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324

BLOOD TYPES IN PACIFIC SALMON

INIarTne Biological Laboratory!

1960 WOODS HOLE, MASS«

SPECIAL SCIENTIFIC REPORT- FISHERIES No. 324

UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE

SPECIAL NOTE

The International North Pacific Fisheries Commission, established in 1953 by the Internationjil Convention for the High Seas Fisheries of the North Pacific Ocean, coordinates the research of the member nations: Japan, Canada, and the United States. The resulting investigations provide data to the Commission for use in carrying out its duties in connec- tion with fishery conservation problems in the North Pacific Ocean. Publication of this scientific report has been approved by the United States Section of the Commission.

United States Department of the Interior, Fred A, Seaton, Secretary Fish and Wildlife Service, Arnie J. Suomela, Commissioner

BLOOD TYPES IN PACIFIC SALMON

by

George J. Ridgway Eind George W. Klontz

Fishery Research Biologists

Bureau of Commercial Fisheries

Contribution No. 12 to research conducted with the approval of the United States Section of the International North Pacific Fisheries Commission.

United States Fish and Wildlife Service Special Scientific Report — Fisheries No. 324

Washington, D. C. January 1960

11

TABLE OF CONTENTS

Page

Introduction 1

Material and methods 3

Results 3

Discussion 6

Acknowledgments 7

Summary 7

Literature cited 7

111

BLOOD TYPES IN PACIFIC SALMON

by

George J. Ridgway and George W. Klontz

U. S. Fish and Wildlife Service

Seattle, Washington

ABSTRACT

Intraspecific differences in erythrocyte antigens (blood types) were shown to occur in four species of Pacific salmon, the sockeye or red salmon (Oncorhynchus nerka). the chinook or king salmon (O. tshawvtscha). the chum salmon (O. keta), and the pink salmon (O. qorbuscha) ■ Antisalmon-erythiocyte sera prepared in rabbits and chickens were used after absorption of species-specific antibodies. Some of these blood types were shown to differ in their frequency of occurrence between different geographic races. In addition, isoimmunizations were conducted on one race of sockeye salmon. Antisera of seven different specificities were prepared and at least eight different patterns of antigenic composition were displayed by the cells tested.

These results indicate that considerable antigenic diversity exists in salmon. Reagents to detect valuable markers for the investigation of geographic races of salmon should be obtained through further research.

INTRODUCTION

One of the most importcint problems in fishery biology is recognition of "races", or reproductively isolated subpopulations , of fishes. Since there may be little or no interbreeding or recruitment between such populations, successful regulation or management of fishery resources must be con- ducted in such a way as to recognize their independence.

Present methods for distinguishing between the races of a particular species of fish are based on tagging by various means, or by seeirching for morphometric or meristic differences between the races (Rounsefell and Everhart 1953).

It has been shown with many animals (Mayr 1942, Dobzhansky 1951), including fishes (Gordon 1947), that reproductively isolated subpopulations of species differ in the frequencies of one or more varieible genes. Therefore, a very useful adjunct to the methods of racial identification, now used in fishery management and research,

would be the recognition and definition of genetically controlled polymorphic charac- ters. One class of such characters which has been found in every animal adequately investigated is intraspecific antigenic variations in the red blood cells. These characters are more simply known as blood types.

Lcumdsteiner (1900) was the first to demonstrate the occurrence of blood types. He found them in man with natural isoagglu- tinins. In the same year Erlich and Mor- ganroth (1900) demonstrated blood types in goats using immune isohemolysins induced by transfusions. Subsequently, many addi- tional instances of intraspecific antigenic differences have been demonstrated. The results are most notable in the case of man (Race and Sanger 1954), cattle (Stormont et al. 1951) and chickens (Briles et al. 1950).

In each of these species, blood types have been used to study reproductively isolated populations. Thus, blood type frequencies have been used to characterize

races of men (Boyd 1950, Mouremt 1954), breeds of cattle (Owen et al. 1947) and inbred lines of chickens (Schultz and Briles 1953).

The studies of Fujino (1958) demon- strating the occurrence of blood types in whales are of particular interest to marine biologists.

Until recently there have been rela- tively few published studies concerning the possible existence of blood types in fishes. Some of these demonstrated marked interspe- cific differences in cellular antigens but provided little or no evidence for intra- specific heterogeneity of such antigens. Noguchi (1903a, b) found that the serum of several species of fishes would agglutinate the red blood cells of other species but no intraspecif ic differences were noted. He did produce isoagglutinins and isohemoly- sins in two species of turtle, Chrysemys picta and Chelopus guttatus, demonstrating that cold blooded vertebrates may possess intraspecif ic antigenic differences, Toth (1932) found no evidence for the existence of blood types in carp from 280 cross- matches. Jensen (1937) tested for natural isoagglutinins in the cod (Gadus morrhua) and also attempted to demonstrate individual differences with the serum of a single rabbit immunized with the red cells of an individual cod. He did not find any conclu- sive evidence for the existence of blood groups in cod.

Suyehiro (1949) reviewed the previous Japanese literature in which there were no instances of the demonstration of blood types in fishes. He was evidently the first to demonstrate individual antigenic differences in fish blood cells since he found a few instances of natural isoagglu- tinins in the eel (Anguilla japonic a) and the gilthead (Sparus swinhonus Gimther). In 249 crossmatches of blood cells and serum from cod (Gadus macrocephalus) he found no evidence for natural isoaggluti- nins. Suyehiro also tested blood samples from 336 fishes of 30 different species with human ABO blood typing sera and found that only 91 were agglutinated. In 21 of the 30 species, individuals varied in their reactivity to the human sera tested.

Gushing and Sprsigue (1953) studied the agglutinative activity of human anti-A and anti-B sera and rabbit antisheep cell

serum for the erythrocytes of a number of species of fish. Considerable £intigenic diversity was noted between species but no individual differences were found within the members of the species tested.

Recent reports have indicated a major breakthrough in the efforts to discover blood types in fishes. Hildemann (1956) applied the method of isoimmunization, which has been so successful in detecting blood types in other animals, to goldfish (Carassius auratus) , One of the isoimmune sera detected six different antigenic types of goldfish. Immune rabbit sera were also prepared by Hildemann, one of which de- tected a single antigenic difference after careful absorption. Gushing (1956) re- ported the existence of individual anti- genic differences in the oceanic skipjack (Katsuwonus pel amis Linnaeus) detectable with natural isoagglutinins, normal bovine serum, and the sera of rabbits immunized with the whole blood of oceanic skipjack, albacore (Germo alalunga Gemlin), or Pacific mackerel (Pneumatophorus japonicus diego Ayres), Gushing and Durall (1957) discovered and analyzed a natural isoagglu- tinin system in the brown bullhead (Icta- lurus n, nebulosus Le Sueur). This system was found to be analogous to the human ABO system in that four antigenic types were found (i.e., some fish possessed antigen 1, some antigen 2, some both antigens, and some had neither), and when an antigen was lacking, its corresponding isoagglut inin was always present. Ridgway, Gushing, and Durall (1958) found quantitative differ- ences in the reactivity of the cells of individual sockeye salmon (Oncorhynchus nerka Walbaum) with natural antibodies from pig sera and demonstrated that there were significant differences between geographi- cally separated populations in the frequency of the different types detected.

Suzuki et al. (1958) have demonstrated the existence of blood groups in species of tunas amd have presented evidence which suggests that differences exist between the blood type frequencies of albacore from the Pacific and Indian Oceeins.

The present report provides further evidence for the existence of blood types in Pacific salmons and additional evidence that some of these characters differ in their frequency of occurrrence in different races of the same species.

MATERIALS AND METHODS

Most blood samples were taken in the field by severing the caudal artery with a sharp knife and collecting the spurting blood in sterile bottles. Some of the samples, including those used in isoimmuni- zations, were taken by cardiac puncture. The clotted samples were maintained on ice or in a refrigerator until used. Samples excessively hemolysed or over ten days old were not used for testing. Blood cells from samples as old as three weeks were used for animal inoculation. Cells for testing were washed three times in 10 to 50 volumes of modified Alsever's solution (Bukantz et al. 1946) and adjusted to a 2- percent concentration in this solution. The use of the Alsever's solution as a suspending medium was found to be essential since salmon red blood cells lysed in saline or phosphate-buffered saline solutions.

Most antisera were prepared by giving intravenous or intraperitoneal injections of 0.5 cc. of a 50 percent suspension of washed cells. Such injections were given to rabbits and salmon three times a week for three weeks and to chickens every three days for three or four injections. Four to twelve days after the last injection the cuiimal was bled and serum collected. In most cases, in order to detect individual differences, several additional stimulations at intervals or two weeks to a month were required. Some rabbits also received sub- cutaneous inoculations of washed suspensions of particulate material from lysed red cells in Freund's adjuvcmt. The sera were preserved by freezing and stored at -30° C. Serum dilutions were made with phos- phate-buffered saline or 1 percent saline solutions.

Absorptions were performed by mixing a 1/2, 1/5 or 1/10 dilution of the heat- inactivated antiserum with an equal volume of washed packed cells, incubating for one hour at room temperature or in the refri- gerator, centrifuging the cells down and decanting the absorbed serum. Usually more than one absorption was required to remove all of the antibody present which would react with the absorbing cells.

Tests were performed by mixing 0.1 ml, of absorbed serum, diluted if necessary, with 0.1 ml. of 2 percent cell suspension

Table 1, — Individual antigenic differences demonstrated by agglutinin absorption tests in the erythrocytes of sockeye salmon from Cultus Lake and Adams River,

(Rabbit antisockeye salmon-erythrocyte serum R19 absorbed and cross-tested with the cells of individual salmon)

		Test	cells
Absorbing		Cultus Lake	Adams River
cells	1	15 19 12 13	15 11 « 13	8

Cultus Lake 1 Cultus Lake 15 Cultus Lake 19 Adams River 15 Adams River 11 Adams River 9 Adams River 13 Saline control

+	+	0	+	0	+	+	+	+	+
0	0	0	+	0	0	0	0	+	0
0	0	0	0	0	0	0	0	0	0
0	0	0	+	0	0	0	0	0	0
0	0	0	0	0	0	0	0	0	0
0	0	0	0	0	0	0	0	0	0

in Alsever's solution in 10 x 75 mm. tubes. Dilutions are expressed as the final dilu- tion of the original serum, taking into account the dilution by the red cell sus- pensions. Readings were made after suit- able incubation periods at room temperature and usually after overnight incubation in the cold. The settling pattern was judged either smooth (S) or rough (R) and the degree of agglutination scored as 0, +_, 1 plus, 2 plus, 3 plus and 4 plus. In order for a test to be considered, the saline control had to be smooth and negative.

RESULTS

The major part of our effort has been directed toward finding blood types in red or sockeye salmon (Oncorhynchus nerka Walbaum). Toward this end we have immunized 26 rabbits and 20 chickens with the washed erythrocytes or stroma from members of this species. For each of the other species of Pacific salmon we have made 2-8 rabbit and chicken anti-erythrocyte sera. Individuals or pools from individuals from the same area were used. These antisera were ab- sorbed with the erythrocytes of individual salmon from other areas and the resulting sera tested for residual activity for the cells of a number of individuals. With most antisera produced, numerous absorptions did not reveal any evidence for antigenic het- erogeneity within this species. However, blood group differences were demonstrated with a few rabbit immune sera. One of these sera (R19) was analyzed by absorption with the cells of sockeye salmon collected in 1955 from Cultus Lake and the Adams River,

Table 2. — Serological differences between races of Fraser River sockeye salmon.

	Number tested	Number reacting wi prepared by absorb R19 serum with	th reagents ing rabbit cells of	Number not reacting
Area (test cells)	Cultus Lake 19	Cultus Lake 1		Cultus Lake 15	with any of the three reagents
Cultus Lake Adams River		17 17	3 11	2 17		12 16	4 0
Chi-square		7.6*	56.6*		3.3	4.5*

* Significantly different at the 95 percent level.

both tributary areas on the Fraser River in British Columbia (table 1). The three samples tested from the Adams River race removed all or nearly all of the antibodies present in the serum. The three samples from the Cultus Lake race each removed all of the antibodies specific for their anti- gens, but left cintibodies which would react with cintigens present on the cells of other individual tested. Thus, three reagents of different specificites were pro- duced.

Chinook salmon from the Columbia River, resulted in the production of reagents of four different specifici- ties. The results indicate a charac- teristic is present in number one which is absent from the other three. Superimposed on this, there appears to be a kind of subtype difference between 2, 3, and 4 which is shared with 1.

Evidence for blood groups in chum salmon (0. keta) is presented in tables 4 and 5. The experiments out- lined in these tables were performed with samples from the Samish River in Washington State. Evidence for the presence of a considerable amount of antigenic heterogeneity in this species and this race is provided by the results pre- sented in table 4, since reagents of four different specificities were obtained by five different absorptions.

Five patterns of reactivity were dis- played by the samples tested; numbers 1, 6, 8, and 9 reacted with the sera absorbed by 2, 4, and 5; numbers 2 and 7 reacted with

These results as well as addi- tional tests made on sajnples col- lected from these areas in 1956 with the above absorbed sera are combined in table 2. Statistically signifi- Ccuit differences in the proportion of individuals reacting positively with these reagents are apparent for the two populations.

Some of the antisalmon red blood cell sera, prepared in rabbits and chickens, were useful in demon- strating blood group differences in species of salmon other than the one used for immunization. This is somewhat analogous to the demonstra- tion of the Rh blood groups in humans by Landsteiner and Weiner (1941), through the use of antirhesus monkey red blood cell sera.

The evidence for blood groups in Chinook or king salmon (0. tsha- wytscha) is presented in table 3. Absorption of serum R13, an anti- sockeye salmon red blood cell serum, by the cells of four different

Table 3. — Demonstration of individual antigenic differences in Chinook salmon erythrocytes using rabbit antisockeye salmon serum (Serum R13 absorbed and cross-tested with four individual samples of Chinook erythrocytes).

Absorb]	■ng	ce	11	Tes	t ce	11	Dilutions of	absorbed	sera
sample	numbe	rs	sample	numbers	1:10	1:20	1:40	1	:80
1(	a)				1 2 3 4		0 0 0 0	0 0 0 0	0 0 0 0		0 0 0 0
2					1 2 3 4		++++ + 0 0	0 0 0 0	0 0 0 0		0 0 0 0
3					1 2 3 4		++++ + 0 0	+ + + 0 0	0 0 0 0		0 0 0 0
4(b)				1		++++	++++	+++		+
					2		++++	+++	0		0
					3		+++ +	+++	++		0
					4		+	0	0		0

(a) This absorbed serum tested against the cells of an additional 12 chinook salmon, none of which reacted. Positive reactions were obtained with the cells of 12 different sockeye salmon.

(b) This absorbed serum tested against the cells of 24 additional chinook salmon. Definite reactions occurred with 16 of them.

Table 4.

the sera resulting from absorption by 1 and 5; number 4 reacted with the sera resulting from absorption with 1, 3, emd 5; number 3 reacted with sera from absorption with num- bers 1, 2, 4, and 5; and number 5 reacted with the sera from absorp- tion with numbers 2 and 4. The re- sults obtained with individuals 2, 4, and 7 demonstrate some of the com- plexities involved in the antigenic differences between individuals of a single species. All gave the same reaction with the reagents prepared, except for the weak reaction of the cells of individual 4 with the re- agent prepared by absorption with the cells of individual 3. Consist- ent with this is the fact that reagents of identical specificity were prepared by absorption with cells of individuals 2 and 4. In contrast, absorption by cells of individual 7 removed the antibodies reactive with the cells of all individuals tested, but the cells of individual 7 failed to react detectably in agglutination tests with reagents prepared by cibsorption with cells of individuals 2, 3, or 4. Such apparent deviations from the principles upon which absorption analyses are generally assumed to rest have been associated, in other spe- cies, with the multiple-allelic control of sets of related but nonidentical specifici- ties. The large amounts of cells required to absorb the antibodies present in this serum which would agglutinate the cells of all members of the species, precluded further investigation of this system.

Absorbing
cell sample
numbers	1	2
1	0	+
2	++	0
3	0	0
4	+++	0
5	+++	+++1
7	0	0

-Demonstration of blood types in chum salmon using chicken antichinook salmon serum

(Serum 202 absorbed and tested with cells of individual chum salmon)

Test cell sample numbers 3 4 5 6 7

+ ++00 +

++ 0 ++++ ++ 0 + 0 + 000

++ 0 +++ ++ 0 ++ +++

+++ +++

++ ++++

The results presented in table 5 are less complicated with apparently a single difference detected between chum salmon sample 18 and the others tested. Superim- posed on this however are rather marked differences in the strength of reactivity of the other cells tested which may indi- cate additional antigenic heterogeneity.

Evidence for the existence of blood group differences in pink salmon (0. gor- buscha) is presented in table 6. A single difference was noted which appeared to be correlated with the area of origin of the samples tested. However, positive state- ments about area differences must await analysis of a larger number of samples.

In addition to rabbit 2ind chicken immunizations, extensive isoimmunizations

Table 5. — Demonstration of blood types in chum salmon using chicken antisockeye salmon sera.

Sera

11

12

13

Test cell sample number 14 15 16 17 18 19 20 21 22 23 24

Serum 210:
Absorbing ce	11
No. 18		+	++++	+++
No. 20		0	0	0
No. 23		0	0	0
No. 24		0	0	0
Serum 220:
Absorbing ce	11
No. 18		++	++++	++
No, 20		0	0	0
No. 23		0	0	0
No. 24		0	0	0

++ ++++ ++++ ++++

0 0 0 0

0 0 0 0

0 0 0 0

n- ++++ +++ +++

0 0 0 0

0 0 0 0

0 0 0 0

0 +++ + +++ +++ ++++ +++

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0	++	+	++	++++	+++	++
0	0	0	0	0	0	0
0	0	0	0	0	0	0
0	0	0	0	0	0	0

Table 6. — Demonstration of blood groups in pink salmon

(Antiserum chicken E213 absorbed with red blood cells from Cordova pink salmon #6)

Sashin Creek

Kodiak Cordova

Sample number Reaction

12 3 4 5 6 7

13 4 5 9

+ + + + +

have been conducted on sockeye salmon (0. nerka). The fish used in all of these experiments were from the Columbia River (where this species is known as the blue- back salmon). In 1955 and 1956, 20 to 30 adult salmon migrating up the Columbia River were captured and isoimmunized over periods of one to two months without any detectable development of isoimmune anti- bodies. Since many of these fish died before an adequate period for the produc- tion of isoimmune antibodies had elapsed, no conclusions about the antigenic het- terogeneity of salmon could be drawn from these experiments.

In order to have fish available for isoimmunization over a longer period, a group of sockeye yearlings were obtained from the Winthrop Hatchery of the U. S. Fish and Wildlife Service in 1956 and reared in salt water at the Deception Pass Marine Research Station of the State of Washington Department of Fisheries. Iso- immunizations involving 100 fish, 50 pairs being cross-immunized, were started in August of 1957. During the period of this experiment the water temperature ranged from 14° C, to 9° C. with a mean of appro- ximately 12* C.

Of these 100 fish, 15 produced iso- immune antibodies after periods of 7 to 14 weeks and 5 to 11 inoculations. Because of the small size of these salmon, due to their being reared in captivity, only small amounts of the isoimmune sera could be collected. In addition, the sera were of low titer, most reacting only to 1 in 4 dilution. Sufficient amounts of serum were obtained from seven of these fish to com- pare the specificity of the reactions against a number of individuals.

The results of these tests are pre- sented in table 7. Inspection of these data reveals that all seven of the sera possessed different patterns of specific-

ity, and at least eight different patterns of antigenic composition were shown by the cells tested. Thus, it would appear that the extent of antigenic variability in salmon is of the same order of magnitude as that found in other animals which have

^ been extensively studied. Further

study and application of this hetero- geneity will depend on the availabil- ity of larger salmon, and experimental

facilities for holding them.

DISCUSSION

The demonstration of blood group dif- ferences in four species of Pacific salmon, as outlined in this paper, along with the demonstration that, in some cases, these characters cEin serve as markers of racial identity, indicates the existence of valu- able tools for the solution of many of the population problems encountered in the mjuiagement and conservation of these impor- tant fishes. However, more research and developmental work must be done. One of the biggest problems is in the production of sufficient type-specific sera to test large numbers of individuals from popula- tions of interest. In all of the immune sera we have produced in rabbits and chickens, most of the antibodies reacted with antigens possessed by all members of the species or genus. The production of type-specific sera in even small amounts required large quantities of cells for absorption.

It is interesting to note that sev- eral of the immune sera were capaible of demonstrating blood group differences in a species other than the one used for immuni- zation, but could not be used to demonstrate differences in the immunizing species. This would appear to indicate that related antigens are fixed in one species and segregating in another. These observations are somewhat analogous to antigenic rela- tionships which have been demonstrated between man and Rhesus monkeys (Landsteiner and Weiner 1941), and recently between cattle asid bison (Owen, Stormont and Irwin 1958), and between tahrs (Hemitragus jam- lahicus) and a variety of other Artiodactyl species (Stormont and Suzuki 1958). Fur- ther extension of these observations in fishes may result in the production of type-specific sera in practical quantities.

Our isoimmunization experiments with sockeye salmon indicate con- siderable antigenic diversity exists in this species. Similar Eind more extensive studies have been con- ducted with rainbow trout and will be presented in another paper. As with other animals, isoimmunization appears to be the most promising ap- proach to the problem of blood group differences in fishes. The species of Pacific salmon are particularly difficult animals on which to conduct isoimmunizations as they invariably die after becoming sexually mature at two to six or seven years of age. It is only shortly before their death (two weeks to three or four months) while they are on their spawning migration that they become readily available. They also grow quite slowly in captivity and only one species, the sockeye, can be readily held in fresh water throughout its lifetime. At least some of these difficulties can be overcome and we hope to continue our attempts to do so.

ACKNOWLEDGMENTS

The authors wish to express their appreciation to Drs, R. S. Weiser, J. E. Gushing and R. D. Owen for their valuable advice and encouragement and to Mary La Rocque and E. D. Ullman for excellent technical assistance.

Table 7. — Comparison of the specificities of isoimmune sera produced in sockeye salmon.

Serum

Cells

14-4

14-6

14-10

15-1

22-10 24-2

13-1	0	0	+
14-3	+	0	0
14-5	0	+	0
14-9	0	0	+
15-1	-	+	+
15-7	0	0	0
22-9	-	-	-
22-10	+	+	0
24-1	+	+	0

+ Agglutination, 0 No agglutination.

Not tested.

13-2

problems involved in its practical utilization are discussed.

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SUMMARY

1. Through the use of absorbed rabbit and chicken antisalmon-erythrocyte sera, blood types were demonstrated to occur in sockeye, chinook, chum, and pink salmon.

2. Some of these types appear to differ in their frequency of occurrence between different geographic races.

3. Isoimmunizations between individuals of race of sockeye salmon indicated the existence of at least eight different antigenic types or combination of types within this race.

4. The usefulness of this kind of research to fishery management and some of the

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INT.DUP..D.C.60- 49613

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