Genetics of resistance to disease in fishes

Aquacuiture. 85 (1990) 83-1O’i Elsevier Science Publishers B.V., Amsterdam

83 -

Printed

in The Netherlands

Genetics of Resistance to Disease in Fishes BERNARD

CHEVASSUS’

and MICHEL

DORSON’

‘Laboratoire de GCnPtique des Poissons and ‘Laboratoire d ‘Ichtyopathologie, INRA, 78350 Jouy-en-Josas (France)

ABSTRACT Chevassus, 83-107.

B. and Dorson. M., 1990. Genetics of resistance

to disease in fishes. Aquaculture. 85:

The first part of this paper deals with the identification and exploitation of genetic variations in resistance to diseases. At the interspecific level, hybridization may in some cases allow the exploitation of the character of resistance of one of the parents. Polyploid hybrids represent a particular advantage for this approach. Variations between popuIations have been treated in many papers. However, the studies are generally limited to F 1 crosses and do not allow analysis of genetic determinism of the observed variations. Significant variations are also observed between individuals of one and the same population. Several mass selection results using this variability have been developed. The second part deals with disease resistance mechanisms. At the quantitative level, the studies concern the variability of antibody levels, neutralizing serum and mucus activities and resistance to stress; several cases of correlation between these characters and resistance to diseases are presented. The Mendelian approach is particularly developed in the field of histocompatibility genetics. However, the link between this variability and disease resistance remains to be established. The final discussion deals with the respective advantages of quantitative. Mendelian and molecular biology approaches in order to improve disease resistance in fish.

INTRODUCTION

In fish the transition from life in a natural environment to aquaculture is accompanied by several changes, among which some have well-established incidences of pathological conditions: increase in culture densities, frequent degradation of environmental quality, mixing of populations of different origin and manipulations. These are all factors increasing the probability of the advent and seriousness of diseases. Confronted with this situation, pathology research has allowed a progressive setting up of a rather large variety of possible interventions: increasingly specific diagnosis methods, sanitary prophylaxis and disinfection, chemotherapy (particularly by antibiotics) and vaccination will continue to supply new possibilities to breeders.

0044-8486/90/%03.50

0 1990 Elsevier Science Publishers

B.V.

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B. CHEVASSUS AND M. DOBSON

However, several constraints often make such interventions difficult and expensive; thus, prophylaxis based on a strict sanitary isolation, which is relatively easy in terrestrial species, is often more difficult in aquatic environments where the water, other fish species or even invertebrates or other vertebrates constitute potential contaminating agents which are difficult to eliminate. Antibiotic therapy, mostly applied orally, has more and more to cope with resistances in selected bacteria due to repeated treatments. At present, vaccination is only operational for two bacterial diseases (vibriosis, yersiniosis) and even in these two cases problems remain because of the time of occurrence of immunocompetence in young fish as well as the duration of conferred immunity following vaccination by immersion or by the oral route which is sometimes insufficient. The efficiency of vaccination against furunculosis is much debated, and studies under way deal above all with possible explanations of this situation and of course with prospects for solving the problem. In the case of viral diseases, a live vaccine against viral haemorrhagic septicaemia is available but preconceived opinions about live vaccines (justified by some recent cases in higher vertebrates) as well as the limited advantages offered by its launching on a small-scale market have restricted its use. Killed vaccine is only efficient via injection and is therefore obviously limited in practice (see De Kinkelin and Michel, 1984, for a comprehensive review of fish vaccination). This is why geneticists and pathologists are interested in the search for intrinsic resistance factors protecting the individual during part or the whole of its biological cycle during the course of aquaculture development. This paper has two parts: ( 1) identification and exploitation of variations of disease resistance between species, populations or individuals, and (2) analysis of resistance mechanisms and the search for indirect selection criteria. Information on this problem may be found in several recent articles, particularly those of Kinghorn ( 1983 ) , Price ( 1985 ) and Ilyassov ( 198’i 1. We have deliberately limited our attention to papers referring to resistance to rather well-defined pathogens or to a precise mechanism able to intervene in disease resistance. Therefore, the following items are not treated: (a) resistance to natural abiotic factors (temperatures, pH, etc. ), to pollutants (heavy metals, chemicals) or ionizing agents; (b) many articles on “survival rates” in various groups without determining more accurately the causes of the observed mortalities; (c) articles on genetic factors responsible for various malformations of favouring the occurrence of tumours (in this field, see particularly the reviews of Gordon, 1985, and Price, 1985).

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IDENTIFICATION AND ANALYSIS OF VARIATXOSS IN DISEASE RESISTANCE

Interspecific variation Pathogens (viruses, bacteria, parasites) present highly variable host specificities, which are often extremely strict but mostly still very little known. The major reasons for this are: absence of a casual or experimental contact between a pathogen and a potential host, possible variation and adaptation to a new host, especially suspected for viruses (e.g., adaptation of infectious haematopoietic necrosis virus to a refractive species, the coho salmon, reported by Hedrick et al., 1987) with the existence, for one and the same pathogen well defined as a species, of strains differing by several traits and especially pathogenicity. If these restrictions are taken into account it can be considered that the relative susceptibilities of fish species in aquaculture to major pathogens are well determined. Cipriano and Heartwell (1986) showed mortalities due to furunculosis of 25,6 and 0%, respectively, in brown trout (Salmo trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Safmo gairdneri) cultured in the same fish farm. Susceptibility of salmonid species to infections by Ceratomyxa Shasta showed important variations (Zinn et al., 1977); mortality was almost total in Salmo gairdneri, Salmo clarkii and Salvelinus fontinalis. It seemed almost non-existent in Salmo salar and reduced in Oncorhynchus nerka. Viral diseases in salmonid species have been examined extensively (Table 1) and they show large differences even between serotypes of one and the same virus. In order to analyse this interspecific variation, the use of hybridization, which is frequent in plants, is limited in fish by the viability of possible hybrids (Chevassus, 1983). Thus, Ord et al. (1976) found that hybrids between rainbow trout female and coho salmon male (Oncorhynchus kisutch) showed the paternal trait of resistance to VHS (viral haemorrhagic septicaemia). However, due to the low viability of these hybrids (Chevassus and Petit, 1975; Chevassus et al., 1983) this result is not useful. On the other hand, the transmission of the resistance trait is not systematic. In the case mentioned above, the hybrids are susceptible to IPN (infectious pancreatic necrosis) (M. Dorson, unpublished results 1987) whereas coho salmon is resistant. In the same way, Pojoga (1972) found that common carp (Cyprinw carpio) x goldfish (Carassius auratw ) crosses are particularly susceptible to dropsy, just like the goldfish. In American catfish several hybridizations are possible and especially between Ictalurus furcatw and Ictaturus punctatus. The viability of the hybrids is good but, like the two parental species, they are susceptible to CCV (channel cattish virus) (Smitherman et al., 1983). However, increased interest for this method is to be expected from the use of polypIoidization (see, among others, Chourrout, 1987; Chevassus, 1987; Nagy, 1987). Several authors have shown that triploid hybrids with high viability

B. CHEVASSIJS AND M. DORSON

86 TABLE 1 Interspecific variation of resistance of the principal following natural challenge (waterborne virus) Species

salmonid

Birnavirus

Rhabdovirus

IPN

IHN

VHS Serotype

Salmo gairdneri S. trutta S. salar Salvelinus fontinalis S. alpinus 5’. namaycush Oncorhynchus kisutch 0. tshawytscha 0. nerka 0. rhodurus

s (1) s (2) R (273) s (1) s (2) R (24) R (1) R (1) s (5) s (5)

S (6) ? s (7) ? ? ? R (8) S (6) S (6) S (6)

species to the main viral diseases

S (9) R (10)

1

Serotype 2

Serotype 3

S (9) S (9)

S (9) S (9)

R (11)

?

R R s R R

? ? ? 7

(2) (2) (2) (IO) (12) ? ?

3

? ?

R (11) R (2) R (2) s (2) ? ? ? ?

( 1) Dot-son, 1983 (review paper). (2 ) M. Dorson, unpublished results 1986. S. trutta appears less susceptible to IPN compared to S. gairdneri. S. namaycush appears less susceptible to VHS compared to S. gairdneri and almost completely resistant to IPN.(3) One clinical case reported, all experimental trials gave negative results (Munro et al., 1983).(4) Silim et al., 1982. (5) Sano, 1973. (6) Pilcher and Fryer, 1980 (review paper). (7) Mulcahy and Wood, 1986. (8) Adaptation of IHN to coho salmon is in progress (Hedrick et al., 1987). (9) De Kinkelin, 1983 (review paper ). ( 10) De Kinkelin et al.. 1974. ( 11) De Kinkelin and Gastric. 1982. S. solar is susceptible only to injection with the virus. (12) Ord, 1975. S = Susceptible, following field or laboratory challenge, to at least one strain of the virus. R = Resistance established (no disease in infected areas nor experimental transmission). ? = No clue available concerning susceptibilities or resistance. The different serotypes have been mentionned only when interesting differences are known. IPN = Infectious pancreatic necrosis. IHN = Infectious haematopoietic necrosis. VHS = Viral haemorrhagic septicaemia.

could be obtained in cases where the diploid hybrids showed low viability; this is particularly the case with salmonid species (Chevassus et al., 1983; Scheerer and Thorgaard, 1983; Arai, 1984) and with cyprinids (Vasilev et al., 1975; Allen, 1983). For salmonid species it has been shown that, despite the existence of a gene assortment in favour of the susceptible species, triploid hybrids in some cases keep the paternal trait of resistance to viral diseases (Table 2). The farming advantage of such triploid hybrids should be examined case by case. Their sterility often constitutes an important advantage (this is the case with the Ctenopharyngodon ide1l.a~ Hypophthalmichthys nobilis cross studied by Allen, 1983) but the same result can be obtained in triploids of pure species. On the

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GENETICSOFRESISI'ANCETODISEASEINFISHES

TABLE 2 Resistance to virus in triploid hybrids between salmonid species: mortalities (results expressed in mortality percent ) Virus

Exp.

Parental RT2

VHS (1)

84 84 85 86 86

A B A B

36 70 68 90 80

(2) (3) (0) (1) (0)

trials

Hybrids”

species’ RT3

cs3

RTxCS

RTxBT

0 (2)

0 (0)

80 (0) 69 (0)

VHS (2)

84 A 85 86A

92 (2) 90 (0) 96 (0)

IPN (1)

84 A 85

72 (20) 85 (12)

92 (19)

81

84

IHN (2)

in standardized

5 (3)

0 (2) 4 (3)

93 (0)

11

68 (22) 62 (18)

7 (1) 27 (1) 0 (0)

4 (1) 33 (2)

76 (14)

21

RT2 = Diploid rainbow trout: RT3 = Triploid rainbow trout: CS2 = Diploid coho salmon. RT x CS = Triploid hybrid rainbow trout female x coho salmon male; RT X BT = Triploid hybrid rainbow trout x brook trout male. ( 1) Dorson and Chevassus. 1985; and unpublished data, 1985. (2 ) Parsons et al., 1986. “Value of uninfected control given in parenthesis.

other hand, their production performances do not always justify their advantage except in areas where the pathogen is a permanent danger (see among others Quillet et al., 1987,1988 in salmonids). The sterility of these hybrids also constitutes a handicap insofar as it is impossible to manipulate the trait of resistance by future crosses (F2 or backcrossing). Such a manipulation could be considered in the case of fertile diploid hybrids (see among others Chevassus, 1979,1983) but as far as we know it has not yet been done. More recently, the development of tetraploidization techniques (see in particular the review of Chourrout, 1987) should allow this area to be extended, as tetraploid hybrids, if they are viable, are fertile a priori as shown by observations made on tetraploids of pure species (Chourrout et al., 1986a; Chourrout and Nakayama, 1987; Diter et al., 1988). It would then be possible to make an introgression of the trait of resistance and to optimize farming performances of the hybrid.

88

B.CHEVASSUSAhDY.DORSON

Another useful approach is that of sperm irradiation in limited doses (around 50 000 rad) and gynogenesis. It is then possible to obtain gynogenetic fish with some residual paternal inheritance. This technique was used by Thorgaard et al. (1985) and Disney et al. (1987) who fertilized rainbow trout ovules by irradiated brook trout sperm. Certain progenies do express some enzymatic functions which are specific to the brook trout and the use of this method to transfer traits of resistance to diseases from one species to another is mentioned by the authors. Variutions between populations

Several data (Table 3) are available on the variation for disease resistance between natural and farmed populations. They relate in particular to resistance to bacteria in salmonid species and many examples are given in Kincaid’s (1981) survey of salmonid strains in American hatcheries. In 11 American hatchery strains of rainbow trout, Cipriano (1983) found mortalities ranging from 0 to 83% in a test of resistance to furunculosis (Aeromonas salmonicida). Brown trout and brook trout have also been subject to many studies in the U.S.A. (Wolf, 1953; Snieszko et al., 1959; Ehlinger, 1964,1977) which, by strain selection, led to a considerable increase in the level of this resistance: Wolf (1953) showed variations between strains in mortality ranging from 5.3 to 97.8% in brook trout and from 35.7 to 65.8% in brown trout (Fig. 1). In Atlantic salmon, Gjedrem and Aulstad (1974) mentioned only slight variations in mortality due to vibriosis in 14 wild populations of Norwegian Atlantic salmon (0.87-8.90% ) whereas it reached 29.7% in a Swedish populations tested under the same conditions. There are also considerable differences in resistance to viral diseases; during 6 years, Okamoto et al. (1987) performed several tests on resistance to IPN in three Japanese hatchery strains of rainbow trout and showed a stable hierarchy between strains (Fig. 2). Silim et al. (1982) found large variations in susceptibility to IPN between three strains of brook trout, with mortalities ranging from 30.9 to 72.3%. With respect to parasites, Zinn et al. (1977), in their work (already mentioned) on Ceratomyxa testedvarious hatchery strains of chinook salmon (Oncorhynchus tshawytscha) and observed variations in mortality ranging from 0 to 100%; the most resistant strains came from zones where the disease is endemic (Columbia Basin). In other species, considerable variations are also shown. Plumb et al. ( 1975) noticed variations in mortality ranging from 12 to 72% among six strains of American catfish (Ictalurus punctatus) after infection with CCV. Among four inbred European lines of common carp of the same cultures, Hines et al. ( 1974) found two presenting a relatively high frequency of either an epidermal epithelioma infection or a swim bladder infection with Aeromonas Liquefaciens. The other two strains were not infected. In this species, numerous comparisons

GENETICS OF RESISTANCE TO DISEASE IN FISHES

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TABLE 3 Examples of genetic variation populations)

Virus

in fah (B = between populations;

W = within

Disease

Species

Level

Ref.

IHN

Sockeye salmon Oncorhynchus nerka Rainbow trout Salmo gardnieri Channel catfish Ictalurus punctatlLs

W

B

Amend and Nelson, 1977; McIntyre and Amend, 1978 Silim et al., 1982; Okamoto et al., 1987 Plumb et al., 1975

Atlantic salmon Salmo salar Rainbow trout

W

Anonymous,

W

Cipriano,

IPN CCV

Bacteria

for disease resistance

Furunculosis

Furunculosis and ulcer disease

Brown trout Salmo trutta

B

B, W

Brook trout Salvelinus fontinalis

1985

1983

Embody and Hayford, 1925: Hayford and Embody, 1930; Wolf, 1953 Snieszko et al.. 1959; Ehlinger. 1964,1977; Cipriano and Heartwell, 1986

Bacterial kidney disease

Coho salmon Oncorhynchus kisutch

W

Suzumoto et al., 1977

Vibriosis

Atlantic salmon Rainbow trout Sockeye salmon

B. W W B, W

Gjedrem and A&tad. Refstie, 1982 Smoker. 1986

Swim bladder inflammation

Common carp Cyprinus carpio

B

Hines et al., 1974

Dropsy

Common carp

B, W

Ilyassov, 1987; Schaperclaus, 1962; Merla, 1959; Pojoga. 1972

Ceratomyxa

B Chinook salmon Oncorhynchus tshawytcha

Zinn et al., 1977

Ichthyophthirius

Xiphophorus maculatus

W

Bone, 1983; Price and Bone, 1985

Bothriocephalus

Common carp

W

Kozinenko

Fungus

Saprolegnia

Sarotherodon aureus

W

Tave et al.. 1983

9

Gastritis

Chanos chanos

B

Smith, 1978

Parasites

et al., 1985

1974

B. CHEVASSIJS

90

AND M. DOBSON

100 00 60 40

20

0 1

2

3

4

5

6

7

0

9

10 G&S

Fig. 1. Between-strains variability of resistance to ulcer disease and furunculosis in brown trout and brook trout: results of a lo-months trial with strains of North-East U.S.A. (from Wolf, 1953). Mortality %

a l

100

200

K N

300

r 5 0.97 r.0.29

400

500

600

Weight (mg)

Fig. 2. Susceptibility to two strains (K and N) of rainbow trout (Salmo gairdneri) fectious pancreatic necrosis virus) (from Okamoto et al., 1987).

to IPN (in-

between populations have been undertaken, especially in the Soviet Union (see Ilyassov, 1987, a review), in particular on infectious dropsy, a disease which may involve several bacterial taxons (Aeromonas, Pseudomonas) or a rhabdovirus (De Kinkelin et al., 1985 ). While many data are available on variations between populations for disease resistance, there are only a few works on the behaviour of these traits of resistance in crosses between populations. Taking the example of Hines et al. (1974) on inbred strains of common carp, all Fl crosses between the four strains are unaffected by the infections noticed in two of the pure strains. In the same way, Pojoga (1972) showed that crosses between a Roumanian hatchery breed

GENETICS OF RESISTANCE TO DISEASE IN FISHES

91

of common carp susceptible to dropsy and a wild resistant breed have good resistance towards the disease. A similar result was obtained by Sijvenyi et al. (1988) in a cross between the Hungarian and the Japanese coloured common carp for the resistance to erythrodermatitis. The work of Plumb et al. (1975) on viral diseases in American catfish also concluded in favour of a resistance in crosses at least equal to the best populations even in crosses between a rather susceptible and a resistant population (mortalities 29,13 and 9%, respectively, for the two populations and their cross). It should, however, be noticed that, in these four studies, between-populations crosses often exhibited significant heterosis for growth and that this factor may play a role in “general resistance” to aggressions which should be estimated. On the other hand, while this “recessive” behaviour of susceptibility to diseases seems to be rather generally accepted in Fl crosses, as far as we know, no precise study has been made on the phenomena involved in F2 and backcrosses. The genetics of these traits of resistance are thus rather little known. Variation within populations A study of the variation within populations, and particularly hereditary segregations, leads to a better knowledge of the genetics of observed differences (Table 3 ) . Significant variations are frequently found between groups of full-brothers: incidence of white spot infection (Ichthyophthyrius multifiliis) in Xiphophorus maculates (Bone, 1983; Price and Bone, 1985), susceptibility to IHN ranging from 52 to 98% in sockeye salmon, Oncorhynchus nerka (Amend and Nelson, 1977). However, a genetic interpretation of this variation is difficult because, in addition to genetic effects that can be selected, it involves dominance and maternal effects. The existence of such maternal effects up to a certain age has in particular been confirmed by the experiment mentioned above on Xiphophorus maculatus (Price and Bone, 1985). Factorial or hierarchical genetic experimental designs allowing a more precise estimation of genetic effects that can be selected (heritability) have also been developed and they lead to various estimations given in Table 4. Although these estimates are inaccurate, they are high enough to expect a response to selection. Such selection work was successfully carried out in several species. In brook trout, Embody and Hayford (1925) selected systematically the surviving fish in a population with furunculosis. The survival rate at the age of 6 months changed from 2% in the initial population to 69% after selection during three generations. Ehlinger (1977) completed his strain selection programme of brown trout and brook trout by a test on family selection which was promising in brown trout but, for practical reasons, was finally interrupted. In a more recent work on this disease, Cipriano and Heartwell (1986), using an indirect criterion, which we will come back to, obtained in one generation, by divergent

92

B.CHEVASSUSANDM.DOBSON

TABLE 4 Between-families

variations and heritability of disease resistance

Disease

Species

Experimental design

Range of survival

Heritability estimate

Ref.

Vibriosis

Atlantic salmon

14 factorial sets (2dX49) 3 factorial sets (4dX49) 3 factorial sets (3dX4?) 13 hierarchical sets (Wx3to19O)

Not indicated

0.115-0.007

1

18-98%

0.32

2

Not indicated

Not indicated (Significant) Not indicated

3

Atlantic salmon Chum salmon Rainbow trout

(Between males) 39-99%

4

IHN

Sockeye salmon

15 hierarchical sets (ldX3?)

2-70%

0.31; 0.27; 0.38

5

IPN

Rainbow trout

50 d x Pool of 9

O-5.5%

Not indicated

6

VHS

Rainbow trout

14 d x Pool of 9

(Between males) 3-72%

0.69 f 0.25

6

(1) Cjedrem and Aulstad, 1974. (2) Anonymous, 1985. (3) Smoker, 1986. (4)Refstie, 1982. (5) McIntyre and Amend, 1977. (6) M. Dorson and B. Chevassus, unpublished data, 1984.

mass selection, two groups of brown trout (Salmo trutta) with in-the-field mortality rates due to furunculosis of 2 and 48%) respectively. In carp, several studies conclude in favour of the efficiency of mass selection to diminish susceptibility to dropsy. The work of Schiiperclaus in Germany (see review in Schliperclaus, 1962) on the selection of breeding fish presenting no apparent sign of the disease, showed an average mortality rate of 11.5% in 65 ponds vs. 57% in 76 ponds stocked with progeny of non-selected breeding fish. Later, a systematic injection of Pseudomonas in clinically healthy breeding fish was recommended in order to increase selection efficiency. In the Soviet Union similar work conducted by Kirpichnikov (see review of Ilyassov, 1987) made it possible in one population to lower the mortality rate from 51 to 10.7% within five generations. APPROACH TO RESISTANCE

MECHANISMS

The fact that a fish or a group of fish resists a pathogen is an extremely complex phenomenon which may have multiple reasons varying from one fish (or one group) to another. The pathogen may not penetrate into the organism

93

GENETICSOFRESISI'ANCETODISEASEINFISHES

because of a barrier or destruction effect of mucous, gastric or intestinal secretions. It may also penetrate but be inactivated by spontaneous (bactericidal serum effect, macrophages, complement, acute phase proteins, killer cells) or induced mechanisms (interferon or antibody production). In addition to its fundamental interest, the study of the mechanisms responsible for this resistance and their determinism offers the possibility of establishing new selection criteria: (a) leading to a quantified individual evaluation of the level of resistance whereas tests with pathogens supply a binary response; this is particularly important in mass selection experiments in which the sole conservation of surviving individuals leads to reducing selection pressure more and more and consequently the efficiency of this selection as it advances; (b) requiring no contact between fish and pathogen, which allows the selected population to be kept free from diseases without using complex schemes such as selection on progeny or collaterals; (c) liable to be involved in resistance to several diseases and therefore to simultaneously improve several levels of resistance. There are two distinct approaches to the work on this problem; one is quantitative, the other is Mendelian. The quantitative approach Several studies have attempted the identification of a quantitative of the characters most likely involved in disease resistance.

variation

Antibody levels. The conventional experiments of Biozzi in mice (Biozzi et al., 1970, 1971, 1984) clearly showed that it is possible by divergent selection to modify both the level of antibodies secreted as a response to a given antigen and the macrophage activity as well as the relationship between these two immunoacting criteria and resistance to various diseases. Several experiments have been devoted to this approach in fish: Refstie (1982) studied the development of serum antibodies against Vibrio anguillarum serum in rainbow trout families after an undefined immunization. Antibody titres remained low so that it was impossible to identify large variations and the calculated heritability (0.15-0.25) was relatively inaccurate. Cossarini-Dunier et al. (1986) studied, in the same species, the level of antibodies induced by the synthetic antigen DNP-KLH. They emphasized the existence of an interindividual variability of antibody titres which is much higher than in mammals. By using inbred lines procreated either by self-fertilization or by gynogenesis, they obtained in some cases a significant reduction in intragroup variance compared to the controls, a phenomenon which is in favour of genetic factors controlling the level of immune response. However, the use of inbred lines is somewhat difficult to interpret; within-line variability may either de-

9. CHEVASSUS AND M. DORSON

94

crease because of the genetic parental relations of the fish or increase because of their inbred character. In carp, Salomoni et al. (1987) also observed a significant difference between two lines in the level of antibodies against sheep erythrocytes (SRBC) where the difference was higher the larger the density of fish. Although they are in favour of genetic effects on the level of antibodies, these approaches do not yet allow these levels of antibodies to be related to the resistance to any disease, as in the case of mammals. On the other hand, this relationship seems to exist for other substances which are naturally present in the serum and mucus. Neutralizing activity of the serum and mucus. Cipriano (1983) distinguished

11 strains of rainbow trout for two serum characters: (a) agglutinating activity against formolized cells of Aeromonus salmonicida ( furunculosis agent); (b) neutralizing activity against a protein fraction of this toxic bacterium for cultured RTG2 trout cells. This characterization was made on healthy fish. It thus concerned activities which were not pathogen-induced. The 11 strains were then tested in a standard test against furunculosis. The observed mortality rates, ranging from 0 to 83%, were not bound to the agglutinating activity of the serum. On the other hand, they presented a high correlation (rz0.90) with the neutralizing activity (Fig. 3). In a later work, already mentioned (Cipriano and Heartwell, 1986)) a batch of brown trout was exposed to furunculosis and presented mortalities of around 25%. The mucus of surviving individuals was used in an immunoprecipitation test against extracts of the bacteria. The diameter of the precipitation halo was used as a divergent individual selection criterion. In an environment contaminated with furunculosis, the progeny of the two couples with the strongest loo T

Mortality 96

y= 142.382-16.611x

60.

R = 0.90

40 -

20 -

neutralizing activity

O-

I 4

5

6

7

a

Fig. 3. Natural anti-Aeromonos neutralizing activity of the serum and post-challenge mortality in 11 strains of rainbow trout (from Cipriano, 1983).

GENETICS OF RESISTANCE TO DISEASE IN FISHES

9.5

reaction presented a cumulative mortality of 2% at the age of 6 months vs. 48% for progeny of fish with a lower reaction. Moreover, 77% of the survivors of this group were infected with the bacteria vs. only 12% for the resistant batch. Finally, there was a significant divergence in response to immunoprecipitation between the two groups. It should be noticed that the precipitating activity of the mucus was also found in non-infected fish and that it acted on other bacteria like Vibrio anguillarum and Aeromonas hydrophila. It is thus clearly distinct from an antibody activity, which is specific and induced. Stress and resistance to diseases. The possible relationship between the susceptibility to stress and diseases has often been pointed out. However, as far as we know, the only study on this problem is not conclusive (Refstie, 1982). Progenies of 13 rainbow trout males were characterized by their mortality in an infection with vibriosis and by the level of cortisol induced by stress (30 min in shallow water). A significant variation for these two criteria was noted between the groups but there was no significant correlation between them (r= 0.05). The Mendelian approach Research on genetic factors with simple Mendelian determinism controlling disease resistance offers and interesting alternative to the quantitative approach insofar as, at least theoretically, it allows rather quick fixing of the best performing genotypes as homozygotes and then continuation of the genetic improvement of the other characters within a “resistant” gene pool. However, Mendelian factors exhibiting a priori a functional link with disease resistance (pleiotropic factors) should be considered rather than genetic markers presenting a more or less close linkage with resistance characters. Genes with visible effect. Such studies have long been limited by the possibility of revealing factors with a Mendelian determinism. The first observations thus concerned primarily factors with a visible effect. In the work of Hines et al. (1974) in carp, the strain which was susceptible to swim bladder infection was that determined by two recessive factors of colouring, “blue” and “grey”. In this species, Merla (1959) (in Schiiperclaus, 1962) also reported that the strains showing the character “absence of scale” were more susceptible to dropsy due to Aeromonas punctata than scaly strains. This trait determined by an allele, N, which is lethal in the homozygous condition, might have a marked depressing effect on the viability of heterozygous fish. In Sarotherodon aureus, Tave et al. (1983) found a dominant lethal mutant, the saddleback phenotype, modifying the body conformation and which was more susceptible to Saprolegnia infection than the wild type. However, a generalization of the largest susceptibility to diseases of these variants with visible effects should only be made with caution, insofar as the studies do not always

B. CHEVASSUS AlriD M. DOBSON

96

accurately indicate whether within-population segregations of the character or line comparison are concerned. In the case of line comparison, the possible development of larger inbreeding effects in mutant lines which are often created and maintained in a low number, may partly account for their lower viability. Protein polymorphism. The development of techniques able to detect protein polymorphism by electrophoresis has led to numerous studies on the viability of different genotypes. Suzumoto et al. (1977) studied the link between the susceptibility of coho salmon to bacterial kidney disease (BKD) and plasmic transferrin polymorphism. It is assumed (Weinberg, 1974) that the availability of iron can regulate bacterial growth and that the transferring can modify plasmic availability of this element by the capacity to bind iron. A comparison between three genotypes, AA, AC and CC, clearly showed a link with susceptibility to bacterial diseases. Mortalities were 34.4, 18.8 and lO.O%, respectively, and the rates of haematologically normal fish after infection were O/36, 3124 and 2/7. The authors also pointed out that the genotype CC, which was the most resistant, was particularly abundant in the population involved, but this was not the case for other hatcheries. Finally, there was no obvious importance of a lack of iron in the modulation of the susceptibility to the disease; complementary injection with Fe3+ ions neither increased the virulence of the disease nor modified the ranking of the three genotypes (Fig. 4 ). Another type of polymorph protein has been the subject of genetic studies in fish; Cossarini-Dunier et al. (1986) and Desvaux et al. (1987) studied Ig-M antibodies, particularly in trout. An interesting preliminary result was the significant reduction in Ig-M diversity in inbred fish obtained by gynogenesis or self-fertilization. It has been shown in higher vertebrates that, even in inbred Mortality

%

n q q

60 50

EKD only BKD + Fe (HL) BKD c Fe (LL)

m 40

0 AA

AC

CC

Fig. 4. Percentage of mortality with three transferrin genotypes (AA, AC, CC) of coho salmon after experimental infection with BKD (bacterial kidney disease) (Fe HL=iron high level; Fe LL = iron low level 1. ( Suzumoto et al., 1977 ) .

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GENETICS OF RESISI’ANCE TO DISEASE IN FISHES

lines, numerous somatic re-organizations led to a large antibody diversity between and within individuals (Pink and Askonas, 1974). The observed reduced diversity by parental effect in fish, which has also been found in amphibia (Brandt et al., 1980), leads one to think that somatic re-organizations are less numerous and perhaps of a different nature, according to the study of Makela and Litman (1980) and Litman et al. (1982,1985) in Heterodontus, one of the elasmobranchii. It would then be possible to reach the germinal register of antibodies and to search for possible links with disease resistance in fish. Histocompatibility. The genetic factors controlling histocompatibility have been widely studied in mammals, birds and amphibia but only a few data are available in fish. Methods of transplantations in fish have long been known. Mori (1931), Goldrich and Nichols (1933), and then Hildemann (1957) (in Avtalion et al., 1988) developed a technique of scale transplantation in the goldfish, CarassiLls auratw; Kallman and Gordon (1957) developed a technique of fin transplantation in Xiphophorus species. The use of this method in the analysis of genetic determinism of histocompatibility has been largely developed by the works of Kallman and his associates. Thus, using inbred lines of Xiphophorus, Kallman (1964a) estimated the minimum number of loci involved and found rather high values, sometimes up to about 10 (Table 5). In another study, Kallman (1964b) showed a clear increase in crossed transplantation tolerance as a function of the increase in relationship between individuals. These methods were then developed in order to confirm the clonal character of progenies of different species presenting an original reproduction mode: gynogenesis in Poecilia formosa (Kallman, 1962) and in a triploid Poeciliopsis (Moore, 1977 ), self-fertilization in Riuulus marmoratus (Kallman and Harrington, 1964; Harrington and Kallman, 1968). Kallman (1962) insisted that this technique allows the simultaneous study of the clonal (genetic identity of individuals) and homozygous character of animals. Even if the two characters are bound in the case of some reproduction systems (self-fertilization, gynogenesis with retention of the second polar body) this is not the case for TABLE 5 Estimation of the maximal number of loci involved in tolerance to fin grafts in Xiphophorus species (from Kallman, 1964a) Host

Donor (Lines)

Species

H. maculatus

H. couchianus

Lines

JP30

JP163

AP

NP

JP30 JP163

* 3

3

6-8 5

10-11 7-8

l

lo- 15 10-15

98

B.CHEVASSUSANDM.WRSON

some other (gynogenesis with premeiotic endomitosis; Cimino, 1972) in which the clonal condition coexists with part of the genome in a heterozygous condition. However, the extension of such studies is limited by the need for inbred lines to investigate the segregation of markers which is possible only in a small number of aquarium species, often with unisexual reproduction. Assuming a genetic determinism based on n loci, each with numerous alleles, the probability of obtaining two histocompatible individuals within one sibship of two unrelated breeding fish varies from 0.25 ( 1 locus or n linked loci) to 0.25” (n independent loci) and is thus low a priori. The development of methods of induced gynogenesis (see Chourrout, 1987, for a review) has considerably increased the range of species that could be investigated. Gynogenesis by retention of the second polar body (the most controlled one at present) does not lead to rapid fixing of homozygosity (see Chevassus, 1987) but to a rapid a priori acquisition of histocompatibility within lines (Fig. 5). This possibility was especially explored by Nagy et al. (1983) who showed that the rejection of scaly grafts was total in goldfish offspring (Carassius auratus or Carassius auratus x Carassius carassius) whereas examples of tolerance appeared from the first generation of gynogenetic goldfish and the second carp generation, In carp, within-line compatibility became total in the fourth generation (Fig. 6). Avtalion et al. (1986) made a similar study in tilapias (Oreochromis aureus) and found in four out of six cases histocompatibility between gynogenetic offspring of the same female. Another promising approach to tissue compatibility is that using the cytotoxicity reaction between lymphocytes which do not carry the same surface 0 . q

Hl H2 Gl

0.01 0.0

0.2

0.4

0.6

0.0

1 .o

Fig. 5. Probability of homozygosity (H) and within-lines graft tolerance (G) after one and two generations of gynogenesis (one locus model). r = Post-reduction rate; Hl = 1- r; HZ = I- r2; G2=32-r+r”).

99

GENETICS OF RESISTANCE TO DISEASE IN FISHES Tolerance 1001

+

%

Hybrid

60 40 20 -

days 0

I

1

0

20

Tolerance

+ +

0

G4G-G2N G2N-G4G

10

20

Fig. 6. Evolution of allograft tolerance in different genotypes of goldfish and common carp (after Nagy et al., 1983). Hybrid= Full-sib family female Carassius auratus x male Carassius carassius. Pure = Full-sib family female Carassius auratus x male Carussius auralus. Gyno=First generation of induced gynogenesis. G I= First generation of induced gynogenesis; G2 = second generation of induced gynogenesis; G4 = fourth generation of induced gynogenesis. N, B, G are different lines. G4-G2 and GZ-G4 = transplantation between lines.

antigenetic determinants (mixed leucocyte reaction: MLR). It allows an in vitro approach which is more easy and rapid than in vivo transplantations (Ellis (1977) in S&no s&r; Caspi and Avtalion (1984) in carp). Using this technique, Gloudemans et al. (1987) showed in carp the segregation of four histocompatibility groups in one sibship. The interpretation by a simple monofactorial model is sufficient but little compatible with the data of Nagy et al. (1983) already mentioned and based on scale grafts. On the other hand, as far as we know, no study has been made in fish on the links between histocompatibility polymorphism and disease resistance. This field, which has been largely expiored in higher vertebrates (see notably Tiwari

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and Terasaki (1985) in man, Lamont et al. (1987) in chicken, Spooner in bovines), probably constitutes an important future topic.

(1986)

DISCUSSIONANDCONCLUSION Three aspects will be discussed: future development of studies on quantitative genetics; use of major genes; introduction of molecular biology methods. The quantitative approach Numerous examples of quantitative variations of disease resistance have been supplied. Such variations result in several types of problems. In theory, the possibility of a response to selection has been questioned a priori by several authors (see notably Moav and Wohlfarth, 1973; Kirpichnikov, 1981). The argument derived from Fisher’s (1930) theorem was that natural selection which acts on the characters bound to survival had probably a priori considerably reduced the additive genetic variability of these characters. As emphasized in the Introduction, this argument is hardly useful for a variety of diseases whose incidence has probably increased since the species have been exposed to new conditions in aquaculture. On the other hand, even if positive results were obtained by selection, the exploration and use of non-additive genetic variability constitute a field which has only been little investigated and may thus be promising. Development of non-specific heterosis effects for a given disease, via a rapid creation of inbred lined by endomitosis constitutes an interesting orientation, In practice, the use of these selection schemes presents some problems. The development of experimental infections in individuals or groups is a complex method which requires specific equipment. On the other hand, the binary character of the response (dead/alive) is a problem to geneticist and especially that of the decrease in the pressure and thereby of selection efficiency as the number of resistant individuals increases. This is why we once again stress the advantage of indirect selection criteria leading to an individual characterization of healthy animals. The use of such criteria eliminates the criticism mentioned before and really allows the integration of characters of disease resistance to be considered in the evaluation of spawners. The development of these criteria will require a close cooperation between geneticists and pathologists, but is certainly promising for the future. Another problem for the incorporation of disease resistance characters in an improvement scheme is the correlation between characters. It may concern either a correlated response between two diseases or an effect on another performance trait. Thus Ehlinger (1977) reported a larger susceptibility to gill diseases in the strain of brook trout selected on resistance to furunculosis. Several authors also mention a low susceptibility to diseases in animals or in groups with the largest growth: Ehlinger (1964) in brook trout, Plumb et al.

GE?iETICS OF RESISTANCE TO DISWSE IN FISHES

101

(1975) in American catfish. However, these data are extremely incomplete and the precise analysis of genetic correlations between characters remains to be done. Another problem seldom mentioned is that of genetic variability of pathogens. Ehlinger (1964) dealt with this problem by testing systematically strains of brown trout and brook trout with a mixture of bacterial strains of geographically different origins. In the already mentioned experiment of Silim et al. (1982) on brook trout, the between-strain variation of resistance to IPN seems more or less marked according to the viral strains used. Such factorial analyses between several strains of pathogens and several strains of fish are worth being developed in order to detect possible interaction effects. Major genes Despite the fact that works on Mendelian genetics in fish have existed for a long time, the link between Mendelian polymorphism and resistance to diseases still remains to be established. The development by geneticists of techniques which make it possible rapidly to reach the clonal condition as well as the elaboration by pathologists of in vitro histocompatibility tests, constitute two recent approaches which should allow advances to be achieved. Taking into account the pioneer work to be done, we think, however, that this approach could hardly contribute to an improvement of disease resistance in the short term. Molecular biology possibilities The possibilities offered by gene transfer which may modulate the resistance to a disease will mainly be discussed. However, attention should be drawn to the advantage of molecular probes for detecting in fish sequences which are homologous to the mammalian sequences controlling, for instance, histocompatibility (HLA or H2 probes) or antibody synthesis (Wilson et al., 1986). As far as gene transfer methods are concerned, we mentioned in the first part of this report the methods of transfer of chromosomal fragments (Thorgaard et al, 1985; Disney et al., 1987). They seem, however, less promising than those using gene transfer by microinjection into the fertilized egg (Chourrout et al., 1986b; Guyomard et al., 1989) which allows one to obtain lines integrating the genes in their chromosomes in a stable way. In the field of resistance to diseases two approaches can be considered: ( 1) gene transfer controlling the synthesis of a product which inhibits an early phase of the biological cycle of the pathogen. It could particularly be the pathogen’s own proteins which are responsible for the adhesion of the pathogen to the cell surface, especially in the case of viral diseases. The synthesis by the host of these proteins may lead to saturation of the cell receptors, thus preventing the fixation of the virus; (2) antisense gene transfer, synthesizing a messenger RNA which is comple-

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mentary to the normal sequence of the virus. In this case a dimer between the RNAm of the virus and that synthesized by the host would be formed and the translation of the viral genome would be inhibited. We will not give details here of the advantages and limits of these two approaches which are likely to be illustrated by numerous works in the future. ACKNOWLEDGEMENT

We thank Marianna Perrier for the English translation.

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