephalus Grey mullet E Diet Chang et al. Ž 1995c. Atherinidae Odontesthes bonariensis Pejerrey E Diet Strussmann et al. Ž continued on next page F. PiferrerrAquaculture 197 ( 001 )

6 Table 1 Ž continued. b c d Family Species Common name Oestrogens Route Refs. Adrianichthyidae Oryzias latipes Medaka DES, E Diet Yamamoto Ž , 1959a,b, 1961, 1963., Onitake Ž 197. E Immersion Egami Ž DES, E Diet Yamamoto and Matsuda Ž 1963., Hishida Ž E Diet Yamamoto Ž EE, Euvestin, Diet Yamamoto Ž Hexestrol E Diet Fineman et al. Ž 1974, EE Injection Papoulias et al. Ž 000. Poeciliidae Poecilia reticulata Guppy E, E Diet Berkowitz Ž 1937, 1938, , Miyamori Ž EB, EP Injection Berkowitz Ž E Immersion Querner Ž E Diet Haskins et al. Ž 1970., Takahashi Ž DES, E, EE, EB Diet Kavumpurath and Pandian Ž 199a, 1993a. Gambusia holbrokii Mosquitofish E Diet Lepori Ž Platypoecilus Õariatus E Immersion Querner Ž Xiphophorus helleri Swordtail E Immersion Querner Ž E Diet Lim et al. Ž 199. Poecilia sphenops Black molly DES, E Diet George and Pandian Ž Synbranchidae Monopterus albus Ricefield eel E, EB Injection Tang et al. Ž 1974., Tao et al. Ž Cyclopteridae Cyclopterus lumpus Lumpfish E Diet Ž through Artemia. Martin-Robichaud et al. Ž andror immersion Moronidae Dicentrarchus labrax Sea bass E, EE Diet Blazquez et al. Ž Serranidae Serranus hepatus Sea bass E Immersion Padoa Ž 1939b. 1 Centrarchidae Micropterus salmoides Largemouth bass E, E Diet Ž through Artemia. Garrett Ž DES, E Diet Al-Ablani Ž F. PiferrerrAquaculture 197 ( 001 ) 9 81

7 Centrarchidae Pomoxis nigromaculatus Black crappie DES, E Diet Al-Ablani Ž Lepomis macrochirus Bluegill DES, E Diet Al-Ablani Ž Percidae Perca flaõescens Yellow perch E Diet Malison et al. Ž 1986., Malison and Garcia-Abiado Ž Sparidae Mylio macrocephalus Black sea bream?? Hibiya Ž 197. Acanthopagrus schlegeli Black porgy E Diet Chang and Lin Ž 1998., Chang et al. Ž 1994, 1995a,b. Sparus auratus Sea bream E, EE Diet Condeça and Canario Ž Cichlidae Oreochromis aureus Tilapia aurea AFemale hormoneb Immersionq diet Yashouv and Eckstein Ž DES, DES-DP Immersion Eckstein and Spira Ž E, E, E Diet Jensen Ž DES, E, EE Diet Hopkins Ž 1977., Hopkins et al. Ž EE Diet Kittler Ž 1986., Melard Ž E, EE Diet Mair et al. Ž DES, EE Diet Rosenstein and Hulata Ž Hemihaplochromis EBA Immersion Muller Ž 1969., Hackmann Ž 1971., multicolor Hackmann and Reinboth Ž Oreochromis mossambicus Tilapia mossambica EE Diet Nakamura and Takahashi Ž E Diet Guerrero and Guerrero Ž DES Diet Varadaraj Ž 1989., Basavaraja et al. Ž DES, E, EE Diet Varadaraj Ž E Immersion Rosenstein and Hulata Ž 199. DES, EE Diet Rosenstein and Hulata Ž Oreochromis niloticus Tilapia nilotica EE Diet Nakamura and Takahashi Ž 1973., Yoshikawa and Oguri Ž DES, E Diet Tayamen and Shelton Ž E Diet Chipunga Ž DES, EE Diet Potts and Phelps Ž DES, E, EE Immersion Gilling et al. Ž DES Diet Abucay and Mair Ž 1997., Tuan et al. Ž continued on next page F. PiferrerrAquaculture 197 ( 001 )

8 36 Table 1 Ž continued. b c d Family Species Common name Oestrogens Route Refs. Tilapia zillii EE Diet Yoshikawa and Oguri Ž O. honrorum EE Diet Obi and Shelton Ž Sarotherodon galilaeus E Diet Chipunga Ž O. spilurus EE Diet Ridha and Lone Ž O. niloticus= Tilapia hybrid E Diet Chipunga Ž O. macrochair O. mossambicus= All-male tilapia E Immersion Rosenstein and Hulata Ž 199. O. urolepis hornorum hybrid Cichlasoma nigrofasciatum Zebra cichlid E Diet George and Pandian Ž Labridae Halichoeres poecilopterus Wrasse EB Injection Okada Ž Zoarcidae Zoarces ÕiÕiparus Eelpout E Christiansen et al. Ž Belontiidae Betta splendens Fighting fish E Diet Kavumpurath and Pandian Ž 199b, 1993b. DES, E, EE Diet George et al. Ž Trichogaster pectoralis Snakeskin gourami DES, E, EE Immersion or diet Pongthana et al. Ž Scophthalmidae Scophthalmus maximus Turbot E Diet Piferrer and Fernandez Ž unpublished. Paralichthyidae Paralichthys oliõaceus Olive flounder E Immersion or diet Tanaka Ž 1988., Bang et al. Ž Pleuronectidae Verasper moseri Barfin flounder E Diet Mori et al. Ž a Research that involves effects of oestrogens on other aspects such as growth, smoltification, gonadal steroidogenesis, etc. is not included. b Systematic classification according to Nelson Ž Note that Oryzias latipes Ž formerly in Family Cyprinodontidae. is now classified as a member of the F. Adrianichthyidae; that Betta splendens Ž formerly in F. Anabantidae. is now in F. Belontiidae; and the genus Platypoeciluss Xiphophorus; the genus Mylios Acanthopagrus; and Serranus hepatuss Hepatus hepatus. c Common name as provided by authors. d Abbreviations used Ž nomenclature of chemical derivatives as in: Anonymous, 1996.: DES, diethylstilbestrol; DES-DP, diethylstilbestrol diphosphate; E 1, oestrone; E, oestradiol-17b; E 3, oestriol; EB, oestradiol benzoate; EBA, oestradiol butyryl acetate; EE, 17a-ethynyloestradiol; EP, oestradiol propionate; Euvestin, diethylstilbestrol dipropionate; Hexestrol, dihydrodiethylstilbestrol; ME, 14,15-methylene oestradiol. F. PiferrerrAquaculture 197 ( 001 ) 9 81

9 F. PiferrerrAquaculture processes account for the existence of the two possible morphological, functional and behavioral phenotypes, male or female. Usually a female genotype gives rise to a female phenotype and a male genotype to a male phenotype Žhermaphrodites are another matter.. However, as pointed out by Adkins-Regan Ž 1987., in some animals, particularly in lower vertebrates, sex differentiation can be so easily disturbed by environmental factors that the resulting phenotypic sex can differ from genetic sex. This sexual lability, along with the diversity of mechanisms of sex determination and differentiation found particularly in fish, may account for the lack of unifying theories regarding sex determination and differentiation for vertebrates as a whole ŽHunter and Donaldson, 1983., i.e., there is no known universal mechanism of sex determination and differentiation that can be applied to all fishes although these mechanisms are better characterized in other groups of vertebrates. Recent reviews on sex determination and differentiation in fish are those of Nakamura et al. Ž and Baroiller et al. Ž Sex determination Here sex determination is defined as the sum of genetic elements that are responsible for the existence of the gonads, i.e., the set of genes responsible for the formation of gonads, in a similar way that another set of genes determines the existence of, say, a pair of kidneys. The sex determining genes will not only be responsible for the presence of the gonads but also for their shape Ž fused organ or paired organ., whether they will differentiate as testes or ovaries, whether or not the ovaries will have an ovarian cavity Ž not all ovaries have an ovarian cavity., etc. The sex determining genes can be spread throughout the genome or mostly concentrated in a pair of chromosomes, in the latter case called sex chromosomes. In any case, the genetic sex of an individual will depend on the set of sex determining genes inherited from both parents. The genetic sex of future fish can then be manipulated by deciding what chromosomes will go into the zygote, which is the basis of genetic sex control or chromosome set manipulation Žsee Section Basically, there are three models for sex determination that can be applied to fish: chromosomal, polygenic and genotype environment interaction sex determination. Chromosomal inheritance relies on the presence of sex chromosomes, where a pair of chromosomes, called heterochromosomes, have accumulated most of the genes responsible for sexual development Ž Bull, 1983; Tave, However, the existence of a single gene responsible for the development of one sex, like Sry is responsible for male differentiation in mammals, has not yet been found in fish Ž Nagahama, Fish normally do not present heterochromosomes ŽVorontsov, 1973; Yamazaki, 1983; Price, 1984; Tave, 1993; Solari, but so far eight chromosomal systems have been proposed based mostly on cytogenetic analyses and data from deliberate crosses. These range from simple systems, such as XXrXY or WZrZZ, the most common in fish, to more complex systems involving more than one pair of sex chromosomes or different numbers of chromosomes depending on the sex Ž Tave, Most species of teleosts of commercial interest produce male:female ratios not significantly different from 1:1 regardless of the broodstock used and environmental conditions, which suggests that they possess either a XXrXY or a ZWrZZ chromosomal system of femalermale sex determination. The sex in which both sex chromosomes are the same is called homoga-

10 38 F. PiferrerrAquaculture metic Ž females in the XXrXY., whereas the sex in which sex chromosomes are different is called heterogametic Ž males in the XXrXY system.. Polygenic Ž also called polyfactorial. sex determination is a system of sex determination where epistatic Ž superior. male and female sex-determining genes are present in the other chromosomes Ž autosomes. as well as in the heterochromosomes ŽWinge, 1934; Hunter and Donaldson, 1983; Price, 1984; Kallman, 1984; Chourrout, The sex of the embryo will thus be the result of the combined relative strength of the male and female factors present in the chromosome sets inherited from each parent. Some variations of these systems have been proposed. Thus, Avtalion and Don Ž proposed the existence of three genotypes Ž WW and WY for females and YY for males. and genetic recombination between sex-determining genes and the centromere to explain the sex ratios observed after three generations of induced gynogenesis in tilapia Ž Oreochromis aureus.. Fish with polygenic sex determination are characterized by sex ratios different from the 1:1 male:female sex ratio typical of species with a pure chromosomal sex determination system. In addition, sex ratios may differ in successive broods originating from the same parents. Finally, environmental sex determination Ž ESD. involves genotype environment interactions Ž however see Section.1... There is one clear case reported among fish, the atherinid Atlantic silverside Menidia menidia, where sex determination is under both genetic and environmental control, depending on the incubation temperature during a critical phase of the larval development ŽConover and Fleisher, 1986; Conover and Heins, 1987a,b., as observed in some reptiles ŽBull, In recent years, several studies have shown that sex in fish can be under the influence of the environment temperature in which they develop. This fact is becoming apparent for an increasing number of species Ž see Baroiller et al., 1999 for review., thus opening the possibility for effective sex control by environmental manipulation..1.. Sex differentiation Sex differentiation relates to the events that occur during development and allow the expression of the genetic sex into the appropriate phenotypic sex. Sex differentiation encompasses all the events that take place in the primordial gonad, including the migration of primordial germ cells Ž PGCs., the establishment of gonadal ridges and the differentiation of the gonads proper into testes or ovaries Ž Brusle and Brulse, Sex differentiation occurs first in females and later in males and is first observed by histological examination of the gonads rather than by any external changes. The earliest signs of female sex differentiation are the entry of oogonia into meiosis andror the proliferation of somatic cells to form the ovarian cavity ŽBrusle and Brulse, 1983; Nakamura et al., In contrast, male sex differentiation occurs later and is characterized by the appearance of spermatogonia, the arrangement of germ cells and somatic cells in lobules and the differentiation of the vascular system of the testis, including the testicular vein, the testicular artery and the sperm ducts. In some species, such as those of the genus Oreochromis, anatomical Ž also called morphological. differentiation of the gonads Že.g., an observable proliferation of somatic cells to begin the formation of the ovarian cavity. precedes cytological differentiation Že.g., the entry of oogonia into meiosis to become primary oocytes.žnakamura and Takahashi, 1973;

11 F. PiferrerrAquaculture Nagahama, In other species such as the medaka, Oryzias latipes, the opposite is true, i.e., cytological differentiation precedes anatomical differentiation ŽSatoh and Egami, Sex differentiation in fish can follow two different pathways. In the first case, the gonadal primordium directly differentiates into either an ovary or a testis. The species that exhibit this pathway are called differentiated species. In the second case, all the animals first differentiate into an ovary-like gonad. Later, approximately half of the fish stop what appears to be female differentiation and testicular differentiation takes over. The species that follow this pathway are referred to as undifferentiated species Ž Yamamoto, Examples of differentiated species include the medaka ŽYamamoto, 1953., the coho salmon, Oncorhynchus kisutch Ž Piferrer and Donaldson, 1989., the common carp, Cyprinus carpio Ž Komen et al., 199. and the European sea bass, Dicentrarchus labrax Ž Blazquez et al., Examples of undifferentiated species include the guppy, Poecilia reticulata Ž Yamamoto, 1969., the hagfish, Eptatretus stouti Ž Gorbman, and the European eel, Anguilla anguilla ŽColombo and Grandi, 1990; The nature of the inducerž. s of sex differentiation is of paramount importance and until recently there was no agreement as to the model that would satisfactorily explain the process of sex differentiation in nonmammalian vertebrates Žsee Hunter and Donaldson, 1983; Adkins-Regan, 1987; Nakamura et al., 1998, for reviews.. Yamamoto Ž 1969., after numerous experiments with the medaka, obtained evidence to support the theory of sex steroids as natural sex inducers, although he was aware of the criticisms of this theory from other colleagues. Based on his own experience of: Ž. 1 the specificity of androgens as masculinizing hormones and oestrogens as feminizing hormones Žhe did not observe paradoxical effects of steroids Ž see below., and Ž. the small doses required for effective sex reversal together with evidence for the selective incorporation of some steroids by developing gonads, provided by Hishida Ž 196, 1965., he was inclined to believe that the sex steroids were in fact the natural sex inducers, although he did not make a categorical statement. Six years later Ž Yamamoto, 1975a. he stated that: A... whether or not natural sex inductors are oestrogens and androgens or allied substances or entirely different other substances is still to be elucidatedb. This equivocal attitude towards sex steroids was probably due to the fact that from 1969 to 1975 some papers appeared, e.g., Satoh Ž and Satoh and Egami Ž 197., which demonstrated that in the medaka the steroid producing cells are only clearly observed after sexual differentiation has taken place. Thus, this observation is difficult to reconcile with the view supporting sex steroids as the natural sex inducers. Later, a Dutch laboratory published a series of papers showing that substances other than steroids Žsee Section below. could also influence the normal course of gonadal development ŽVan den Hurk et al., 1980; Van den Hurk and Slof, 1981; Van den Hurk and Lambert, 198; Van den Hurk and Leeman, 1984; Van den Hurk and Van Oordt, Therefore, sex steroids appeared not to be as specific as sex-reversing substances as initially thought. Steroidogenesis, however, has been demonstrated at very early stages of development before or coinciding with sex differentiation in the rainbow trout, O. mykiss ŽVan den Hurk et al., 198; Antilla, 1984; Fitzpatrick et al., 1993; Feist and Schreck, 1996., coho salmon, O. kisutch Ž Fitzpatrick et al., 1988; Feist et al., and

12 40 F. PiferrerrAquaculture Nile tilapia, Oreochromis niloticus Ž Hines et al., Recently, research in this area has focused on the function of key steroidogenic enzymes during the process of sex differentiation in fish and other vertebrates. In this regard, it was shown that treatment with a specific aromatase inhibitor caused genetic female salmon to develop into functional males Ž Piferrer et al., 1994a.. After a series of experiments involving aromatase gene expression during sex differentiation Ž Guiguen et al., 1999., or the immunolocalization of several key steroidogenic enzymes Ž Nakamura et al., 1998., there is now strong evidence indicating that endogenous oestrogens are responsible for ovarian differentiation and that aromatase plays a central role in this process Žsee Gonzalez and Piferrer, 000; Nagahama, 000, for reviews.. Together, these new findings are the best evidence obtained so far to support the idea that sex steroids are in fact natural inducers of sex differentiation in fish. This does not necessarily imply that they are responsible for it, since there is also evidence that suggests that sex steroids are involved downstream in the cascade of events resulting in sex differentiation ŽSolari, In any case, there is no doubt that sex steroids and their actions are involved in the process of sex differentiation in fish. Sex steroids act through specific receptors in target cells. Androgen and oestrogen receptors have been characterized in fish ŽFitzpatrick et al., 1994; Chang et al., 1999; Todo et al., with similar characteristics of the androgen and oestrogen receptor of other vertebrates Ž see Thomas, 000, for review.. During sex differentiation sex steroids act mainly as morphogenic factors. However, later in the life cycle, during sexual maturation, they act mainly as activational factors. These are the two classically recognized functions of sex steroids in development Ž Adkins-Regan, Recent research suggests that the effects of environmental factors on the resulting phenotype are exerted by modifying aromatase enzyme activity Ž Pieau et al In any case, it seems clear that environmental factors affect the phenotype by interfering in the process of sex differentiation, by promoting or inhibiting the expression of certain genes, but they are not involved in the process of sex determination. Thus, even in the case of ESD still there is a genetic basis that determines the presence of the sexes. As pointed out by Hayes Ž 1998., Aonly the direction of differentiation Ž the switch. is controlled environmentallyb. Therefore, it seems more appropriate to speak of environmental sex differentiation rather than environmental sex determination, as is customary to do. Thus, I propose to use the term AEnvironmental Sex DifferentiationB to refer to those cases in which the environment influences the process of sex differentiation... Control of sex differentiation Since sex steroids are involved in the natural process of sex differentiation, the basis for controlling this process is the administration of exogenous sex steroids to sexually undifferentiated fish. Thus, by appropriate sex steroid therapy it is possible to alter the normal course of sex differentiation towards the desired phenotype. Although occasionally effects of exogenous sex steroid treatments have been reported to be transitory Ži.e., masculinized or feminized fish regress to their original phenotype.žolito and Brock, 1991; Lim and Wong, 1996., effects are usually permanent in a large number of teleosts.

13 F. PiferrerrAquaculture This implies that the obtained sex is maintained after steroid withdrawal and no further treatments are required to ensure the desired gonadal sex. The manipulation of the mechanisms that control sex differentiation in fish began with the administration of steroid hormones during early development ŽBerkowitz, 1937; Castelnuovo, 1937; Padoa, once oestradiol-17b Ž E., the major oestrogen, had been synthesized and became available for research in However, in those early experiments only intersex fish were produced. Yamamoto Ž first achieved complete sex reversal and later established the criteria for effective steroid treatment ŽYamamoto, Most studies where the sex steroids were administered for the purposes of sex control were based on these criteria, which later became the working principle in hormonal sex control in modern fish farming. Yamamoto Ž 1969., postulated that for effective sex reversal, sex steroids should be given prior to any sign of gonadal differentiation at a dose that is related to both the species treated and the nature of the steroid. Further, he stated that hormone administration should be continued until after the time when normal sex differentiation takes place. This criterion implies the existence of a period when fish gonads are most sensitive to the action of steroids. Sexual differentiation towards male or female would be achieved as a consequence of the effects of androgens or oestrogens, respectively. These steroids mimic the effects of the endogenous sex steroids, thus redirecting gonadal development towards the sex that typically produces the type of hormone administered. Hence, oestrogens are used for feminization purposes and androgens are used either to masculinize or to sterilize, depending on whether they are administered at relatively low or high concentrations, respectively Žsee, however, the paragraph on Aparadoxical feminizationb after androgen treatment in Section Although excessive exposure to oestrogens can in some cases also result in sterilization Ž Eckstein and Spira, 1965; Blazquez et al., 1998., oestrogens are not purposely used to sterilize fish due to their deleterious effects when administered at high doses or for long periods of time Ž see Sections 6.3 and 6.4 below.. 3. Feminization strategies There are essentially two methods for the feminization of fish: hormone therapy and through the induction of gynogenesis. Hormonal feminization by means of sex steroids has reached the point in some species, e.g., salmonids, where it has been tested on a production basis. Hormonal feminization can be achieved in two different ways, through what is called the direct and the indirect methods. It is important to mention that oestrogens are also required for sex control in cases where the ultimate purpose is the production of all-male stocks Ž see Section 3.3 below.. In any case, endocrine Ž hormonal. sex control affects the process of sex differentiation, not sex determination. Hence, administration of sex steroids in species with chromosomal sex determination like the salmon and trout, allows the masculinization or the feminization of fish Žchange of phenotype. without changing their sex chromosome composition Žno change of genotype.. This is, in fact, the basis of the indirect methods of feminization Ž see below. and masculinization Ž not shown..

14 4 F. PiferrerrAquaculture Direct feminization This method can be applied to any species of fish regardless of the sex determination system and regardless of which sex is the homogametic or heterogametic sex, and involves the use of oestrogen treatment during the early stages of development. This method of feminization, which usually includes the use E, a naturally occurring piscine oestrogen, or a synthetic oestrogen Ž or even compounds with oestrogenic activity., produces the desired gonadal sex in the same generation that receives the treatment. Ž Fig. 1A.. This fact, along with its simplicity, are the major advantages of direct feminization and explain why it is frequently used in most species where all-female stocks are sought Ž Table 1.. Furthermore, there is no treatment-associated mortality at the optimal dose and the fish produced are phenotypically indistinguishable from normal females. However, this approach has the inconvenience of treatment success variability. Also, fish treated in this way cannot become part of a breeding program, since half of the females produced are of male genotype. These females, when mated with normal males will produce offspring with altered sex ratios. In addition, there may be some Fig. 1. Endocrine methods to produce all-female stocks. Diagram showing the direct method of feminization using oestrogens Ž A., suitable for any system of sex determination, and the indirect method of feminization by androgen treatment Ž B., suitable only for species with female homogamety.

15 F. PiferrerrAquaculture public concern about marketing fish that have been treated during early development with sex steroids. 3.. Indirect feminization This method is suitable for those species in which females are the homogametic sex Ž i.e., the XXrXY femalermale sex determination system.. The approach first involves the masculinization of genotypic females Ž XX. from a batch of mixed sex fish ŽXX and XY. and then the fertilization of normal ova Ž X. with the sperm produced by these new males Ž AneomalesB., which carry only the X chromosome Ž Fig. 1B.. To identify the neomales in the F 1, a progeny test consisting of individual crosses using the sperm of the androgen-treated fish and ova from untreated females is performed ŽHunter et al. 198, 1983; Blazquez et al., Crosses including only neomales will result in an F of all-female offspring Ž genotypically and phenotypically. whereas those including normal males will result in mixed male and female offspring. When using this method, masculinization is not an objective per se but is an essential step needed for indirect feminization. Species in which crosses of masculinized females with regular females have produced an all-female progeny include, among others, the medaka ŽYamamoto, 1958., the goldfish, Carassius auratus Ž Yamamoto and Kajishima, 1969., the rainbow trout Ž Johnstone et al., 1979a., the Atlantic salmon, Salmo salar ŽJohnstone and Youngson, 1984., the chinook salmon, O. tshawytscha Ž Hunter et al., 1983., Nile tilapia Ž Mair et al., 1991., the guppy Ž Kavumpurath and Pandian, 1993c., the Siamese fighting fish, Betta splendens Ž Kavumpurath and Pandian, 1994., the airbreathing catfish, Clarias lazera Ž Liu et al., and the yellow perch, Perca flaõescens ŽMalison and Garcia-Abiado, The indirect method, however, requires more than one generation for the production of fish with the desired gonadal sex. Once an all-female stock Žgenotypic and phenotypic. has been achieved, it is relatively easy to maintain by masculinizing every year a small portion of the offspring to provide more neomales for future crosses to obtain 100% female offspring, thus closing the production cycle. The untreated part of the stock is grown out and marketed as females or retained as broodstock. A shortcut in the production of all-female stocks by the indirect method has been made possible by the development of DNA sex-specific probes that allow for identification of the genetic sex in some species. These probes, initially developed for chinook salmon, have also been applied to coho, chum Ž O. keta. and pink salmon Ž O. gorbuscha.. However, the existing probes cannot identify the genetic sex of other salmonids such as the Atlantic salmon or the rainbow trout Ž Devlin et al., 1991; Du et al., This DNA-based methodology has found its actual application in the production of all-female stocks of chinook salmon using the indirect method eliminating the need for progeny testing Ž Devlin et al., Although initially tedious and time-consuming, the indirect method has the advantage that the marketed fish have never been treated with steroids. This method has already been used on a commercial scale in the culture of rainbow trout in the United Kingdom Ž Bye and Lincoln, and chinook salmon in Canada ŽDonaldson, 1986; Solar et al.,

16 44 F. PiferrerrAquaculture In species with polygenic or environmental sex differentiation mechanisms, crosses between sex-reversed fish and regular fish usually produce a range of sex ratios that makes it very difficult to establish the indirect method of feminization. This is the case in the European sea bass Ž Blazquez et al., Other uses of oestrogens Oestrogens are also important for the production of all-male stocks in female homogametic species such as O. niloticus and O. mossambicus Ž Fig.., or in male homogametic species such as O. aureus Ž Fig. 3.. In the first case, a sexually undifferentiated stock is treated with oestrogens to produce heterogametic Ž XY. females Ži.e., genotypic males phenotypic femaless neofemales.. These neofemales Ž F. 1 are separated by progeny testing from normal Ž XX. females and are crossed with normal males. The males of the resulting progeny Ž F. are crossed with females Ž XX.. One third of the crosses should give rise to a progeny Ž F. 3 consisting of 100% XY males. This proves the viability of the homogametic YY males Ž AsupermalesB. in the F Ž Fig.., implying that the presence of two Y chromosomes is not lethal for the animal. Production of AsupermalesB has succeeded in several species, including medaka and goldfish Ž Yamamoto, 1964, 1975b., rainbow trout ŽJohnstone et al., 1979a; Chevassus et al., 1988., Nile tilapia Ž Scott et al., 1989., O. mossambicus Ž Varadaraj and Pandian, 1989., guppy Ž Kavumpurath and Pandian, 199a., Siamese fighting fish ŽKavumpurath and Pandian, 199b., amago salmon, O. rhodurus Ž Onozato, and airbreathing catfish Ž Liu et al., However, in other species, e.g., the zebra cichlid, Cichlasoma nigrofasciatum, YY males are inviable Ž George and Pandian, Nevertheless, a Fig.. Endocrine methods to produce all-male stocks. Diagram showing the indirect method using estrogen treatment to produce an all-male stock in a female homogametic species.

17 F. PiferrerrAquaculture Fig. 3. Endocrine methods to produce all-male stocks. Diagram showing the indirect method of masculinization by oestrogen treatment to produce an all-male stock in a male homogametic species Žmodified from George and Pandian, successful oestrogen treatment is the first important step for maximizing the number of neofemales produced in the F1 and hence the chances of obtaining supermales in the F for all-male production in the F 3. In the case of male homogametic species Ž Fig. 3. such as the black molly, Poecilia sphenops, and O. aureus, the methodology involves the treatment of sexually undifferentiated fish Ž F. with oestrogen. This results in ZW females in the F Ž 0 1 i.e., genotypic phenotypic females not affected by oestrogen treatment. and homogametic ZZ females Ži.e., genotypic males phenotypic femaless AneofemalesB, as a result of oestrogen treatment.. After progeny testing, ZZ females are separated from ZW females and crossed with ZZ males to obtain 100% ZZ males in the F Ž Desprez et al., 1995; George and Pandian, Crosses involving sex-reversed fish with regular fish can be useful in elucidating the sex determination mechanism of a species. Broods of fish are masculinized or feminized and crossed with regular fish. If either all-female or mixed sexes are obtained in the F in the first case Ž as in Fig. 1B., and a variable number of males are obtained in the second case Ž as in Fig.., this indicates that the species in question most probably has female homogamety. On the other hand, if masculinized fish do not produce all-female broods and feminized fish produce broods with all males or mixed sexes Ž as in Fig. 3., then this suggests that the species in question has male homogamety Feminization by gynogenesis Feminization of fish without the use of steroids can be achieved in some cases by induced gynogenesis Ž Refstie et al., Briefly, gynogenesis is a special type of parthenogenesis where an egg is stimulated to divide by a genetically inactive spermato-

18 46 F. PiferrerrAquaculture zoon Ž Thorgaard, Thus, embryonic development is induced without the genetic contribution of the sperm. To inactivate the sperm, usually UV irradiation is used but other types of irradiation Ž gamma, X-rays. or even sperm from another species can also be utilized. Gynogenetic fish produced in this way are haploid, and generally do not survive beyond yolk absorption. Restoration of diploidy by temperature or pressure shock during the second meiotic division of the egg or the first mitotic division of the embryo increases the survival of gynogens. Diploidy restoration at metaphase II of meiosis leads to a level of inbreeding in excess of 50%, depending on the extent of recombination Ž Purdom, Thus, gynogenetic diploids may be fully homozygous or slightly heterozygous, depending on whether diploidy restoration has been achieved by blocking the first mitotic division of the embryo or by blocking the extrusion of the second polar body, respectively. Gynogenesis may also be used as a method for rapid development of inbred broodstock in fish without the delays associated with several generations of sib mating Ž Purdom, Induced gynogenesis, however, results in increased mortality Ž viability in the range of 1 40% survival with respect to diploid controls at hatch., associated with both increased homozygosity and treatment during early embryonic development Ž Felip et al., Reviews on chromosome set manipulation, including induced gynogenesis in fish and the derived advantages for aquaculture are, among others, those of Thorgaard Ž 1983., Chourrout Ž 1987., Ihssen et al. Ž 1990., Solar et al. Ž and Benfey Ž 1999., Arai Ž Diploid gynogens have both chromosome sets inherited from the mother and none from the father. Provided that females of the species in question are the homogametic sex, as for example in coho and chinook salmon Ž Hunter et al., 198, 1983, respectively., gynogenetic fish are expected to be female, because of the maternal inheritance of all of their chromosomes, including sex chromosomes. In species with types of sex determination mechanisms other than female homogamety, including polygenic and environmental sex differentiation, gynogenesis will produce variable sex ratios as a consequence of the influence of autosomal factors in the first case and of the strength of the environmental factors in the second Ž Muller-Belecke and Horstgen-Schwark, Gynogenesis has been induced in a large number of species for purposes of both basic and applied research Ž Solar et al., In about 0 of them including the rainbow trout ŽChourrout and Quillet, 198., coho salmon Ž Refstie et al., 198., Nile tilapia Ž Penman et al., 1987., common carp Ž Komen et al., and the catfish, Ictalurus punctatus ŽGoudie et al., 1995., it has served to corroborate or demonstrate female homogamety. Due to the significantly lower viability of gynogenetic fish, females obtained through induced gynogenesis are not suitable for direct production. In addition, usually a proportion of these females exhibit abnormal ovaries Ž Piferrer et al., 1994c., probably also a consequence of inbreeding. Nevertheless, gynogenetic fish of a female homogametic species can be hormonally masculinized and their sperm used to fertilize normal eggs to obtain an all-female progeny Ž Donaldson, Sex reversing gynogenetic females could be a means of counteracting the effect of inbreeding Ž Gall, Furthermore, progeny testing would not be necessary with gynogenesis and thus the establishment of all-female stocks could be accomplished in less time than is required by using the indirect method of hormonal feminization. This approach has been demon-

19 F. PiferrerrAquaculture strated to be feasible in, for example, grass carp, Ctenopharyngodon idella ŽJensen et al., 1983., O. mossambicus Ž Pandian and Varadaraj, 1990., rainbow trout ŽSchmelzing and Gall, 1991., marbled sole, Limanda yokohamae Ž Aida and Arai, and silver barb, Puntius gonionotus Ž Pongthana et al., Hormonal treatments 4.1. Methods for hormone therapy in fish Several methods are currently available for the administration of hormones to fish. Crim Ž classified these methods as acute and chronic. Acute hormone administration methods include: Ž. a intramuscular injection Žnormally into the epaxial muscle; Schreck, 1973., or injection into the body cavity of the material in solution ŽOlivereau and Olivereau, or as a suspension Ž Pankhurst et al., 1986., Ž b. administration in the aquarium water Ž Van den Hurk and Van Oordt, 1985., and Ž c. by immersing fish in a static bath containing the hormone Ž Goetz et al., 1979., commonly used in species in which the process of sex differentiation occurs shortly after hatching, like in most salmonids. Methods for chronic therapy include: Ž. a dietary treatment ŽGoetz et al , Ž b. silastic capsules Ž Shelton, 198., Ž c. cholesterol pellets ŽHiggs and Donaldson, and Ž d. osmotic pumps Ž Down et al., For the purposes of controlling sex, only three methods have been used Ž Table 1.: Ž 1. Dietary treatment, including live food such as Artemia as the vector, Ž. injection and Ž. 3 immersion. However, under commercial practice, the choice of methodology must depend on economic and practical considerations. Thus, it is clear that the two methods currently feasible for delivery of sex hormones to large numbers of fish are immersion treatment in a static or recirculating bath, and dietary treatment for acute and chronic administration of hormones, respectively. Bath treatments can vary greatly in time, ranging from brief dips to exposures of indefinite duration. However, usually the duration of the immersion treatments is in the range of hours and hence this method is classified under acute treatments. Because freshwater fish drink little or no water, during the immersion treatment the drug has to enter by way of the gills or the integument ŽHunn and Allen, 1974; Allen and Hunn, Immersion is suitable for those species in which the labile period Ž see below. coincides with embryogenesis or occurs during the larval stage, while dietary treatments are more appropriate for species in which the labile period coincides with external feeding. However, there are instances when a particular approach may not work. For example, immersion treatments were unable to feminize O. mossambicus Ž Rosenstein and Hulata, In contrast, they were successful for the feminization of Nile tilapia Ž Gilling et al., The use of live feed such as Artemia as a vehicle for steroids has been investigated in the largemouth bass, Micropterus salmoides ŽGarrett, and in the lumpfish, Cyclopterus lumpus Ž Martin-Robichaud et al., This is a promising alternative to immersion treatments since some species could be treated with steroid-enriched Artemia shortly after hatching without the disturbance of replacing water after immersion or transferring larvae to immersion baths. Avoiding these potential disturbances is very important, particularly in marine species with very small,