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Ž .Aquaculture 197 2001 205–228www.elsevier.nlrlocateraqua-online

Genetic improvement of aquaculture finfish speciesby chromosome manipulation techniques in Japan

Katsutoshi Arai)

Laboratory of Breeding Science, DiÕision of Marine Biosciences, Graduate School of Fisheries Sciences,Hokkaido UniÕersity, Hakodate, 041-8611, Japan

Received 1 October 2000; accepted 31 December 2000

Abstract

The purpose of this review is to introduce recent advances in research, development andapplication of chromosome manipulation techniques in Japan. Triploids have been induced andutilized to improve growth. In most cases, triploids are produced as all-female populations byusing spermatozoa of artificially sex-reversed males, so as to assure their complete sterility. Incontrast, triploid males show better gonadal development and sometimes generate functionalspermatozoa, which give rise to inviable aneuploids in most species. Although induced tetraploidscan be useful for mass production of triploids by mating with normal diploids, tetraploid lineshave only been produced in rainbow trout by a few institutes belonging to prefectural govern-ments. In most cases, the techniques to inhibit the first cleavage often result in very low survival

Ž .and mosaicism. In the loach Cobitidae , polyploid lines such as hexaploids have been developedby using natural tetraploid individuals as an intermediate step. Cloned fish can be produced by thesecond cycle of gynogenesis in the eggs of completely homozygous diploids, which wereproduced by inhibiting first cleavage after induction of gynogenetic and androgenetic develop-ment. The second cycle of androgenesis, using spermatozoa of completely homozygous males, canalso generate clonal lines. The most serious technical problem of cloning is the extremely lowsurvival of homozygous gynogenetic and androgenetic diploids, probably due to the expression ofdeleterious recessive genes and side effect of treatments. However, cloned populations have beenrealized in commercially important ayu Plecoglossus altiÕelis, amago salmon Oncorhynchus

Ž .masou ishikawae, coho salmon O. kisutch, hirame Japanese flounder Paralichthys oliÕaceus,fancy carp Cyprinus carpio, and red sea bream Pagrus major. In hirame, a practical method formass production of clones was proposed and better performance has been reported in a heterozy-

) Tel.: q81-138-40-5535; fax: q81-138-40-5537.Ž .E-mail address: [email protected] K. Arai .

0044-8486r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 01 00588-9

( )K. ArairAquaculture 197 2001 205–228206

gous clone, produced by hybridization between two different homozygous clonal lines. RepeatedŽ .meiotic polar body gynogenesis may be more practical than cloning from homozygous gynogens

as a method to generate isogenic lines. In meiotic gynogenesis, the proximal region of chromo-somes should be homozygous, whereas the distal region should be heterozygous due to high ratesof gene–centromere recombination. Consequently, similar genotypes are predicted in the secondand later generations of gynogenetic progeny. Their isogenic nature has been confirmed byminisatellite, microsatellite, and other DNA analyses. Gynogenesis, androgenesis and cloning canbe used for elucidation of genetic sex determination. The involvement of environmental factorshas been indicated from the sex ratios of chromosomally manipulated populations of severalspecies. Finally, the regulation of chromosomally manipulated fish by the guidelines and theintegration of such techniques with molecular genetics for further gene mapping and transgenicsare discussed. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Polyploid; Gynogenesis; Androgenesis; Clone; Sex determination

1. Introduction

There has been an explosive growth in both research and application of chromosomeŽmanipulation techniques to various finfish species in the last 2 decades Hussain, 1996;

.Arai, 1997a,b; Pandian and Koteeswaran, 1998 . In Japan, pioneering works concerningploidy manipulation and induction of unisexual development began in the late 1970s andearly 1980s in some universities and national institutes. The results were presented inannual meetings of the Japanese Society of Fisheries Science and were soon recognizedas a breakthrough for the rapid improvement of genetic traits of fish and shellfishspecies, in which conventional selection has been considered difficult due to their longlife cycle. Due to such backgrounds, research projects collectively referred to asAPromotion of Regional BiotechnologyB had been allotted to each prefectural govern-ment under the initiative of the Fisheries Agency of the Japanese Government since1984, so as to develop systematic techniques of chromosome manipulations for a fishspecies, which is especially important for the fisheries industry of each prefectureŽ .Fisheries Agency, 1997 . Since 1991, such projects have been taken over by more

Ž .advanced ones in order to apply techniques directly to aquaculture Seki, 1999 . Fromthese projects as well as other activities, sterile triploids, all-female populations andclonal lines have been produced for several finfish species over the last 1.5 decades in

Ž .all areas of Japan Arai, 1997a,b; Seki, 1999 . Besides such practical research, pilotstudies on the production of higher polyploidies using natural tetraploidy as a stepping

Žstone, as well as of clones using unreduced eggs, have been conducted Arai et al., 1993,.1995, 1999; Matsubara et al., 1995; Zhang and Arai, 1996; Arai and Mukaino, 1997 .

The significance of meiotic gynogenesis has also been revalued as a more practicalŽ .method to fix desirable genotypes than cloning Arai, 1997a,b .

In the present review, the current status of technologies and applications of chromo-some manipulations developed in Japan are outlined, and mention was made of theguidelines for use in manipulated fish, and the possible development of the technologyby integration with molecular techniques.

( )K. ArairAquaculture 197 2001 205–228 207

2. Application of polyploid techniques for aquaculture

2.1. Induction techniques and performances of triploid fish

The most popular method of chromosome manipulation is the production of fish withŽ .extra set s of chromosomes, i.e., polyploidy. In finfish, triploids can be easily induced

Ž .by blocking the second polar body release with high temperature heat , low temperatureŽ .cold , or high hydrostatic pressure shocks, just after fertilization with normal spermato-

Ž .zoa Fig. 1 . Chemical treatments using cytochalasin B and other kinds of agents are notpopular in triploidization of finfish. For practical induction, optimum treatment condi-

Ž .tions such as magnitude temperature or pressure level , duration, and timing, must bedetermined by trial and error approaches so as to yield 100% triploidy without seriouslydecreasing survival rates. Effective protocols have been developed for a large number of

Žspecies in the last 2 decades as seen in several reviews Ueno, 1989; Hussain, 1996;.Arai, 1997a,b; Pandian and Koteeswaran, 1998 .

In Japan, application-oriented studies of triploids are now in progress in about 26finfish and seven shellfish species by research organizations, such as AFisheries Experi-

Ž .mental StationB belonging to prefectural governments Fisheries Agency, 1997 . Espe-cially in rainbow trout, Oncorhynchus mykiss, biological characteristics and aquacultureperformances of induced triploids have been intensively studied for commercial applica-

Ž . Žtions in Hokkaido Okada, 1985 , Shiga Kobayashi, 1992, 1997; Kobayashi and. Ž .Fushiki, 1997; Kobayashi et al., 1993, 1995, 1998a , Gifu Usuda, 1988 and other

active prefectures. Similar approaches have been made in ayu, Plecoglossus altiÕelis, inŽ . Ž .Fukuoka Inada, 1997 , Gifu Mori, 1990; Kuwada et al., 1992a,b , Wakayama

Ž .Tsujimura et al., 1991 and other active prefectures. According to these works, thepre-maturation growth of induced triploids is similar to that of diploids in separaterearing conditions, but is a little inferior to that of diploids in communal rearingconditions due to strong inter-individual competitions. However, during the period offinal maturation, there is a consensus that triploids show better growth, survival andmeat quality than diploids. Such advantages of induced triploids can be well explainedby the sterility expressed in females, which may reduce energy costs of reproduction. Incontrast, triploid males exhibit more gonadal development than females and showsecondary sex characters. Thus, males are semi-fertile and exhibit no improved growth.To assure better growth in induced triploids, all-female triploids have been produced in

Ž .rainbow trout by fertilization with sex-reversed physiological males neomales . Aneffective protocol of androgen treatment to masculinize genetic females has been

Ž .developed in rainbow trout Okada et al., 1979 . Similar procedures to produce andutilize all-female triploids for aquaculture were also developed in other salmonids.

Ž . Ž .Current status of seed production individual number and aquaculture metric ton ofall-female triploid salmonids produced is shown in Table 1. In ayu, sex-reversedphysiological XX males and XX intersexes with both ovary and functional testis have

Ž .been experimentally produced by androgen treatments Tsujimura et al., 1991 . Thecommercial production of all-female triploid ayu is operated by Kanagawa PrefectureŽ .Fisheries Agency, 1997; Hara, 1999 .


Ž .Fig. 1. A schematic diagram of inducing polyploidy triploidy and tetraploidy and different types of gynogens.1PB: first polar body; 2PB: second polar body.

In contrast to the promising perspective of all-female triploids in freshwater speciesŽsuch as rainbow trout, triploids in marine fish species, such as hirame Japanese

.flounder Paralichthys oliÕaceus, red sea bream Pagrus major and Japanese parrot fish


Table 1Seed production and aquaculture of all-female triploid salmonids monids in 1996 fiscal year in Japan

Ž .Translated and compiled from the publication of Fisheries Agency 1997 .

Species Seed Prefectures Aquaculture PrefecturesŽ .production metric ton

Ž .=1000 fish

Rainbow trout 4801.2 Hokkaido, Nagano, 516.8 Nagano, Niigata,Niigata, others Hokkaido

Amago salmon 180.0 Gifu 6.7 GifuMasu salmon 460.0 Hokkaido, Miyagi 23.0 Isikawa, Miyagi

aYamame salmon 18.3 Ibaraki 2.9 Ibaraki

a Landlocked form of masu salmon.

Oplegnathus fasciatus, gave no significant improvement in growth and other character-Ž .istics Tabata, 1991; Murata, 1998 . In hirame, a continuous growth through life was

observed both in diploids and triploids and normal diploids were found to continueŽ .growth even in the spawning season Tabata, 1991 . Thus, the advantage of triploidy due

to reallocation of energy costs from gonadal to somatic growth is rather doubtful in thisŽ .species Tabata, 1991 . There is also no significant difference in growth between

Ž .diploids and triploids of red sea bream and Japanese parrot fish Murata, 1998 .However, the lack of improvement of growth in triploid red sea bream may relate to theexclusive occurrence of males in this species.

2.2. Potential of allotriploid fish

In intergeneric crosses between chum salmon O. keta females and Japanese charSalÕelinus leucomaenis males, the resultant progeny cannot survive beyond the hatching

Ž .stage due to inviable embryogenesis Arai, 1984 . They are not aneuploid, but trueŽ .diploid hybrids allodiploid with an intermediate karyotype between the two species

Ž . Ž . ŽArai, 1984 . In contrast, triploid hybrids allotriploid including two genomes sets of.homologous chromosomes from maternal chum salmon and one genome from paternal

Ž .charr exhibit normal development and significantly improved survival Arai, 1984 . Theaddition of an extra-maternal genome neutralizes developmental instabilities occurringbetween maternal and paternal genomes andror between maternal cytoplasm and

Žpaternal exotic genome. In most inter-specific salmonid crosses, triploid hybrids allo-.triploids often show better survival and fewer developmental abnormalities than their

Ždiploid counterparts Arai, 1986, 1988; Yamano et al., 1988; Oshiro et al., 1991; Deng.et al., 1992 . In salmonids, all-female allotriploids are expected to show improved

growth in the maturation season due to the sterility expressed by the females, asconfirmed in all-female autotriploid rainbow trout and other species. Stable seedproduction and aquaculture of all-female allotriploid rainbow trout=amago salmon O.masou ishikawae and rainbow trout=Japanese char are conducted in Aichi PrefecturalInlandwater Fisheries Experimental Station, and these allotriploids are commercially

Ž .utilized Fisheries Agency, 1997 .


2.3. Tetraploid for mass production of triploid and other ploidies

Tetraploid lines are important for the mass production of sterile triploids by hy-bridization between diploid and tetraploid fishes. Tetraploidy is also useful, as it is thesource for the production of other kinds of polyploids such as penta-, hexa-, hepta- andoctaploids. For these purposes, production of tetraploid finfish has been attempted by

Ž .inhibiting the first cleavage after normal fertilization Fig. 1 since the early 1980s. Todate, in Japan, production of live, feeding, and spawning tetraploid fish has beenrealized only in rainbow trout. The Nagano Prefectural Fisheries Experimental Stationhave produced tetraploid populations of rainbow trout. According to the progress reportfor the project of AProduction of triploid using tetraploid rainbow trout,B which included

Ža series of results obtained in 1991–1995 Nagano Prefectural Fisheries Experimental.Station, 1996 , they successfully generated a very small number of tetraploid founder

Ž 2 .fish by pressure shock 650 kgrcm , 6-min duration, beginning 50–608C h ; productionrates of the 50–150-g size tetraploid trout were 0.006–0.065% of the total eggs used,and rates of mature tetraploid trout were 0.006–0.007% of the initial number of eggs.Similar yields of tetraploids were achieved by double heat shocks, but no effect ofnormal single heat shock was reported. After the maturation of induced tetraploids, theyproduced a 100% tetraploid line by crosses between them in 1993, and then tetraploidlines were generated by crosses between second generation of tetraploids in 1994–1995.In the same years, production of almost 100% triploids was also made by reciprocalcrosses between tetraploid and diploid rainbow trout. As reported in the pioneering

Žworks by Chourrout and his colleagues Chourrout et al., 1986; Chourrout and Nakayama,. Ž1987 , the following results were obtained by the Nagano station Nagano Prefectural

. Ž .Fisheries Experimental Station, 1996 : a occurrence of diploid–tetraploid mosaicismŽ .determined by erythrocytes size; b lower yields of resultant progeny in diploid

female= tetraploid male cross than tetraploid female=diploid male cross, as a result ofŽ .the difficult egg penetration of diploid spermatozoa with larger head size; c production

Ž .of more males than females female 3: male 13 in progeny due to more XY and YYŽ .than XX spermatozoa of a founder tetraploid; d significantly better growth of triploids

Ž .tetraploid female=diploid sex-reversed male than normal diploids and tetraploidsŽ . Ž .tetraploid female= tetraploid male in 2-year-old fish; and e better growth of all-

Ž .female triploids tetraploid female=diploid sex-reversed male than induced all-femaletriploids, produced by the second polar body-inhibition after fertilization in 2-year-fish.

During the project, sex-reversed tetraploid males were also produced by 17 a-methyl-Žtestosterone 0.5 mg MTrkg food, 60-day oral administration after the beginning of

.feeding . Thus, mass production of all-female triploids was made by crosses betweenŽ .normal diploid females and sex-reversed probably XXXX tetraploid males. All-female

triploids were also produced by crosses between tetraploid females and sex-reversedŽ . ŽXX diploid males. The latter triploids exhibited better hatching rates 72.0–86.4%; no

. Ž .data of fertility than the former ones 29.3–37.3% , probably due to a lack of theŽproblems of large sperm head size in tetraploids Nagano Prefectural Fisheries Experi-

.mental Station, 1996 . Since 1996, the research has been oriented to produce all-femaleallotriploids using sex-reversed tetraploid rainbow trout males and diploid females of

Ž .other species Nagano Prefectural Fisheries Experimental Station, 1997, 1998 . Accord-


Ž .ing to Fisheries Agency 1997 , similar projects using tetraploid rainbow trout are inprogress in Yamagata, Fukushima, Ibaraki, Tochigi, Niigata, Yamanashi, and GifuPrefectures.

2.4. Mosaicisms occurred in polyploids

In most fish species, viable tetraploid fish are still difficult to produce by inhibition ofŽ . Ž . Ž .the first cleavage, probably due to a mosaicism, b aneuploidy, c reduced cell

Ž . Ž . Žsurface area, d wrong cytological events, andror e high homozygosity Pandian and.Koteeswaran, 1998 . As mentioned in the foregoing paragraphs, however, artificial

induction of live tetraploid finfish is possible at least in rainbow trout. This evidencemeans that fish appear to tolerate genomic changes from diploid to tetraploid status. Thetetraploidization is very difficult, but once viable founders are obtained, it is possible toproduce and keep tetraploid lines by normal breeding procedures.

Ž .Recently, Yamaki et al. 1999a found that a 2-year-old amago salmon female, whichdeveloped from a chromosome manipulation to inhibit the first cleavage, but exhibitedonly diploid DNA content in erythrocytes, produced both diploid and triploid progenyby fertilization of its eggs with haploid spermatozoa of a normal diploid male. Althoughthe DNA content, flow-cytometrically measured in erythrocytes, showed that this fishwas diploid, the occurrence of triploid progeny indicated the involvement of diploideggs. Consequently, this amago salmon was considered a mosaic including diploiderythrocytes and tetraploid germ cells. Occurrence of such a somatic diploid-germ linetetraploid mosaic fish in tetraploid-oriented manipulation suggests possible productionof tetraploid lines, by crosses between mosaics including diploid gametes.

2.5. Production of higher polyploidies in the loach

The loach Misgurnus anguillicaudatus is a small fish belonging to the familyCobitidae, the order Cypriniformes, and has been utilized for traditional Japanese food.

Ž .This species includes polyploid forms; diploid individuals 2ns50 are most commonŽ .in Japanese populations, although a small number of natural triploids 3ns75 have

Ž .been found in wild population Arai et al., 1991a; Zhang and Arai, 1999a . Tetraploidindividuals with 100 chromosomes were found among specimens obtained from fish

Ž .dealers Arai et al., 1991a . Despite the origin of these tetraploid individuals beingunknown, they are regarded as genetically true tetraploids with four sets of homologous

Ž .chromosomes 4ns100 , because viable gynogenetic and androgenetic progeny havebeen produced from their eggs and spermatozoa, respectively, without any manipula-

Ž .tions to duplicate chromosomes Arai et al., 1991b, 1993, 1995 . The author and hiscolleagues have made a series of pilot studies to open new possibilities of polyploidbreeding using natural polyploid variants. They have realized higher polyploids such aspentaploid, hexaploid, and heptaploid, using natural tetraploids as a stepping stoneŽ .Matsubara et al., 1995; Zhang and Arai, 1996; Arai et al., 1999 . An outline of theprocedures for production of higher polyploid loaches is shown in Fig. 2.


Fig. 2. A schematic diagram of pedigree to produce higher polyploidies in the loach Misgurnus anguillicauda-Ž . Žtus, using diploid gametes of natural tetraploid 4ns100 or unreduced triploid eggs of triploid 3ns75,

. Ždiploid female=tetraploid male as a stepping stone. Open and closed circles mean a genome haploid set of. Ž . Ž .chromosomes from normal diploid 2ns50 and natural tetraploid 4ns100 , respectively.


In 1990, the first tetraploid line was produced by a natural tetraploid pair, and thegeneration of triploid stock was carried out by reciprocal crosses between normal diploid

Ž .and natural tetraploid individuals Arai et al., 1991b, 1993; Zhang and Arai, 1996 . Asmall number of pentaploid loach were also induced by inhibiting the second polar bodyextrusion after crossing a tetraploid female and diploid male. In 1993, founder te-traploids reached maturity and second generation progeny were produced by normalcross fertilization between tetraploid individuals. Using diploid gametes of tetraploids, ahexaploid line was successfully created by inhibiting the second polar body extrusionjust after tetraploid female= tetraploid male cross in 1993. The sex ratio in tetraploidand hexaploid lines did not deviate from 1:1 and induced tetraploid gynogens were

Žall-female, suggesting an involvement of the male heterogametic system Arai et al.,.1999 . In 1997, second generation hexaploid progeny were successfully produced by

normal fertilization of hexaploid pairs, and crosses between hexaploid and tetraploid andthose between hexaploid and diploid gave rise to pentaploid and tetraploid progeny,

Ž .respectively Arai et al., 1999 . Eggs of hexaploids were significantly larger than thoseŽof tetraploids, whose eggs were also larger than those of normal diploids Arai et al.,

.1999 . Now, comparisons of biological performances such as growth and survivalŽ . Ž .among these hexaploid 6n female=6n male , pentaploid 6n=4n and tetraploid

Ž . Ž . Ž .6n=2n , as well as those among diploid 2n=2n , triploid 2n=4n, 4n=2n andŽ .tetraploid 4n=4n are under way in communal rearing conditions.

2.6. Polyploidization using unreduced eggs in the loach

Occurrence of atypical reproduction was recognized in triploid progeny derived fromŽ .natural tetraploid loach Fig. 2 . The triploid loaches produced by hybridization between

normal diploids and natural tetraploids showed sterility in males, but the females laidŽsimultaneously both large-sized triploid and small-sized haploid eggs Matsubara et al.,

.1995; Zhang and Arai, 1996 . In such triploid loaches, triploid eggs were produced by aquasinormal meiosis started from hexaploid oogonia, occurring by pre-meiotic endomi-tosis, whereas haploid eggs were produced from bivalents after eliminating univalents intriploid oocytes, in which equal number of bivalents and univalents had been formedŽ .Zhang et al., 1998 . Identical DNA fingerprints of the triploid gynogens, induced from

Žlarge-sized eggs, gave evidence that triploid eggs were unreduced Arai and Mukaino,.1997 . Thus, the reproduction of such triploid loaches is apparently different from that

reported in induced triploid loaches, in which the females were sterile, but on average,the male generated 1.3n spermatozoa and viable aneuploids when backcrossed to natural

Ž . Ž .tetraploids Zhang and Arai, 1999b and normal diploids Arai and Inamori, 1999 .Occurrence of unreduced eggs has been observed in natural triploid loaches found in

Ž .some localities in Japan Zhang and Arai, 1999a . A normal diploid loach generatingŽ .unreduced diploid eggs was also found Zhang and Arai, 1999a . Thus, the production of

different types of polyploids is possible by using such unreduced triploid and diploideggs. Hexaploids were produced by the inhibition of the second polar body extrusionafter triggering gynogenetic development in unreduced triploid eggs, and tetraploidswere successfully generated by fertilizing unreduced triploid eggs with normal haploid

Ž .spermatozoa Matsubara et al., 1995; Zhang and Arai, 1996 .


3. Application of gynogenetic and androgenetic techniques to aquaculture

3.1. Production of cloned line by gynogenesis

Gynogenesis is a form of all-female inheritance. One of the practical goals of inducedgynogenesis is the production of genetically identical population, i.e., clonal lines; theseclonal lines may be important for the improvement of fish stocks. In finfish, ultravioletŽ .UV irradiation has been used to inactivate spermatozoa, and such UV-irradiatedsprmatozoa are routinely used to trigger gynogenetic development without contribution

Ž .of the paternal genome Fig. 1 . Heterospecific spermatozoa are often used because anyspermatozoa that survived UV treatment would produce inviable hybrids. In inducedgynogenesis of the loach, spermatozoa of other species such as carp, goldfish or minnow

Ž .were used Arai et al., 1991b; Suwa et al., 1994 . Recessive colour mutants such asŽorange and albino were also used as mother fish to verify gynogenesis Suzuki et al.,

.1985 . Gynogenetic haploids, thus induced, are inviable and die during embryonicdevelopment before or soon after hatching, or during early larval stages before thebeginning of feeding, due to characteristic abnormalities referred to as Ahaploid syn-drome.B However, a few haploids with severe malformation were reported to survive in

Ž .the induced gynogenesis of tilapine fish Varadaraj, 1993 . Recently, a live haploid–di-ploid mosaic charr, without any abnormalities in external appearance, was found in afarm population; this individual included haploid liver, spleen, and blood cells, but

Ž .mostly diploid brain cells Yamaki et al., 1999b .Viable gynogenetic diploids can be induced by activation of eggs with UV-irradiated

spermatozoa and subsequent diploidization of the maternal chromosome set, by inhibit-Ž .ing either the second polar body extrusion or the first cleavage Fig. 1 . The former type

Ž .of gynogenetic diploid gynogen is termed Ameiotic gynogenetic diploid,B Apolar bodygynogen,B or Ameiogynogenesis,B whereas, the latter is termed Amitotic gynogeneticdiploid,B Acleavage gynogen,B or Amitogynogenesis.B Meiotic gynogenesis has been

Žsuccessfully induced in a large number of finfish species Arai, 1997a,b; Pandian and.Koteeswaran, 1998 . In meiotic gynogens developed from a female which is heterozy-

gous for given gene loci, the loci on distal portions of chromosomes are consideredheterozygous because of gene–centromere recombination, while those on proximal

Ž .portions must be homozygous Thorgaard et al., 1983 . These gene–centromere recom-Ž .binations have been verified by allozyme markers in ayu Taniguchi et al., 1988 , hirame

Ž . Ž . ŽTabata, 1991 , loach Suwa et al., 1994 , ocellated puffer Takifugu rubripes Kakimoto. Ž .et al., 1994a , marbled sole Limanda yokohamae Kakimoto et al., 1994b and other

Ž .finfish species Hussain, 1996 . Thus, completely homozygous, inbred lines cannot beproduced by this type of gynogenesis.

In contrast, the mitotic gynogens are completely homozygous for all the loci, and areabsolutely necessary as mother fish for producing cloned fish by second cycle gynogene-

Ž .sis in the next generation. Meio-gynogenetic progeny clones are produced fromgenetically identical eggs of the induced mitotic gynogen. However, survival rates are

Žusually very low among mitotic gynogens Arai, 1997a,b; Pandian and Koteeswaran,.1998 . In 1994 and 1995, the author attempted to produce meiotic and mitotic gynogens

Ž . Žin the loach Arai, 1997b . In the induction of meiotic gynogenesis, about 60% 13 out


. Ž . Ž .of total 22 of females experiments gave relatively high proportion 10–53% ofnormally feeding 7-day-old fry. Mean survival rate of the meiotic gynogen was

Ž .19.3 "s.d. 15.5 % to 7 days after fertilization. In the induction of mitotic gynogenesis,Ž . Ž .about 90% 20 out of total 29 of females experiments resulted in very low percentages

Ž .less than 10% of feeding gynogens. Rates of the normal feeding fry from 11 out of the29 females were less than 1%. Only three females gave reasonable survival ratesŽ . Ž10–26% . Thus, mean survival rate of mitotic gynogens was very poor 4.1"s.d.

.5.7% when compared with the results of meiotic gynogens.Another problem of surviving mitotic gynogens is drastically reduced fertility with

small numbers of mature fish, low fecundity, and very poor egg quality. No gynogeneticprogeny were produced from the surviving gynogenetic homozygous loaches, due to

Žpoor egg quality and very low survivals of the embryonic stages Arai, unpublished.data . Another constraint for the development of clonal lines is contamination by

spontaneously occurring meiotic gynogens, which show better survivals than inducedmitotic gynogens. Such spontaneous meiotic gynogens in presumed batches of mitoticgynogens would generate aclonal progeny.

3.2. Cloned fish in Japan— examples and performances

In Japan, over the last decade, clonal lines have been successfully established inŽ .several commercially important species such as ayu Han et al., 1991 , amago salmon

Ž . Ž .Kobayashi et al., 1994 , and hirame Tabata, 1997; Yamamoto, 1999 . Recently,production of clonal lines in red sea bream by the Fisheries Laboratory, at KinkiUniversity, was reported at the Kinki branch of the Japanese Society of Fisheries

Ž . Ž .Science Kato, 1999a and also by commercial aquaculture Kato, 1999b . In theseŽexamples, verification of the clonal nature has been made by tissue grafting Han et al.,

. Ž .1991; Kobayashi et al., 1994 , DNA fingerprinting Han et al., 1992; Hara et al., 1993Ž .or RAPD-PCR using an autosequencer Kobayashi et al., 1998b . The pioneer works on

heritabilities of physiological and morphological quantitative traits, such as growth,haematocrit, plasma glucose and meristic characters, have been made by Taniguchi and

Ž .his co-workers for the ayu Taniguchi et al., 1994; Del Valle et al., 1994, 1995, 1996 .At present, studies of the protocols, biological characteristics, andror aquacultural

Žperformances of cloned fish are in progress for the ayu Wakayama, Kochi, and. Ž .Fukuoka Prefectures , hirame Hokkaido, Hyogo, Tottori, and Yamaguchi P. , masu

Ž . Ž .salmon Aomori and Kanagawa P. , amago salmon Shiga and Gifu P. , coho salmon O.Ž . Ž . Žkisutch Miyagi P. , Atlantic salmon Salmo salar Akita P. , rainbow trout Aomori,

. ŽFukushima, Saitama, Tokyo, Nagano, Shizuoka, Gifu and Shiga P. , fancy carp Saitama. Ž .P. , nigorobuna crucian carp Carassius carassius grandculis Shiga P. , honmoroko

Ž . Ž .minnow Gnathopogon caerulescens Shiga P. , goldfish C. auratus Aichi P. and redŽ . Ž .sea bream Kumamoto P. Fisheries Agency, 1997 .

Among these approaches, the most advanced leadings were seen in hirame in theŽ .Tottori Prefecture, by Yamamoto 1999 . He produced cloned hirame populations by

meiotic gynogenesis of genetically identical eggs of the first generation of completelyhomozygous mitotic gynogens. The survival rates were very low as reported in mitoticgynogens of other species, but the large numbers of eggs produced by each individual in


this species increases the probability of successful production of live, feeding, andŽ .spawning gynogens. Yamamoto 1999 also produced a second generation of these

homozygous clones by crossing females and sex-reversed males within a clone. As thegenetic female of the hirame is easily differentiated from the physiological male by theirresponse to environmental factors, thus a large-scale production of clonal fish in a tankby natural spawning procedure was suggested. Genetically identical but heterozygousclones were also developed by hybridization between parental fish from differenthomozygous clonal lines, and such heterozygous clones often showed better survival,growth, disease resistence, and reproductive potential, probably due to masking of

Ždeleterious recessive genes which are expressed in homozygous clones Yamamoto,.1999 . Consequently, he recommended that homozygous clones should be used as

broodstock, but heterozygous clones should be used for commercial aquaculture. SimilarŽ .results were also independently obtained by Tabata 1997 from the Hyogo Prefectural

Fisheries Experimental Station.

3.3. Isogenic line established by repeating meiotic gynogenesis

Application of clones for aquaculture is one of the goals of chromosome manipula-tion. The genetically identical nature of cloned fish is also important for basic biologicalsciences such as genetics, physiology and immunology. As mentioned in Section 3.1,production of cloned fish is difficult due to the low survivals of the first generationmitotic gynogens as well as their reproductive performance. The production of meioticgynogens is easier than mitotic gynogens, but it is very difficult to establish completelyhomozygous lines by meiotic gynogenesis due to gene–centromere recombinationsŽ .Thorgaard et al., 1983 . However, it may be possible to produce AisogenicB lines by

Ž .two, three or more cycles of meiotic polar body gynogenesis, because the distal andproximal loci on homologous chromosomes would be fixed in the heterozygous andhomozygous conditions, respectively. This means that repeating meiotic gynogenesiswould give rise to an isogenic line, in which gene loci of proximal and distal portionwould be fixed to the same homozygous and heterozygous geneotypes, respectively.Such an isogenic line may be regarded as a kind of heterozygous clone.

In the loach, nearly identical DNA fingerprint patterns were observed among theŽ .second generation progeny of meiotic gynogenesis Fig. 3 . Although the average band

Ž .sharing index BSI among wild individuals from diffferent localities was 0.149"0.055Žin the combination of probe 33.6 and restriction enzyme Hae III Arai and Mukaino,

.1997 , BSI among the second generation of meiotic gynogenetic loach was very close toŽ . Ž .1 Arai and Ishimoto, 1996 . A decrease in the number of DNA fragments )2 kb was

also observed in the fingerprinting pattern; in probe 33.6, 42 fragments were obtainedfrom the founder female, 38.5 fragments in the first generation, and 37 fragments in the

Ž .second generation of meiotic gynogens. The same result BSIs1 was also reportedŽamong progeny in the third generation of meiotic gynogens of red sea bream Taniguchi

. Ž .and Takagi, 1997 . High similarity isogenic nature among the second generation ofmeiotic gynogenesis was also suggested in goldfish by immunogenetic methods. Nakan-

Ž .ishi et al. 1988 reported that rejection of transplanted scales did not occur in thesegynogenetic progeny even 4 months after allografting.


Fig. 3. Fixation of genotypes by repeating meiotic gynogenesis in the loach M. anguillicaudatus. Note almostidentical DNA fingerprints in the second generation of meiotic gynogens.

Ž .Kobayashi 1997 , who previously belonged to the Shiga Prefectural FisheriesExperimental Station and is now at Kinki University, reported the successful establish-ment of isogenic Asuper growth strainB in rainbow trout by repeating meiotic gynogene-


Žsis. He produced the first generation of meiotic gynogens from a normal female bodyŽ . Ž Ž .3. .weight 2.8 kg, condition factor 19sbody weight g r body length cm =1000 in

1986 and found that 2.5-year-old survivors exhibited extremely rapid growth, character-Ž . Ž .ized by high condition factors 22–27 and heavy weight approximately 3.5 kg . He

considered that the expression of such traits might result from homozygosity ofŽ .recessive gene s located on proximal region of chromosomes by gynogenesis. In 1989,

he produced second generation progeny by meiotic gynogenesis and found the stableŽ .inheritence of high condition factor 22.11"0.42 in these progeny in 1992. Thus, the

Asuper growth strainB of rainbow trout had been kept by meiotic gynogenesis, and itsclonal nature was verified by completely identical RAPD-PCR fragment patterns amongindividuals of the fifth generation progeny of this strain.

As mentioned above, meiotic gynogenesis seems to be a more practical method to fixdesirable genotypes than normal cloning procedures for rapid improvement of aquacul-ture strains, when production of clones is quite difficult due to very low survival andfecundity of homozygous mitotic gynogens.

3.4. Cloning by gynogenesis of spontaneous unreduced eggs

Production of clone can be made by induction of gynogenetic development ofspontaneously occurring unreduced eggs, which have been generated in some hybrids

Ž .and unisexuals Vrijenhoek et al., 1989 . Clonal nature of the diploid eggs of hybridbetween two different medaka species, Oryzias latipes and O. curÕinotus, was evi-denced by immunological responses of mutually transplanted scales in the progeny of a

Žhybrid female crossed with a male of an inbred line of O. latipes Sakaizumi et al.,.1993 . Such unreduced eggs were also observed in triploid individuals produced by

Žhybridization between normal diploid and natural tetraploid loach Matsubara et al.,.1995; Zhang and Arai, 1996 . Triploid eggs were produced by premeiotic endomitosis in

Ž .the triploid loach Zhang et al., 1998 . Most gynogenetic triploid progeny, which wereinduced by fertilization of unreduced triploid eggs with UV-irradiated heterospecificspermatozoa, gave DNA fingerprints identical to the somatic cells of the triploid mother,

Ž .and thus were considered to be clones Arai and Mukaino, 1997 . These results suggestpossible methods for the production of clonal lines using hybrids or triploids in somefish species. Recently, a diploid loach which laid both normal haploid and unreduced

Ž .diploid eggs was identified by Zhang and Arai 1999b , and subsequent gynogeneticprogeny, induced by activation with UV-irradiated heterospecific spermatozoa, are nowbeing reared for further studies. If such a trait is heritable, selective breeding of thesefish could be used to establish clonal line. An unusually high frequency of spontaneousdiploidization in a maternal genome was also reported in an ornamental fancy carp in

Ž . ŽIsrael Cherfas et al., 1991 . The heritable nature of such a trait occurrence of diploid. Ž .eggs was confirmed by studies of the progeny Cherfas et al., 1995 .

3.5. Androgenesis as a mehod to produce indiÕiduals from spermatozoa

Androgenesis is defined as all-paternal inheritance. Viable androgenetic diploids canbe generated by inhibiting the first cleavage to double paternally derived chromosomes,


after fertilization of genetically inactivated eggs with normal spermatozoa. Gamma andX-ray inactivation are the usual methods used to inactivate egg nuclei. These methods

Žare especially effective in salmonids, which have large eggs and opaque chorion Arai et.al., 1979; Parsons and Thorgaard, 1984 . However, since special facilities are required to

manage radioactivity safely, these methods are not practical for routine induction ofandrogenesis. It was reported that UV-irradiation was also effective in the inactivation of

Ž . Žthe egg nucleus in the loach Arai et al., 1992 and carp Kondoh et al., 1989; Kondoh.and Sato, 1990; Bongers et al., 1994 . Androgenetic diploids, produced by blocking the

first cleavage with temperature or pressure shock, exhibited very low survival asŽobserved in mitotic gynogens Parsons and Thorgaard, 1985; Masaoka et al., 1995;

.Bongers et al., 1995; Nagoya et al., 1996 . Better survival rates were recorded inandrogenetic diploids, which had been produced by using diploid spermatozoa of

Ž . Ž .induced Thorgaard et al., 1990 or natural tetraploid fish Arai et al., 1995 . Androge-netic individuals were also obtained by fertilization of genetically inactivated eggs withfused spermatozoa, but the yields were very low due to low efficiencies of sperm fusionŽ .Araki et al., 1995 .

Once completely homozygous androgenetic diploid fish are obtained, however, it ispossible to establish clonal lines from eggs of androgenetic females by meiotic gynogen-esis, or from spermatozoa of androgenetic males by a second cycle of androgenesis.Production of clonal lines from homozygous androgenetic diploid fish has been reported

Ž . Ž .in rainbow trout Young et al., 1996 , common carp Bongers et al., 1995 and in amagoŽ .salmon Nagoya et al., 1996 . Recently, a clonal line of fancy carp was also produced by

induction of androgenesis using spermatozoa of the first generation of androgeneticŽ .diploids Niigata Prefectural Inlandwater Fisheries Experimental Station, 1998 . As

Ž .mentioned by these researchers, androgenesis may be useful for the following points: 1rapid cloning through androgensis due to shorter generation times of males than females

Ž .in many fish species, 2 maintenance of inbred lines or endangered species in the formof cryopreserved spermatozoa and reconstitution of individuals through androgenesis,Ž .3 monitoring of the effect of cytoplasmic factors on performance by using androgeneti-

Ž .cally produced nucleo-cytoplasmic hybrids of one species, and 4 elimination ofcontamination by spontaneous diploids from accidental polar body inhibition, whichgive rise to the failure of cloning in gynogenetic technique. The sex of androgeneticprogeny is predicted to segregate into XX females and YY supermales in a species thathas a male heterogametic sex-determining system. Sex of androgenetic products isconsidered further in Section 4.

4. The use of chromosome manipulations to investigate sex-determining systems

Chromosome manipulation can be regarded as a powerful approach to examinesex-determinating systems. Such a function is especially important in fish speciesbecause most of them have no morphologically distinguishable sex chromosomesŽ . Ž .Yamazaki, 1983 . In species with male heterogametic system XX female–XY male ,all female progeny are predicted in induced gynogenesis due to no genetic contributionof paternal chromosomes. Such a system can be confirmed by the all-female progeny

()

K.A

rairA

quaculture197

2001205

–228

220

Table 2Sex-determining systems evidenced from sex ratios in gynogenetic diploids studied in Japan

Ž . Ž .Fish species common name Female % Sex determination Other factors influencing sex Reference

a aControl G1 G2

Ž . Ž .M. anguillicandatus loach 60.4 94.9 – XX–XY – Suzuki et al. 1985bw x w x Ž .35.9 208C 100 208C – XX–XY high temperature Nomura et al. 1998

w x w x8.6 258C 78.8 258C –w x w x0 308C 95.2 308C –

Ž . Ž .C. auratus goldfish 58.8 100 – XX–XY presence of varieties with Oshiro 1987ZW –ZZ system within species

44.8 69.2 –0 –

Ž .C. carassius grandculis 57.1 100 – ? high temperature? Fujioka 1997Ž .nigorobunarcrucian carp

90 –63.6 –50 –37.6 –

c cŽ . Ž . Ž . Ž .G. caerulescens 44.7 22.0–73.3 87.2 61.8–100 – XX–XY minor sex-determining gene s ? Fujioka 1998Ž .honmorokorminnow

Ž .Rhodeus ocellatus ocellatus 52.6 13.3 12.5 ZW –ZZ 100% male in 3n, sublethality Kawamura 1998Ž .rosy bitterling in superfemale WW

Ž .P. oliÕaceus 36 48–93 50–88 XX–XY unstable sex differentiation in Yamamoto 1999Ž .hiramerJapanese flounder genetic female; sex reversal by

high and low temperaturedw x w x w x46–68 E 93–100 E 97–100 E2 2 2

ew x w x w xca. 50 It ca. 100 It ca. 100 ItŽ . Ž .L. yokohamae marbled sole 50 100 – XX–XY – Kakimoto et al. 1994b

Ž . Ž .T. rubripes ocellated puffer 66.6 100 – XX–XY unknown reason for Kakimoto et al. 1994agynogenetic male

65 96.545 95.8

77.3

a Ž . Ž .G1 Meiotic gynogens , G2 Mitotic gynogens .bRearing temperature.c Range of rearing temperatures.d Treatment with estradiol-17 b.e Ž .Intermediate temperature 17.5–22.58C .


Ž .derived from the cross between a normal female XX and a sex-reversed gynogeneticŽ .male XX . Table 2 summarizes the type of sex-determining system, evidenced from sex

ratios observed in gynogenetic progeny of the fish species studied in Japan. In speciesŽ .examined, male heterogamety XX–XY appears the most common, with female

Ž . Ž .heterogamety ZW–ZZ only observed in the rose bitterling Kawamura, 1998 . How-ever, even in species with male heterogametic system, various proportions of gynogenticmale have been recorded. Causes for frequent occurrence of gynogenetic males have

Ž .been exhaustively studied in hirame see review in Yamamoto, 1999 . This author foundŽ .that a low dose of ‘estrogen’ estradiol-17b, E made gynogenetic diploids all-female2

by inhibiting spontaneous reversal of sex-differentiation from genetic female to physio-logical male, and thus concluded that there is an XX–XY system in this species.Temperature was also found to affect sex differentiation. When the genetic females were

Ž . Ž .reared under high 25.0–27.58C or low 158C temperature conditions, physiologicalmales developed. Thus, the sex of hirame can be modified by temperature manipula-tions. Such an involvement of temperature on sex ratio has also been suggested in loachŽ . Ž . Ž .Nomura et al., 1998 and nigorobuna Fujioka, 1997 Table 2 . However, an involve-ment of minor multilocus or multiallelic genes is also suggested in some speciesŽ .Oshiro, 1987; Fujioka, 1998 .

Androgenesis is also useful to get insight into sex-determining system. In amagosalmon, an approximately 1:1 ratio was observed, indicating an equal occurrence of XX

Ž .females and viable YY supermales Nagoya et al., 1996 . Supermales are especiallyŽ . Ž .useful for the production of all-male XY population by mating normal females XX

Ž .and supermales YY .

5. Regulation of the use of chromosomally manipulated fish in Japan

Chromosome manipulation is considered a practical means of rapid genetic improve-ment of cultured fish, and is generally not so risky for the environment because it can beregarded as an extension of conventional selective breeding. To minimize ecological andgenetic impacts when such manipulated fish escape or are released, the Fisheries Agencyof Japan organized a committee to prepare guidelines for the commercial use of

Ž .chromosomally manipulated fish CMF . The purpose of the guidelines was to indicateŽ .basic requirements for using CMF and other aquatic organisms in the fisheries

industry, including aquaculture and seed production. Risk assessment of CMF must beperformed in advance by the users. More detailed provisions and texts of the guidelinesfor the requirement of investigating items in the original species and manipulatedorganisms have been already been reviewed and commented upon by Taniguchi et al.Ž .1993 . In Japan now, chromosomally manipulated fish can only be used after approvalby the Government. Such fish and shellfish currently in use under the authorization ofthe Government are shown in Table 3.

6. Final remarks for further integration with molecular techniques

As mentioned in previous sections, sterile all-female triploids have been utilized forŽ .commercial aquaculture of several species Fisheries Agency, 1997 and cloned hirame


Table 3Authorized use of chromosomally manipulated fish according to the guidelines of Fisheries Agency, Japan

Ž .Translated and compiled from the publication of Fisheries Agency 1997 .

Year, month Species Manipulation Application PrefectureŽ .applicant

1994, December amago salmon all-female 3n seed, aquaculture Gifuyamame all-female seed, aquaculture YmagataŽ .landlocked masusalmonrainbow trout all-female 3n seed, aquaculture Naganorainbow trout all-female seed, aquaculture Shiga

1995, August hirame feminization seed, aquaculture TottoriŽ .Japanese floundermasu salmon all-female 3n seed, aquaculture Hokkaidocoho salmon all-female seed, aquaculture Miyagi

1996, June oyster 3n seed, cage culture Hiroshimain the sea inapproved area

amago salmon all-female seed, aquaculture Gifurainbow trout= all-female allo-3n seed, aquaculture Aichiamago salmon

1997, July ayu all-female 3n seed, aquaculture Kanagawarainbow trout= all-female allo-3n seed, aquaculture AichiJapanese charhirame complete homozygotes seed, aquaculture Tottori

Ž .were already in commercial use in 1998 Yamamoto, 1999 . Thus, chromosomemanipulation techniques have now been developed in a number of fish species and arenow approaching to the final goal of practical use: establishment of excellent clonallines exhibiting high growth rate andror disease resistance. However, chromosomemanipulation should not be viewed as an obsolete technology to be discarded, becausefurther advancement and improvements of the technology are predicted, particularlywhere they are used in conjunction with other genetic technology such as DNA markersand transgenic.

Recently, gene mapping has been actively conducted in some aquaculture species toidentify chromosome regions associated with quantitative traits by the development ofdetailed linkage analyses based on hypervariable DNA markers such as microsatellites,

Žminisatellites, and AFLPs Moore et al., 1999; Sakamoto et al., 1999; Danzmann et al.,.1999 . If DNA markers are shown to be closely linked to quantitative traits, marker-as-

Ž .sisted selection MAS can be used for the genetic improvement of finfish. Chromoso-mally manipulated fish are very useful for such a gene mapping. For example, a detailedgene map was reported in rainbow trout by using doubled haploid trout produced byandrogenesis from a hybrid between the two different androgenetically generated

Ž .homozygous lines Young et al., 1998 . Such approaches using heterozygous clone as aparental fish for backcross to a homozygous clone may be promissing for rapid genemapping of fish. Analyses of haploid gynogens has been shown to be an efficient

Ž .method for linkage mapping of tilapia fish Kocher et al., 1998 . Gene–centromere


recombination analyses by using meiotic gynogens and DNA markers have also beenŽ .used to map genes efficiently Estoup et al., 1993 . Thus, integration of chromosomally

manipulated fish and hypervariable markers should make it possible to identify quantita-tive traits on fish genome.

Chromosome manipulation is also considered a method to establish transgenetic linesefficiently, as well as to minimize the risk of escape of transgenics to the wild. Inducedgynogenesis is capable of fixing transgenic genes in the homozygous conditions. Suchan approach has not been reported in Japan, but such developments have already been

Ž .made in mud loach M. mizolepis by Korean scientists Nam and Kim, 1999 . All-femaletriploids can be used for effective biological containment of transgenic fish, so as toprotect wild population from contamination with genetically modified fish.

Acknowledgements

ŽThe author would like to express his sincere gratitude to Dr. C.-S. Lee The Oceanic. ŽInstitute and E.M. Donaldson Emeritus Researcher, West Vancouver Lab., Fisheries

.and Ocean, Canada for inviting him to the AIP workshop. H. Okada, N. KoideŽ . ŽHokkaido Fish Hatchery , K. Katsura Yamagata Prefectural Fisheries Experimental

. Ž .Station , M. Satoh Niigata Prefect. Inlandwater Fish. Exp. Stn. , I. Denda, Y. SawamotoŽ . Ž .Nagano Prefect. Fish. Exp. Stn. , T. Kuwada Gifu Prefect. Fish. Exp. Stn. , Y. FujiokaŽ . Ž . ŽShiga Prefect. Fish. Exp. Stn. , T. Kobayashi Kinki University , K. Tabata Hyogo

. Ž .Prefect. Fish. Exp. Stn. , E. Yamamoto Tottori Prefect. Fish. Exp. Stn. , Y. InadaŽ . ŽFukuoka Marine Technology Center , I. Nakayama, T. Masaoka National Research

.Institute of Aquaculture . The author is also grateful to all the above mentioned personswho gave me valuable comments, suggestions and publications during various stages ofthe preparation of this manuscript.

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