Hydrobiologia 420: 4547, 2000. A. M. Sol -Cava, C. A. M. Russo & J. P. Thorpe (eds), Marine Genetics.

e 2000 Kluwer Academic Publishers. Printed in the Netherlands.

45

Biotechnology and sh culture

F. Foresti
Departamento de Morfologia, Instituto de Biocincias, Universidade Estadual Paulista, 18618-000 Botucatu, So Paulo, Brazil Key words: aquaculture, chromosome manipulation, ploidy

Abstract Biotechnology can currently be considered of importance in aquaculture. The increase in the production of aquatic organisms over the last two decades through the use of biotechnology indicates that in a few generations biotechnology may overtake conventional techniques, at least for the commercially more valuable species. In the last few years, genetics has contributed greatly to sh culture through the application of the more recent techniques developed in biotechnology and in genetic engineering. At present, the most commonly used methods in sh biotechnology are chromosome manipulation and hormonal treatments, which can be used to produce triploid, tetraploid, haploid, gynogenetic and androgenetic sh. These result in the production of individuals and lineages of sterile, monosex or highly endogamic sh. The use of such strategies in sh culture has as a practical objective the control of precocious sexual maturation in certain species; other uses are the production of larger specimens by control of the reproductive process and the attainment of monosex lines containing only those individuals of greater commercial value. The use of new technologies, such as those involved in gene transfer in many species, can result in modied individuals of great interest to aquaculturists and play important roles in specic programmes of sh production in the near future.

Introduction The role of aquaculture in increasing sh production is well recognised today (Donaldson, 1997). Although the basis of sh genetic improvement is roughly similar to that employed for animals, and appreciable progress has been made in recent years in the elds of selection, inbreeding, hybridisation and sex control, the application of such technologies to aquaculture has been limited. One important difference between sh and terrestrial animals for cultivation and genetic improvement is that, usually, sh have higher levels of genetic variation, and hence more scope for selection, than most mammals or birds.

Chromosome manipulation Fish species are generally very tolerant of articial manipulation of their chromosomes during early development and this property has been exploited for the production of inbred lines, monosex populations, and

the control of ploidy. A variety of different techniques has been applied to obtain polyploids, gynogenetics and androgenetics, sex-reversed individuals and transgenics (Purdom, 1993; Toledo-Filho et al., 1996). Methods used include interspecic hybridisation or the control of sex by the administration of sex steroids to larvae or juveniles and even surgical or autoimmune castration. The articial modication of the chromosome set of an organism permits the production of monosex and sterile individuals, while gynogenesis and androgenesis provide methods for the rapid production of inbred populations which can be used in crossbreeding programs. Manipulation of chromosome number to give polyploid individuals is common in aquaculture. The retention of the second polar body during the second meiotic division in the oocyte results in triploid individuals, which have two chromosome sets from the mother and one from the father. Unlike mammals, where chromosomal rearrangements of this magnitude are usually fatal, many sh with three sets of chromo-

46 somes survive quite readily. In addition to triploids, individuals with four sets of chromosomes (tetraploids) or those with both of their chromosome sets derived from a single parent can be produced. Triploid sh are of interest because their sterility is useful in aquaculture and sheries management (Lutz, 1998a). Sterility caused by genetic and physiological factors leads to different characteristics in male and female sexual development. A number of studies have shown that triploid rainbow trout are sterile, whether spontaneously occurring in a population or articially induced through heat or pressure shocks. Male triploid rainbow trout, however, typically undergo considerable changes in secondary sex characteristics and gonad development at the time of maturation, affecting the carcass appearance and reducing meat quality. Female triploids, on the other hand, normally have minimal gonad development and maintain carcass quality throughout the period of maturation of their diploid counterparts. The performance of triploid sh in production situations has been found to be comparable to that of diploid individuals, and the use of triploid females should be considered when they are to be grown past the time of normal sexual maturation. Gynogenesis is the production of offspring with genes from the mother only. In practice, in sh and some other organisms, it is possible to produce offspring from a mature female with no paternal genetic contribution (Purdom, 1993). The technique for producing gynogenetic individuals requires the inactivation of the male genome and the diploidisation of the female genetic material in the zygote, in a process induced by physical or chemical agents. Gynogenesis can be achieved easily in rainbow trout and other species of sh by fertilising eggs with irradiated sperm and inducing polar body retention through the same type of treatments (pressure or temperature shocks) used to produce triploids (Parsons, 1998). In the same way, if the eggs are irradiated and then fertilised by normal spermatozoa, androgenetic individuals can be produced under certain conditions, with only a set of parental chromosomes. These haploids are then changed to diploids by heat shock treatment before the rst cell division. The technique for inducing gynogenesis has been usefully applied to different species of sh (Lutz, 1997a,b; 1998b). Gynogenetic individuals are used to produce all-female sh populations. The reasons for inducing gynogenesis range from the production of monosex populations to the development of partially or completely inbred organisms. Partially inbred offspring (from meiotic gynogenesis) may be useful in genome studies for examining the map position of different loci in relation to the centromeres of their chromosomes. If completely inbred animals (from mitotic gynogenesis) survive to maturity, their eggs can be subjected to a second round of gynogenesis, producing true clones in large numbers. In a successful long-term program based on genetic selection and hybridisation for the development of inbred pure lines of carp in Hungary (Bakos, 1979; Bakos & Gorda, 1995), the use of such a technique resulted in very productive hybrids (Nagy et al., 1979). Although the rst gynogenetic individuals produced in a line typically perform rather poorly due to inbreeding effects, in trout they can be hormonally sex-reversed to functional males that produce only Xbearing sperm. This sperm is then used to fertilize eggs from normal females, producing all-female populations (Donaldson, 1997). All-female rainbow trout and salmon have shown a variety of advantages in aquaculture, including faster and more uniform growth and better carcass yield, particularly in stocks with high rates of male precocity; they have found favour with aquaculturists throughout the world.

Concluding remarks and notes on emerging technologies As seen in the above discussions, genetic technology is rapidly being applied in aquaculture. Hormonal treatments that regulate the action of genes and modify the sex of offspring are now widely used in sh culture programs. Chromosome manipulation, resulting in sterile polyploid individuals or monosex inbred lines, constitutes an important tool. DNA markers are being used to study stock identication and population differences, in gene mapping studies, and potentially as aids to selective breeding programs. Gene transfer work in many species of sh, stimulated by the possibility of producing rapidly-growing individuals through the introduction of foreign growth hormone genes, has produced modied sh in numerous laboratories around the world. Evidence has been documented of the expression of foreign genes introduced into embryonic sh, as well as transmission of the introduced genes by transgenic individuals to their progeny (Iyengar et al., 1996; Dunham, 1999). Once this technique has proved to be effective, and difculties overcome in the optimal genome incorporation and expression of genetic

47 characteristics, a variety of genes with the potential for growth promotion or disease resistance will be of great interest to aquaculturists. To date, a large number of species of sh have been genetically modied using different DNA constructs, reecting the wide application of this emerging technology in sh culture. However, as might be expected, considerable concern has been expressed from philosophical and ethical points of view. Also, the potential negative impact that transgenic sh could have should be considered (Kapuscinski & Hallerman, 1991); this includes negative market impacts (Berkowitz & Kryspin-Sorensen, 1994) and possible interaction of transgenic sh with wild populations. Regulation has been proposed in many countries attempting to control the production and use of transgenic animals (Hallerman & Kapuscinski, 1995). These concerns will cause signicant delay in the direct adoption of gene transfer technologies in sh culture. In the meantime, other less controversial biotechnological methodologies could play a role. While these biotechnologies are being developed and adopted by aquaculturists, new developments in molecular genetics point to the evolution of stem cell tissue cultures and even the growing of totipotent cells, such as egg blastomeres in culture, as future possibilities. Thus, the production of cells containing new gene arrangements, some of them introduced by chromosome manipulation or by gene transfer, could be undertaken in tissue culture to give new combinations of genetic material. Furthermore, the organisation of gene banks based on collections of frozen sperm and cells or on puried DNA molecules, obtained from different sh species, should be considered and could play an important role in the future. References
Bakos, J., 1979. Crossbreeding Hungarian races of common carp to develop more productive hybrids. In Pillay, T. V. R. & W. Dill (eds), Advances in Aquaculture. Fishing News Books, Farnham, Surrey, UK: 633635. Bakos, J. & S. Gorda, 1995. Genetic improvement of common carp strains using intraspecic hybridization. Aquaculture 129: 183 186. Berkowitz, D. B. & I. Kryspin-Sorensen, 1994. Transgenic sh: safe to eat? A look at the safety considerations regarding food transgenics. Biotechnology 12: 247252. Donaldson, E. M., 1997. The role of biotechnology in sustainable aquaculture. In Bardach, J. E. (ed.), Sustainable Aquaculture. John Wiley & Sons, New York. Dunhan, R. A., 1999. Utilization of transgenic sh in developing countries: potential benets and risks. J. Word aquacult. Soc. 30: 111. Hallerman, E. M. & A. R. Kapuscinki, 1995. Incorporating risk assessment and risk management into public policies on genetically modied nsh and shellsh. Aquaculture 137: 917. Iyengar, A., F. Muller & N. Maclean, 1996. Regulation and expression of trangenes in sh a review. Transgen. Res. 5: 147166. Kapuscinski, A. R. & E. M. Hallerman, 1991. Implications of introduction of transgenic sh into natural ecosystems. Can. J. Fish. aquat. Sci. 48: 99107. Lutz, C. G., 1997a. Tinkering with chromosomal complements: gynogenesis an introduction. Aquacult. Magaz. 23: 6771. Lutz, C. G., 1997b. Gynogenesis part II: examining recent work. Aquacult. Magaz. 23: 7378. Lutz, C. G., 1998a. Triploidy: potential and problems. Aquacult. Magaz. 24: 9295. Lutz, C. G., 1998b. Gynogenesis part III. Aquacult. Magaz. 24: 75 78. Nagy, A., K. Rajki, J. Bakos & V. Csnyi, 1979. Genetic analysis in carp (Cyprinus carpio) using gynogesesis. Heredity 43: 3540. Parsons, J., 1998. Status of genetic improvement in commercially reared stocks of rainbow trout. World Aquacult. 29: 4447. Purdom, C. E., 1993. Genetics in sh breeding. Chapman & Hall, London, 277 pp. Toledo-Filho, S. A., F. Foresti & L. F. Almeida-Toledo, 1996. Biotecnologia gentica aplicada piscicultura. Cadernos de Gentica 3, CCS/USP, So Paulo, Brazil.