2. REPORT OF THE TECHNICAL SESSIONS

2.1 Panel 1. Genetic bases of species improvements in aquaculture

The scope of this panel was to scrutinize know facts, equipment and methods used and gaps in knowledge, mainly at the population level of three groups of aquatic organisms of interest to aquaculture, viz. finfish, crustaceans and molluscs.

2.1.1 Population genetics of coldwater and warmwater finfish

Sufficient genetic variation to permit improvement programmes to be carried out probably exists in most species of fish. This has been shown both by allozyme studies (measuring the level of heterozygosity, the proportion of polymorphic loci and/or the mean number of alleles per locus), and by employing quantitative traits. Also, it is a well established fact, that the electrophoretic analyses have revealed that the predominant part of this variation is expressed between individuals at the intrapopulation level whereas only a minor portion is expressed at the interpopulation level even though, particularly in salmonids, this is large enough to establish breeding programmes. Needless to say, also quantitative traits have been shown to be stock specific, like e.g. growth rate, migratory behaviour and disease resistance.

The most important gap in our knowledge is the lack of “coordination” between monogenic (biochemical) data and polygenic (quantitative) traits. Thus few polymorphisms may be correlated with a polygenic character like, e.g. growth and age at maturation. A major obstacle, leading to reduced variability and fitness, is that basic genetic principles have been ignored in designing aquaculture production systems. This applies equally to monitoring natural stocks and with regard to selection programmes. Another problem may be the general lack of electrophoretically detectable variations, even though there may be ample supplementary data proving extensive variability.

2.1.2 Population genetics of crustaceans

As with finfish, there is already substantial information on the population-genetic structure of crustaceans. This information is derived both from classical studies in the polychromatisms of many groups and from more recent electrophoretic studies. The genetic variation detected is considerable and, again, comparable to that of fishes. Thus, there appears to be sufficient variation to support genetic improvement programmes. The techniques employed to detect and utilize this variation are by and large identical to those employed for finfish, viz. electrophoresis of allozymes.

A major gap with regard to the potential for utilizing these genetic resources is the lack of control over crustacean reproduction processes. Ignorance of basic genetic principles employed elsewhere in animal husbandry has led to pronounced inbreeding and subsequent genetic drift.

2.1.3 Population genetics of molluscs

Most knowledge on the population genetic status of mollusc species has been gathered among oysters and blue mussels. A large amount of genetic variability exists in most of these bivalves, but in contrast to the genetic equilibrium generally displayed in allozyme gene frequencies of fish and crustaceans, bivalves frequently appear to show excessive heterozygote deficiency. This is likely to be the result of the bivalve reproduction system. Electrophoresis is also the most common tool here for the evaluation of the genetic variation, but there are also promising results from studies on mitochondrial DNA, e.g. in blue mussels. A technique for culture of mussel glochidia and juveniles is proposed.

Gaps in knowledge occur with regard to the phenotypical plasticity of many bivalves. This renders taxonomic studies difficult. Electrophoretic data often provide additional criteria, with the help of which new species have even been detected. Sampling may be very laborious when applying mito-chondrial DNA studies, and, if insufficient material is available, cloning will be necessary.

2.2 Panel 2. Selective breeding and intraspecific hybridization

2.2.1 Critical review of methodology for finfish

The establishment of selective breeding and intraspecific hybridization programmes and the determination of genetic variation are important considerations in aquaculture.

The initial step should be the establishment of breeding goals, preferably in response to industry needs. The usual aims are improved growth rates, feed conversion efficiency, survival, meat quality and the adjustment of age at first maturation. While individual selection is less expensive and permits intensive selection, there is an increased danger of inbreeding which should be avoided by appropriate methods like systematic crossing between selected lines. The most efficient selection, particularly when selecting for several traits, is to combine family and individual information in a selection index.

2.2.2 Critical review of methodology for shellfish

Conventional guidelines have been proposed for mollusc selective breeding programmes. It was suggested that any programme for shellfish improvement should be tailored to the different rearing conditions of molluscs.

The reported results of selected breeding of molluscs are variable and the consequences of heterozygosity are unclear. It would seem advisable to concentrate on those traits which are under artificial control in the hatchery and nursery. Excessive inbreeding should be avoided by the use of adequate numbers of breeding individuals.

2.2.3 Selective breeding and intraspecific hybridization of coldwater finfish

Salmonids are the only coldwater fish where genetic parameters have been extremely investigated. All breeding programmes should begin with a comparison of strains. The environment where growth and selection takes place should approximate farming conditions. A parallel investigation of husbandry methods and improvement should maximize commercial gains. The greatest gains have been made in growth and in timing of maturation, notably involving additive gene effects.

Selection based on feed conversion efficiency and apparent protein digestibility does not appear promising and heterosis appears to be of little importance. Measurements of immune response of various strains suggest promising group differences but these have not yet been tested for correlation with respect to disease tolerance.

2.2.4 Selective breeding and intraspecific hybridization of warmwater finfish

Intensive selection can lead to the reduction of the effective size of the population resulting in gene losses. Adequate methods should be developed in order to maintain sufficient effective size under selection. A gene bank such as for Hungarian landrace carp can maintain basic populations for genetic work. As hybridization becomes more popular, a register for different genotypes using genetic markers should be established.

Although productivity in carp, catfish and tilapia has been improved by intraspecific hybridization, information on heritability of quantitative characteristics is inadequate. Genotype-X environment interaction requires further investigation and so do observed differences in food conversion ratios. Tolerance to disease and environmental stress are complex factors of importance for future research programmes.

2.2.5 Selective breeding and intraspecific hybridization of molluscs

There is great variation in heritability value studies conducted on oysters, mussels and pearl oysters due to difficulties of design and maintenance of large-scale experiments for quantitative genetics. Information on correlation of various traits is lacking.

Some success has been reported for selection for disease resistance, growth, tolerance of thermal stress, and oyster shell weight and colour, but few results have been tested in commercial operations. Several studies indicate improvement in growth and survival as a result of intraspecific hybridization and heterosis, but there is a need to understand its genetic, physiological and biochemical bases.

2.2.6 Selective breeding and intraspecific hybridization of crustaceans

Very little work has been done on intraspecific variation in crustaceans but genetically diverse populations exist. Work on the freshwater prawn Macrobrachium rosenbergii has shown distinct populations but the races are uniform for economically important carcass characteristics. A base population from which individual selection for size will be conducted, has been constructed from interracial hybrids. However, non-random association among loci in the base population (gemetic disequilibrium) may confound the interpretation of selection response.

2.3 Panel 3. Interspecific hybridization

Summarising the different documents presented under Panel 3, it was agreed that the advantage of using interspecific hybrids is limited, in so far as significantly better growth and feed conversion rates are concerned. But, it is obviously correct that, in some cases, valid interspecific hybrids combine valuable characters from both of the parental species.

It was stated by several authors and mostly agreed by the audience that interspecific hybridization is not a reliable tool for aquacultural practice, except in some special cases such as producing unisex specimens and other related genetic manipulations such as the production of gynogenetic and androgenetic lines.

Although interspecific hybrids may represent an alternative for stocking natural habitats without the risk of destroying natural gene pools by introgression of genes from the stocked populations, some caution should be exercized against their broad and indiscriminant use for such a purpose. This is because in some cases interspecific hybrids are known to be fertile and thus potentially capable of breeding with related natural species. Stocking a natural habitat with artificially produced hybrids may also destroy the existing ecological structure in a given environment.

Some other ideas, at the present of mostly theoretical or laboratory/-experimental value, such as those suggested by Dr. Longwell's review, are: (i) molecular changes in chromosome structure; (ii) introgression by directed substitution or addition of a chromosome or of parts of it; (iii) sub-chromosome mediated gene transfer; and (iv) hybridization by cell fusion. These ideas which go beyond the topic of interspecific hybridization will be discussed under Panel 4.

But, even if such methods of genetic engineering become generally available, they would not be readily transferable to aquaculture practice because of a lack of facilities for carrying out such highly sophisticated methods under field conditions.

2.4 Panel 4. Genetic manipulations

Genetic manipulations in fish and shellfish are aimed at the same goal as selection, crossbreeding or hybridization, i.e. the genetic improvement of commercial performances. Although they may act as partners to these classical methods they may also offer unique solutions for specific problems like the control of reproduction. Despite some preliminary attempts with self-fertilization or gene transfer, the genetic manipulations essentially consist of modifications of the chromosome set as gynogenesis, androgensis, auto- and allo-polyploidy.

2.4.1 Critical review of methods

Haploid gynogenesis and androgenesis. The genetic inactivation of sperm required for the induced gynogenesis is obtained by different techniques of which ultraviolet irradiation is more practical and ensures full inactivation of the genome. Genetic markers are recommended for confirmation of maternal inheritance. Egg inactivation for androgenesis is obtained by gamma-irradiation of the ovule. In shellfish, physical or chemical agents can efficiently activate the egg, but ultraviolet inactivated sperm is also very effective.

Suppression of meiosis II. Triploids and heterozygous gynogenetics have now been attained in the major commercial fish by cold, heat and pressure treatment applied early after insemination. These three agents might be fully efficient in all species but, in practice, thermal shocks are easily implemented. Additional studies, however, are needed to elucidate whether the action of these agents actually results in equivalent chromosome complements. In shellfish, triploid production is fully feasible but for reasons not yet established gynogenesis has not been demonstrated. Because shellfish eggs are more vulnerable to chemical action, agents such as cytothalasin-B can be used.

Suppression of first mitosis. The success of mitotic inhibition until now has been restricted to several species. Both pressure and heat shocks appear to be effective. Homozygous diploids and tetraploids are better suited as broodstock rather than for production. Therefore, the practical advantage of thermal shocks, i.e. economically treating large numbers of eggs, is not so evident. Tetraploids, at least in salmonids, are fertile.

2.4.2 Performances for aquaculture

Heterozygous gynogenetics. The gynogenetics diploidized by inhibition of meiosis II are in general poorly viable and in fact, are not as inbred as it had been a priori supposed. Use for inbreeding is however foreseeable, particularly by alternating generations of gynogenesis and within-line matings. Application of inbred lines will be of multiple utility, for example for the development of bioassay animals for physiological and endocrinological investigations and in selective breeding programmes.

Diploid gynogenesis with meiotic II suppression followed by sex reversal, represents a shortcut to start the monosexing instead of progeny testing to identify sex-reversed individuals; this approach can be used when sex determination models involve strict heterogametic systems. Gynogenesis can be a tool for analysing sex determination but must be considered along with other information.

2.4.3 Sex control of exotic fish

The utilization of exotic fish in aquaculture may require additional consideration beyond genetic improvement. Escape or direct stocking of exotics from aquaculture facilities demands consideration of sex control as a security against unwanted reproduction and potential naturalization. The various techniques of sex control used in aquaculture for enhancement purposes will also provide security for prevention of naturalization. Security should be viewed as a two-step process: (i) assessment of environmental risk; and (ii) modification of reproductive potential. The specific acceptable risk will determine the complexity of applied sex control. For example, direct induction of triploidy may be adequate in some instances while other situations may require greater security and thus complexity, such as triploid monosex populations. The capacity to provide reproductive control for exotics should be utilized as a component of protocol for introductions of exotic fish.

2.4.4 Triploidy

Triploid fish and shellfish are viable. Although their performances are usually not better than those of diploids until the onset of sexual maturity, they surpass them as adults. These better yields are related to their lower investment in gonadogenesis: triploid females have under-developed ovaries and therefore a much higher carcass index than diploid females. Triploid males have partially developed testes and also a good carcass index, but because some maturity is evident, secondary sex characteristics may develop. Recent data suggests that triploids produced by tetraploid × diploid crosses may be more vital during non-reproductive stages of life history.

Triploid hybrids. Hybrids may combine the desirable properties of two different species; triploid hybrids seem to reduce concomitant genomic incompatability. Disease resistance in rainbow trout × coho salmon triploid hybrids and salinity tolerance in Atlantic salmon × arctic char triploid hybrids are examples of characters that have been combined in interspecific crosses. Triploid hybrids are likely to be more sterile than diploid hybrids, and they are viable in some cases where the diploid hybrid is not.

2.4.5 Tetraploidy

Tetraploids may be an effective means of producing triploids by back-crossing to diploids in fish or shellfish, although tetraploid shellfish have not as yet been produced. Meiotic tetraploids, i.e. inhibition of both polar bodies, may be a useful approach in shellfish. Storing diploid sperm produced by tetraploids may be an effective means to reconstitute gene pools via and rogenesis.

2.4.6 Homozygous diploids

Completely homozygous diploids produced by mitotic gynogenesis or androgenesis are viable. For inbreeding, they represent a better and more promising avenue than meiotic gynogenesis.

2.4.7 Conclusions

Genetic programmes involving manipulated genotypes should be considered according to the predictability of gains, i.e. on an a priori basis. For example, triploidy seems to be a plus in all situations, producing large animals. On the other hand, the early schemes of improvement involving gynogenetic lines should not be particularly encouraged until simpler and cheaper methods have been tested such as mass and strain selection or ordinary cross-breeding.

2.5 Panel 5. Case studies of large-scale breeding programmes

Seven review papers were presented, mainly dealing with salmonids, common carp, European and American catfish, oysters and abalones. Subjects treated included genetic variability, individual selection, intra- and inter-specific hybridization, genotype-X environment interactions and chromosome manipulations.

Genetic variability for traits under investigation was demonstrated in most cases. Individual selection was instrumental in increasing the growth rate and other production traits in channel catfish, tolerance to a parasitic infestation in oysters, and to temperate tolerance in abalones. No response to individual selection was found for growth rate in common carp or tilapia. Crossbreeding between strains resulted in increased growth rate in common carp and channel catfish. Tolerance of oysters to parasite infestation was not affected by strain crossing. Hybridization between species is in commercial use with tilapias, and with catfish a promising interspecific hybrid has been isolated. Genotype-X environment interactions were demonstrated in common carp by differential growth of Chinese and European stocks under different aquacultural management. Gynogenesis is applied to common carp for the production of inbred lines for improved crossbreeding performance. In abalones, induction of triploidy (by first polar body retention) resulted in increased temperature tolerance.

Practical application of these breeding plans to commercial fish farming is restricted to common carp culture in Hungary and Israel, channel catfish farming in the USA, and salmonid culture in Norway. Wider application of available knowledge in other areas and to a broader range of cultured species is recommended.

2.6 Panel 6. Conclusions and recommendations

The purpose of this session was to examine the conclusions of the five preceding sessions and to approve their recommendations, see Chapter 3.

Panel 1: The necessity of preserving natural genetic resources was approved but it was emphasized during the discussions that few data were available concerning the real consequences of mixing genetically differentiated populations. Therefore, long-term surveys of the genetic structures of natural populations should be complemented by studies on disturbed populations.

Panel 2: Participants agreed that the efficiency of genetic improvement programmes was well demonstrated by several contributions. However, the question of the relative efficiency of the different methods should still be documented by theoretical and experimental studies. In the same way, the creation of control populations and studies on the physiological basis of quantitative traits should be encouraged.

Panel 3: The rather general lack of heterosis after interspecific hybridization was recognized by the participants. However, the interest of this method for creating original genotypes for example by the introduction of a specific trait, or production of monosex populations, was underlined, in connection with the methods discussed in Panel 4.

Panel 4: Participants agreed that genetic methods were interesting for controlling sex and reproduction for intensive aquaculture, as well as for the experimental introduction of exotic species.

On the other hand, they underlined that other applications such as developing inbred lines or gene transfers are still experimental and that their potential interest for aquaculture has not been properly evaluated. Such methods, although promising, should not be presently considered as alternatives to classical genetic improvement programmes based on selection or crossbreeding. However, their interest in providing experimental material for other scientific fields (e.g. pure lines for physiology or pathology) was underlined.

Panel 5 and general discussion: The question of preservation of genetic resources was discussed. Two types of problems were mentioned: (i) technical aspects including control of cryopreservation techniques, and procedures for maintaining synthetic populations; and (ii) theoretical difficulties by population genetics. In connection with this question, the Symposium recognized the initiative taken by the ICES Working Group on Genetics in promoting the development of an international register of available strains of fish and shellfish. It wished to record its support for this initiative which should enhance the growth of genetic benefits to aquaculture.