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The multiple hemoglobins of coho salmon : Oncorhynchus kisutch Giles, Michael Arthur 1973

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THE MULTIPLE HEMOGLOBINS OF COHO SALMON, Onoorhynahus kisutch. by M i c h a e l A r t h u r G i l e s B.Sc. Hon., U n i v e r s i t y c£ M a n i t o b a , 1965 M . S c . j U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Zoology We.accept t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA February, .197 3 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT S t u d i e s were c o n d u c t e d t o d e t e r m i n e the o n t o -g e n e t i c changes i n the number and r e l a t i v e c o n c e n t r a t i o n o f the e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e h e m o g l o b i n polymorphs o f coho s a l m o n , Oncorhynchus kisutoh. and the i n f l u e n c e o f c e r t a i n e n v i r o n m e n t a l f a c t o r s upon the e x p r e s s i o n o f the h e m o g l o b i n v a r i a n t s . In a d d i t i o n some of the oxygen e q u i l i b r i u m c h a r a c t -e r i s t i c s o f the h e m o g l o b i n o f f r e s h w a t e r f r y and a d u l t coho were i n v e s t i g a t e d u s i n g b o t h h e m o l y z a t e s and whole b l o o d . T h r o u g h o u t the l i f e c y c l e o f coho salmon s e v e n -t e e n t o n i n e t e e n d i s t i n c t h e m o g l o b i n components were i d e n t i f i e d i n m i c r o - s t a r c h - g e l e l e c t r o p h e r o g r a m s p r e p a r e d i n b o r a t e b u f f e r a t pH 8.5. These components formed t h r e e main e l e c t r o p h o r e t i c h e m o g l o b i n p a t t e r n s a s s o c i a t e d w i t h d i f f e r e n t s t a g e s o f the l i f e c y c l e . U n h a t c h e d embryos and a l e v i n s p o s s e s s e d t w e l v e a n o d i c and one c a t h o d i c components. A l l e x c e p t t h r e e a n o d i c components had d i s a p p e a r e d from the b l o o d o f f r e e - s w i m m i n g f r y f o u r t e e n weeks a f t e r h a t c h i n g . T h i s t h r e e - c o m p o n e n t p a t t e r n was r e t a i n e d u n t i l the b e g i n n i n g o f the p r e s m o l t p e r i o d , a p p r o x -i m a t e l y e l e v e n months a f t e r h a t c h i n g . A t t h i s s t a g e , f i v e new c a t h o d i c components, one new a n o d i c component and one a n o d i c component w h i c h had p r e v i o u s l y been v i s i b l e i n a l e v i n e l e c t r o -pherograms a p p e a r e d . In p r e s m o l t s and s m o l t s t h e s e a d d i t i o n a l s e v e n components a c c o u n t e d f o r l e s s than 201 o f the t o t a l hemo-g l o b i n o f the b l o o d w h i l e the t h r e e components o b s e r v e d i n f r y b l o o d a c c o u n t e d f o r the r e m a i n d e r . F o l l o w i n g m i g r a t i o n t o sea - i i -water the r e l a t i v e concentration of these seven components gradually increased to 45 to 50% of the hemoglobin over a two-month period. No further change i n either the number or r e l a t i v e concentrations of the hemoglobin components was observed during the remaining phases of the l i f e c y c l e . Since i t was apparent that changes i n hemoglobin pattern were temporally associated with changes i n the chara-c t e r i s t i c s of the environment occupied by the juvenile coho the e f f ects of water temperature, dissolved oxygen concen-t r a t i o n and s a l i n i t y upon the physical development and electro-phoretic hemoglobin pattern of underyearling coho were examin-ed. Exposure to freshwater temperatures of 1.4 to 15.0 C, dissolved oxygen concentrations of 2.2 to 9.7 ppm and s a l i n i t -ies of 0 to 30 °/oo for periods of 49 to 60 days had no i n f l u -ence upon the electrophoretic hemoglobin pattern of either 3 1/2-month-old fry or 11-month-old presmolts. Presmolts reared for 60 days i n freshwater at 15 C and i n 10 °/oo s a l -i n i t y at 9.2 C grew at a highly accelerated rate and were equal or greater i n size than 16 1/2-month-old postsmolts which had been residing i n sea water for one month. These large presmolts retained the hemoglobin pattern c h a r a c t e r i s t i c of normal presmolts of the same age. Postsmolts maintained i n aerated freshwater rather than sea water underwent changes i n the electrophoretic hemoglobin pattern c h a r a c t e r i s t i c of sea-water residents. The foregoing observations suggest that age rather than physical size or environmental factors i s the main determinant i n the expression of the polymorphic hemoglobins of coho salmon. - i i i -The oxygen equilibrium c h a r a c t e r i s t i c s of adult coho hemoglobin and hemoglobin components A6-8 (fry hemoglobin) i s o l a t e d from adult hemolyzates by ion-exchange chromatography were investigated. Adenosine triphosphate concentrations rang-ing from 0.0 to 0.76 moles/mole hemoglobin had no influence upon the oxygen equilibrium of adult hemolyzates whereas at a concentration of 7.56 moles/mole P J - Q increased by 1 to 2 mm Hg. Since erythrocyte ATP concentrations of freshwater adult coho ranged between 0.8 and 1.3 moles/mole hemoglobin this organic phosphate i s probably not a modifier of oxygen a f f i n i t y i n coho salmon. The hemoglobin of adult coho was r e l a t i v e l y insen-s i t i v e to variations i n pH and temperature with 0 =-0.172 at 9.8 C over the pH range of 6.95 to 8.20 and A l o g P50 = 0.019 A T between 5 and 15 C. The Bohr e f f e c t of f r y hemoglobin was non-line a r so that 0 = -0.033, -1.729 and -0.182 i n the pH ranges of 6.82 to 7.08, 7.08 to 7.50 and 7.50 to 8.50, r e s p e c t i v e l y . The estimate of A l o g P50 was 0.056 for fry hemoglobin. Thus A T at 9.8 C the oxygen a f f i n i t y of fry hemoglobin exceeded that of adult hemoglobin at pH greater than 7.3 but was lower at values of pH less than 7.3. At pH 7.4, the P ^ Q of f r y and adult coho hemoglobin was 8.4 and 17.9, r e s p e c t i v e l y . In neither case was a Root e f f e c t observed. Heme-heme in t e r a c t i o n was s i m i l a r for both adult and fry hemoglobin and the value of n always exceeded 1.0. The estimate of n was generally less than 2.0 at pH greater than 7.0 - i v -and tended to decrease as the pH or the e q u i l i b r a t i o n temperature increased. Studies with f r y and adult whole blood e q u i l i b r -ated w i t h . 0 . 2 and 3 . 4 mm Hg of carbon dioxide generally confirmed the q u a l i t a t i v e differences observed between the oxygen e q u i l -i b r i a of f r y and adult hemolyzates. The estimates of P 5 0 at 9 . 3 C and P ^ Q of 0 . 2 and 3 . 4 mm Hg were 5 . 5 and 1 2 . 5 mm Hg respect-2 i v e l y for f r y blood and 1 0 . 7 and 1 5 . 6 mm Hg, r e s p e c t i v e l y , for the blood of freshwater adults. - v -TABLE OF CONTENTS Page Introduction 1 General Methods 6 PART ONE - ONTOGENETIC CHANGES IN THE MULTIPLE HEMOGLOBINS OF COHO SALMON Introduction I 12 Methods I 14 Results I 15 Discussion I 23 Summary I 29 PART TWO - INFLUENCE OF WATER TEMPERATURE, DISSOLVED OXYGEN CONCENTRATION AND SALINITY UPON GROWTH, DEVELOPMENT AND ELECTROPHORETIC HEMOGLOBIN PATTERN IN UNDERYEARLING COHO SALMON Introduction II 31 Methods II 32 Results II 39 Discussion II 64 Summary II 74 PART THREE - OXYGEN EQUILIBRIUM CHARACTERISTICS OF THE HEMOGLOBIN AND WHOLE BLOOD OF COHO • FRESHWATER ADULTS AND FRY Introduction III 75 - v i -Page Methods III 77 Results III 87 Discussion III 111 Summary III 132 Major Findings of Thesis 134 Literature Cited 139 Appendix 15 2 - v i i -LIST OF TABLES Table Facing Page I Relative concentrations of the hemoglobin components of coho salmon from the f r y to adult stages of the l i f e cycle. 20 II Environment regimes to which underyear-l i n g coho were exposed. 37 III Growth and hematocrit of presmolt coho reared i n the a r t i f i c i a l creek. 40 IV Ef f e c t of water temperature upon growth and hematocrit of underyearling coho. 43 V Eff e c t of dissolved oxygen concentration upon growth and hematocrit of underyear-l i n g coho. 44 VI Ef f e c t of s a l i n i t y upon growth and hema-t o c r i t of underyearling coho. 45 VII E f f e c t of environmental factors upon the re l a t i v e concentration of hemoglobin comp-onents A6-8 i n presmolt coho. 50 VIII E f f e c t of temperature upon the r e l a t i v e concentrations of hemoglobin components A l , A3, C l , C3, C4, C5 and C6 of pre-smolt coho. 52 IX Ef f e c t of dissolved oxygen concentration upon the r e l a t i v e concentrations of hemo-globin components A l , A3, C l , C3, C4, C5 and C6 of presmolt coho. 53 X Eff e c t of s a l i n i t y upon the r e l a t i v e concen-trations of hemoglobin components A l , A3, C l , C3, C4, C5 and C6 of presmolt coho. 54 XI Effect of extended freshwater residence at high and low dissolved oxygen conditions upon the hemoglobin pattern of coho post-smolts. 63 XII Summary of the oxygen equilibrium char-a c t e r i s t i c s of coho hemoglobin. 88 XIII Hematological parameters of the blood of freshwater adult coho blood. 92 - v i i i -Tab l e F a c i n g Page X I V I n f l u e n c e o f a d e n o s i n e t r i p h o s p h a t e u p o n t h e o x y g e n e q u i l i b r i u m o f a d u l t h e m o g l o b i n . 97 XV I n f l u e n c e o f t e m p e r a t u r e u p o n t h e o x y g e n e q u i l i b r i u m o f f r y and a d u l t h e m o g l o b i n . 99 XVI I n f l u e n c e o f pH and CC^ u p o n t h e o x y g e n e q u i l i b r i u m o f f r y a n d a d u l t h e m o g l o b i n . 105 X V I I Summary o f t h e o x y g e n a f f i n i t i e s o f t h e b l o o d o f v a r i o u s s a l m o n i d s . 122 XVIII C o r r e l a t i o n between r e l a t i v e amount of hemoglobin a p p l i e d t o s t a r c h - g e l and the e s timate of tha r e l a t i v e c o n c e n t r a t i o n of hemoglobin components A6-8. 153 - i x -L I S T OF FIGURES F i g u r e F a c i n g Page 1 S c h e m a t i c r e p r e s e n t a t i o n o f t h e o n t o g e n y o f t h e m u l t i p l e h e m o g l o b i n s o f c o h o s a l -mon. 17 R e l a t i o n b e t w e e n i n s t a n t a n e o u s g r o w t h r a t e a n d w a t e r t e m p e r a t u r e i n p r e s m o l t c o h o s a l m o n . 48 R e l a t i o n b e t w e e n h e m o g l o b i n c o n c e n t r a t i o n a n d h e m a t o c r i t i n c o h o s a l m o n o f v a r i o u s a g e s . 94 I n f l u e n c e o f a d e n o s i n e t r i p h o s p h a t e u p o n t h e o x y g e n e q u i l i b r i u m o f a d u l t c o h o hemo-g l o b i n . 96 5 I n f l u e n c e o f t e m p e r a t u r e u p o n t h e o x y g e n e q u i l i b r i u m o f c o h o f r y h e m o g l o b i n . 100 I n f l u e n c e o f t e m p e r a t u r e u p o n t h e o x y g e n e q u i l i b r i u m o f t h e b l o o d o f f r e s h w a t e r a d u l t c o h o . 102 7 I n f l u e n c e o f pH u p o n t h e o x y g e n e q u i l i b r i u m o f f r y h e m o g l o b i n . 106 8 I n f l u e n c e o f pH u p o n t h e o x y g e n e q u i l i b r i u m o f a d u l t h e m o g l o b i n . 107 9 C o m p a r i s o n o f t h e B o h r e f f e c t o f f r y a n d a d u l t h e m o g l o b i n . 109 10 I n f l u e n c e o f c a r b o n d i o x i d e u p o n t h e o x y g e n e q u i l i b r i u m o f t h e w h o l e b l o o d o f c o h o f r y a n d a d u l t s . 110 - x -LIST OF PLATES Plate Facing Page 1 Electrophoretic hemoglobin components of coho from the embryonic to the f r y stages of the l i f e c y c l e . 18 Electrophoretic hemoglobin components of coho from the f r y to adult stages of the l i f e c y c l e . 19 Electropherograms of the hemoglobin of presmolts exposed to various water temperatures for si x t y days. 55 Electropherograms of the hemoglobin of f r y exposed to various water temper-atures for f i f t y days. 56 Electropherograms of the hemoglobin of presmolts exposed to various dissolved oxygen concentrations for s i x t y days. 58 Electropherograms of the hemoglobin of fry exposed to various water temperatures for f i f t y days. 58 Electropherograms of the hemoglobin of presmolts exposed to various s a l i n i t i e s for s i x t y days. 59 Electropherograms of the hemoglobin of fry exposed to various s a l i n i t i e s for forty-nine days. 60 Electropherograms of the hemoglobins of coho postsmolts maintained i n aerated ( 9.7 ppm 0~ ) and unaerated (2.2 ppm 0^) freshwater ana of normal postsmolts i n seawater 62 / - x i -P l a t e F a c i n g Page 10 E l e c t r o p h e r o g r a m s o f t h e h e m o l y z a t e s o f a d u l t b l o o d a nd o f c o m p o n e n t s A6-8 i s o l a t e d f r o m a d u l t h e m o l y z a t e s b y a n i o n -e x c h a n g e c h r o m a t o g r a p h y . 89 ACKNOWLEDGEMENTS I would l i k e to express my appreciation and gratitude to the Fisheries Research Board of Canada, West Vancouver, B.C. for providing experimental f a c i l i t i e s and f i n a n c i a l support throughout this i n v e s t i g a t i o n . Also I would l i k e to express i n d i v i d u a l thanks to Drs. J.C. Davis, W.S. Hoar, J.D. Randall, and W.E. Vanstone for t h e i r guidance and helpful c r i t i c i s m s during the preparation of this manuscript. - 1 -INTRODUCTION The major function of hemoglobin i n the blood of vertebrates and most invertebrates possessing this r e s p i r a -tory pigment is the extraction from the environment and sub-sequent delivery to the tissues of molecular oxygen. In many instances hemoglobin may perform secondary functions which include the transport of carbon dioxide from the t i s -sues to the respiratory surface (Guyton, 1961), and acting as one component in the buffer system of the blood (Rossi-Bernardi and Roughton, 1967; Reeves, 1972). The character-i s t i c s of the reversible combination of oxygen with hemo-globin and the effects of carbon dioxide, pH, temperature and certain a l l o s t e r i c effector substances are prime consid-erations i n the analysis of the in vivo transport of oxygen-Substantial v a r i a t i o n i n these c h a r a c t e r i s t i c s has been ob-served i n the hemoglobin from a wide range of animals (Prosser and Brown, 1961). In f i s h the oxygen equilibrium c h a r a c t e r i s t i c s of hemoglobin are f u n c t i o n a l l y adapted to maintain e f f i c i e n t oxygen transport under the prevalent environmental conditions. Thus f i s h which inhabit hypoxic water generally possess hemo-globin with a high oxygen a f f i n i t y (Krough and Leitch, 1919; Black, 1940; Riggs, 1970). Willmer (1934) asserted that in --f i s h residing i n water at high carbon dioxide tensions the oxygen a f f i n i t y of the hemoglobin i s r e l a t i v e l y i n s e n s i t i v e to variations i n carbon dioxide but additional evidence makes this generalization somewhat questionable (Lenfant,Johansen - 2 -and G r i g g , 1 9 6 6 ) . T h i s v a r i a t i o n i n o x y g e n a f f i n i t y w i t h c h a n g e s i n PCC^ o r pH i s t e r m e d t h e B o h r s h i f t a nd i t , i n c e r t a i n a c t i v e f i s h , a c t s t o m i t i g a t e somewhat, t h e i n c r e a s e i n o x y g e n a f f i n i t y o f h e m o g l o b i n w h i c h o c c u r s a t r e d u c e d temp-e r a t u r e s ( B l a c k , K i r k p a t r i c k and T u c k e r , 1966 a , b; B l a c k , T u c k e r and K i r k p a t r i c k , 1966a) t h e r e b y m a i n t a i n i n g h i g h o x y g e n t e n s i o n s and r a t e o f r e l e a s e o f o x y g e n t o t h e t i s s u e s . I n o t h e r i n s t a n c e s t h i s d i f f i c u l t y i s r e s o l v e d by a r e l a t i v e l y t e m p e r a -t u r e - i n s e n s i t i v e h e m o g l o b i n ( L e n f a n t , et at., 1 9 6 6 ) . I n t u n a , Thunnus thynnus, t h e d e e p e r b o d y t e m p e r a t u r e s a r e m a i n t a i n e d above a m b i e n t and t h e o x y g e n a f f i n i t y i s c o m p l e t e l y t e m p e r -a t u r e - i n d e p e n d e n t ( R o s s i - F a n e l l i a n d A n t o n i n i , 1 9 6 0 ) . One o f t h e m o s t o u t s t a n d i n g f e a t u r e s o f t h e h e m o g l o b i n o f c e r t a i n f i s h i s t h e p r e s e n c e o f a R o o t e f f e c t ( R o o t , 1931) w h i c h i s t h e d e c r e a s e i n o x y g e n c a p a c i t y a s s o c i a t e d w i t h d e c r e a s e s i n pH o r e l e v a t e d P C ^ . R e c e n t e v i d e n c e s u g g e s t s t h a t t h i s e f f e c t may be an i m p o r t a n t f e a t u r e i n t h e f u n c t i o n i n g o f t h e gas g l a n d o f t e l e o s t s w i m b l a d d e r s ( S t e e n , 1 9 6 9 ) . On t h e m o l e c u l a r l e v e l f i s h h e m o g l o b i n s a r e u n u s u a l i n t h e p r e v a l e n c e o f m u l t i p l e f o r m s o f t h i s p r o t e i n o c c u r r i n g i n most s p e c i e s . A l t h o u g h e a r l y o b s e r v a t i o n s s u g g e s t e d t h a t o n l y two t o f o u r e l e c t r o p h o r e t i c a l l y d i s t i n c t p o l y m o r p h s ex-i s t e d i n most f i s h b l o o d ( B u h l e r and S h a n k s , 1 9 5 9 ; H a s h i m o t o and M a t s u u r a , 1 9 5 9 a ; Schumann, 1959) more r e c e n t i n v e s t i g a t i o n s e m p l o y i n g i m p r o v e d e l e c t r o p h o r e t i c a l t e c h n i q u e s h a v e demon-s t r a t e d t h a t two t o n i n e t e e n h e m o g l o b i n p o l y m o r p h s may be p r e s e n t ( V a n s t o n e , R o b e r t s and T s u y u k i , 1964; Yamanaka, - 3 -Yamaguchi and Matsuura, 1965; Kock. et al., 1967; Grigg, 1969; Tsuyuki and Ronald, 1971). Subunit analysis of the multiple hemoglobins of A t l a n t i c salmon and certain species of P a c i f i c salmon have indicated that the high degree of polymorphism i n salmonids is probably a r e f l e c t i o n of the t e t r a p l o i d genetic condition believed to occur i n these f i s h (Wilkins, 1970; Tsuyuki and Roberts, 1971). Ontogenetic variations in the number and r e l a t i v e concentrations of the multiple hemoglobins have been describ-ed for the lamprey Petromyzon -planerii ( A d i n o l f i , C h i e f f i and S i n i s c a l c o , 1959), rainbow trout Salmo gaivdnevi i r i d e u s Cluchi and Yamagami, 1969), herring Clupea harengus (Wilkins and l i e s , 1966), coho salmon Oncorhynchus kisutoh and sockeye salmon 0. nerka (Vanstone et al.3 1964) and A t l a n t i c salmon Salmo salar (Kock, Bergstron and Evans, 1964a, b; Wilkins, 1968; Westman, 1970). The detailed ontogenetic changes i n the el e c t r o p h o r e t i c a l l y d i s t i n c t hemoglobin polymorphs have been described for the l a t t e r species only and demonstrate that i n some f i s h these changes may be extremely complex. Westman (1970) observed eight d i s t i n c t electrophoretic hemoglobin patterns involving thirteen components i n A t l a n t i c salmon 3.5 to 95.0 cm i n length. Although considerable overlap was reported, the p a r t i c u l a r hemoglobin pattern exhibited by these salmon was correlated with the size and presumably the age of the f i s h . The question arises as to the function of the mul t i -ple hemoglobins in f i s h and to the control of t h e i r synthesis. Ultimately the genotype l i m i t s the number and structure of the various polymorphs. Many examples e x i s t , however, in - 4 -both plants and animals in which environmental factors i n -fluence the expression of certain genes (Gardner, 1960). Baldwin and Hochachka (1970) have demonstrated that two el e c t r o p h o r e t i c a l l y d i s t i n c t forms of acetylcholinesterase are synthetized i n the brain of rainbow trout kept at 12 C. At 2 and 17 C only one polymorph was present with the slower migrating form occurring at the lower temperature. The two forms of the enzyme were fu n c t i o n a l l y d i f f e r e n t with the Km of the "cold" form being much lower than that of the "warm" form ( i b i d . ) . A si m i l a r process apparently occurs i n the regulation of the synthesis of lactate dehydrogenase i s o -enzymes i n trout (Hochachka and Somero, 1968). Thus although these salmonids are capable of synthetizing d i f f e r e n t i s o -enzymes i t i s apparent that environmental temperatures regu-late the expression of the appropriate genes. Coho salmon are anadromous f i s h and therefore reside i n at least three d i s t i n c t environments during the course of the i r development: the stream-bed as embryos and a l e v i n s , the stream as f r y and smolts and the sea as g r i l s e and matur-ing adults. Vanstone et al. (1964) observed three d i s t i n c t e l e ctrophoretical hemoglobin patterns associated with the f r y , smolt and seawater postsmolt stages of the coho l i f e - c y c l e . Although the precise timing of the t r a n s i t i o n from one pat-tern to another was not described these observations i n d i c a t -ed that the d i f f e r e n t hemoglobin patterns could be associated with either environmental or ontogenetic changes or a combina-tion of both f a c t o r s . The present investigation was conducted, therefore, to define p r e c i s e l y the ontogenetic changes i n the - 5 -electrophoretic hemoglobin pattern of coho salmon from the embryonic to the adult stages of the l i f e - c y c l e and to test the hypothesis that at certai n stages of development environ-mental temperature, dissolved oxygen concentration and s a l i n i t y exert some form of control upon the number or r e l a -t i v e concentration of the hemoglobin polymorphs. A second aspect of this thesis was to investigate some of the function-a l c h a r a c t e r i s t i c s of the hemoglobins representative of f i s h at d i f f e r e n t stages of development i n r e l a t i o n to t h e i r re-spective environmental regimes. - 6 -GENERAL METHODS EXPERIMENTAL ANIMALS A l l f i s h studied were coho salmon, Onoorhynohus kisuteh, the majority of which had been raised i n the aquarium f a c i l i t i e s of the Fisheries Research Board of Canada i n West Vancouver, B.C. Adult coho were trapped during th e i r spawning migration at the Big Qualicum River on Vancouver Island, B.C. and transported i n large aerated tanks to the aquarium. These f i s h were held i n 1.8 or 3.0 m diameter tanks provided with aerated well water or Cypress Creek water, u n t i l sexually mature and then a r t i f i c i a l l y spawned by removing the eggs from the female through a ven-t r a l s l i t i n the abdomen and thoroughly mixing the eggs and sperm in a p l a s t i c bucket. The f e r t i l i z e d eggs were buried under gravel in troughs, 4.9 m by 0.4 m by 0.2 m and provided with flowing water at a temperature of 3.4-8.0 C. After hatching, the alevins were c o l l e c t e d as they emerged from the gravel and placed i n 1.8 or 3.0 m diameter aquaria. These f i s h were fed to s a t i a t i o n , f i v e times d a i l y on a diet com-posed of beef l i v e r , 4500 g; beef heart, 4500 g; canned s a l -mon, 4500 g; s a l t , 110 g; and Pablum (Meade-Johnson Canada L t d ) , 40 g. After the f i s h had begun to grow, the feeding was gradually reduced to once d a i l y . The f r y were placed i n an a r t i f i c i a l creek (104 m by 0.6 m by 0.2 m), containing - 7 -r o c k y r i f f l e s , p o o l s and a q u a t i c v e g e t a t i o n and s u p p l i e d w i t h f l o w i n g w a t e r f r o m C y p r e s s C r e e k . The f o l l o w i n g s p r i n g , a f t e r a p p r o x i m a t e l y one y e a r i n f r e s h w a t e r , t h e s m o l t s w e re t r a n s f e r r e d t o 3 m a q u a r i a a n d g r a d u a l l y e x p o s e d t o i n c r e a s i n g c o n c e n t r a t i o n s o f s e a w a t e r ( s a l i n i t y , c a . 27-30 °/oo). T h e s e f i s h w e r e m a i n t a i n e d i n s e a w a t e r f o r a p p r o x i m a t e l y 19 m o n t h s , a t w h i c h t i m e t h e y w e r e c o n s i d e r e d t o be a d u l t s and were t r a n s -f e r r e d t o f r e s h w a t e r w h e r e t h e y became s e x u a l l y m a t u r e . S i n c e t h i s p r o c e d u r e h a d b e e n i n i t i a t e d two y e a r s b e f o r e t h e e x p e r i -ment b e g a n , f i s h o f v a r i o u s a g es w e r e a v a i l a b l e a t any p a r t i c -u l a r t i m e . I n t h e autumn o f 1 9 7 1 , a d u l t coho w e r e c a u g h t a t t h e e n t r a n c e t o G r e a t C e n t r a l L a k e on V a n c o u v e r I s l a n d , B r i t i s h C o l u m b i a , d u r i n g t h e i r s p a w n i n g m i g r a t i o n and t r a n s p o r t e d , l i v e , t o t h e a q u a r i u m f a c i l i t i e s i n West V a n c o u v e r . T h e s e f i s h w e r e u s e d i n t h e m a j o r i t y o f s t u d i e s on t h e o x y g e n e q u i l i b r i a o f a d u l t c oho b l o o d . The a d u l t s a l m o n w e r e n o t f e d d u r i n g t h e i r r e s i d e n c e i n f r e s h w a t e r . The p h o t o p e r i o d r e g i m e i n t h e a q u a r i u m b u i l d i n g was e q u i v a l e n t t o t h e n a t u r a l p h o t o p e r i o d a t 45 d e g r e e s n o r t h l a t i -t u d e w h e r e a s t h e f i s h m a i n t a i n e d i n t h e o u t d o o r f a c i l i t i e s e x p e r i e n c e d t h e n a t u r a l p h o t o p e r i o d o f 49.5 d e g r e e s n o r t h l a t i t u d e . Thus a l l e x p e r i m e n t a l coho and n e w l y e m e r g e d a l e v i n s w h i c h w e r e m a i n t a i n e d i n t h e a q u a r i u m b u i l d i n g e n c o u n t -e r e d a s l i g h t l y d i f f e r e n t p a t t e r n o f l i g h t a n d d a r k n e s s t h a n t h e m a j o r i t y o f t h e s t o c k s o f s a l m o n . - -8 -PREPARATION OF BLOOD FOR ELECTROPHORESIS The b l o o d o f s m a l l f i s h , ( a l e v i n s t o s m o l t s t a g e s ) , was c o l l e c t e d i n h e p a r i n i z e d m i c r o h e m a t o c r i t t u b e s , ( I . D . 0.55, o r 1.2 mm., C l a y - A d a m s I n c . ) , f r o m t h e s e v e r e d c a u d a l p e d u n c l e . B l o o d was o b t a i n e d f r o m l a r g e r f i s h , ( g r i l s e a n d a d u l t s ) , b y a o r t i c p u n c t u r e i n t h e t a i l r e g i o n w i t h an 18 o r 20 gauge n e e d l e f i t t e d w i t h a h e p a r i n i z e d (40 U.S.P. u n i t s / m l o f b l o o d ) s y r i n g e . The m i c r o h e m a t o c r i t t u b e s w e r e s e a l e d w i t h S e a l -e a s e ( C l a y - A d a m s I n c . ) , a n d c e n t r i f u g e d a t 3000 r.p.m. f o r 10 m i n u t e s i n a c l i n i c a l c e n t r i f u g e ( B u c h l e r I n s t r u m e n t s I n c . ) . H e m a t o c r i t s w e r e m e a s u r e d a n d t h e p l a s m a and w h i t e b l o o d c e l l s r e m o v e d by s u c t i o n w i t h a f i n e l y d r a w n c a p i l l a r y t u b e . The m i c r o h e m a t o c r i t t u b e was f i l l e d w i t h 1.01 N a C l f r o m a f i n e l y d r a w n c a p i l l a r y . The o p e n e n d was s e a l e d b y f l a m e and t h e e r y t h r o c y t e s c e n t r i f u g e d t h r o u g h t h e s a l i n e . A f t e r r e p e a t i n g t h i s w a s h i n g p r o c e d u r e t w i c e , t h e p a c k e d e r y t h r o c y t e s w e re p l a c e d i n a d e s i c c a t o r c o n t a i n i n g i c e . The d e s i c c a t o r was e v a c u a t e d , t h e n f i l l e d w i t h c a r b o n mon-o x i d e gas ( r e s e a r c h g r a d e , M a t h e s o n o f Canada L t d . ) . T h i s g a s s i n g p r o c e d u r e was r e p e a t e d t w i c e a n d f o l l o w i n g t h e f i n a l f l o o d i n g w i t h c a r b o n m o n o x i d e , t h e d e s s i c a t o r v a l v e was c l o s e d and a few pounds p r e s s u r e o f gas t r a p p e d i n t h e v e s s e l . A f t e r 20 m i n u t e s e q u i l i b r a t i o n , t w i c e t h e p a c k e d e r y t h r o c y t e v o l u m e o f c o l d , c a r b o n m o n o x i d e g a s s e d d i s t i l l e d w a t e r was p l a c e d i n e a c h m i c r o h e m a t o c r i t t u b e a n d t h e open e n d o f t h e t u b e f l a m e d s h u t . The e r y t h r o c y t e s w e r e h e m o l y z e d b y r e p e a t e d - 9 " centrifugation through the d i s t i l l e d water i n a cold room at 0T2 C. A f i n a l centrifugation at 3000 r.p.m. for 20 minutes at 0-2 C served to separate the carboxyhemoglobin solut i o n from the c e l l debris. Larger blood samples were prepared i n centrifuge tubes i n e s s e n t i a l l y the same manner. ELECTROPHORESIS Eighteen grams of hydrolyzed starch (lots 261-1 and 283-1, Connought Medical Research Laboratories), were suspend-ed i n 150 ml of 0.023 m borate b u f f e r , pH 8.5, i n a 500 ml vacuum fl a s k and heated with constant swirling over a naked flame u n t i l the suspension was clear (Smithies, 1959). The gel was degassed under vacuum during the l a s t minute of heating, pour-ed into plexiglass moulds, 14.0 by 3.7 by 2.5 cm, covered with a sheet of mylar and allowed to set at 0-2 C for 2-3 hours. S l i c e s of g e l , 1.6 mm thick were removed from the cooled gel and transverse s l o t s cut at the mid-line as described by Tsuyuki et al.3 (1966). The hemoglobin solution was introduced into the sl o t s from f i n e l y drawn c a p i l l a r y tubes and the gel covered with a s t r i p of mylar. The electrophoresis chamber contained 0.3 m borate b u f f e r , pH 8.5, i n both compartments, which were separated by a distance of 10.5 cm. A constant po t e n t i a l difference of 200 volts was applied for 90 minutes. During electrophoresis the starch-gels were maintained at 0-2 C i n a cold room and the current did not exceed 2.5 milliamps per g e l . - 10 -STAINING AND IDENTIFICATION OF HEMOGLOBIN BANDS Immediately after electrophoresis, the starch gels were stained i n a 0.1! solution of Amido Schwartz B, (w/v), in acetic acid: methanol: water, (1:5:5, v/v/v), for approxi-mately 10 minutes and then cleared by r e p e t i t i v e washing i n a s i m i l a r solution without the s t a i n . The i d e n t i t y of the hemoglobin bands was ascertained by staining c e r t a i n gels with 3,3'-dimethoxybenzidine dihydrochloride, (Owen, Silbermann and Got, 1958; O'Brien, 1961). p r i o r to staining with Amido Schwartz. Gels were stored for future analysis i n p l a s t i c bags containing a small amount of clearing s o l u t i o n . No deterioration i n the gel or the p r e c i p i t a t e d proteins was ob-served over a 2 1/2-year period. DETERMINATION OF THE RELATIVE CONCENTRATION OF HEMOGLOBIN COMPONENTS The cleared starch gels were positioned on glass plates and covered with a sheet of Mylar i n such a manner that no a i r bubbles were trapped around the g e l . The starch-gel sandwich was placed over a 30 by 30 cm piece of flashed opal glass which was illuminated from below by four 25 cm-long 40 watt l i g h t bulbs. The extraneous l i g h t was eliminated and the gels were photographed using only the l i g h t transmitted through the g e l . The photographic negatives of the starch gels were scanned at 660 mu i n a G i l f o r d Model 2400 spectrophotometer f i t t e d with a l i n e a r transport carriage and a 0.05 by 2.36 - w -mm s l i t p l a t e . The changes i n absorbance were recorded on a variable span chart recorder coupled with a disc integrator (Disc Instruments Inc., Model 2 0 1 - B ) . The chart area represented by each hemoglobin compon-ent was expressed as a percentage of the t o t a l area of a l l the bands. This l a t t e r area was an estimate of the r e l a t i v e amount of hemoglobin applied to each starch g e l . Since i t was p o s s i -ble that the estimate of the r e l a t i v e concentration of the hemoglobin components was influenced by the r e l a t i v e amount of hemoglobin applied to the g e l , a Spearman rank c o r r e l a t i o n c o e f f i c i e n t test was conducted on six electropherograms from each of six groups of presmolt coho. The indi v i d u a l f i s h within each group were a l l of the same age and had been reared under the same environmental conditions. No c o r r e l a t i o n be-tween the r e l a t i v e concentrations of the hemoglobin components and the r e l a t i v e amount of hemoglobin applied to the gel was observed at the 99% confidence l e v e l . PART ONE ONTOGENETIC CHANGES IN THE MULTIPLE HEMOGLOBINS OF COHO SALMON INTRODUCTION Despite the widespread occurrence of hemoglobin polymorphism i n f i s h , r e l a t i v e l y l i t t l e work has been done on the ontogeny of multiple hemoglobins in these animals. Observations as to the electrophoretic pattern of hemoglobins at c e r t a i n d i s t i n c t phases of the l i f e - c y c l e have been made for the lamprey Petromy zon p l a n e r i i ( A d i n o l f i , C h i e f f i and S i n i s c a l c o , 1959), rainbow trout Salmo gairdneri ivideus, (Iuchi and Yamagami, 1969), and coho and sockeye salmon, Onoorhynahus kisutah and 0. nevka3 (Vanstone, Roberts and Tsuyuki, 1964). In a l l of the foregoing studies frequent observations of the multiple hemoglobin pattern was not made although in most cases i t appeared that there was a general progression from the patterns observed i n juvenile f i s h to the pattern observed i n mature f i s h . Fish between these two general age groups had a hemoglobin pattern containing e l e -ments from both groups. The detailed ontogeny of the multiple hemoglobins has been described for only two species of f i s h . Wilkins and l i e s (1966) have demonstrated that the blood of the herrin g , Clupea harengus, contains four hemoglobin polymorphs which, based upon d i f f e r e n t r e l a t i v e concentrations, form up to nine electrophoretical patterns during the l i f e - c y c l e of thi s - 13 " f i s h . S i m i l a r l y , A t l a n t i c salmon, Salmo salar, exhibit up to nine anodic and eight cathodic hemoglobin components during th e i r l i f e - c y c l e (Wilkins, 1968). E a r l i e r studies on t h i s salmon had only demonstrated two components (Schumann, 1959), but, using improved electrophoretical techniques, f i r s t t h i r teen (Koch, Bergstrom, and Evans, 1964 a,b) and then seventeen d i f f e r e n t hemoglobin polymorphs were observed (Koch, Wilkins, Bergstrom and Evans, 1967). No single electrophore-t i c a l technique however, could separate a l l seventeen compon-ents. Westman (1970), employing a micro starch-gel method, has demonstrated that the thirteen hemoglobin polymorphs of A t l a n t i c salmon which can be separated with t h i s technique form eight d i f f e r e n t e lectrophoretical patterns depending upon the size and presumably the age of the f i s h . This l a t t e r study did not include salmon less than 3.5 cm i n length. In view of the complex ontogenetic changes i n the multiple hemoglobins of certain salmonids i t appeared probable that the observations of Vanstone et al. . (1964) were not s u f f i c i e n t l y detailed to outline the complete ontogeny of the multiple hemoglobins of coho salmon. To this end the present studies were undertaken using an improved micro starch-gel electrophoresis method (Tsuyuki et al.3 1966) to separate the hemoglobin components of coho salmon from the embryonic to adult stages of the l i f e - c y c l e . This information was also required p r i o r to the investigation of the effects of certa i n environmental factors upon the multiple hemoglobin pattern and ontogenetic changes i n the oxygen equilibrium of the blood of - 14-coho salmon. METHODS I In order to observe early changes in hemoglobin pattern just after hatching, i t was necessary to determine the precise date of hatching. This was accomplished by r e -moving a sample of eggs of the 1971 brood year, from the gravel about a week p r i o r to the expected date of hatching. These eggs were placed i n p l a s t i c screen boxes, returned to the incubation trough and observed twice dai l y for evidence of hatching. Thus the time of hatching was ascertained to within 16 hours. The majority of eggs hatched within one day and these young alevins were then divided into ten groups of 10-12 f i s h and placed into i n d i v i d u a l cages composed of a 4-inch piece of 2-inch diameter PVC p l a s t i c pipe with both ends capped with p l a s t i c screen. A few pieces of gravel were also placed i n the cages which were then returned to the incu-bation trough. Thus, coho alevins of known age could be ea s i l y obtained at l a t e r dates. The ages of older f i s h are known to within 2-3 weeks, since the precise time of hatching was only determined for the 1971 brood year. Blood samples were generally obtained as described in general methods. The blood from unhatched embryos was ob-tained by cutting the outer egg case and expelling the embryo onto a piece of tissue paper. The t a i l region was c a r e f u l l y dried with tissue and the caudal peduncle severed. Blood was co l l e c t e d i n a f i n e l y drawn microhematocrit tube containing - 15 heparin, (50 USP units per ml), i n 1.0% NaCl. The use of heparinized saline during blood c o l l e c t i o n was continued u n t i l the stage when the yolk sac had been adsorbed since the blood tended to c l o t more quickly i n these young f i s h . The erythro-cytes were then washed, hemolyzed and electrophoresed as de-scribed previously. The gels were stained and photographed and the photographic negatives scanned i n a densitometer as described i n the general methods. RESULTS I A summary of the ontogenetic changes i n the multiple hemoglobins of coho salmon i s presented i n Figure 1. For reference purposes the hemoglobin components have been l a b e l l e d as migrating either toward the anode (A) or the cathode (C) from the central o r i g i n . The number following the anodic or cathodic designation represents the r e l a t i v e p o s i t i o n of the component i n order of increasing distance from the o r i g i n toward either the anode or cathode. In t h i s respect the ele c t r o p h o r e t i c a l m o b i l i t i e s of a l l the components observed throughout the l i f e - c y c l e were ranked p r i o r to the assignment of each label (Figure 1). The code designating the various hemoglobin components presented i n Figure 1 w i l l be used i n the discussion of the photographic plates 1 to 10. Twelve closely-spaced anodic and one diffuse cathodic carboxyhemoglobin bands were observed i n coho embryos approxi-mately two days p r i o r to hatching, (Plate 1-A). Blood sampled within 14 hours and 15 days a f t e r hatching, (Plate 1-B,C) had - 16-. -i d e n t i c a l electrophoretic patterns to that observed p r i o r to hatching, suggesting that the environmental changes experienc-ed by the embryos during hatching and early inter-gravel r e s i -dence were not associated with changes i n the composition of the hemoglobin. Thirty days after hatching, bands A6, 7, and 8 became r e l a t i v e l y more dense (Plate 1-E), while the slower migrating anodic bands, A l , 2, 4, became quite f a i n t . The remaining anodic components also decreased i n r e l a t i v e concentration. At six-weeks post-hatch when the f i s h were emerging from the gra v e l , bands A6-8 represented the large majority of the hemo-globin (Plate 1-F). At this stage, a second very f a i n t cathodic component corresponding to C3 (Figure 1) appeared. The apparent dark band at the o r i g i n i n Plate 1-F i s an a r t i f a c t representing a small amount of c e l l debris inadvertently applied with the hemoglobin s o l u t i o n . Fourteen weeks after hatching, the free-swimming f r y stage was well established and components A6-8 comprised v i r t u a l l y a l l the hemoglobin (Plate 1-G, Figure 1). The electropherogram presented i n Plate 1-H represents the hemo-globin pattern of a f r y from the 1970 brood year at a s i m i l a r time, thus demonstrating that f i s h from both brood years had i d e n t i c a l hemoglobin patterns at this stage of their l i f e c y c l e . Coho fry remain i n fresh water for approximately one year and then migrate to sea during the period from A p r i l to June as smolts. Salmon undergo several biochemical and behavioural changes during the s m o l t i f i c a t i o n process (Baggerman, 17 " FIGURE 1: Schematic representation of the r e l a t i v e electrophoretic m o b i l i t i e s of the multiple hemoglobin components of coho salmon during the a l e v i n , f r y and adult stages of the l i f e c y c l e . No attempt has been made to reproduce the r e l a t i v e concentrations of the various components observed at d i f f e r e n t ontogenetic stages. 0 I 1 I  I I I1 I 3 Q IP Bl CO N ID < < < CO N CD < < < S H B \ C s \ 0 \ i i to I I ro O o m to u. > UJ to N - O fl) CO N ID l O ^ - O J — < < < C . < < < < < < < < < 1 CVJ I o DC O UJ Q O LU Q O X ( - 1=8 PLATE 1: Electropherograms of the multiple hemoglobins of coho salmon from the embryo to the f r y stages of development. The blood samples were obtained 2-3 days before hatching (A) and > 14 hours (B), 6 days (C), 15 days (D), 30 days (E), 42 days (F), and 98 days (G) a f t e r hatching from the 1971 brood stock. Electropherogram H i l l u s t r a t e s the electrophoretic hemoglobin pattern of a 6-month-old coho fry from the 1970 brood stock. The electropherograms are enlarged to 1.4 X actual s i z e . (-H • 1 • D G H - 19 PLATE 2 Electropherograms of the multiple hemoglobins of coho salmon from the fry to adult stages of development. The blood samples were obtained from a 4 1/2-month-old f r y (A), 11 1/2-month-old (B), 14-month-old (C), 15-month-old (D) presmolts, 15 1/2-month-old smolts (E), a 16 1/2-month-old postsmolt i n seawater for one month (F), a 2-year-old seawater g r i l s e (G) and a 3-year-old spawning female (H). The electropherograms are enlarged to 1.4 X actual s i z e . I f X I CD - 20 -TABLE I: The r e l a t i v e concentration of the components of the multiple hemoglobins of the blood of coho salmon from the fry to the freshwater adult stages of the l i f e c y c l e . The age i s given from the time of hatching. RELATIVE CONCENTRATION OF HEMOGLOBIN SAMPLE DEVELOPMENTAL AGE YEAR COMPONENTS (%) DATE STAGE HATCHED A6-8 A3 Al Cl C3 C4 C5 C6 1/6/71 F.W. Fry 3 1/2 mos. 1971 100. 29/6/71 F.W. Fry 4 1/2 mos. 1971 94. 3 2. 3 1. 4 1. 7 0. 3 0 0 0 2/2/71 F.W. Presmolt 11 1/2 mos. 1970 90. 5 0. 8 2. 0 3. 8 1. 0 2.0 0 0 2/3/71 F.W. Presmolt 12 1/2 mos. 1970 *86. 7 1. 1 1. 7 4. 9 1. 9 1.6 1.4 0.7 16/3/71 F.W. Presmolt 13 mo s. 1970 *80. 4 1. 5 2. 6 5. 5 3. 7 3.2 1.9 1.2 22/4/71 F.W. Presmolt 14 mos. 1970 78. 0 0. 6 4. 6 6. 9 4. 1 4.3 1.0 0.6 12/5/71 F.W. Presmolt 15 mos. 1970 80. 4 1. 6 3. 7 5. 4 3. 5 3.4 ' 0.8 1.2 1/6/71 F.W. Smolt 15 1/2 mos. 1970 78. 4 0. 7 5. 4 4. 9 4. 9 4.8 0.6 0.5 29/6/71 S.W. Postsmolt 1 1/3 y r s . 1970 68. 7 1. 5 6. 2 8. 2 6. 3 6.2 1.5 1.4 3/8/71 S.W. Postsmolt 1 1/2 y r s . 1970 50. 9 3. 3 10. 5 13. 3 9. 0 9.1 0.9 3.1 Oct/Nov.70 S.W. G r i l s e 1 3/4 y r s . 1969 *55. 4 5. 8 8. 8 9. 3 8. 7 6.7 2.8 2.5 10/1/72 S.W. Gr i l s e 2 y r s . 1970 61. 7 4. 7 8. 3 7. 7 8. 5 6.2 2.3 0.8 10/1/72 F.W. Adult 3 y r s . 1969 53. 1 2. 7 10. 1 6. 9 10. 8 11.3 1.4 3.6 F.W. - Fish r e s i d i n g i n fresh water. S.W. - Fish r e s i d i n g i n s a l t water. Average values of in d i v i d u a l electropherograms of hemoglobins from 6 f i s h . - 21 " 1965; G i l e s , 1969, Hoar, 1965; Vanstone and Markert, 1968), which may begin several months before the onset of seaward migration. In the present study, the smolts were simply . adapted to seawater in early June and did not undergo a sea-ward migration as such. It was impossible, therefore, to use seaward migration as a c r i t e r i o n for completion of the smolt-ing process. Since the f i s h used i n this part of the study had hatched i n mid-February, 1970, t h e i r approximate age from hatching can be determined from the dates on which the blood was sampled. In addition to the electropherograms, the r e l a t i v e concentration of the hemoglobin components from densi-tometric scans of the photographic negatives are presented (Table 1). Plate 2-A presents the t y p i c a l electrophoretic pattern of hemoglobin from 4 1/2-month-old f r y . Components A6-8 comprised over 941 of the t o t a l hemoglobin (Table 1) while A3, A l , C l , and C3 (Figure 1), account for the remainder. Component A3 was the most prominant of the minor components (2.3%). Components C4-6 (Figure 1) were not present at th i s age. By early February components A6-8 had decreased s l i g h t l y i n r e l a t i v e concentration and component C4 had appeared (Plate 2-B). Components C5 and C6 had appeared by early March when the r e l a t i v e concentration of components A6-8 had decreased to approximately 87% (Table 1). No substantial changes in either the electrophoretic pattern or the r e l a t i v e d i s t r i b u t i o n of the components occur-red during the period from mid-March to early June (Plates 2-C, 22 -D,E). At this time the f i s h were adapted to seawater. After approximately 30-days i n seawater (Plate 2-F) components A6-8 had decreased to approximately 68% of the t o t a l hemoglobin concomitant with increases i n the remaining seven components. Although there was considerable v a r i a t i o n , the minor hemoglo-bin components were normally ranked i n the following order of decreasing r e l a t i v e concentration; CI, C3, A l , C4, A3, C5, and C6. In older saltwater f i s h CI tended to decrease in r e l a t i v e concentration and ranked fourth i n this s e r i e s . This reduction in the faster-moving anodic bands continued u n t i l by August components A6-8 comprised only 551 of the t o t a l hemoglobin, (Table 1). The d i s t r i b u t i o n of hemoglobin components in f i s h a f t e r four to f i v e months in seawater remained r e l a t i v e l y un-changed throughout the remainder of the l i f e cycle (Plate 2-G, H, Table 1). Although Figure 1 indicates that nineteen hemoglobin components can be separated with micro starch-gel electrophoresis i t should be pointed out that the designation of components A4 and C2 of the alevin blood as d i s t i n c t from A3 and CI, respect-i v e l y , of adult blood may be erroneous. Component CI of the 15-month-old presmolt presented i n Plate 2-D was quite s i m i l a r in p o s i t i o n to C2 of alevin blood (Plate 1-A, B,C). In most cases, however, CI was much nearer the o r i g i n of the electropherogram (Plate 2-D to H) and was therefore considered to be d i s t i n c t from C2. The uniqueness of A3 and A4, however, i s much more questionable. The leading edge of A3 overlapped the t r a i l i n g - 23 -'edge of A4 and minor differences i n gel preparation could have caused small changes i n electrophoretic mobility of the hemoglobin components. It i s quite p o s s i b l e , therefore that the nineteen components i l l u s t r a t e d i n Figure 1 should be reduced to seventeen or more probably eighteen. DISCUSSION I The analysis of the electrophoretical data indicates J;b.a£, yfye (multiple hemoglobins of coho salmon are composed of seventeen to nineteen d i f f e r e n t components which undergo a r e l a t i v e l y complex series of ontogenetic changes. Although no v a r i a t i o n in the number or d i s t r i b u t i o n of hemoglobin com-ponents was observed i n f i s h of the same age minor v a r i a t i o n in the r e l a t i v e proportions of the components did occur. Various uncertainties are encountered i n the i n t e r -p r e t a t i o n of electropherograms of organic compounds, e s p e c i a l -ly those composed of two or more sim i l a r subunits. Multiple electrophoretic zones can arise from protein-buffer i n t e r -action (Cann and Goad, 1965; Cann, 1966) or from the binding of inorganic or organic compounds such as adenosine triphosphate or 2,3-diphosphoglycerate to a portion of the protein (Chanutin and Curnish, 1964). This l a t t e r factor may be of some impor-tance i n ontogenetic studies of hemoglobin since in many animals the concentrations of certain organic phosphates i n the erythro-cyte" do change during the l i f e cycle (Mission and Freeman, 1972; Wood, 1972). In this respect removal of over 90% of the t o t a l phosphate from hemolyzates of adult coho blood did not decrease - 24* " the number or electrophoretic m o b i l i t i e s of the hemoglobin components (Plate 10, Part I I I ) . Subunit d i s s o c i a t i o n or aggregation may occur under the stresses imposed by electrophoresis which would lead to spurious results when interpreting the electropherograms. Although th i s factor was not investigated i n the present study, i t has been demonstrated that the molecular weights and subunit composition of A t l a n t i c salmon hemoglobins are unchanged during a second electrophoretic run, after an i n i t i a l electrophoretic separation (Wilkins, 1970). There i s no reason to assume that P a c i f i c salmon hemoglobins are less stable than A t l a n t i c salmon hemoglobins i n thi s respect. A l s o , the uniformity of r e l a t i v e concentrations and d i s t r i -bution of hemoglobins observed i n f i s h of s i m i l a r age argues against the occurrence of a s i g n i f i c a n t amount of subunit d i s s o c i a t i o n . The oxidation of the iron molecule from the ferrous to f e r r i c state would obviously change the electrophoretic mobility of the hemoglobin molecule since one to four p o s i t i v e ionic charges would be added to the molecule. Hemoglobin can be oxidized to the f e r r i c state (methemoglobin) more r e a d i l y when i t i s i n the deoxygenated state (Benesch, Benesch, and Macduff, 1964; Mahler and Cordes, 1966). Since oxyhemoglobin and carboxyhemoglobin have i d e n t i c a l electrophoretic m o b i l i t i e s (Tsuyuki and Ronald, 1971) carbon monoxide was employed to s t a b i l i z e the hemoglobin p r i o r to electrophoresis. The d i s -s o c i a t i o n constant of carbon monoxide from hemoglobin i s 25 -approximately 200 times less than that of oxygen and the former gas therefore forms a much more stable complex with the respiratory pigment. Methemoglobin, when present i n s i g n i f i c a n t amounts (1-5%) forms secondary bands v i s i b l e adjacent to each hemoglobin component (Plate 10, Part I I I ) . No such secondary bands were observed i n the present hemo-globin samples which were less than 24 hours o l d . The seventeen to nineteen hemoglobin components of coho salmon exhibit a complex series of ontogenetic changes although only four d i s t i n c t electrophoretic patterns were ob-served compared to the eight d i f f e r e n t patterns described for A t l a n t i c salmon (Westman, 1970). A s t r i k i n g s i m i l a r i t y was evident between the hemoglobin electrophoretic pattern of newly hatched coho alevins and the eight to nine anodic com- , ponents of trout a l e v i n hemoglobin reported by Iuchi and Yamagami (1969). While 25-day-old trout alevins possessed three anodic and three cathodic components { i b i d . ) , the reduction in the number of anodic components of coho alevin blood did not begin u n t i l 30 days after hatching and an additional 10 weeks was required to develop the f r y hemoglobin pattern consisting of three anodic components (A6-8). Although the difference, i n the timing of the change i n hemoglobin pattern may r e f l e c t species differences or d i f f e r e n t rearing conditions i t i s i n t e r e s t i n g to note that i n both species, the change i n pattern was associated with the timing of the emergence of the alevins from the gravel. Vernidub (1966) during an extensive study of - 26> -c y t o l o g i c a l changes i n the blood of A t l a n t i c salmon during the embryonic to free-swimming stages, demonstrated the occurrence of two general periods of erythropoeisis. The f i r s t ended just p r i o r to hatching and was characterized by large numbers of normoblasts and basophilic erythrocytes, which were derived from blood islands on the p e r i b l a s t . The second period of erythropoeitic a c t i v i t y began 200 degree days af t e r hatching at a length of 21 to 22 mm and continued u n t i l after 700 degree days at a length of 33 to 37 mm, 95% of the red blood c e l l s consisted of mature erythrocytes, derived from the new source. VernidUb suggested that middle kidney was the erythropoeitic organ during the second phase of erythrocyte production. The close correspondence between the timing of erythropoeitic a c t i v i t y i n the alevins of A t l a n t i c salmon and the timing of changes in the multiple hemoglobin pattern of rainbow trout and coho salmon suggest that these two processes are i n some way linked and may be c h a r a c t e r i s t i c of salmonids at this stage of development. The f i n a l major changes i n hemoglobin pattern of coho salmon was associated with the period of smolting, and resulted i n an electrophoretic pattern of five anodic and f i v e cathodic components. During the period of January to mid-February f i v e cathodic and two anodic hemoglobin compon-ents appeared i n presmolt hemoglobin while the r e l a t i v e con-centration of components A6-8 decreased to 80%. At this time components A3, A l , C l , C3, C4, C5, and C6, accounted for 1.5, 2.6, 5.5, 3.7, 3.2, 1.9 and 1.2 %, r e s p e c t i v e l y , of the t o t a l - 27 " hemoglobin. This pattern remained e s s e n t i a l l y unchanged during the next 2 1/2 months of freshwater residence. Following transfer to sea water, however, the r e l a t i v e proportion of A6-8 began to decrease and was only 51% of the t o t a l hemoglobin of coho postsmolts a f t e r approximately 2 months i n sea water. Coupled to this decrease i n the con-centration of A6-8 was an increase in the seven minor components of smolt hemoglobin. Thereafter l i t t l e v a r i a t i o n i n the d i s t r i b u t i o n of the ten hemoglobin components was ob-served even when the mature coho were returned to freshwater p r i o r to spawning. Thus throughout the entire l i f e cycle of the mu l t i -ple hemoglobins of coho under three periods of r e l a t i v e l y rapid change i n composition while the number and r e l a t i v e concentration of the components remain almost constant be-tween these three periods. The r e l a t i v e proportion of anodic components decreased during the l i f e cycle but the change was intermittent rather than continuous as observed i n A t l a n t i c salmon (Westman, 1970). The molecular basis for hemoglobin polymorphism i n salmonids appears to reside in the presence of eight struc-t u r a l genes coding for eight d i f f e r e n t polypeptide chains (Tsuyuki and Ronald, 1971; Wilkins, 1971). In A t l a n t i c salmon, these eight polypeptides appear to be divided into two groups of four "<*-like" and four "non « - l i k e " chains. Two chains of each group are involved i n the formation of the nine anodic hemoglobins and the remaining pair of each 28 -group form the seven to eight cathodic components and these sets of complementary pairs are mutually exclusive (Wilkins, 1971). Each polymorphic form of hemoglobin i s a tetrameric combination of two "non-«-like" and two "<*-like" chains. B a s i c a l l y , the same system has been found to occur i n the f i v e species of P a c i f i c salmon (Tsuyuki and Ronald, 1971), although the l a t t e r workers used a d i f f e r e n t method of no-ta t i o n for each subunit of the hemoglobin molecule. In the l a t t e r study i t was found on the basis of t r y p t i c d igests, that hemoglobins from d i f f e r e n t species with i d e n t i c a l electrophoretic m o b i l i t i e s were composed of d i f f e r e n t subunits. Since both of these investigations only include blood from the juvenile to adult stages, i t is possible that additional subunits may be discovered which occur only i n the embryonic stages as i s observed i n c e r t a i n mammals (Ingram, 1963). At the present time, the r e s u l t s observed i n this study are compatible with the concept of eight s t r u c t u r a l genes coding for the hemoglobin subunits i f the previously discussed l i m i t a t i o n s on the possible tetrametric combinations are applied. In such a system, a t o t a l of eighteen unique tetrameric combinations are p o s s i b l e . Since there were serious doubts as to the d i f f e r e n t i a t i o n of components A3 and A4, i t is probable that only eighteen instead of nineteen multiple forms of hemoglobin occur during the l i f e cycle of coho salmon. - 2 9 -SUMMARY I 1) The ontogenetic changes i n the multiple hemoglobin of coho salmon, Onoorhynahus kistuoh, from the embryonic to freshwater adult stages of the l i f e cycle were investigated using micro starch-gel electrophoresis to separate the hemo-globin polymorphs. 2) A t o t a l of twelve to thirteen anodic and f i v e to six cathodic hemoglobin polymorphs were observed during the l i f e cycle of coho salmon. 3) The hemoglobin of embryos, just p r i o r to hatching, and of alevins for a period of approximately two to three weeks after hatching, is composed of twelve anodic and one cathodic component. A l l except three of the anodic components (A6-8) disappeared during subsequent development to the free-swimming fry stage. 4) At the age of approximately eleven to twelve months, fi v e new cathodic and one new anodic component, C l , C3, C4, C5, C6, and A3, r e s p e c t i v e l y , appeared i n the hemoglobin of presmolt coho. The uniqueness of A3 and Cl may be questionable. Com-ponent Al of alevin hemoglobin also reappeared, r e s u l t i n g in a ten-component pattern. In presmolt coho components A6-8 comprised over 80% of the t o t a l hemoglobin. 5) Following transfer to seawater, a further reduction i n the r e l a t i v e concentration of A6-8 was observed and a f t e r approximately two months i n seawater, A6-8 accounted for - 30 -approximately 50 to 55% o£ the hemoglobin. The remaining two anodic and f i v e cathodic components accounted for the remainder of the hemoglobin. Generally, no s i g n i f i c a n t change i n the electrophoretic pattern or r e l a t i v e concentrations of components was observed during the portion of the l i f e cycle following the f i r s t two months of marine existence. PART TWO INFLUENCE OF WATER TEMPERATURE, DISSOLVED OXYGEN CONCENTRATION AND SALINITY UPON GROWTH, DEVELOPMENT AND ELECTROPHORETIC HEMOGLOBIN PATTERN IN UNDERYEARLING COHO SALMON INTRODUCTION II During the ontogenetic studies of the multiple hemo-globins of coho salmon i t was observed that major changes in the electrophoretic pattern were associated with periods of change in behaviour and habitat of the f i s h . The alevin hemoglobin pattern was transformed into the f r y pattern upon emergence from the stream bottom and the presmolt pattern appeared during a period when the f i s h are known to be under-going a variety of physiological and biochemical changes i n preparation for marine residence (Baggerman, 1965; Vanstone and Markert, 1968; G i l e s , 1969). The f u l l expression of the postsmolt or adult hemoglobin pattern was not complete u n t i l the f i s h had been in sea water for at least two months, again implicating environmental factors i n the e l i c i t a t i o n of a change i n hemoglobin pattern. Although the. foregoing considerations suggest that environmental factors may be responsible for ontogenetic variations in the coho hemoglobin polymorphs i t may s t i l l be argued that the size or the age of the f i s h i s the im-portant c r i t e r i o n i n the control of the timing of the hemo-globin changes. Growth i n f i s h i s dependent upon environmental conditions and i t i s conceivable that both factors, may exert a combined action i n control of hemoglobin polymorphism. Since the juvenile stage of the coho salmon l i f e cycle i s characterized by considerable changes i n both the number and r e l a t i v e concentrations of the hemoglobin poly-morphs as well as variations i n the pattern of growth the following experiments were conducted to determine the effects of temperature, dissolved oxygen concentration and s a l i n i t y upon the growth and hemoglobin pattern of 3 1/2-month old f r y and 11-month old presmolt coho. METHODS II EXPERIMENTAL ANIMALS Eleven-month old presmolts from the 1970 brood stock were anesthetized i n 2-phenoxyethanol and measured for fork length on January 28, 1971. Fish i n the 8.5 to 8.9 cm length class (mean length, 8.6 ± 0.1 cm; mean weight, 6.6 ± 0.4 g) were removed and allowed to recover i n aerated fresh water for one day p r i o r to exposure to the d i f f e r e n t environmental conditions. Eighteen f i s h were placed i n each fi b r e g l a s s aquarium, maintained under the conditions described i n the following methods. Fish were transferred d i r e c t l y to the experimental conditions with no period of adaptation. On June 6, 1971, 3 1/2-month old coho fry of the 1971 brood stock were separated randomly into groups of approximately t h i r t y i n d i v i d u a l s , without the use of anesthetic. With certain exceptions, ind i v i d u a l groups of t h i r t y f i s h were transferred d i r e c t l y to aquaria main-tained at the constant environmental conditions described i n the following Section. Fry to be exposed to s a l i n i t i e s of 20 and 30 °/oo were maintained i n d i l u t e seawater, s a l i n i t y 10°/oo for two days and then transferred to 20°/oo seawater. Fish to be exposed to f u l l - s t r e n g t h seawater were maintained in 20°/oo seawater. In addition, f i s h to be exposed to water temperatures of 15 °C were exposed to increasing water tem-peratures from approximately 9.5 to 15 °C over a 2 day p e r i -od. BLOOD SAMPLING The presmolt coho were sampled at approximately 2-week intervals over a period of 60 days. Three f i s h were removed from each treatment and anesthetized with 2-phen-oxyethanol. The ind i v i d u a l lengths and weights were measured and duplicate blood samples co l l e c t e d i n microhematocrit tubes from the severed caudal peduncle. Hematocrits were measured and the hemoglobin samples prepared and electro-phoresed as described i n the General Methods Section. Coho fr y were treated i n an i d e n t i c a l manner, except that a single sample of 4-6 f i s h was taken from each experimental environ-ment after 49-50 days of treatment. The electropherograms were photographed and scanned i n a densitometer as described previously. In some cases, photographic prints of the gels 3:4 -are presented rather than the densitometer scans. WATER SUPPLY Well water (hardness (hardness 18-40 mg/1 CaCO^) was used as a source of fresh water i n a l l experiments. During the experimental period the fresh water temperature was 9 to 10 °C and dissolved oxygen concentration was 2.2 ppm be-fore aeration. Water aspirators were f i t t e d to the valve outlets to aerate the water and compressed a i r was supplied to the aquaria when necessary to maintain high oxygen ten-sions. Seawater ( s a l i n i t y ca. 30°/oo, range 27-32 °/o o , temperature 8.4 °C) was supplied to some aquaria as required. The sea water was generally more than 90% saturated with oxy-gen. TEMPERATURE CONTROL Insulated, 60-1 fi b r e g l a s s aquaria were f i t t e d with thermo-switches (micro-set Model 62542, Precision S c i e n t i f i c Co.) which controlled the operation of electronic relays (Lapine S c i e n t i f i c Co.). To maintain temperatures above ambient, st a i n l e s s s t e e l immersion heaters, (250-750 watts) were placed i n the aquaria and connected to the rel a y s . To maintain temperatures below ambient, 4m aluminum cooling c o i l s were placed i n the aquaria and connected through a pump to a reservoir of ethylene glycol-water mixture main-tained at -10°C with a cooling unit (Bendin Westinghouse L t d . ) . The pump was. controlled through the relay and when the water - 3.5 -temperature rose above the desired temperature, the pump was activated to run coolant through the c o i l s . Aerated well water was supplied to these aquaria at the rate of 200 1/minute. Each aquaria was f i t t e d with a submersible r e c i r c u l a t i o n pump ( L i t t l e Giant Pump Co.) to provide a c i r c u l a r water flow and maintain uniform tempera-tures throughout the tank. OXYGEN CONCENTRATION CONTROL Three, 225-1 fi b r e g l a s s aquaria were supplied with flowing well water which was maintained in c i r c u l a r flow by-means of submersible r e c i r c u l a t i o n pumps. The valves c o n t r o l -l i n g water inflow were f i t t e d with water as p i r a t o r s . The a i r intake of the aspirators was connected to a gas flow-meter (Gilmont Instruments'Inc.) and the a i r flow through the meter was controlled by a needle valve. By a t r i a l and error method, the flow of a i r to each aspirator required to produce desired dissolved oxygen concentrations was determined and the flows were maintained at these l e v e l s . Oxygen concentrations were normally determined every two days, using the modified Winkler method (Strickland and Parsons, 1968) or by measuring the p a r t i a l pressure of oxygen with a Radiometer thermostated oxygen micro-electrode. When coho f r y were exposed to low oxygen concentrations (2.2-3.2 ppm) considerable gulping behaviour was observed at the water surface. To prevent this behaviour from i n t e r f e r -ring with the experiment, these f i s h were placed i n 2 5 cubic 35 -centimeter boxes constructed of netting on a sta i n l e s s s t e e l frame, positioned 5 cm below the surface. Since this gulping behaviour was not observed i n presmolt coho, these f i s h were not confined i n the net enclosures. SALINITY CONTROL Two constant head devices, one for seawater and one for well water, were constructed to permit a uniform water pressure to be maintained. A controlled flow of water was drawn from each header and supplied i n various rat i o s to 250 l i t r e f i b r e g l a s s aquaria to produce the desired s a l i n i -t i e s . Each aquaria was provided with a r e c i r c u l a t i o n pump to maintain a c i r c u l a r flow and uniform mixing of the two water supplies. The temperature of the seawater and the well water d i f f e r e d by approximately 1.2-1.6 C, but no attempt was made to adjust this d i f f e r e n t i a l since the maximum d i f -ference i n temperature between the three experimental tanks was 1.0 C. The oxygen concentrations, water temperature and s a l i n i t i e s maintained through the experimental period of January 29, 1970 to March 29, 1971, are presented i n Table I I . The control f i s h represent presmolt coho which were maintained i n aerated well water (dissolved oxygen concen-t r a t i o n , 9.7 ppm = 87% a i r saturation) and thus serve as controls for both the s a l i n i t y and oxygen concentration experiments. - 37 -TABLE I I : A summary o f t h e w a t e r t e m p e r a t u r e a n d d i s s o l v e d o x y g e n c o n c e n t r a t i o n s m a i n t a i n e d i n t h e v a r i o u s e n v i r o n m e n t a l r e g i m e s e m p l o y e d i n t h i s s t u d y . V a l u e s g i v e n a r e t h e mean t 1 s t a n d a r d d e v i a t i o n o f m e a s u r e m e n t s r e c o r d e d t h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d . ENVIRONMENTAL VARIABLE WATER TEMPERATURE (C) OXYGEN CONCENTRATION (ppm) Temperature + + 1.3 0.1 10.0 0.9 4.1 + 0.2 9.6 + 0.9 8.0 + 0.1 9.1 + 0.6 10.1 + 0.1 9.4 + • 0.6 15.0 + 0.0 9.3 + 0.7 Dissolved Oxygen 4. 4. Concentration 9.7 i 0.2 3.1 0.3 9.8 + 0.2 5.8 + 0.3 9.7 + 0.2 7.9 + 0.6 S a l i n i t y 1/3 SW 9.2 + 0.5 9.9 + 0.6 2/3 SW 8. 7 + 0.9 9.9 + 0.6 3/3 SW 8.5 + 0.1 9.2 + 0.8 EFFECT OF LONG-TERM EXPOSURE TO LOW-OXYGEN LEVELS On February 5, 1971, a group of coho presmolts from the"8.5-8.9 cm length class were placed i n a 225-1 f i b r e -glass aquaria provided with flowing aerated well water. The aeration was gradually reduced over a 20-day period to a f i n a l dissolved oxygen concentration of 2.2 ppm and the f i s h maintained under these conditions throughout the smolting period u n t i l August 3, 1971. A second group of f i s h was maintained under s i m i l a r conditions except that the water was aerated to oxygen levels at least 85% of a i r saturation. On August 3, 1971, blood was obtained from one f i s h of each group and the hematocrits and electrophoretic hemoglobin pattern compared to a fish.which had undergone normal smolti-f i c a t i o n and was l i v i n g in seawater. Unfortunately, soon thereafter the water supply to the freshwater tanks was i n t e r -rupted and the f i s h died within a few minutes. The results therefore, represent the blood of a single f i s h from each experiment. CALCULATIONS EMPLOYED IN THIS STUDY The instantaneous rate of growth was calculated using the re l a t i o n s h i p : . l o g W„ - log W Growth rate = —. — ± — — — - X 100 T - t where log W„ and log W, are the natural logarithms of the 6e T &e t & wet weights of f i s h at time t and some l a t e r time T and both T and t are i n days (LeBrasseur and Parker, 1964; B r e t t , Shelbourne and Sloop, 1969). These instantaneous rates of growth were calculated on the mean weights of each sample of f i s h since i t was not possible to determine rates of growth of in d i v i d u a l f i s h over the experimental period. 3 25 The weight-length r e l a t i o n s h i p , W = <*L " , where W i s the wet weight i n mg and L i s the fork length i n cm was used to determine the « intercept. This value is quite si m i l a r to the so-called condition factor —, but i t has be L found that the slope, b, of a double logarithmic p l o t of weight on length i s approximately 3.20 to 3.25 i n P a c i f i c salmon (LeBrasseur and Parker, 1964; Vanstone and Markert, 1968, G i l e s , 1969). The value of °= i s given in mg and was calculated from the lengths and weights of in d i v i d u a l f i s h rather than from the mean length of each sample. RESULTS II GROWTH AND HEMATOCRIT OF UNTREATED PRESMOLT COHO The changes i n mean length, weight and « intercept 3 2 5 of the weight-length r e l a t i o n s h i p W = <*L ' as well as hematocrit of coho salmon reared i n the a r t i f i c i a l creek environment from February 16, 1971 to June 1, 1971 and i n seawater from June 1, 1971 to June 29, 1971 are presented i n Table I I I . The mean instantaneous rates of growth in - 4o: TABLE III Growth parameters and hematocrit of.juvenile coho salmon reared i n an a r t i f i c i a l stream during the presmolt to smolt stages of the l i f e cycle and of presmolts i n seawater. The « intercept was calculated for i n d i v i d u a l f i s h from the weight-length r e l a t i o n -3 2 5 ship W = =L " , where W i s the wet weight i n mg and L i s the fork length i n cm. The instantaneous growth rates were calculated at log W - log W X 100 from the mean wet weight of 6 to 9 f i s h . T - t With the exception of growth r a t e , a l l data are presented as the mean ± 1 standard.deviation. a INSTANTANEOUS DATE N LENGTH WEIGHT INTERCEPT GROWTH RATE HEMATOCRIT (cm) (g) (mg) (% I DAY) (% RBC) 16/2/71 6 8. .70 + 0. ,7 7. .5 + 2. ,05 6. .42 + .49 31, .3 + 3. ,3 2/3/71 6 9. , 20 + 0. .7 8. .5 + 1. ,70 6. ,23 + .34 0. , 894 34, .8 + 5. , 2 16/3/71 6 9. ,00 + 0. .4 8. .3 + 1. .20 6. .50 + .30 -0. ,170 28. . 7 + 1. , 8 29/3/71 6 9. .50 + 0. .8 9. ,6 + 2. .60 6. .25 + .27 1. .119 39. .1 + 3. ,0 11/Mil 9 10. ,40 + 0. .7 11. .5 + 2. .5 5. .67 + .49 0, .683 37. .9 + 6. ,0 12/5/11 6 11. .10 + 0. ,8 13. ,5 + 2. .8 5. .44 + .31 0, .385 29. .2 + 4. ,0 1/6/71 6 11. ,60 + 1, .0 16. .1 + 4. ,0 5, .35 + .14 1. .381 33. . 7 + 3. ,2 29/6/71* 6 11. ,90 + 1. ,3 17. ,0 + 5. .3 5. ,38 + .35 0. .194 33. ,2 ±10. ,8 In seawater since June 1, 1971. - 41. -weight have been calculated for each sampling period. The mean length and weight of coho presmolt i n -creased from 8.7 cm and 7.5 g to 9.5 cm and 9.6 g, respect-i v e l y , during the period of January 16, to March 29. This represents a mean instantaneous growth rate of 0.614%/day during this period. The « intercept remained r e l a t i v e l y constant, ranging from 6.23 to 6.50 mg. Hematocrits averag-ed about 301 with the exception of the March 29 sample which was 39.11. From March 29, to June 1, the f i s h continued to grow at a s l i g h t l y accelerated rate. The mean growth rate during this period was 0.816%/day but was highly v a r i a b l e , ranging from 0.385 to 1.381%/day. The value of «, however, s t e a d i l y declined from 6.25 to 5.35 mg during this two-month period. This indicates that the f i s h were becoming more streamlined, a phenomenum observed i n salmon p r i o r to seaward migration ( G i l e s , 1969; Vanstone and Markert, 1968). Hematocrits also declined s l i g h t l y over this period. On June 1, 1971, after obtaining a f i n a l sample of freshwater coho smolts the f i s h were transferred to seawater. Although the growth rate had declined to 0.194%/day by June 29, no change was observed i n either the hematocrit or the value of the a intercept. Thus the most consistent change in the pattern of growth observed i n coho salmon during the progression of the smolting period was the decrease i n the value of « in the 3 25 weight relat i o n s h i p W = «L * . This streamlining e f f e c t was 42 -u t i l i z e d as a physiolo g i c a l indicator i n determining whether or not the experimentally manipulated v a r i a t i o n i n ce r t a i n envi-ronmental factors resulted i n an acceleration of the smolting process in underyearling coho. A decrease i n the value of « as well as maintenance or acceleration of growth rate would be ind i c a t i v e of acceleration of the smolting process. If the value of « remained r e l a t i v e l y unchanged but variations i n growth rate were related to the environmental factors occurred, then i t could be concluded that the environmental variable was influencing the rate of growth but not the rate of s m o l t i f i c a -t i o n . EFFECT OF WATER TEMPERATURE, DISSOLVED OXYGEN CONCENTRATION AND SALINITY UPON GROWTH, HEMATOCRIT AND HEMOGLOBIN PATTERN OF COHO PRESMOLTS AND FRY The fork lengths wet weights, « in t e r c e p t s , instan-taneous growth rates and hematocrits are presented for coho presmolts and f r y reared i n fresh water at f i v e temperatures (Table IV) and three dissolved oxygen concentrations (Table V) and i n three d i l u t i o n s of sea water (Table V I ) . In general, water oxygen saturation ranged between 80 to 90% i n the con-t r o l l e d temperature experiments and 95 to 100% i n the various d i l u t i o n s of sea water. The temperature of the water contain-ing the three levels of dissolved oxygen was 9.7 to 9.8 C. The d e t a i l s of the conditions maintained i n each experimental environment are presented in Table I I . - 43 TABLE IV Growth parameters and hematocrit of 3 1/2-month old coho fry and 11-month old presmolts exposed to fresh water of various temperatures. The « intercept was calculated for i n d i -3 2 5 vidual f i s h from the weight-length r e l a t i o n s h i p W = «L , where W i s the wet weight i n mg and L i s the fork length i n cm. The instantaneous growth rate calculated as l o g e WT .-. l o g e -W^. ^  ^ T - t from the mean wet weight of 2 to 3 f i s h . With the exception of growth r a t e , a l l data are presented as the mean ± 1 standard deviation, for a sample size N of 3. WATER TREATMENT a INSTANTANEOUS AGE TEMPERATURE TIME N LENGTH WEIGHT INTERCEPT GROWTH RATE HEMATOCRIT GROUP (C) (DAYS) (cm) (K) (mg) (% / DAY) (% RBC) Presmolt 1.3 19 3 8.8±0.2 6.9±0.7 5.89±0.34 0.234 48.112.3 Presmolt 4.1 19 3 9.1±0.2 8.210.1 6.17±0.28 1.142 33.6±1.3 Presmolt 8.0 19 3 9.1±0.4 8 . 8±1 . 5 6.6810.22 1.514 32.7±1.5 Presmolt 10.2 19 3 9.2+0.3 8.8±0.7 6.85±0.33 1.514 35.6±2.3* Presmolt 15.0 19 3 9.9±0.3 10.9±2.1 6.36±0.60 2.640 32.9±7.7 Presmolt 1.3 33 3 8.3±0.1 6.1±0.2 6.17±0.16 -0.239 32.5±2.8 Presmolt 4.1 33 3 9.4±0.3 9.111.3 6.27±0.27 0.973 39.6±1.4 Presmolt 8.0 33 3 9.8±0.1 11.2±0.5 6.73±0.37 1.603 32.6±1.5 Presmolt 10. 2 33 3 10.1±0.3 12.010.9 6.62±0.11 1.812 35.7±5.7 Presmolt 15.0 33 3 10.6±0.3 13.8±1.1 6.50±0.14 1.932 33.1±6.1 Presmolt 1.3 47 3 8.810.3 7.611.3 6.35±0.59 0. 300 3 5 . 8 1 8 . 5 Presmolt 4.1 47 3 9.4±0.2 9.7±1.2 6.63±0.47 0.819 30.1±5.7 Presmolt 8.0 47 3 10.2±0. 2 12.510.7 6.53±0.41 1.146 28.9±4.6 Presmolt 10. 2 47 3 10.7±0.4 13.511.5 6.09±0.30 1.310 3 2 . 5 1 3 . 8 Presmolt 15.0 47 3 11.5±0.3 17.211.8 6.12±0.17 2.038 33.2±3.6 Presmolt 1.3 60 3 8.7±0.3 7.5±0.5 6.52±0.40 0.213 33.7±5.2 Presmolt 4.1 60 3 9.8±0.1 9.810.3 5.98±0.23 0.659 38.411.1 Presmolt 8.0 60 3 10.9±0.7 14.5±2.8 6.08±0.12 1.312 39.2±4.2 Presmolt 10. 2 60 3 11.3±0.4 16.2±1.8 6.10±0.08 1.497 37.4±2.3 Presmolt 15.0 60 3 11.9±1.1 19.5±6.0 6.10±0.19 1.806 34.5±7.5 Fry 1.3 49 6 4.0±0.1 0.6±0.1 7.31±0.78 37.5±6.3 Fry 4.1 49 6 4.3±0.2 0.8±0.1 7.39±0.28 32.215.4 Fry 8.0 49 6 5.1±0.4 1.510.3 7.72±0.71 34.3±6.3 Fry 10. 2 49 6 5.7±0.5 2.5±0.7 8.80±0.99 35.0±5.5 Fry 15.0 49 6 6.3±0.8 3.711.2 9.29±1.02 26.4±2.6 Two measurements only: values given are mean ± 1/2 difference between two observations. - 44 -TABLE V Growth parameters and hematocrit of 3 1/2-month old coho fry and 11-month old presmolts exposed to fresh water contain-ing various concentrations of dissolved oxygen at 9.7 to 9.8 C. The °= intercept was calculated for in d i v i d u a l f i s h from the weight 3 25 length rela t i o n s h i p W = «L ' , where W i s the wet weight i n mg and L i s the fork length i n cm. The instantaneous growth rate calculatea as e 1 e—t x 1 0 0 £ r Q m t h e m e a n w e t w e i g h t T - t of 2 to 3 f i s h . With the exception of growth r a t e , a l l data are presented as the mean ± 1 standard deviation, for a sample size N of 3. DISSOLVED TREATMENT AGE OXYGEN TIME M LENGTH GROUP (ppm) (DAYS) 1N (cm) Presmolt 3.1 19 3 9.0±0.3 Presmolt 5.8 19 3 9.2±0.5 Presmolt 7.9 19 3 9.6±0.1 Presmolt 9.7 19 3 9.2±0.1 Presmolt 3.1 33 3 9.210.3 Presmolt 5.8 33 3 9.9±0.3 Presmolt 7.9 33 3 9.7±0.5 Presmolt 9.7 33 3 9.8±0.1 Presmolt 3.1 47 3 9.8±0.4 Presmolt 5.8 47 3 10.4±0.1 Presmolt 7.9 47 3 10.4±0.1 Presmolt 9. 7 47 3 10.3±0.2 Presmolt 3.1 60 3 10.0±0.3 Presmolt 5.8 60 3 10.6±0.5 Presmolt 7.9 60 3 10.7±0.1 Presmolt 9.7 60 3 11.0±0.3 Fry 2.2 49 4 4.2±0.4 Fry 5.5 49 6 4.7+0.3 Fry 8.1 49 6 5.8±0.6 Fry 9.3 49 6 5.7±0.5 INSTANTANEOUS WEIGHT (g) INTERCEPT (mg) GROWTH RATE (% / DAY) HEMATOCRIT (% RBC) 7.8±0.3 6.22+0.37 0.879 34.013.0 8.7±0.9 6.3510.40 1.454 36.4H.4 10.3±0.1 6.5510.05 2.343 32.4H.4 7.9+0.1 5.8010.72 1.514 36.216.9 8.5H.1 6.22+0.37 0.767 31.713.1 11.HI. 2 6.3510.36 1.575 34.314.0 10.0±2.1 6.1610.46 1.259 39.213.1 10.2±0.1 6.1710.06 1.812 28.914.2 9.8±1.5 5.9210.13 0.841 39.311.4 12.8±0.5 . 6.3610.21 1.197 34.411.0 12.5±0.9 6.1910.31 1.146 33.519.1 12.3±1.1 6.20i0.26 1.310 34.413.6 10.2±0.9 5.7310.10 0. 726 41.414.7 12.3±1.7 5.6610.09 0.871 41.815.4 13.4±0.2 6.0610.17 1.186 38.513.8 14.2±0.9 5.8110.19 1.497 35.814.5 0.6±0.2 6.0810.57 39.012.1 1.0±0.2 6.2H0.41 35.813.5 2.4±0.5 7.88H.24 30.4+3.6 2.510.7 8.8010.99 35.015.5 45 -TABLE VI Growth parameters and hematocrit of 3 1/2-month old coho fry and 11-month old presmolts exposed to water of various s a l i n i t i e s . The water temperature varied from 8.5 to 9.2 C. The « intercept was calculated for in d i v i d u a l f i s h from the weight-3 25 length r e l a t i o n s h i p W = <*L ' , where' W i s the wet weight i n mg and L i s the fork length i n cm. The instantaneous growth rate cal-log WT - log W culated as X 100 from the mean wet weight of 2 T - t to 3 f i s h . With the exception of growth r a t e , a l l data are pre-sented as the mean ± 1 standard deviation for a sample size N of 3. AGE GROUP SALINITY (°/oo) TREATMENT TIME (DAYS) N LENGTH (cm) WEIGHT Cg) a INTERCEPT (mg) ' INSTANTANEOUS GROWTH RATE (% / DAY) HEMATOCRIT (% RBC) Presmolt 0 19 3 9.210.1 7.9±0.9 5.80±0.72 0.946 36.2±6.9 Presmolt . 10 19 3 9.4±0.1 9.2±0.4 6.57±0.20 1. 748 30.Oil.1 Presmolt 20 19 3 9.7±0.3 11.6±1.2 6.71±0.14 2.698 28.7±2.0* Presmolt 30 19 3 9.2±0.4 9.0±1.2 6.60±0.32 1.632 33.6±3.3 Presmolt 0 33 3 9.0±0.1 10.2+0.1 6.17±0.06 1.319 28.9±4.2 Presmolt 10 33 3 9.9±0.3 10.6±1.1 6.23±0.05 1.436 30.7±5.1 Presmolt 20 33 3 9.8±0.3 10.8±1.4 6.55±0.16 1.492 28.3±3.5 Presmolt 30 33 3 9.9±0.4 10.7±1.8 6.49±0.40 1.464 27.1±1.5 Presmolts 0 47 3 10.3±0.2 12.3±1.1 6.20±0.26 1.112 34.4±3.6 Presmolts 10 47 3 11.0±0.6 15.7±1.7 6.49±0.40 1.844 30.8±4.7 Presmolts 20 47 2* 11.0±0.5 14.6±2.3 5.95±0.09 1.689 34.4±4.4 Presmolts 30 47 3 10.7±0.4 15.7±1.4 7.1110.40 1.844 33.1±3.4 Presmolts 0 60 3 11.0±0.3 14.2±0.9 5.81±0.19 1.277 35.8±4.5 Presmolts 10 60 3 11.7±0.4 18.0±2.2 6.04±0.16 1.67 2 39.6H.7 Presmolts 20 60 3 11.2±0.5 14.4±1.8 5.57±0.17 1.300 36.1±4.7 Presmolts 30 60 3 10.8±0.4 14.9±1.6 6.43±0.13 1.190 34.1±5.9 Fry 10 50 6 5.8±0.4 2.3±0.5 7.51±0.37 33.9±7.3 Fry 20 50 6 5.9±0.8 2.6±0.9 7.97±0.56 29.6±3.3 Fry 30 50 6 4.7±0.3 1.18±0.3 7.72±0.57 32.1±3.6 Two measurements only: values given are mean ± 1/2 difference between two observations. - 46j -In the presmolt coho the <= intercept of the weight-length r e l a -tionship did not exhibit a consistent r e l a t i o n s h i p to any of the environmental variables examined during the period of 19 to 47 days of treatment and generally exceeded 6.0 i n those f i s h reared at various water temperatures for the entire 60-day experimental period. The intercept of presmolts exposed to 3.1 and 5.8 ppm dissolved oxygen and to 20°/oo s a l i n i t y , how-ever, did decrease su b s t a n t i a l l y a f t e r 60 days to 5.73, 5.66, and 5.57mg, res p e c t i v e l y . The f i n a l sample (60-days treatment time) of the presmolts exposed to the various environmental regimes was obtained on March 29, 1971. A comparison of these f i s h with presmolts reared i n the a r t i f i c i a l creek (Table I I I ) demonstra-tes that, with the exception of the presmolts reared i n fresh water at 1.3 C and i n f u l l - s t r e n g t h sea water, the « intercept was consistently less i n the experimental f i s h at this time. The differences i n the food rati o n and da i l y photoperiod experienced by these two groups of f i s h probably accounts for this d i s p a r i t y . The « intercept of coho f r y was generally greater than 7.3 mg indi c a t i n g that the f r y were considerably less streamlined than the presmolts. The value of « increased with elevated water temperature (Table VI) and with increasing dissolved oxygen concentration (Table V ) , but was unaffected by variations i n s a l i n i t y . The values of 6.08 and 6.21 mg recorded i n fry reared at 2.2 and 5.5 ppm dissolved oxygen suggest that these f i s h were growing r e l a t i v e l y faster i n length than i n weight, although growth i n general was retarded. - 47 -The instantaneous growth rate of presmolt coho, expressed as percentage increase i n weight per day, was markedly dependent upon the water temperature. From 4.1 to 15.0 C the mean instantaneous growth rate calculated over the 60-day experimental period increased by 0,112%/day for each degree increase in water temperature (Figure 2). The maximum mean growth rate recorded was 2.104%/day i n presmolts reared i n freshwater at 15C. Presmolts reared in freshwater at 10.2 C (Table IV) and at 9.7 C with 7.9 to 9.7 ppm dis-solved oxygen (Table V) and i n water of 10 to 30 °/oo s a l i n i t y a l l exhibited mean growth rates of 1.5 to 1.7%/day over the experimental period. Dissolved oxygen concentrations of 3.1 and 5.8 ppm reduced the rates of growth to 0.8 and 1.3 %/day respectively. Although growth rates of the coho f r y could not be calculated i t i s evident that f i s h reared for 49 to 50 days at temperatures of 10.2 C, dissolved oxygen concentrations of 8.1 and 9.3 ppm and s a l i n i t i e s of 10 and 20 °/oo a l l exhibited wet weights of 2.3 to 2.6 g (Tables IV, V, VI). Growth was sub s t a n t i a l l y reduced at dissolved oxygen concentrations of 2.2 and 5.5 ppm and at a s a l i n i t y of 30 °/oo. The severity of the stress imposed upon these f r y was evident from the 85 to 50% m o r t a l i t i e s recorded i n f r y held at 2.2 ppm oxygen and °/oo s a l i n i t y , respectively. - 48 -FIGURE 2 Mean instantaneous growth rates of presmolt coho held i n aerated well water at various temperatures for 60 days. The points represent the mean of the measurements obtained afte r 19, 33, 47, and 60 days of exposure and the v e r t i c a l bars represent ± 1 standard deviation of this mean. INSTANTANEOUS GROWTH RATE (%/day) o O — — — ro ro rb <n o J> do rb oS J I I I I I I I 1 I I ' i - 49 -No consistent r e l a t i o n s h i p between any of the en-vironmental regimes and blood hematocrit was observed in either coho presmolts or f r y (Tables IV, V, V I ) , although occasional s i g n i f i c a n t differences did occur (presmolt at 1.3 C a f t e r 19 days treatment, Table IV). Although f r y reared at 2.2 ppm dissolved oxygen exhibited a mean hematocrit of 39.0% those exposed to 5.5 and 9.3 ppm had almost i d e n t i c a l hematocrits. Although no s p e c i f i c response to any of the environ-mental regimes was observed i n the .hematocrits of coho pre-smolts the average hematocrit of the grouped f i s h did exhi-b i t a change during the 60-day experimental period. The means of the grouped hematocrits after 19, 33, and 47 days of t r e a t -ment were 33.5, 32.5, and 33.4%, r e s p e c t i v e l y , whereas af t e r 60 days exposure, this value increased to 37.4%. This l a t t e r increase coincided with si m i l a r increases i n hematocrit observed i n the coho presmolts reared i n the a r t i f i c i a l creek during the same time period (Table I I I ) . The r e l a t i v e concentration of hemoglobin components A6-8 of presmolts reared i n the outdoor a r t i f i c i a l creek and of presmolts exposed to various regimes of temperature, d i s -solved oxygen concentration and s a l i n i t y for periods of 19, 33, and 47 days are presented i n Table VII. It i s evident that with the possible exception of the f i s h reared at 1.3 and 4.1 C no v a r i a t i o n related to the experimental treatments occurred i n the r e l a t i v e concentration of these three components. - 5D -TABLE V I I Relative concentration of hemoglobin components A6-8 of the blood of presmolt coho exposed to water of various d i s -solved oxygen concentrations, temperatures and s a l i n i t i e s for periods of 19, 33 and 47 days and of coho presmolts maintained i n an a r t i f i c i a l creek during the same period. A l l data are presented as the mean ± 1 standard deviation for a sample of 3 to 6 f i s h . TREATMENT T r e a t m e n t Time ( D a y s ) * D i s s o l v e d O x y g en SAMPLE S I Z E RELATIVE CONCENTRATION OF HEMOGLOBIN COMPONENTS A6-8 (%) 19 33 47 3.1 ppm 3 86.7 ± 6.2 89.3 + 2.0 88. 5 + 1.8 5.8 ppm 3 83.0 ± 1.8 86.8 + 6.3 85.2 + 1.6 7.9 ppm 3 85.2 ± 5.9 89. 0 + 1.5 87.1 + 0.5 T e m p e r a t u r e 1.3 C 3 90.5 ± 3.1 91.4 + 0.5 86.4 + 3.1 4.1 C 3 87.2 ± 1.8 89.5 + 4.9 86.4 + 2.4 8.0 C 3 84.7 ± 2.2 88.0 + 0.7 83.6 + 0.3 10. 2 C 3 89 . 8 * * * 86.3 + 2.6 84.4 + 2.5 15. 0 C 3 84.0 ± 3.2 86.3 + 0.2 83. 7 + 3.7 S a l i n i t y 10 °/oo 3 84.7 ± 5.1 86.0 + 1.5 84.1 + 1.2 20 /oo 3 86.8 ± 4.5 89.0 + 0.6 83.5 + 2.7 30 °/oo 3 83.4 ± 5.4 87.9 + 0.9 86.6 + 1.7 C o n t r o l s 3 87.9 ± 2.4 87.9 + 0.9 85.1 + 1.4 A r t i f i c i a l C r e e k F i s h * * 6 84.9 ± 3.3 86.7 + 3.5 80.4 + 3.3 The s a m p l i n g d a t e c o r r e s p o n d i n g t o t h e t r e a t m e n t t i m e s w e r e : 19 d a y s - 1 6 / 2 / 7 1 ; 33 d a y s - 2 / 3 / 7 1 ; 47 da y s - 1 6 / 3 / 7 1 . ** F i s h f r o m t h e a r t i f i c i a l c r e e k w e r e s a m p l e d on t h e same d a t e s as t h e t r e a t e d f i s h . * * * Two f i s h o n l y : Range 8 8.5 t o 91.2%. - 51 The presmolts exposed to water temperatures of 1.3 to 4.1 C exhibited s l i g h t l y elevated concentrations of A6-8 when com-pared to f i s h reared at higher temperatures, although the difference was only 2%. Components A6-8 generally comprised a higher per-centage of the t o t a l hemoglobin i n the experimental f i s h than i n presmolts in the a r t i f i c i a l creek at the same time, although the differences were less than 5 to 6%, (Table V I I ) . This difference i s d i f f i c u l t to explain since the experimental f i s h experienced a s l i g h t l y accelerated photoperiod which should accelerate the smolting process. The r e l a t i v e concentrations of hemoglobin components A3, A l , CI, C3, C4, C5,, and C6 of presmolt coho likewise did not exhibit any changes related to any of the eleven experi-mental treatments (Tables V I I I , IX, X). These components were ranked i n order of decreasing concentration C1:C3:C4: A1:A3:C5:C6. Although small variations occurred i n in d i v i d u a l f i s h the general .ranking order of the minor components of the experimental f i s h was e s s e n t i a l l y i d e n t i c a l with that of un-treated coho presmolts as presented i n Table I. The electropherograms of the: multiple hemoglobins of coho presmolts afte r 60 days exposure and of coho f r y f o l -lowing 49 to 50 days exposure to the eleven environmental regimes are presented i n Plates 3, 4, 5, 6, 7, and 8. These electropherograms are indistinguishable from the respective electropherograms of untreated presmolts (Plates 2A,B) and fry (Plate 1F,G) observed i n Part I of this t h e s i s , again - 5 2 ' TABLE VIII The r e l a t i v e concentrations of the hemoglobin components A3, A l , Cl,C3, C4, C5, and C6 of the blood of coho presmolts reared i n aerated well water at f i v e temperatures for periods of 33 and 47 days. The values presented represent the mean ± 1 standard deviation for a sample of 3 f i s h . WATER EXPOSURE TEMPERATURE TIME (C) (DAYS) A3 1.3 33 0.9 ±0.7 4.1 33 0.2 ±0.3 8.0 33 0.8 ±0.2 10.1 33 0.5 ±0.3 15.0 33 1.2 ±1.0 1.3 47 1.3 ±0.2 4.1 47 1.4 ±0.3 8.0 47 1.3 ±0.2 10.1 47 1.1 ±0.3 15.0 47 0.9 ±0.4 Al RELATIVE Cl CONCENTRATION (%) C3 C4 C5 C6 0.9 ±0.2 1.1 ±0.9 1.3 ±0.1 1.3 ±0.4 1.4 ±0.8 1.9 ±1.0 1.7 ±0.6 2.3 ±0.5 2.0 ±0.5 2.1 ±0.6 2.8 ±0.8 4.6 ±1.2 5.4 ±0.3 5 . 8 ±1.3 4.9 ±0.6 4.1 ±0.8 3.9 ±0.2 4.6 ±0.2 5.5 ±1.3 5.5 ±2.0 1.6 ±0.5 1.7 ±0.4 2.0 ±0.6 2.8 ±0.2 2.3 ±0. 1 2.5 ±0.7 2.6 ±0.7 3.3 ±0.3 2.7 ±0.5 3.1 ±0.2 1.3 ±0.2 1.9 ±0.6 1.4 ±0.4 2.3 ±0.6 2.4 ±0.1 2.3 ±0.7 2.2 ±1.4 2.9 ±0.4 2.9 ±0.9 2.6 ±0.9 0.6 ±0.3 1.2 ±0.7 0.8 ±0. 1 0.6 ±0.4 1.0 ±0.2 0.6 ±0.3 0.8 ±0.1 1.2 ±0.2 0.7 ±0.3 1.2 ±0.6 0.6 ±0.4 0.3 ±0.6 0.5 ±0. 1 0.3 ±0.3 0.8 ±0.2 0.9 ±0.6 1.0 ±0.5 0.8 ±0.4 0.8 ±0.7 0.9 ±0.3 - 53 -TABLE IX The r e l a t i v e c o n c e n t r a t i o n s o f t h e h e m o g l o b i n c o m p o n e n t s A 3 , A l , C l , C 3 , C4, C 5 , and C6 o f t h e b l o o d o f c oho p r e s m o l t s r e a r e d i n w e l l w a t e r a t t h r e e c o n c e n t r a t i o n s o f d i s s o l v e d o x y g e n f o r p e r i o d s o f 33 and 47 d a y s . The v a l u e s p r e s e n t e d r e p r e s e n t t h e mean ± 1 s t a n d a r d d e v i a t i o n f o r a s a m p l e o f 3 f i s h . OXYGEN EXPOSURE CONCENTRATION TIME (ppm) (DAYS) 3.1 33 5.8 33 7.9 33 3.1 47 5.8 47 7.9 47 RELATIVE CONCENTRATION ( I ) A l C l C3 C4 C5 C6 1.0 ±0.9 2.0 ±1.1 1.8 ±0.3 1.3 ±0.1 1.8 ±0.02 1.5 ±0.9 3.5 ±2.6 3.5 ±1.8 2.7 ±0.6 2.9 ±0.3 3.5 ±0.3 4.1 ±0.9 1.6 ±0.8 2.9 ±1.3 1.6 ±0.4 2.1 ±0.5 2.9 ±0.5 2.5 ±0.4 2.0 ±0.8 2.4 ±1.0 1.9 ±0.2 2.1 ±0.6 2 . 5 ±0.3 2.3 ±0.3 1.5 ±0.4 1.1 ±0.9 1.2 ±0.5 0.7 ±0.4 1.3 ±0.9 0.8 ±0.2 0.3 ±0.2 0.7 ±0.2 0 . 8 ±0.7 0.9 ±0.6 0.8 ±0.4 0.6 ±0.3 - 54^  TABLE X The r e l a t i v e c o n c e n t r a t i o n s o f t h e h e m o g l o b i n components A3, A l , C I , C3, C4, C5, and C6 o f t h e b l o o d o f coho p r e s m o l t s r e a r e d a t t h r e e w a t e r s a l i n i t i e s f o r p e r i o d s o f 33 and 47 d a y s . The v a l u e s p r e s e n t e d a r e the mean ± 1 s t a n d a r d d e v i a t i o n f o r a sample o f 3 f i s h . EXPOSURE S A L I N I T Y TIME (°/oo) (DAYS) A3 RELATIVE CONCENTRATION (%) . A l CI C3 C4 C5 C6 10 33 0.7 1.8 5.8 3.0 1.1 1.1 0.5 ±0.4 ±0.5 ±0.5 ±1.2 ±0.2 ±0.7 ±0.5 20 33 0.7 1.5 3.2 3.1 1.7 0.6 0 . 3 ±0.8 ±0.1 ±1.4 ±0.4 ±1.3 ±0.7 ±0.6 30 33 0.5 .1. 7 2.8 2.8 1.9 1.5 1.0 ±0.4 ±0.5 ±0.6 ±0.9 ±0.6 ±0.2 ±0.4 10 47 0.9 2.3 4.2 3.5 3.0 1.0 1.1 ±0.3 ±0.3 ±0.7 ±0.3 ±0.7 ±0.1 ±0.3 20 47 1.4 2.9 4.4 3.2 2.9 1.1 0.6 ±0.2 ±0.8 ±1.4 ±0.2 ±0 . 6 + 0.2 ±0.4 30 47 0.6 1.8 3.4 2.7 2.6 1.2 1.1 ±0.1 ±0.1 ±0.2 ±0.6 ±0.8 ±0.3 ±0.2 - 5.5, PLATE 3 E l e c t r o p h e r o g r a m s o f t h e m u l t i p l e h e m o g l o b i n s o f p r e s m o l t c o h o s a l m o n w h i c h h a d b e e n e x p o s e d t o a e r a t e d w e l l w a t e r m a i n t a i n e d a t 1.3 C ( A ) , 4.1 C ( B ) , 8.0 C ( C ) , 10.2 C ( D ) , and 15.0 C ( E ) , f o r 60 d a y s . The e l e c t r o p h e r o g r a m s f r o m two f i s h a r e p r e s e n t e d f o r e a c h t e m p e r a t u r e . 56 -PLATE 4 Electropherograms of the multiple hemoglobins of coho f r y which had been exposed to aerated well water maintained at 1.3 C (A), 4.1 C(B), 8.0 C(C), 10.2 C(D), and 15.0 C '(E) , for 49 days. The electropherograms from two f i s h are presented for each tem-perature . (+) (-) 57 -PLATE 5 Electropherograms of the multiple hemoglobins of presmolt coho which had been exposed to well water containing dissolved oxygen concentrations of 3.1 (A), 5.8 (B), and 7.9 (C) ppm for 60 days. The electropherograms of blood samples from two f i s h are presented for each dissolved oxygen concentration. (+) .5-8 -PLATE 6 Electropherograms of the multiple hemoglobins of coho f r y which had been exposed to well water containing dissolved oxygen concentrations of 2.2 (A), 5.5 (B), and 8.1 (C), ppm for 49 days. The electropherograms of blood samples from two f i s h are presented for each dissolved oxygen concentration. - 59 PLATE 7: Electropherograms of the multiple hemoglobins of presmolt coho which had been exposed to s a l i n i t i e s of 10 (A), 20 (B), and 30 (C) °/oo for 60 days. The electropherograms of blood samples from two f i s h are presented for each s a l i n i t y . (+) -60, PLATE 8 Electropherograms of the multiple hemoglobins of coho f r y which had been exposed to s a l i n i t i e s of 10 (A), 20 (B), and 30 (C) °/oo for 50 days. The electropherograms of blood samples from two f i s h are presented for each s a l i n i t y . (+) (-) - 6 i -demonstrating that the environmental regimes to which these f i s h were exposed did not influence the electrophoretic pattern of the hemoglobin polymorphs. EFFECT OF EXTENDED FRESHWATER RESIDENCE AND HYPOXIA UPON JUVENILE COHO. Plate 9 and Table XI present the electrophoretic patterns and r e l a t i v e concentrations of the multiple hemo-globins of seventeen month-old coho salmon which had been reared for 5 1/2 months i n fresh water at low (2.2 ppm) and high (9.7 ppm) dissolved oxygen concentrations and of normal seawater postsmolt of the same age. For reasons noted prev-i o u s l y , the re s u l t s represent the observations made on August 3, 1971 on one f i s h from each environment. It i s evident that the seawater and high-oxygen freshwater postsmolts are s i m i l a r in respect to s i z e , hema-t o c r i t and number and r e l a t i v e concentration of the ten hemo-globin components. Components A6-8 comprise approximately 1% more of the hemoglobin in the freshwater postsmolt but i t is d i f f i c u l t to attach much significance to this r e l a t i v e l y small difference without a large sample s i z e . The coho which had been reared under hypoxic conditions, however, was much smaller and has a much higher hematocrit than the former f i s h . This increase i n hematocrit had occurred by the time the f i s h were fourteen months old when the mean hematocrit of three f i s h was 50.4%. The most s i g n i f i -cant effect of the hypoxic environment, however, was upon the - 62 -PLATE 9 Electropherograms of the multiple hemoglobins of 1 1/2-year-old coho presmolts which had been maintained i n aerated ( 9,7 ppm well water (A), unaerated (2.2 ppm C^) well water (B), or had been transferred to aerated ( 9.3 ppm C^) 2 months previously (C) . Two electropherograms are presented for the same blood sample from each f i s h . (+) (-) - 6 3 -TABLE X I C o m p a r i s o n o f t h e s i z e , a i n t e r c e p t o f t h e w e i g h t - l e n g t h 3 25 r e l a t i o n s h i p W = a t ' , h e m a t o c r i t and r e l a t i v e c o n c e n t r a t i o n o f t h e h e m o g l o b i n c o m p o n e n t s o f t h e b l o o d o f p r e s m o l t coho s a l m o n m a i n t a i n e d i n a e r a t e d (9.7 ppm C^) and u n a e r a t e d (2.2 ppm C^) w e l l w a t e r and o f p o s t s m o l t s o f t h e same age w h i c h h a d b e e n r e -s i d i n g i n s e a w a t e r f o r a p p r o x i m a t e l y 1 month. O n l y one f i s h f r o m e a c h e n v i r o n m e n t was e x a m i n e d . a LENGTH WEIGHT INTERCEPT (cm) (g) (mg) RELATIVE CONCENTRATION OF HEMATOCRIT HEMOGLOBIN COMPONENT (%) (! RBC) A6-8 A3 A l C l C3 C4 C5 C6 F r e s h w a t e r (9.7 ppm 0 2 ) 13.5 26.4 5.59 27.0 57.9 2.6 7.1 8.6 9.3 7.8 3.7 3.1 F r e s h w a t e r (2.2 ppm 0 2 ) 9.0 7.3 5.78 54.8 81.2 2.3 2.7 4.2 3.1 3.1 2.2 1.3 SG3. WcltOT* ( 9.2 ppm 0 2 ) 14.1 30.7 5.65 33.3 50.9 3.3 10.5 13.3 9.0 9.1 0.9 3.1 " 64' r e l a t i v e concentrations of the 'hemoglobin polymorphs. Com-ponents A6-8 comprised over 81% of the t o t a l hemoglobin which i s only 4% less than the r e l a t i v e concentration ob-served at the beginning of the adaptation to the hypoxic conditions. Similar lack of change occurred i n the d i s t r i -bution of the seven remaining hemoglobin components.. These observations suggest that exposure of 11 1/2-month old pre-smolt coho to extremely hypoxic water resulted i n a v i r t u a l cessation of growth and ontogenetic hemoglobin development as well as a marked increase in hematocrit. . DISCUSSION II One of the major requirements in the design of experiments to determine the influence of some factor upon a p a r t i c u l a r reaction i s to maintain adequate control of a l l the remaining factors which may influence the reaction. In b i o l o g i c a l systems, e s p e c i a l l y i n cases were l i v i n g animals are used, this i d e a l i s seldom r e a l i z e d . In f i s h complex interactions between season, a var i e t y of environmental f a c t o r s , s i z e , r a t i o n size and composition,and feeding schedule influence the observed rates of growth and develop-ment (LeBrasse.ur and Parker, 1964; Paloheimo and Di c k i e , 1966; LeBrasseur, 1969; Atherton and Aitken 1970; Brett and Higgs, 1970; Brett, 1971). When such animals are subjected to en-vironmental manipulations i t i s necessary to determine whether or not some uncontrollable factor may be l i m i t i n g the r e -sponse to the f i s h to the variable under study. This pro-blem i s expecially applicable to the present study since i t encompassed two possible e f f e c t s of environment upon hemo-globin polymorphism i n coho salmon. F i r s t c e r t a i n environ-mental factors could d i r e c t l y influence the composition of the hemoglobin polymorphs. Secondly, the environment could influence the growth of the f i s h and result i n si z e - r e l a t e d changes in the hemoglobin pattern.If, therefore, the growth responses of the coho to variations i n ce r t a i n environmental factors were abnormal because of some uncontrolled variable the i n t e r p r e t a t i o n of any changes i n hemoglobin pattern would be d i f f i c u l t , i f not impossible. The effects of water tempera-ture, dissolved oxygen concentration and s a l i n i t y upon the patterns of growth of coho presmolts and fry observed i n the present study, were generally i n agreement, however, with s i m i l a r information published for this and other species of salmonids. In juvenile freshwater sockeye salmon, the rate of growth increased by 0.159%/day/degree C over the range of 5 to 15 C and i s maximal at 15C (Brett, Shelbourn and Shoop, 1969). Although the coho presmolts exhibited, maximum growth at 15C the effect of elevated water temperature upon the rate of growth was somewhat lower (0.112%/day/degree C). Hermann, Warren, and Doudoroff (1962) reported that dissolved oxygen concentrations below 5.6 ppm severely cur-t a i l e d growth and food consumption i n juvenile freshwater coho at 20C. The present re s u l t s are i n general accord with 66 -t h e s e o b s e r v a t i o n s s i n c e t h e r a t e s o f g r o w t h o f p r e s m o l t c o h o a t 5.8, 7.9, and 9.7 ppm o x y g e n were e s s e n t i a l l y t h e same, w h e r e a s a t 3.1 ppm t h e r a t e was o n l y 56% o f t h a t ob-s e r v e d a t t h e f o r m e r c o n c e n t r a t i o n s . I n coho f r y , h o w e v e r , g r o w t h was r e d u c e d a t 5.5 ppm o x y g e n a n d more s e v e r e l y a t 2.2 ppm. O v e r 85% o f t h e f r y d i e d a t t h i s l a t t e r c o n c e n t r a t i o n . Hermann et al. ( 1 9 6 2 ) r e p o r t e d m o r t a l i t i e s o f 30 t o 100% f o r f r y o f a p p r o x i m a t e l y t h e same a g e . M o r t a l i t y d e c l i n e d t o z e r o a f t e r J u l y a n d A u g u s t and t h i s d e c l i n e was c o r r e l a t e d t o a d e c r e a s e i n t h e r a t e o f o x y g e n c o n s u m p t i o n as t h e f r y p r o g r e s s e d t h r o u g h t h e i r f r e s h w a t e r s t a g e ( i b i d . ) . T h e s e o b s e r v a t i o n s p r o b a b l y e x p l a i n t h e d i f f e r e n c e i n e f f e c t o f o x y g e n c o n c e n t r a t i o n s o f a p p r o x i m a t e l y 5.5 t o 5.8 ppm u p o n g r o w t h i n p r e s m o l t s and f r y r e c o r d e d i n t h e p r e s e n t e x p e r i -m e n t s . A d i s p a r i t y e x i s t s b e t w e e n t h e e f f e c t o f s a l i n i t y u p o n t h e g r o w t h o f coho p r e s m o l t s r e p o r t e d h e r e a n d f r y by O t t o ( 1 9 7 1 ) who r e p o r t e d t h a t s a l i n i t i e s e x c e e d i n g 10°/oo i n h i b i t e d g r o w t h and s a l i n i t i e s o f 30°/oo r e s u l t e d i n mor-t a l i t i e s o f 30 t o 50%. G r o w t h i n p r e s m o l t coho a t a l l s a l i n i t i e s up t o 30°/oo e x c e e d e d t h a t i n f r e s h w a t e r c o n t r o l s and was m a x i m a l a t 20°/oo. The a v e r a g e r a t e o f g r o w t h a t 20°/oo s a l i n i t y i n c l u d e d one v a l u e ( 1 9 - d a y s a m p l e ) w h i c h was a p p r o x i m a t e l y d o u b l e t h a t o b s e r v e d i n t h e r e m a i n i n g t h r e e s a m p l e s . I f t h i s v a l u e i s e l i m i n a t e d f r o m t h e c a l c u l a t i o n s t h e n t h e mean i n s t a n t a n e o u s r a t e o f g r o w t h a t a s a l i n i t y o f 10°/oo e x c e e d s t h a t a t 20°/oo t h e r e s p e c t i v e v a l u e s b e i n g 1.675 and 1'.494%./day. In f r y growth was approximately equal i n fresh water and at s a l i n i t i e s of 10 and 20 °/oo but r e -duced at 30 °/oo s a l i n i t y . Although no data are available concerning the amount of potential food carried to the f i s h i n the seawater i t i s probable that organisms i n the sea water were a supplementary source of food to the presmolts and may account for the d i s p a r i t y i n growth of these f i s h i n fresh and sea water. Saunders and Henderson (1969) reported that the rate of growth of A t l a n t i c salmon parr were the same in fresh water as i n s a l i n i t i e s of 7 and 15 °/oo, but were reduced at s a l i n i t i e s exceeding 22 °/o o . These observations are i n f a i r l y close agreement with the present r e s u l t s . No sa t i s f a c t o r y explanation can be offered for the d i s p a r i t y of these results with those of Otto (1971) although i t i s p o s s i -ble that the frequent anesthetization and measurement for length and weight conducted i n the l a t t e r study may have adversely effected the a b i l i t y of coho to adapt to higher s a l i n i t i e s . In general, then, there i s no evidence to suggest that any abnormal responses to variations in the temperature, dissolved oxygen concentration or s a l i n i t y occurred i n the rates of growth of the juvenile freshwater coho salmon, A comparison of the = intercept of the weight-length r e l a t i o n -3 25 ship W = <*L' ' of presmolt coho from the a r t i f i c i a l creek and from the eleven experimental environments after 60 days of treatment suggests that with one possible exception, l i t t l e 68 -or no acceleration of the streamlining process occurred i n the treated f i s h . Presmolts reared at 3.1 ppm dissolved oxygen exhibited reductions i n the « intercept over the experimental period but this was coupled to an extreme reduction i n growth. A similar occurrence was observed i n the 60-day sample of presmolts reared at 5.8 ppm dissolved oxygen. At 20 °/oo s a l i n i t y « decreased to 5.57 mg which would correspond to the value i n 14-month old presmolts in the a r t i f i c i a l creek. Thus, although exposure of presmolt coho to 20 °/oo s a l i n i t y for a sixty day period did acceler-ate the streamlining process the value of « did not decrease to that observed i n normal f i f t e e n to sixteen-month old smolts. The foregoing considerations lead to two major con-clusions based on the observation that no s i g n i f i c a n t changes occurred i n the hemoglobin pattern of coho fry or presmolts exposed to f i v e water temperatures, three dissolved oxygen concentrations and three s a l i n i t i e s . F i r s t the t r a n s i t i o n from the fry hemoglobin pattern composed of three anodic components to the presmolt pattern of f i v e anodic and f i v e cathodic com-ponents i s not a d i r e c t r e s u l t of fluctuations of water temperature and dissolved oxygen concentrations i n the envi-ronment. Several major biochemical and physiological changes are known to occur during this period of t r a n s i t i o n (Baggerman, 1960; Conte et aZ.,,1966; Vanstone and Markert, 1968; G i l e s , 1969) and i t appears l i k e l y that factors influencing erythro-poretic a c t i v i t y may be responsible for the observed s h i f t s i n hemoglobin pattern. Since f i s h erythrocytes are nucleated they may be capable of increasing the r e l a t i v e concentration of the minor hemoglobin components A l , A3, CI, C3-6 in the red blood c e l l s of fry which contain only components A6-8. This would make the stimulation of erythropoiesis, possibly a new c e l l l i n e , t o t a l l y unnecessary. Fantoni et al. (1969) have demonstrated that new erythroid c e l l l i n e s are activated during the ontogenetic t r a n s i t i o n in synthesis of f e t a l to adult hemoglobin i n mammals. Mammalian erythrocytes, however are non-nucleated and therefore incapable of synthetizing new proteins. A second aspect of the present findings was that while the various environmental regimes to which presmolt coho were exposed did not r e s u l t i n any d i r e c t changes i n the hemoglobin electrophoretic pattern, t h e i r influence upon growth did i n certai n instances r e s u l t i n f i s h which were p h y s i c a l l y larger than untreated postsmolts i n which hemoglobin meta-morphosis had begun. Presmolts reared for 60 days in f r e s h -water at 15 C and i n 1 0° / o o . s a l i n i t y at approximately 9 C were equal to or larger than 16 1/2-month old postsmolts which had been i n seawater for one month. In addi t i o n , c e r t a i n i n d i v i -duals from the 20°/oo s a l i n i t y treatment and the 10C fresh-water treatment were as large as the postsmolts. As discussed previously, the increase in size i n the treated f i s h was not associated with a decrease in the « intercept of the weight-length r e l a t i o n s h i p which is c h a r a c t e r i s t i c of normal smolts and postsmolts. These observations suggest that physical size i s not a s i g n i f i c a n t factor i n determining the timing of the presmolt to postsmolt s h i f t in the multiple hemoglobin pattern of coho salmon. It i s also s i g n i f i c a n t that larger presmolts which had been reared i n dilu t e d sea water did not exhibit hemoglobin pattern changes since a combination of size and exposure to sea water may have been necessary to e l i c i t a response. The elimin-ation of this p o s s i b i l i t y i s reinforced by the observation that coho postsmolts maintained i n fresh water exhibited si m i l a r changes in hemoglobin pattern to normal seawater postsmolts of the same age. Since environmental and si z e - r e l a t e d factors do not control the t r a n s i t i o n from the hemoglobin pattern of juvenile freshwater coho to that of seawater postsmolts i t would appear that the changes are related to the age of the f i s h . The mechanism of this age-related control i s unknown for both f i s h and higher . vertebrates. In mammals the t r a n s i t i o n from f e t a l to adult hemoglobin synthesis apparently involves the production of an erythropoiesis-stimulating substance and the a c t i v a t i o n of d i f f e r e n t erythroid c e l l l i n e s (Fantoni et al. 3 1969-, Jonix and Nijhof, 1969). Since a s i m i l a r process i s apparently op-erative i n the change from alevin to fry hemoglobin i n A t l a n t i c salmon (Vernidub, 1966) i t i s probable that the production of the adult hemoglobin pattern during the early period of marine residence involves the stimulation of a separate c e l l l i n e . Present information regarding the s i t e s of production of erythrocytes and factors c o n t r o l l i n g red c e l l production in f i s h i s i n s u f f i c i e n t to resolve the foregoing questions. The ontogenetic variations i n coho hemoglobin with age rather than size appears to c o n f l i c t with observations made upon certai n other salmonids i n which such changes i n hemo-globin pattern were related to length (Koch et al.s 1964; Wilkins and l i e s , 1966; Westman, 1970). In a l l of the fore-going studies, however, the precise age of the f i s h was apparent-l y unknown. Under normal rearing conditions the size of the f i s h i s usually related to i t s age and i t would seem reasonable to conclude that the apparent d i s p a r i t y could be resolved by trans-posing the length measurements to thei r respective ages. When the length-related changes i n the hemoglobin polymorphs of sea-run and landlocked A t l a n t i c salmon are compared (Westman, 1970) i t i s evident that the landlocked f i s h generally exhibit more rapid changes than those which migrate to sea. Since land-locked salmon grow at a slower rate i t i s probable that the differences i n the rates of hemoglobin changes would be elimina-ted or at least reduced by r e l a t i n g the hemoglobin changes to age rather than length. In only one instance did an environmental factor influence the hemoglobin pattern of juvenile coho salmon. Fish reared i n freshwater containing 2.2 ppm dissolved oxygen throughout the entire period of smolting and early marine r e s i -dence did not exhibit the changes in the hemoglobin pattern normally associated with the early postsmolt period. Although chronologically postsmolts these f i s h ceased growing after being acclimated to the low oxygen concentrations. The r e l a -t i v e concentrations of the ten hemoglobin components of thi s - J.2 -seventeen-month old f i s h remained e s s e n t i a l l y undistinguish-able from those of eleven-month old presmolts. The hemato-c r i t of the low-oxygen f i s h increased to approximately 501 suggesting that erythropoeisis was not impaired by t h i s treatment. Two possible explanations could account for the e f f e c t of long-term exposure to low oxygen tensions. F i r s t , since growth v i r t u a l l y ceased i t i s possible that other aspects of t h e i r p h y s i o l o g i c a l development were also retarded and the f i s h remained p h y s i o l o g i c a l l y presmolts. Although the <= intercept of the weight-length relationship did decrease to about 5.8 mg this e ffect was not the r e s u l t of growth and probably represents an i n a b i l i t y to s a t i s f y the animals meta-b o l i c requirements. In thisxconnection, i t i s well established that small juvenile sockeye salmon, Oncorhynchus nerka7may remain i n freshwater for an additional year before smolting and embarking upon the i r seaward migration (Foerster, 1968). Although this occurrence i s less frequent i n coho salmon i t would seem reasonable to expect these smaller f i s h to return to t h e i r presmolt physiological condition. Secondly, i t i s possible that the hypoxic conditions exerted some dir e c t i n -fluence upon the erythropoeitic organs, thereby retarding the production or erythroid c e l l s capable of synthetizing adult hemoglobins. Such a system could involve the production of s p e c i f i c erythropoeitins for the s e l e c t i v e stimulation of erythrocytes containing presmolt or adult hemoglobins. Further experimentation involving the exposure of marine coho to hypoxic conditions and the i n j e c t i o n of postsmolt plasma into presmolts i n order to elucidate the actual mechanism involved. No relat i o n s h i p between hematocrit and water tempera-ture, dissolved oxygen concentration or s a l i n i t y was observed i n either coho presmolts or f r y . In view of the severity of some of the treatments this r e s u l t was somewhat unexpected. Grigg (1969) observed increased hematocrits i n bullheads acclimated to 9 C when compared to those acclimated to 24 C. Hematocrits tended to increase with increasing water temperature in the common carp, Cyprinus oarpio (Houston and DeWilde, 1968) and rainbow t r o u t , Salmo gairdnerij, (DeWilde and Houston, 1967). Cameron (1970) demonstrated that higher temperatures, hemato-c r i t s were raised i n f i e l d populations of p i n f i s h , Lagodon rlaomboides3 stripped mullet, Mugit aephalus, but that these increases were not observed during acclimation to temperatures of 10 and 25 C under laboratory conditions. It must be noted that i n a l l the foregoing studies extensive overlap occurred i n hematocrit values at the various acclimation temperatures and that the differences observed were generally in the order of 2-6%. It i s quite l i k e l y that the small sample size and con-commitant high v a r i a b i l i t y may have obscured small effects of the environmental variables upon hematocrit i n the present studies. SUMMARY II 1) Coho presmolts (11-months old) and f r y (3 1/2-months old) were exposed to f i v e d i f f e r e n t water temperatures from 1 to .15 C, three dissolved oxygen concentrations from 2.2 to 8.1 ppm and three s a l i n i t i e s from 10 to 30 °/o o . Changes i n growth, hematocrit and hemoglobin pattern were recorded. 2) Growth increased with increasing water temperatures to a maximum of 15 C i n both groups of f i s h . 3) • Growth was i n h i b i t e d i n f r y at oxygen concentrations of 5« 5 pp m and more strongly at 2.2 pp m oxygen. i n presmolt coho, growth was severely reduced at 3.1 ppm oxygen but not at 5.8 ppm or higher. 4) Growth was optimal at a s a l i n i t y of 20 °/oo and decreased at 30 °/oo i n both groups of f i s h although the decrease was much more evident i n the fry than i n the presmolts. 5) No change i n hematocrit or hemoglobin pattern occurred i n response to any of the treatments i n either fry or presmolts. 6) Postsmolts maintained i n oxygenated freshwater developed the hemoglobin electrophoretic pattern c h a r a c t e r i s t i c of sea-water postsmolts of the same age. 7) The hemoglobin pattern of juvenile coho reared i n hypoxic freshwater (2.2 ppm dissolved oxygen) throughout the presmolt and early postsmolt period f a i l e d to exhibit the changes c h a r a c t e r i s t i c of normal postsmolts and remained i n d i s t i n g u i s h able from the pattern exhibited by twelve-month old presmolts. - 75 -PART THREE OXYGEN EQUILIBRIUM CHARACTERISTICS OF THE HEMOGLOBIN AND WHOLE BLOOD OF COHO FRESHWATER ADULTS AND FRY INTRODUCTION III Although the oxygen e q u i l i b r i a of f i s h blood have been studied for some time, almost nothing i s known of onto-genetic variations in oxygen a f f i n i t y , Bohr and Root s h i f t s and heme-heme in t e r a c t i o n for these animals. This i s especial -ly surprising since s i g n i f i c a n t differences i n the oxygenation c h a r a c t e r i s t i c s of the embryonic or f e t a l and the adult hemo-globins have been reported for mammals (Prosser and Brown, 1961), birds (Mission and Freeman, 1972) and amphibians (Riggs, 1951; Wood, 1971). The f e t a l hemoglobin of the skate, Raja binooulata, (Manwell, 1958) and the spiny dogfish Squalus suekleyi, (Manwell, 1963) exhibits a higher oxygen a f f i n i t y and, for the l a t t e r species, a higher heme-heme inter a c t i o n than adult hemoglobin. The p o s s i b i l i t y of ontogenetic v a r i a -tions i n the oxygen equilibrium of the blood of free-swimming juveniles was not investigated i n these species. Somewhat more i s known regarding the oxygenation c h a r a c t e r i s t i c s of the ind i v i d u a l components of the multiple hemoglobins of f i s h . Work on such diverse species as the Japanese e e l , Anguilla japonica, (Yamaguchi et al., 1962 b; Hamada et al., 1964; Yoshioka et a l . , 1968; Itada, T u r i t z i n , and Steen, 1970), rainbow trout Salmo ivideus (Binotti et al., - 76 -1971), chum salmon Onoorhynohus keta, (Hashimoto, Yamaguchi and Matsuura, 1960) and loach (Yamaguchi et al., 1962 a ) , has demonstrated that i n general the e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h -able components of f i s h hemoglobin d i f f e r i n one or more of the i r oxygenation c h a r a c t e r i s t i c s . This d i v e r s i t y i n the functional c h a r a c t e r i s t i c s of the hemoglobin polymorphs lends support to the concept that each component may serve to more e f f e c t i v e l y meet the oxygen requirements of the f i s h under d i f f e r e n t p h y s i o l o g i c a l and environmental conditions. Willmer (1938) asserted that the lack of a large Bohr s h i f t i n the blood of ce r t a i n f i s h i s an adaptation to high environmental carbon dioxide tensions irregardless of oxygen a v a i l a b i l i t y , whereas Krough and Leitch (1919) suggested that f i s h l i v i n g at- low environmental tensions possessed hemo-globin with a high oxygen a f f i n i t y i n order to saturate the blood with oxygen at the g i l l s and a s i g n i f i c a n t Bohr s h i f t to f a c i l i t a t e the maintenance of a high blood:tissue oxygen gradient. The large Bohr ef f e c t observed i n the blood of active f i s h which do not encounter low environmental oxygen tensions supposedly offsets the increase i n oxygen a f f i n i t y which occurs when environmental temperature i s reduced, thereby maintaining a high blood:tissue oxygen gradient (Black, Kirkpatrick and Tucker, 1966a,b; Black, Tucker and K i r k p a t r i c k , 1966a). Although the foregoing interpretations may apply to ce r t a i n species of f i s h there are many cases i n which they do not apply (Hashimoto et al., 1960; Lenfant, Johansen and Grigg, 1967), thereby making generalizations somewhat d i f f i c u l t . The situations further com-p l i c a t e d by the possible action of various a l l o s t e r i c effectors - 77 -which may modify one or more of the functional characteris-t i c s of the hemoglobin. Since coho salmon are anadromous f i s h , they encoun-ter a wide range of environmental conditions. It has been demonstrated i n this thesis that the hemoglobin components of f ry and postsmolts are considerably d i f f e r e n t (Part I),but that the electrophoretic hemoglobin pattern of juvenile freshwater f i s h i s e s s e n t i a l l y unaffected by variations i n en-vironmental temperature, dissolved oxygen concentration and s a l i n i t y (Part I I ) . The following experiments were undertaken to determine whether ontogenetic changes i n hemoglobin compo-s i t i o n were associated with changes i n the oxygen equilibrium c h a r a c t e r i s t i c s of the blood and to interpret the functional significance of the ontogenetic variations i n r e l a t i o n to the transport of oxygen by the blood under normal environmental conditions. METHODS III FISH EMPLOYED Migrating adult coho were trapped at the Department of Fisheries of Canada f a c i l i t i e s at Robertson Creek during August and September, 1971, and transported to our aquarium f a c i l i t i e s i n West Vancouver where they were maintained i n 10-foot diameter c i r c u l a r aquaria provided with flowing aerated well water. Although these f i s h were used i n the majority of the following experiments, a few migrating adult coho were obtained from the Big Qualicum River on Vancouver Island. No - 78 -difference i n the hemoglobin pattern was observed i n f i s h of either sex 0 r l o c a l i t y . PREPARATION OF HEMOGLOBIN SOLUTIONS The f i s h were removed from the aquarium and k i l l e d by a blow on the head. Approximately 50 ml of blood was c o l -lected by a o r t i c t a i l puncture into a 50 ml syringe f i t t e d with a 1 1/2-inch 18 gauge needle and containing 1 ml of sodium heparin solution (1000 U.S.P. units/ml, Connaught Medical Research Laboratories, Toronto, Ontario). The blood was well mixed and samples removed for determination of hematocrit con-centration and ATP concentration. The remaining blood was cen-trifuged at 3000 rpm i n a Phillips-Drucker centrifuge and the plasma and buffy layer of white blood c e l l s removed by aspi r a -t i o n . The packed erythrocytes were washed twice with 1.0% NaCl. After the f i n a l wash the erythocytes were cooled i n an ice bath, hemolyzed with 5 times the erythocyte volume of ice cold 0.001 M t r i s - H C l , pH 7.5 and frozen at -40 C for 1 hour. The frozen suspension was then slowly thawed at room temperature and centrifuged at 37,000 X g for 1 hour i n a re f r i g e r a t e d Sorvall Model RC-2 centrifuge. The supernatant was removed and subjected to u l t r a f i l t r a t i o n through a PM-30 DIAFLOW u l t r a f i l t r a t i o n mem-brane (Amicon Corp., Lexington, Massachusetts) at 50 p . s . i . pressure of nitrogen. This f i l t r a t i o n resulted i n a solution containing 8 to 22.7 gl hemoglobin. In order to further reduce the phosphate concentration of the f i l t e r e d adult hemoglobin solution 1.0 M NaCl i n 0.001 M - 79 -tr i s - H C l b u f f e r , pH 7.5, was added to y i e l d a f i n a l NaCl concentration of 0.1 molar. Approximately 1.2 to 2.0 g of the hemoglobin in a volume of 5 to 15 ml were applied to a 2.5 X 40 cm water jacketed chromatographic column of G25-fine Sephadex (Pharmacia Canada Ltd.) equilibrated with 0.1 M NaCl i n 0.001 M t r i s - H C l b u f f e r , pH 7.5, maintained at 2 C (Berman, Benesch and Benesch, 1971). The hemoglobin was eluted with the e q u i l i b r a t i o n buffer at a pressure head of 30 cm of water and c o l l e c t e d in 3 to 10 ml fractions i n a re f r i g e r a t e d f r a c t i o n c o l l e c t o r (Buchler Instruments Inc.). After concentrating the eluted hemoglobin to approximately 10 g% with the Diaflow molecu-l a r f i l t e r t r i s - H C l buffer (1.0 M t r i s - H C l containing 0.1 moles / l of NaCl), pH 7.5, was added to y i e l d a f i n a l t r i s - H C l con-centration of 0.1 molar. The t o t a l phosphate concentration of this solution was estimated to be approximately 0.038 ymoles /ml or 0.1 moles phosphate/mole hemoglobin employing the method of B a r t l e t t (1959). The hemoglobin concentration was reduced to approximately 4.0 g% by d i l u t i o n with 0.1 M NaCl i n 0.1 M t r i s - H C l . The pH of this d i l u t i o n buffer was previously adjusted to y i e l d a f i n a l pH near the desired value and f i n a l pH adjust-ments were made with 0.1 N HC1 or NaOH. Hemoglobin components A6-8 were iso l a t e d from the o r i g i n a l concentrated hemolyzate with a modification of the method of B i n o t t i et al. (1971). S u f f i c i e n t 1.0 M t r i s - H C l , pH 8.6, was added to the hemolyzate to yie.ld a f i n a l buffer concentration of 0.1 molar and the pH adjusted to 8.5. A r e -fri g e r a t e d chromatographic column (8.0 X 5.0 cm, h X d) of DEAE-Sephadex, A-50, was equi l i b r a t e d at 2 C with 0.1 M t r i s -- 80 -HC1, pH 8.5 employing a p o l y s t a l t i c pump (Buchler Instruments Inc.) to maintain a constant flow rate of 90 ml/hr through the column. Up to 4.0 grams of hemoglobin i n a volume of 50 ml were applied to the column and eluted overnight with 0.1 M t r i s -HC1 bu f f e r . Under these conditions only components A6-8 of the adult hemoglobin were retained on the exchange res i n as determined by electrophoresis. These three components were then eluted with 1:1 (v:v) mixture of 0.1 M t r i s - H C l , pH 8.5 and 0.1 M KH2P04. Sol i d NaCl was added to the elutant to y i e l d a f i n a l NaCl concentration of 0.1 molar and the pH adjusted to 7.5 with 0.1 N HC1. The hemoglobin was concentrated to approx-imately 10 g% by pressure f i l t r a t i o n and chromatographed on the G25-Sephadex column as described previously. Following a f i n a l concentration by pressure f i l t r a t i o n the hemoglobin was bu f f e r -ed with 0.1 M t r i s - H C l containing 0.1 moles/1 NaCl at the appro-priate pH. The t o t a l phosphate of thi s solution was approxi-mately 0.2 moles/mole hemoglobin as determined by the method of B a r t l e t t (1959). MEASUREMENT OF INTRACELLULAR ATP CONCENTRATION The concentration of adenosine triphosphate, (ATP), in the blood was determined by measuring the decrease in op-t i c a l density at 340 mu when diphosphopyridine nucleotide (DPN) is oxidized from i t s reduced form (DPNH), according to the f o l l -owing set of reactions; (Sigma Technical B u l l e t i n 366-UV, 1967): - 81 -phosphoglycerate ATP + 3 phosphoglycerate —> ADP + 1,3-diphos-phoglycerate kinase r • ' • g 1 y c e r a 1 d e hy d e : : 1,3-diphosphoglycerate + DPNH < phosphate dehydrogenase glyceraldehyde-3-phosphate + DPN + P^ The method i s not s p e c i f i c for ATP but measures the t o t a l concentration of the nucleoside triphosphates. Since ATP comprises the vast majority of the organic phosphate in f i s h blood (Rapoport and Guest, 1949; G i l l e n and Riggs, 1971) the contribution of these other nucleoside triphosphates to the enzyme reaction i s probably n e g l i g i b l e . Analysis were per-formed on duplicate or t r i p l i c a t e samples (0.5 to 1.0 ml) of whole blood using the enzyme k i t d i s t r i b u t e d by the Sigma Chemical Company. MEASUREMENT OF HEMOGLOBIN CONCENTRATION Total hemoglobin concentration was assayed by measur-ing the absorbance at 540 my afte r conversion of a l l hemoglobin to the cyanmethemoglobin form with an aqueous alkaline solution containing ferricyanide and cyanide reagent (Uni-Tech Chemical Manufacturing Co.). Duplicate or t r i p l i c a t e 20 ml samples of whole blood or hemoglobin solution were added to the 5 ml of cyanide-ferricyanide reagent and the absorbance measured using the reagent as a blank. The absorbances were related to hemo-globin concentration by comparison to a low range Hemochrome-- 82 -Fe standard (Uni-Tech Chemical Manufacturing Co.) treated i n the same manner. Methemoglobin concentration was determined on duplicate samples of blood or hemoglobin solutions by measur-ing the decrease i n absorbance at 635 my upon the addition of sodium cyanide to a hemoglobin solution (Evelyn and Malloy, 1938). COLLECTION OF WHOLE BLOOD FOR MEASUREMENT OF OXYGEN EQUILIBRIA: Blood was removed from the t a i l as described previous-l y (Methods I ) . The blood was well mixed to ensure uniform d i s t r i b u t i o n of erythrocytes and aliquots delivered into 25 or 50 ml pear-shaped b o i l i n g flasks which contained a few grains of sodium heparin (160,000 U.S.P. units/gram). Coho fry were anesthetized with 2-phenoxyethanol and blood c o l l e c t e d i n l i g h t l y h e parinizedcapillary tubes from the severed caudal peduncle. The blood was expelled into 5 ml culture tubes containing a few grains of sodium heparin and immersed i n an ice bath. A few additional drops of sodium heparin solution were added to the culture tubes during the c o l l e c t i o n period. The blood from a t o t a l of 113 fry was col l e c t e d on January 17, 1972. This required about 75 minutes and yielded 11.5 ml of blood. One-half ml of 1.0% NaCl was added to bring the volume to 12.0 ml. The blood was well mixed and divided into aliquots to be used i n the determination of oxygen e q u i l i b r i a . - 83 -DETERMINATION OF OXYGEN EQUILIBRIA Oxygen e q u i l i b r i a of whole blood and hemoglobin solutions were determined by a modification of the method of Edwards and Martin (1966). The blood or hemoglobin solution to be studied was divided into 2 aliquots and placed i n 25 or 50 ml pear-shaped b o i l i n g flasks which served as tonometers. The flasks were clamped to a variable speed wrist-action shaker and p a r t i a l l y submerged i n a water bath at the appropriate e q u i l i b r a t i o n temperature. A i r and nitrogen gas were saturated with water vapor by bubbling through a series of flasks which were half f i l l e d with water and maintained at the desired e q u i l i b r a t i o n temperature by submersion in the water bath. Each gas was then delivered to one of the tonometers at the rate of 200 ml/minute and the blood or hemoglobin solution shaken u n t i l the tonometer receiving nitrogen had an oxygen tension of less than 0.5 mm Hg and the tonometer receiving a i r had an oxygen tension exceeding 120 mm Hg. In order to determine the effect of carbon dioxide upon the oxygen equilibrium of whole blood, gas m i x t u r e s ( c e r t i -fied'grade Matheson of Canada Ltd.) containing 0.447 ± .009 % (v:v) C0 2:balance N2 and 0.452 ± .009! C0 2:balance a i r were substituted for the pure nitrogen or a i r i n the e q u i l i b r a t i o n procedures. Since such gas mixtures may become s t r a t i f i e d after standing for some time the cylinders were r o l l e d back and forth along the f l o o r for 10 minutes p r i o r to use. After e q u i l i b r a t i o n a portion of the oxygenated blood was drawn into a 1.0 ml disposable p l a s t i c syringe marked i n divisions of 0.01 ml and f i t t e d with a 1 1/2-inch long - 84 -21 gauge needle (Becton-Dickenson Ltd.). The a i r was expelled from the syringe and the volume of oxygenated blood recorded. An appropriate volume of deoxygenated blood was drawn into the syringe to y i e l d the desired percentage of oxygenated blood. Approximately 0.05 ml of mercury was then drawn into the syringe anaeobically and the needle sealed by i n s e r t i n g i t into a rubber stopper. The mercury was run back and forth in the syringe to thoroughly mix the blood for 2 to 3 minutes. The p a r t i a l pressure of the blood i n the syringe was then measured using a water-jacketed Radiometer PC^ electrode, type E5046, i n conjunction with a Radiometer acid-base analy-zer, type PHM 71b or PHM 72b. The c i r c u l a t i n g coolant in the water jacket was the same temperature as the blood e q u i l i b r a -t i o n temperature. Two oxygen equilibrium curves could be determined within a 4-5 hour period. The oxygen content of the hemoglobin at various mixing rat i o s was calculated from the following r e l a t i o n s h i p : P 0 ? Content (ml 02/100 ml solution) = S(C H b) + C b(S- -p=0 s where S is the proportion of oxyhemoglobin in the mixture, C j ^ i s the oxygen capacity of the hemoglobin i n ml O2 / I O O ml solution or whole blood and i s calculated by multiplying the hemoglobin concentration i n g% by the oxygen capacity i n ml O^/g hemoglobin, C^ i s the oxygen content of the buffer or plasma i n ml O2 / I O O ml buffer or plasma at a i r saturation, Pg i s the p a r t i a l pressure of oxygen i n mm Hg at a i r saturation and P O 2 i s the measured p a r t i a l pressure of oxygen i n the mixture. The percent saturation of the hemoglobin was then calculated as the r a t i o of oxygen content to oxygen capacity of the - 85 -hemoglobin i n vol % mu l t i p l i e d by a factor of 100. The s o l u b i l i t y of oxygen i n plasma was taken as 0.79 ml 02/100 ml plasma at 10 C (Stevens, 1968) and the s o l u b i l i t y i n the 0.1 M t r i s - H C l buffer was measured as 0.75 vol % at the same temperature. No correction was made i n this l a t t e r value for the decrease i n oxygen s o l u b i l i t y caused by the dissolved hemoglobin since the maximum hemoglobin concentra-tion was only 0.64 m molar which would have a n e g l i g i b l e e f f e c t on the oxygen content. DETERMINATION OF OXYGEN CAPACITY The method of Tucker (1967) was employed to deter-mine the oxygen content of air-saturated blood and hemoglobin sol u t i o n s . A water-jacketed cuvette of about 2 ml capacity containing a magnetic s t i r r i n g bar was f i t t e d with a Radiometer P0 2 electrode. A p a r t i a l l y degassed solution of 6 g of po-tassium ferricyanide and 3 g of saponin i n a l i t r e of water was delivered into the cuvette and the oxygen tension measured. A 50 y l sample of the oxygenated blood was delivered into the cuvette anaerobically. The hemoglobin was converted to cyan-methemoglobin thereby e f f e c t i n g the release of the bound oxygen into the ferricyanide reagent. The increase i n P02 of the ferricyanide solution was measured and the oxygen content of the hemoglobin calculated by the following formula: . AP02 X 100V X oc Oxygen Content (vol !) = 760 X v - 86 -AP0 2 i s the increase i n P 0 2 of the ferricyanide reagent i n mmHg recorded afte r the addition of v y l of blood or hemo-glob i n , V i s the volume of the cuvette i n y l and « i s the s o l u b i l i t y c o e f f i c i e n t of oxygen in the ferricyanide reagent in ml 0 2 dissolved per ml ferricyanide reagent at an oxygen p a r t i a l pressure of 760 mm Hg. In most cases the oxygen capacity was related to the ml 0 2 bound by one gram of oxyhemoglobin. Hemoglobin, and methemoglobin concentrations and hematocrits where appropriate were measured before and after the determination of the oxygen e q u i l i b r i a . CALCULATION OF P 5 0 AND n Since the foregoing transformation of the mixing r a t i o s of oxygenated and deoxygenated hemoglobin to percent saturation of the hemoglobin based on the r a t i o Of oxygen content to oxygen capacity of the hemoglobin at various oxygen p a r t i a l pressures seldom resulted in measurements of the P 0 2 at h a l f - s a t u r a t i o n of hemoglobin (P^Q)> this value was calcu-lated from the H i l l approximation, ^QQ^y• = kPn, where Y is the percent saturation, k i s a constant, P i s the oxygen p a r t i a l pressure and n i s an estimate of the heme-heme i n t e r -action (Manwell, 1960; Prosser and Brown, 1 9 6 1 ) . The value of n i s determined from the least squares f i t of a double loga r i t h -mic plot of the various values of i'Qo-y a n (^ ^* T n e v alu e ° * P r n i s then calculated frojn this estimate of n when log - 87 -H i l l ' s approximation frequently f a i l s to describe the oxygen equilibrium curve at the high or low percentage saturations (Wyman,1948; Antonini et al.3 1964; Wyman, 1964) which could lead to s i g n i f i c a n t errors i n the calculated values of P J . Q and n. For this reason, estimates of P ^ Q and n for each equilibrium curve were calculated using a l l the points from 1 to 951 saturation and also from 20 to 80% saturation of the hemoglobin with oxygen. These data along with the e q u i l i b r a -t i o n conditions and hemoglobin concentration are provided i n Table XII, for each oxygen equilibrium curve constructed i n this study. The estimates of P ^ Q and n calculated over the 20 to 80% range i n oxygen saturation were u t i l i z e d during the discussion of results since they are probably the more accurate of the two sets of estimates. RESULTS III ELECTROPHORETIC IDENTITY OF THE HEMOGLOBIN SOLUTIONS The electropherograms of the solutions of the entire complement of adult coho hemoglobins and of the anion exchange f r a c t i o n eluted with a 1:1 (v:v) mixture of 0.1 M t r i s - H C l , pH 8.5, and 0.1 M K^PO^ are presented i n Plate 10. Both hemoglo-bin solutions were approximately 60 hours old from the time the adult salmon were k i l l e d and were representative of the solutions employed i n the oxygen e q u i l i b r i a studies. 88 -TABLE XII The e q u i l i b r a t i o n conditions, hemoglobin concentra-tions and calculated values of oxygen capacity, oxygen a f f i n i t y and heme-heme interactions for a l l the oxygen e q u i l i b r i a measured i n this t h e s i s . The l a t t e r two parameters were calculated over two ranges of saturation of hemoglobin with oxygen to demonstrate the e f f e c t of including very high or low saturation values i n the ca l c u l a t i o n of heme-heme i n t e r a c t i o n . Hb 20-80% Saturation 1-95% Saturation E q u i l i b r a t i o n Oxygen P50 P50 Temperature pH [Hb] MetHb Capacity (C) (%g) (% o£ Hb) (ml 02/g Hb) (mm Hg) (mm Hg) 1 Fry-Hb 9.8 6.82 3.60 7.0 0. 78 34. 3 1.99 34.2 2. 04 2 Fry-Hb 9.8 7.08 3. 72 5.7 0. 71 31. 8 2.17 31.6 2.17 3 Fry-Hb 9.8 7.44 3.50 6.5 0.94 8.4 1.69 8.6 1.44 4 Fry-Hb 9.8 7.50 4.04 8.0 0.92 6.0 1. 86 6.2 1.98 5 Fry-Hb 9.8 7.90 3.50 6.5 0.90 4.9 1.62 4.8 1. 54 6 Fry-Hb 9.8 8.50 3. 72 5.7 0. 80 3.9 1. 75 4.0 1.67 7 Fry-Hb 15.2 7.38 4.20 8.6 0. 85 17.0 2.17 17.1 2.12 8 Fry-Hb 15.2 7.90 4.20 7.7 0.77 9.9 2.12 10. 2 1.80 9 Fry-WB 9.3 * 6. 79 0.0 1.23 5.5 1. 38 5.5 1.52 10 Fry-WB 10.0 * 6.05 1.23 4.4 1.64 4.2 1.90 11 Fry-WB 9.3 6.63 0.0 1. 23 12.5 1.62 12.6 1.58 12 Adult-Hb 9.8 6.95 4.10 3.3 1.12 23.9 2.67 24.0 2.37 13 Adult-Hb 9.8 7.43 4.10 5.3 1.09 17.9 1.98 17.7 1.99 14 Adult-Hb 9.8 8.20 4.10 4.4 1.08 14.0 1.40 13. 8 1. 54 15 Adult-Hb 10.0 8.00 3. 70 4.8 15.6 1.44 15.6 1.44 16 Adult-Hb 10.0 7.48 3. 70 5.9 1.17 17.6 1. 51 17.4 1.57 17 Adult-Hb 10.0 7.48 3. 70 5.9 1.12 18.8 1.63 18.0 1.56 18 Adult-Hb 10.0 7.48 3. 70 5.9 1.15 16.6 1.63 16. 7 1.65 19 Adult-Hb 10.0 7.48 3. 70 5.9 1.11 19. 7 2.03 19. 7 1.94 20 Adult-WB 9.3 ** 12.10 2.4 1.14 15.6 1. 75 15.0 1.64 21 Adult-WB 9.3 * 11.60 2.4 1.16 10. 7 1.26 10. 3 1. 34 22 Adult-WB 5.0 * 12.33 0.0 1.17 9.8 1. 39 9.6 1.51 23 Adult-WB 10.0 * 8.95 0.0 1.19 12 .5 1.60 12.4 1. 74 24 Adult-WB 14. 8 * 12.38 0.0 1.05 14.8 1. 79 14.6 1. 74 25 Adult-WB 9.9 * 12.46 0.0 1.13 12.8 1.68 12.4 1.64 Adult WB-SW 11.2 * 4.60 - - • 1.20 11.4 1.38 11.1 1.71 Eq u i l i b r a t e d with a i r and pure nitrogen E q u i l i b r a t e d with a i r and nitrogen containing 0.45% carbon dioxide by volume. - 89 -PLATE 10: A comparison of the electrophoretic m o b i l i t i e s of the hemoiyzates of the blood of freshwater adult coho salmon ( l e f t ) and the hemoglobin components A6-8 (right) prepared from the adult hemoiyzates by ion-exchange chromatography. The protein bands adjacent to the black dots are t e n t a t i v e l y i d e n t i f i e d as the met-derivatives of the hemoglobin component immediately anodic to t h e i r p o s i t i o n s . (+) - 90 -A l l ten components normally present in adult hemoglobin were observed i n the p u r i f i e d adult hemolyzates. Eight additional hemoglobin bands were also observed and are marked with a black dot (electropherogram A l , Plate 10). These additional components were a l l approximately equi-distant from the component immediately anodic from t h e i r p o s i t i o n s . Since these additional components were not observed i n fresh hemoglobin samples (Part I ) , and oxidation of the ferrous iron to the f e r r i c form would increase the p o s i t i v e charge on the hemoglobin which would therefore tend to be more cationic the eight bands marked with a dot i n the adult hemoglobin electropherogram were designated as methemoglobin. The second f r a c t i o n eluted from the anion exchanger was i d e n t i f i e d as hemoglobin components A6^8 (electrophero-gram A2, Plate 10). No traces of components A l , A3, CI, C3, C4, C5, or C6 were present. Two methemoglobin bands were observed and are marked with black dots. The slower migrating of these l a t t e r bands was not present i n electropherogram A l , of Plate 10, i n d i c a t i n g that oxidation of the hemoglobin components A6, 7 and 8 was more extensive following i s o l a t i o n by ion exchange. Since the electropherograms of the m u l t i -ple hemoglobins of f r y blood (Part I ) , were e s s e n t i a l l y i d e n t i c a l to the electropherogram A2 (Plate 10) t h i s l a t t e r f r a c t i o n was termed f r y hemoglobin i n the presentation of the following r e s u l t s . Recovery of this " f r y hemoglobin f r a c t i o n " from the hemolyzates of freshwater adult coho blood was estimated - 91 -to exceed 94! on the basis that components A6-8 accounted for 50! of the hemoglobin of the adult coho (Part I ) . BLOOD PARAMETERS AND ERYTHROCYTE ATP CONCENTRATION OF ADULT FRESHWATER COHO SALMON Table XIII presents the hematocrit, hemoglobin concentration, percent methemoglobin, red c e l l ATP concen-t r a t i o n and molar r a t i o of ATP to hemoglobin of the blood of several of the adult freshwater coho s a c r i f i c e d i n this study. A molecular weight of 66,000 was assumed for hemo-globin in determining this l a t t e r r a t i o . Individual hematocrits of the adult coho ranged from 23.0 to 50.4! while t o t a l hemoglobin concentration varied from 6.2 to 12.4 g!, with the respective means being 39.3% and 10.6 g%. Methemoglobin level s ranged from approxi-mately 2.0 to 7.5! of the t o t a l hemoglobin i n adult fresh-water coho during the period of October, 1971 to January, 1972, as determined by the method of Evelyn and Malloy (1938). Although the f i s h were becoming extremely ragged and covered with fungus by December, the data do not indicate a trend of increasing methemoglobin levels with length of freshwater residence. ATP concentrations in the blood tended to decrease with the age of the adult coho during their period of fresh-water residence (Table XIII) although considerable v a r i a b i l i t y was observed among the data. The concentration of ATP i n whole blood generally exceeded 1.0 ymoles/ml and was as high-- 92 -TABLE XIII Hematological parameters and ATP concentrations of the blood of some of the freshwater adult coho used i n the preparation of hemoglobin components A6-8 and adult hemolyzates. DATE SEX HEMATOCRIT (% RBC) HEMOBLOGIN (g*) METHEMOGLOBIN (% of to t a l ) ATP rymoles/ccN blood } ATP:Hb (moles/mole) 15/10/71 u* 32.9 9.3 7.5 1. 50 1.26f 19/10/71 p** 23.0 6.2 4.7 1.03 1.06 24/10/71 F 43.3 11.7 1.9 1. 70 0.96 24/10/71 F 38.9 11.2 4.8 1.58 0.94 29/10/71 F 40.3 11.3 1. 73 1.01 11/11/71 F 43. 7 12.38 30/11/71 F 50.4 12.1 1.43 • 0.78 30/11/71 M+ + 42.9 11.8 0.97 0.54 29/12/71 F 33.9 8.6 7.1 1.11 0.86 29/12/71 F 43.4 11.0 6.9 1.39 0.84 * sex unknown, female. + assuming a molecular weight of hemoglobin of 66,000. A. a. male. - 93 -as 1.73 umoles/ml. No ATP was detected i n any of the samples of plasma examined and the whole blood ATP concentrations are therefore a direct estimate of red c e l l ATP concentrations. The molar r a t i o of ATP to hemoglobin declined from 1.26 to approximately 0.85 during the period of mid-October to late-December. No inferences can be made regarding the p o s s i b i l i t y of differences i n erythrocyte ATP concentration between adult male and female coho since only 1 male was s a c r i f i c e d i n this study. A l i n e a r r e l a t i o n s h i p was observed between hemo-globin concentration and hematocrit (Figure 3) described by the equation: Hb = 0.514 + 0.255 Hct for which r = 0.97, n = 13 and hemoglobin concentration i s i n grams per 100 ml blood. The s o l i d t r i a n g l e of Figure 3 repre-sents the pooled blood of 113 coho fry anesthetized with 2-phenoxyethanol p r i o r to sampling and was included i n the cal c u l a t i o n of the foregoing equation. The s o l i d diamond-shaped points representing the blood of 2-year-old seawater g r i l s e provided by Dr. J . C. Davis were not included in the cal c u l a t i o n of the hemoglobin-hematocrit r e l a t i o n s h i p . It was e v i d e n t h o w e v e r , that l i t t l e , i f any change occurred in this relationship through the free-swimming stage of the coho salmon l i f e c y c l e . - 94 -FIGURE 3 The relati o n s h i p between blood hematocrit and hemoglobin concentration i n coho salmon. The s o l i d c i r c l e s represent f r e s h -water adult coho; the open squares , the pooled blood of 113 coho f r y ; the s o l i d , triangle the blood of a 2-year-old seawater g r i l s e and the s o l i d diamonds, the blood of 2-year-old seawater g r i l s e measured by D r . J . C. Davis. A l l the points with the exception of the l a t t e r g r i l s e were used to calculate least squares f i t of the regression l i n e . Hb = 0.514 + 0.255 Hct f o r which r = 0.97 and n = 13. HEMATOCRIT ( % R B C ) - 95 -INFLUENCE OF ATP UPON THE OXYGEN EQUILIBRIUM OF ADULT COHO HEMOGLOBIN The oxygen equilibrium c h a r a c t e r i s t i c s of adult coho hemoglobin were determined at four concentrations of adenosine triphosphate. The e q u i l i b r a t i o n temperature and pH were 10 C and 7.48, resp e c t i v e l y . ATP concentrations of 0.0, 0.08, and 0.76 moles/mole hemoglobin resulted i n essen-t i a l l y i d e n t i c a l oxygen e q u i l i b r i a (Figure 4) i n which P50 range from 16.6 to 18.8 mm Hg and heme-heme int e r a c t i o n from 1.51 to 1.63 (Table XIV). Increasing the ATP concentration to 7.56 moles/mole hemoglobin resulted in a s l i g h t decrease in oxygen a f f i n i t y (P ^ Q = 19.7 mm Hg) and a r e l a t i v e l y large increase in heme-heme in t e r a c t i o n (n = 2.03). The equilibrium curves recorded at a l l four ATP concentrations coincided at oxygen saturations exceeding 70%. The increase i n heme-heme int e r a c t i o n observed at 7.56 moles ATP/mole hemoglobin was therefore a r e f l e c t i o n of the s h i f t to the right of the lower portion of the equilibrium curve. No change i n oxygen capacity was observed i n r e l a -tion to ATP concentrations. Oxygen capacity averaged 1.14 ml 02/g hemoglobin which i s somewhat less than the th e o r e t i c a l capacity of 1.3 ml 02/g hemoglobin. Since the only e f f e c t of ATP upon the oxygen e q u i l i b r i a of solutions of adult hemo-globin occurred at ATP concentrations which were over f i v e times the maximum concentration observed i n whole blood i t was concluded that ATP did not subs t a n t i a l l y a l t e r the oxygen e q u i l i b r i a . ATP, therefore, was not included i n the buffer - 96 -FIGURE 4 The effect of ATP upon the oxygen equilibrium curve of solutions of adult freshwater coho hemoglobin. The hemoglobin solutions contained 3.70 G% hemoglobins, and 5.9% methemoglobin and were equ i l i b r a t e d at pH 7.48 and 10C. The molar r a t i o of ATP to hemoglobin employed were 0 (open c i r c l e s ) , 0.076 (crosses), 0.76 ( s o l i d c i r c l e s ) , and 7.6 (open squares). IOO- I 10 20 30 40 50 PARTIAL PRESSURE OF OXYGEN (mm Hg) - 97 -TABLE XIV The influence of various concentrations of adenosine triphosphate upon the oxygen capacity, oxygen a f f i n i t y and heme-heme in t e r a c t i o n of hemoiyzates from freshwater adult coho salmon. ATP OXYGEN CONCENTRATION CAPACITY P ( m o l e / m o l e Hb) (ml.0 2/gHb) (mm Hg) * 0 1.17 17.6 1.51 0.08 1.12 1.8.-8 1.63 0.76 1.15 16.6 1.63 7.56 1.11 19.7 2.03 - 98 -solutio n i n the remaining experiments. EFFECT OF EQUILIBRATION TEMPERATURE UPON OXYGEN EQUILIBRIA The oxygen equilibrium curves of solutions of f r y hemoglobin equilibrated at 9.8 and 15.2 C and pH of approxi-mately 7.4 and 7.9 are presented i n Figure 5, while the oxygen capacity and calculated values of P^Q and n for each set of e q u i l i b r a t i o n conditions are presented i n Table XV. At pH 7.90 an increase i n temperature from 9.8 to 15.2 s h i f t e d the curve to the right and P 5 Q increased by 5.0 mm Hg from 4.9 to 9.9 mm Hg. At pH of approximately 7.4, however, the same increase i n temperature resulted i n an increase i n PJ-Q of 8.6 mm Hg. These l a t t e r equilibrium curves were determined at e q u i l i b r a t i o n temperatures of 9.8 and 15.2 C and a pH of 7.44 and 7.38 r e s p e c t i v e l y . If the former curve is corrected to pH 7.38 (see Bohr e f f e c t s , following s e c t i o n ) , the P 5 Q would be 9.6 mm Hg, which would then re s u l t i n a change i n P,-Q of 7.4 mm Hg between fry hemoglobin solutions e q u i l i -brated at 9.8 and 15.2 C. Thus the same temperature increase of 5.4 C resulted i n a much larger reduction i n oxygen a f f i n i t y at pH 7.38 than at pH 7.90. The oxygen capacity of the fry hemoglobin appeared to decrease at higher temperatures (Table XV) but these data must be viewed with caution since d i f f e r e n t hemoglobin prepara-tions were employed at the two e q u i l i b r a t i o n temperatures. - 99 -TABLE XV The ef f e c t of d i f f e r e n t e q u i l i b r a t i o n temperatures upon the oxygen capacity, oxygen a f f i n i t y and heme-heme in t e r a c t i o n of f r y hemoglobin solutions (components A6-8) at d i f f e r e n t values of pH and of fry and adult whole blood equilibrated with air. and pure nitrogen. pH EQUILIBRATION TEMPERATURE (C) OXYGEN CAPACITY (ml 02/g Hb) P50 (mm Hg) n Fry Hemoglobin* 7.44 9.8 0.94 8.4 1.69 Fry Hemoglobin 7. 38 15.2 0.85 17.0 2.17 Fry Hemoglobin 7.90 9.8 0.90 4.9 1.62 Fry Hemoglobin 7.90 15. 2 0.77 9.9 2.12 Fry Blood** 10.0 1.23 4.4 1.64 Adult Blood 5.0 1.17 9.8 1.39 Adult Blood 10. 0 1.19 12.5 1.60 Adult Blood — 14.8 1.05 14.8 1. 79 Fry refers to hemoglobin solutions i n t r i s - H C l buffer Oxygen e q u i l i b r i a determined using whole blood. - 100 -FIGURE 5 The effect of e q u i l i b r a t i o n temperature and pH upon the oxygen equilibrium of solutions of hemoglobin components A6-8 (fry hemoglobin). The oxygen e q u i l i b r i a were measured at pH 7.90 and 9.8 C (open c i r c l e s ) , pH 7.44 and 9.8 C ( s o l i d squares), pH 7.90 and 15.2 C ( s o l i d c i r c l e s ) , and pH 7.38 and 15.2 C (open squares). Hemoglobin concentration and percentage methemoglobin were 3.50 g ! and 6.5! at the e q u i l i b r a t i o n temperature of 9.8 C. At 15.2 C the respective values were 4.20 g! and 8.6! at pH 7.38 and 4.20 g! and 7.7! at pH 7.90. 1 0 0 - 1 PARTIAL PRESSURE OF OXYGEN (mm Hg) - 101 -The estimate of heme-heme i n t e r a c t i o n , n, increased at 15.2 C r e l a t i v e to 9.8 C and was independent of pH (Table XV). At 9.8 C, n, was 1.69 and 1.62 at pH 7.44 and 7.90, r e s p e c t i v e l y , while at 15.2 C, n was 2.17 and 2.12 at pH 7.38 and 7.90, re-spectively . The oxygen e q u i l i b r i a of whole blood from adult coho l i v i n g at 10 C were determined at 5.0, 10.0 and 14.8 C. The blood was equilibrated with a i r and pure nitrogen. The oxygen a f f i n i t y of this blood was r e l a t i v e l y unaffected by changes i n the e q u i l i b r a t i o n temperature since the calculated values of P 5 Q at these temperatures were 9.8, 12.5, and 14.8 mm Hg, respectively (Table XV). Thus P^ Q increased approxi-mately 2.5 mm Hg for each 5 degrees r i s e in temperature. Heme-heme int e r a c t i o n increased as the e q u i l i b r a t i o n temperature was elevated such that n was 1.39, 1.60 and 1.79 at 5.0, 10.0 and 14.8 C, respectively (Table XV). The res u l t of this increase i n subunit cooperativity was that with i n -creasing temperature the e q u i l i b r a t i o n curves were sh i f t e d to the right to a greater extent at intermediate oxygen satura-tions of 25 to 65% than at high saturations (Figure 6 ) . Thus at a P 0 2 of 25 mm Hg, the oxygen saturation at 5.0 and 14.8 C di f f e r e d by 6!, whereas at 15 mm Hg, this difference increased to 25%. Since the oxygen capacities recorded at the three e q u i l i b r a t i o n temperatures were s i m i l a r , the amount of oxygen bound to the hemoglobin of adult coho salmon at oxygen p a r t i a l pressures exceeding 25 mm Hg was e s s e n t i a l l y unrelated to tem-perature over the range of 5.0 to 14.8 C. - 102 -FIGURE 6 The e f f e c t of e q u i l i b r a t i o n temperature upon the oxygen equilibrium of the whole blood of freshwater adult coho salmon The blood was equilibrated with a i r and pure nitrogen at 5.0 C (s o l i d c i r c l e s ) , 10.0 C ( s o l i d t r i a n g l e s ) , and 14.8 C ( s o l i d squares), for which the hemoglobin concentrations were 12.3, 9.0 and 12.4 g!% re s p e c t i v e l y . Methemoglobin was below detect-able l i m i t s . 100-1 PARTIAL PRESSURE OF OXYGEN (mm Hg) - 104 -EFFECTS OF pH AND CARBON DIOXIDE UPON OXYGEN EQUILIBRIA The oxygen e q u i l i b r i a of f r y hemoglobin over the pH range of 6.82 to 8.50 are presented (Figure 7) along with the respective estimates of oxygen capacity, P^Q and heme-heme interaction (Table XVI). A l l experiments were performed at an e q u i l i b r a t i o n temperature of 9.8 C. The oxygen capacity of fry hemoglobin was not decreased over the pH range of 7.44 to 8.50 but an apparent reduction i n capacity was observed below this range. Oxygen a f f i n i t y , however, was strongly pH-dependent and the estimate of P^Q increased from 3.9 to 34.3 mm Hg as the pH decreased from 8.50 to 6.82. The heme-heme inter a c t i o n was r e l a t i v e l y constant at 1.62 to 1.85 i n the pH range of 7.44 to 8.50 but increased to 2.17 and 1.99 at pH 7.08 and 6.82, respectively (Table XVI). The oxygen e q u i l i b r i a of adult hemoglobin at pH 6.95, 7.43 and 8.20 are i l l u s t r a t e d in Figure 8 and the estimates of oxygen capacity, oxygen a f f i n i t y and heme-heme inte r a c t i o n are presented in Table XVI for e q u i l i b r a t i o n temperatures of 9.8 to 10.0 C. The oxygen capacity of adult hemoglobin was unaffected by variations in pH and remained at approximately 85% of the theoretical capacity of 1.3 ml O^/g Hb. Heme-heme i n t e r a c t i o n , however increased from approxi-mately 1.4 at pH 8.00 to 8.20 to 2.67 at pH 6.95. Thus i n both f r y and adult hemoglobin subunit cooperativity increased as the pH of the buffer was lowered. The oxygen a f f i n i t y of adult hemoglobin was only s l i g h t l y reduced as pH was lowered - 105 -TABLE XVI The oxygen capacity, oxygen a f f i n i t y and heme-heme i n t e r -action of solutions of f r y hemoglobin (components A6-8) and adult hemoglobin at various pH and of f r y and adult whole blood at two tensions of carbon dioxide. The abbreviations Hb and WB ref e r to hemoglobin solutions and whole blood, respectively. EQUILIBRATION TEMPERATURE PC02 pH (C) (mm Hg) Fry-Hb 8.50 9.8 0.23 Fry-Hb 7.90 9.8 0.23 Fry-Hb 7.50 9.8 0.23 Fry-Hb 7.44 9.8 0.23 Fry-Hb 7.08 9.8 0.23 Fry-Hb 6.82 9.8 0.23 Adult-Hb 8.20 9.8 0.23 Adult-Hb 8.00 10.0 0.23 Adult-Hb 7.48 10.0 0.23 Adult-Hb 7.43 9.8 0.23 Adult-Hb 6.95 9.8 0.23 Fry-WB 9.3 0.23 Fry-WB -- 9.3 3.35 Adult-WB -- 9.3 0.23 Adult-WB -- 9.3 3.35 OXYGEN CAPACITY P 5 0 n (ml 02/g/Hb) (mm Hg) 0. 80 3.9 1.75 0. 90 4.9 1.62 0.92 6.0 1.86 0.94 8.4 1.69 0. 71 31.8 2.17 0.78 34. 3 1.99 1.08 14.0 1.40 1.13 15.6 1.44 1.17 17.6 1. 51 1.09 17.9 1.98 1.12 23.9 2.67 1. 23 5.5 1. 38 1. 23 12.5 1.62 1.18 10.7 1.26 1.14 15.6 1. 75 - 106 -FIGURE 7: The ef f e c t of pH upon the oxygen equilibrium of solutions of hemoglobin components A6-8 (fry hemoglobin). The e q u i l i b r a t i o n temperature was 9.8 C and the hemoglobin concen-t r a t i o n and levels of methemoglobin ranged from 3.5 to 4.0 g % and 5.7 to 8.0 % res p e c t i v e l y . The oxygen e q u i l i b r i a were measured at pH 8.5 (open c i r c l e s ) , 7,90 (open t r i a n g l e s ) , 7.50 (small s o l i d c i r c l e s ) , 7.44 (open squares), 7.08 (large s o l i d c i r c l e s ) and 6.82 ( s o l i d t r i a n g l e s ) . 1 0 0 PARTIAL PRESSURE OF OXYGEN I mm Hg) - 107 -FIGURE 8 The e f f e c t of pH upon the oxygen equilibrium of solutions of freshwater adult coho hemoglobin equilibrated at 9.8 C. The oxygen e q u i l i b r i a determined at pH 6.95 ( s o l i d t r i a n g l e s ) , 7.43 (s o l i d squares) and 8.20 ( s o l i d c i r c l e s ) . Hemoglobin concentra-tion was 4.1 g % and methemoglobin ranged from 3.3 to 5.3 %. PERCENT OXYHEMOGLOBIN ro 4> o oo o o o o o o J l I ! I 0> o - 108 -and the P^Q increased by approximately 10 mm Hg over the pH range of 8.20 to 6.95. The Bohr s h i f t (0 = A. log P 5 0M P H) w a s determined for fry and adult hemoglobin (Figure 9 ) . The Bohr ef f e c t of adult hemoglobin was approximately l i n e a r over the pH range of 6.95 to 8.20 (0 = 0 . 1 7 2 ) . Although this r e l a t i o n s h i p in f r y hemoglobin was always negative over the pH range of 6.82 to 8.50, i t was strongly non-linear so that the calculated values of 0 were -0.033, -1.729, and -0.182 i n the pH ranges 6.82 to 7.08, 7.08 to 7.50 and 7.50 to 8.50, r e s p e c t i v e l y . The oxygen e q u i l i b r i a of the whole blood of f r e s h -water adult coho and the pooled blood of 113 eleven-month-old fr y at 9.3 C and carbon dioxide tensions of 0.2 and 3.4 mm Hg are presented i n Figure 10. With the foregoing increase in PCC^the oxygen a f f i n i t i e s of adult and fry blood were decreased by 4.9 and 7.0 mm Hg respectively although no changes were observed i n the respective oxygen capacities (Table X V I ) which were 90 to 95% of the maximum the o r e t i c a l capacity. Both groups of blood exhibited increases in the value of n as the P C 0 2 was r a i s e d . The pH values of Figure 9 corresponding to the log PJ.Q of adult blood at carbon dioxide tensions of 0.2 and 3.4 were estimated to be 9.0 and 7.85, r e s p e c t i v e l y . Simi-l a r calculations yielded the respective pH values of 7.7 and 7.4 for fry blood. - 109 -FIGURE 9 The Bohr ef f e c t of solutions of hemoglobin components A6-8 (fry hemoglobin), ( s o l i d c i r c l e s ) and freshwater adult hemoglobin (s o l i d squares) at e q u i l i b r a t i o n temperatures of 9.8 to.10.0 C. - 110 -FIGURE 10 The influence of carbon dioxide upon the oxygen e q u i l i b r i a of the blood of coho fry ( c i r c l e s ) and freshwater adults (squares). The blood was equilibrated with a i r and pure> nitrogen (open squares and c i r c l e s ) or with a i r and nitrogen containing 0.45 % carbon d i -oxide by volume ( s o l i d squares and c i r c l e s ) a t 9.3 C. Hemoglobin con-centration was 6.6 to 6.8 gl for f r y blood and 11.6 to 12.1 g% for adult blood. No methemoglobin was formed during these experiments. PERCENT OXYHEMOGLOBIN ro .$> cr> co o o O O o o - I l l -DISCUSSION III , The recovery of hemoglobin components A6-8 from hemolyzates of adult coho blood was estimated to exceed 901. The majority of the loss occurred during the concentration of these components with the Amicon molecular f i l t e r follow-ing t h e i r e l u t i o n from the ion-exchange column. About 0.2 and 0.4 ml of the concentrated hemoglobin was retained i n the f i l t e r . Since washing the f i l t e r would have defeated the pur-pose of the procedure, this volume of hemoglobin solution was discarded. These losses were not s p e c i f i c for any p a r t i c u l a r component, therefore, no change i n the r e l a t i v e proportions of the three components occurred. Under no circumstances were components A l , A3, CI ,.C3 ,C4 ,C5 or C6 present in any of the preparations of components A6-8. It may be somewhat questionable whether the r e l a -tive proportions of the three components A6-8 are i d e n t i c a l i n the blood of fry and adult coho. Some v a r i a t i o n i n the r e l a t i v e proportions of A6:A7:A8 were v i s i b l e i n electrophero-grams of hemoglobin from coho of a l l ages although t h i s v a r i -ation did not exhibit a consistent pattern with age. Such in d i v i d u a l v a r i a t i o n i n the r e l a t i v e concentration of hemoglo-bin components has been observed in other salmonids (Hashimoto and Matsuura, 1960b; Westmann, 1970) and i s generally much larger than that observed for coho blood. This d i f f i c u l t y i s further complicated by the formation of methemoglobin during the i s o l a t i o n of components A6-8. From the observed changes in electrophoretic behaviour of the cathodic and slower - 112 -migrating anodic hemoglobin components, i t would appear l i k e l y that the oxidized form of component A8 would migrate to the same p o s i t i o n as unoxidized component A6 during e l e c -trophoresis. This would account for the apparent increase i n the r e l a t i v e concentration of component A6, following i s o l a -t i o n of the f r y hemoglobin components from adult hemoiyzates. E f f i c i e n c y of acid-soluble phosphate reduction from the hemoiyzates by the combined procedures of pressure f i l t r a t i o n and column chromatography was estimated to be 91-96%. This es-timate was based on the percentage reduction i n t o t a l phosphate bound as ATP alone. Since i t has been demonstrated that approxi-mately 55-80% of the acid soluble phosphate of erythrocytes i s i n the form of ATP (Rapoport and Guest, 1941; G i l l e n and Riggs, 1971), the estimate of the e f f i c i e n c y of phosphate removal i s probably too low. In absolute terms, 0.1 and 0.2 moles of phosphate per mole of hemoglobin were retained i n the f i n a l preparations of adult and fry hemoglobin, r e s p e c t i v e l y . The higher phosphate concentration i n the f r y hemoglobin i s a re-f l e c t i o n of the 0.05 M phosphate concentration in the buffer employed to elute hemoglobin components A6-8 from the ion exchange column. There can be no doubt that the a b i l i t y of the f r y hemoglobin to combine with oxygen was impaired following ion exchange chromatography. Whether or not this decrease in oxygen capacity i n these preparations influenced the c h a r a c t e r i s t i c s of oxygenation of the functional hemoglobin, i s questionable. A comparison of the oxygen equilibrium - 113 -curves of solutions of f r y hemoglobin prepared by ion-exchange chromatography with those of the whole blood of f r y under s i m i l a r conditions may provide some insight into t h i s problem. At 10 C f r y whole blood equilibrated with a i r and pure n i t r o -gen exhibited a P,-Q of 4.4 mm Hg and n of 1.64. Assuming that the pH of the plasma i s s i m i l a r to that of trout blood under si m i l a r conditions of e q u i l i b r a t i o n (Cameron and Randall, 1972) the pH of the plasma would be approximately 8.2. Siggaard-Andersen (1963) presents equations r e l a t i n g intra-erythrocyte pH to plasma pH which i f applied to the present data y i e l d an intra-erythrocyte pH of 7.6 to 7.8. The estimates of P^Q and n of fry hemoglobin solutions at e q u i l i b r a t i o n conditions were 4.0 to 4.9 and 1.6 to 1.8, r e s p e c t i v e l y . Thus although the oxygen capacity of fry hemoglobin i n solution was much less than that i n whole blood, 0.7 to 0.9 and 1.23 ml 02/g Hb res p e c t i v e l y , i t would appear that both had s i m i l a r oxygena-tio n c h a r a c t e r i s t i c s when subjected to simi l a r e q u i l i b r a t i o n conditions. The most probable explanation for the foregoing d i s p a r i t y l i e s i n the methods employed i n the estimation of methemoglobin and t o t a l hemoglobin. In the method of Evelyn and Malloy (1939) methemoglobin concentration is estimated by the decrease i n absorption at 635 my upon the addition of sodium cyanide to a sample of blood or hemoglobin i n phosphate buffer at pH 6.7. Total hemoglobin, however was determined spectrophotometrically as the cyanmet-derivative i n an alka-l i n e s o l u t i o n . Since the small amounts of brown p r e c i p i t a t e - 114 -which formed i n the f r y hemoglobin solutions following ion-exchange and subsequent adjustment of pH to. values from 6.8 to 8.5 were p a r t i a l l y redissolved at a higher pH, i t i s l i k e l y that the estimates of methemoglobin may have been too low and the estimates of t o t a l dissolved hemoglobin too high. This would re s u l t i n reduced estimates of oxygen capacity. Since hemoglobin has a tetrameric structure from 0 to 4 of the iron atoms per molecule of hemoglobin could be oxidized to the f e r r i c form, r e s u l t i n g i n fi v e e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h -able bands for each component. The electropherograms of both adult and f r y hemoglobin solutions employed in this study suggest that only one met-derivative of each hemoglobin component was formed during the preparative procedures. Since oxidation of some one to three of the heme groups of the four incorporated i n the hemoglobin may a l t e r the oxygen a f f i n i t y of the remaining non-oxidized groups (Riggs, 1970) high levels of methemoglobin are to be avoided. The methemoglobin levels recorded i n this study are similar to those reported for other studies on f i s h hemoglobins (Aggarwal and Riggs, 1969; G i l l e n and Riggs, 1971). The oxygen equilibrium curve of adult coho hemoglo-bin was only s l i g h t l y modified by the presence of ATP and these ef f e c t s were only observed at ATP concentrations which were 5 to 10 times those observed i n the adult erythrocytes. ATP has been found to be the most common acid-soluble organic phosphate i n teleost erythrocytes and accounts for well over half of this group of compounds i n these c e l l s (Rapoport and Guest, 1941; G i l l e n and Riggs, 1971). V i r t u a l l y no information i s available - 115 -regarding ontogenetic changes i n ATP in f i s h erythrocytes although decreases i n the concentration of this compound in coho salmon during the period of freshwater residence p r i o r to spawning were observed i n the present study. During the l a t t e r part of t h i s period the molar r a t i o of ATP to hemoglo-bin was approximately 0.8 which is s i m i l a r to the r a t i o of 0.76 observed i n the Rio Grande c i c h l i d ( G i l l e n and Riggs, 1971) and of 0.65.observed i n 2-year old sockeye g r i l s e ( G i l e s , unpublished observations). Certain organic phosphates are known to modify the oxygen e q u i l i b r i a of a wide v a r i e t y of animals. In humans, 2, 3-diphosphoglycerate sharply decreases the oxygen a f f i n i t y of adult hemoglobin but not of f o e t a l hemoglobin. I n o s i t o l hexaphosphate (IHP) s i m i l a r l y influences the hemoglobin of developing chicks and of certain t u r t l e s (Benesch and Benesch, 1969). Wood (1971) observed that the oxygen a f f i n i t i e s of both l a r v a l and adult hemoglobins of the salamander, Decamp to don ensatus, were reduced by ATP and that the difference in oxygen a f f i n i t y of the blood at these two l i f e stages was accounted for by higher erythrocyte ATP concentrations i n the adult. G i l l e n and Riggs (1971) demonstrated that the oxygenation c h a r a c t e r i s t i c s of the hemoglobin of Cichlasoma cyanoguttatum were strongly influenced by ATP which generally lowered the oxygen a f f i n i t y , increased the Bohr ef f e c t and caused variations in the heme-heme i n t e r a c t i o n . In a d d i t i o n , these effects were pH dependent. The log P^Q of the hemoglobin at a molar r a t i o of ATP to hemoglobin of approximately 10 was increased by 0.75 at pH 6.7 and by 0.47 at pH 7.2 in comparison to phosphate-free - 116 -hemoglobin s o l u t i o n s . Complex interactions between blood pH, state of oxygenation and 2,3-diphosphoglycerate (2,3-DPG) have been reported for human hemoglobin (Brewer and Eaton, 1 9 7 1 ) . Since increases i n red c e l l pH have an antagonistic e f f e c t upon oxygen a f f i n i t y to increases i n red c e l l 2,3-DPG concentration i t i s possible that they serve as part of a regulatory mechanism i n the unloading of oxygen i n the c a p i l l a r y beds. SUch a mechanism implies that the hemoglobin exhibits a s i g n i f i c a n t Bohr e f f e c t . The small Bohr e f f e c t observed in adult coho hemoglobin may therefore account i n part to the lack of s e n s i t i v i t y to ATP. Such arguments must be considered with caution, however, since 50% of the adult hemoglobin i s composed of components which when i s o l a t e d exhibit a very marked Bohr s h i f t i n the pH range of 7.1 to 7.5. Also evidence is accumulating to suggest that organic phosphates may not be involved i n the regulation of hemoglobin oxygen a f f i n i t y in certai n mammals and b i r d s . Thus the decrease i n P ^ Q of 17 mm Hg observed i n chicks just after hatching does not correspond to variations i n the levels of erythrocyte ATP or i n o s i t o l hexaphosphate (IHP) both of which may modify oxygen a f f i n i t y of the hemoglobin of unhatched chicks (Mission and Freeman, 1 9 7 2 ) . Although the f o e t a l and adult hemoglobin of sheep exhibit a Bohr s h i f t and lamb erythrocytes have a much greater 2,3-DPG concentration than adult red c e l l s this organic phosphate does not have a s i g n i -f i c a n t e f f e c t upon the oxygen a f f i n i t y of sheep of either age (Baumann, Bauer, and Rathschlag-Schaefer, 1 9 7 2 ) . It would appear therefore that i n vertebrates modification of - 117 -the oxygen equilibrium by organic phosphate compounds i s a common but not universal phenomenum. The oxygen e q u i l i b r i a of hemoglobin components A6-8 were s i g n i f i c a n t l y d i f f e r e n t from those of the whole hemolyzate of adult coho blood. The oxygen a f f i n i t y of adult blood was only s l i g h t l y decreased by increasing temperature ; A l0& p50 ( = 0.016 - 0.021) whereas much larger decreases AT were observed i n solutions of hemoglobin components A6-8 A log P r 0 ( — — — = 0.056 at pH 7.90). In addition the Bohr ef f e c t observed i n adult hemoglobin was approximately l i n e a r from pH 6.95 to 8.20 and quite small, 0 = 0.172, whereas that of com-ponents A6-8 was non-linear so that 0 = 0.033, -1.729, and -0.182 i n the pH ranges of 6 .'82 to 7.50, 7.08 to 7.50, and 7.50 to 8.50, r e s p e c t i v e l y . These differences i n Bohr s h i f t resulted i n components A6-8 possessing a higher a f f i n i t y for oxygen above pH 7.3, but a lower a f f i n i t y below this pH than the complete hemolyzate of adult blood at 9.8 to 10 C. The heme-heme i n t e r a c t i o n of both hemoglobin solutions was generally less than 2.0 and exhibited s i m i l a r increases as the pH was lowered or the e q u i l i b r a t i o n temperature r a i s e d . Although only components A6-8 could be successfully i s o l a t e d from hemolyzates of freshwater adult blood the data indicated that the hemoglobin components of adult blood must be d i v i s i b l e into at least two groups based on differences in oxygen a f f i n i t y and Bohr e f f e c t s . B i n o t t i et al. (1971) reported that at 20 C component I of t r o u t , Salmo i r i d e u s , hemoglobin had a P,-Q - 118 -of approximately 17.0 mm Hg and no Bohr effect whereas com-ponent IV had a large non-linear Bohr ef f e c t and a P^Q at pH 7.4 of approximately 42 mm Hg. At pH 7.9, the oxygen a f f i n i t y of both components was equal. In addition, com-ponent IV demonstrated a decrease i n oxygen capacity (Root s h i f t ) , below pH 7.0, whereas no such effect was observed i n component I over the pH range of 6.8 to 7.6. The heme-heme int e r a c t i o n of component I decreased as pH was r a i s e d , whereas that of component IV increased as pH was raised from 6.12 to 7.1 and thereafter remained r e l a t i v e l y constant. Hashimoto et al. (1960) observed that hemoglobin component S of the blood of adult chum salmon had a higher oxygen a f f i n i t y below pH 7.8 than the second component (F) and was r e l a t i v e l y un-A log P 5 Q affected by v a r i a t i o n i n e q u i l i b r a t i o n temperature ( A T = 0.006 for component S and 0.017 for component F ) , s a l t and buffer concentration and pH when compared to component F. The Bohr ef f e c t of component F was li n e a r from pH 7.0 to 7.8 and very lar g e , 0 = -2.35. In ad d i t i o n , the oxygenation character-i s t i c s of the whole hemolyzate were generally compatible with those expected i f no i n t e r a c t i o n occurred between the two hemoglobin components. A Root e f f e c t was observed in component F below pH 7.26 but not i n component S. Similar differences i n oxygenation c h a r a c t e r i s t i c s have been observed i n the multiple hemoglobins of such diverse species as the Japanese e e l , Angxtilla japonica (Yamaguchi, et al.3 1962 ; Hamada et al. 3 1964; Yoshioka et al.3 1968; Itada et al. 3 1971), and the loach (Yamaguchi et alj 1963). To date no s a t i s f a c t o r y explanation for the wide 119 -d i v e r s i t y i n oxygenation c h a r a c t e r i s t i c s of the multiple hemoglobins of f i s h has been made. The problem of i n t e r -pretation i s made even more complex since i t appears that i n some f i s h the oxygen e q u i l i b r i a of whole hemolyzates are not simply the average of the e q u i l i b r i a of the in d i v i d u a l components (Yamaguchi et al.3 1963). There has been some speculation that the hemoglobin polymorphs may represent a molecular adaptation to s a t i s f y d i f f e r e n t p h y s i o l o g i c a l re-quirements (Hashimoto et al.3 1960; B i n o t t i et al.3 1971). In the instances where a Root s h i f t i s observed i n one hemo-globin component but i s lacking in other components this speculation may be j u s t i f i a b l e since the Root s h i f t appears to be an important factor i n the functioning of the swim bladder i n certa i n f i s h . It has not, however, been e s t a b l i s h -ed that the other functional c h a r a c t e r i s t i c s of the various hemoglobin polymorphs which have of necessity been studied in hemoglobin solutions rather than in intact c e l l s are a c t u a l l y representative of the oxygenation c h a r a c t e r i s t i c s of the whole blood. S i m i l a r l y i t has not been demonstrated "that a l l hemoglobin polymorphs occur within the same erythrocyte i n f i s h blood although human erythrocytes do contain both hemoglobin Al and A2 (Matioli and Neiwisch, 1965). Such information i s v i t a l to the discussion of possible molecular interactions between the various hemoglobin polymorphs and may explain the observations of Yamaguchi et al. (1963). From data of Cameron and Randall (1972) for trout blood, the pH of the plasma of coho salmon at 9.3 C was calculated to be 8.27 at a PC09 of 0.2 mm Hg (air - 120 -equili b r a t i o n ) and 7.872 at a P C G ^ of 3.4 mm Hg. Applying the equations r e l a t i n g erythrocyte pH to plasma pH for human blood (Siggaard-Andersen, 1963) the respective values of i n t r a -erythrocyte pH at these two C C ^ p a r t i a l pressures would be approximately 7.81 and 7.52. Relating the measured increases i n log P^Q to these calculated values of i n t r a c e l l u l a r pH yiel d s Bohr s h i f t estimates of -1.228 and -0.569 for the blood of f r y and adult coho salmon, r e s p e c t i v e l y , when the P C G ^ i s raised from 0.2 to 3.4 mm Hg. It i s evident that the Bohr s h i f t estimates calcu-lated from f r y and adult hemoglobin s o l u t i o n s , while being q u a l i t a t i v e l y s i m i l a r were qua n t i t a t i v e l y much d i f f e r e n t from those observed i n whole blood. In the case of coho f r y the difference can be resolved by assuming that the estimates of i n t r a c e l l u l a r pH at 0.2 and 3.4 mm Hg were both 0.15 units too high. The estimates of red c e l l pH were based upon equations developed for human blood and i t i s quite possible that such r e l a t i o n s do not hold for f i s h blood. The d i s p a r i t y i n Bohr s h i f t estimates for adult coho hemoglobin solutions and whole blood are, however, much greater and more d i f f i c u l t to resolve. The P^Q recorded i n whole blood at 0.2 mm Hg P C C ^ would correspond to the value occurring at pH 9.0 in hemoglobin solutions.^ Such a pH i s ph y s i o l o g i c a l l y u n r e a l i s t i c and i t must be concluded that the oxygenation c h a r a c t e r i s t i c s of adult hemoglobin in intact red c e l l s are extremely d i f f e r e n t from those observed i n - 121 -hemoiyzates. Although increases i n oxygen a f f i n i t y are often observed following hemolysis (Black, Tucker and K i r k p a t r i c k , 1966b) and can often be explained on the basis of changes i n the concentrations of a l l o s t e r i c e f f e c t o r substances or i n -creases i n pH decreases i n oxygen a f f i n i t y are much more d i f -f i c u l t to i n t e r p r e t . In this connection Hashimoto et al. (1960) demonstrated that the type and concentration of buff e r -ing substance as well as the presence of various s a l t s may profoundly influence the oxygenation c h a r a c t e r i s t i c s of hemoiy-zates and that these effects may be d i f f e r e n t i n the various hemoglobin polymorphs. A d d i t i o n a l l y , oxygen-linked NH2 groups may be involved i n the formation of carbamino complexes with hemoglobin (Albers, 1970; Riggs, 1970) which would r e s u l t i n an additional factor i n the Bohr s h i f t of whole blood but not hemoiyzates. The present data do not provide any insight into the causes of the variations in the oxygen equilibrium character-i s t i c s observed i n hemoglobin solutions. In any case the q u a l i t a t i v e relationships observed i n these c h a r a c t e r i s t i c s between fry and adult hemoglobin appear to be maintained in whole blood. Estimates of heme-heme in t e r a c t i o n in fry blood and hemoiyzates were sim i l a r and the q u a l i t a t i v e change i n n with changes i n pH were s i m i l a r for both adult and fry hemoglo-bin solutions and whole blood. A b r i e f summary of the oxygen a f f i n i t i e s and effects of carbon dioxide reported i n the l i t e r a t u r e for the whole blood of various salmonids along with the relevant observations from the present study i s presented in Table XVII. Although - 122 -TABLE XVII A summary of the oxygen a f f i n i t y of the blood of various salmonids recorded at d i f f e r e n t e q u i l i b r a t i o n tempera-tures and carbon dioxide tensions, ' FISH EQUILIBRATION CONDITIONS 50 PCO^ TEMPERATURE REFERENCE (mm Hg) (mm ' Hg) (C) Rainbow t r o u t , Salmo gaivdnevi 9. 0 0. 3 6 Eddy £j Hughes 24. 0 3. 0 6 (1970) II It 9. 0 0. 3 10 Cameron (1971) 18. 5 3. 0 10 14. 0 0. 3 15 20. 0 3. 0 15 Trout* 11. 0 0. 3 15 Krough § Leitch 19. 0 4. 5 15 (1919) Brook t r o u t , 5. 0 0. 3 5 Black, K i r k p a t r i c k , Salvelinus 26. 0 10. 0 5 and Tucker (1966,a) f o n t i n a l i s 12. 0 0. 3 15 43. 0 10. 0 15 Landlocked salmon, Salmo salav sebago 8. 0 0. 3 5 Black, K i r k p a t r i c k , 19. 0 0. 3 25 and Tucker (1966,b) 31. 0 10. 0 5 27. 0 10. 0 25 A t l a n t i c salmon, Salmo salav 7. 5 0. 3 5 Black, Tucker and 27. 0 10. 0 5 Kirkpatrick (1966,a) 10. 0 0. 3 15 36. 0 10. 0 15 Coho salmon, Oncorhynchus kisutch 11-month-old f r y 5. 5 0. 2 9.3 Present study 12. 5 3. 4 9.3 2-year-old g r i l s e 12. 0 0. 2 11. 2 Davis (unpublished 20. 0 3. 4 11. 2 observations) spawning adults 10. 7 0. 2 9.3 Present study 15. 6 3. 4 9.3 9. 8 0. 2 5.0 12. 5 0. 2 10.0 14. 8 0. 2 14.8 * Species not reported. - 123 -the age of the f i s h was normally not given i t i s probable that a l l except the coho f r y , were over two years of age. It i s immediately apparent that atPC0 2 of Onto 1 mm Hg the oxygen a f f i n i t y of coho f r y blood i s much higher than that of other salmonids. The large Bohr s h i f t of f r y blood calcu-lated previously i s p a r t i a l l y a r e f l e c t i o n of thi s high a f f i n -i t y at a PC02 of 0.4 mm Hg since increasing PC02 to 3.4 mm Hg only increased P ^ Q by 7 mm Hg. This i s not an es p e c i a l l y large decrease i n oxygen a f f i n i t y when compared to simi l a r observations for other salmonids (Table XVII). The oxygenation c h a r a c t e r i s t i c s of the blood of coho postsmolts (seawater g r i l s e and freshwater adults) are similar to those of other salmonids. The Bohr s h i f t of -0.569 observed i n adult coho salmon at 9.3 C i s only s l i g h t l y d i f f e r e n t from that of-0.53 reported for rainbow trout over the temperature range of 6 to 20 C (Eddy, 1 9 7 1 ) . The decrease in the oxygen a f f i n i t y of adult coho blood at-elevated tempera-tures i s comparable to that observed i n A t l a n t i c salmon, rainbow trout and brook trout (Table XVII). Although an apparent d i s -p a r i t y exists between the P ^ Q recorded at 9.3 and 10.0 C for spawning adult blood equilibrated at 0.2 mm Hg PC02 the former estimate of oxygen a f f i n i t y was obtained from whole blood con-taining 2.4% methemoglobin whereas the l a t t e r contained no detectable methemoglobin. A d d i t i o n a l l y since the seawater g r i l s e had been reared at higher temperatures than the spawning adults, the oxygen a f f i n i t i e s of the two groups of blood are not s t r i c t l y comparable. In this connection Grigg (1969) demon-strated that thermal history influences the oxygen equilibrium - 124 -i n the brown bullhead, Iatalurus nebulosus and that this change i n oxygen a f f i n i t y did not involve a change i n the composition of the seven hemoglobin polymorphs. Differences i n the p h y s i o l o g i c a l state of the blood may also have been responsible for the d i s p a r i t i e s since spawning salmon are undergoing progressive degeneration during t h e i r period of freshwater residence. In either case the d i s p a r i t i e s i n P^Q were generally less than 2 mm Hg. It should be pointed out that a Root s h i f t was not observed i n either hemoglobin solutions or whole blood of coho salmon at any developmental stage examined. This observation was surprising since the blood of rainbow trout (Eddy, 1971), brook Trout (Black, Kirkpatrick and Tucker, 1966a), and A t l a n t i c salmon (Black, Tucker and K i r k p a t r i c k , 1966a) a l l exhibit a substantial decrease i n oxygen capacity with increasing tensions of carbon dioxide. Hashimoto et al. (1960a) observed a Root e f f e c t i n hemolyzates of chum salmon blood and demonstrated that i t was component F and not com-ponent S which exhibited the Root s h i f t . Although the Root s h i f t i s thought to function in the maintenance of hydrostatic pressure in the swim bladder of certain physoclist teleosts this function has not been demonstra-ted i n physostomous f i s h without a well developed gas gland (Steen, 1969). S i m i l a r l y the role of the Root s h i f t i n the transport of gas to the t i s s u e c a p i l l a r y beds of f i s h has not been i d e n t i f i e d although i t could permit the establishment of very high blood:tissue oxygen gradients. The Bohr s h i f t also - 125 -increases the tension of oxygen unloading by hemoglobin but can-not, under p h y s i o l o g i c a l conditions, produce blood:tissue oxygen gradients of the magnitude t h e o r e t i c a l l y possible with a Root s h i f t . The presence of a Root e f f e c t i n the blood of f i s h could r e s u l t i n d i f f i c u l t i e s i n achieving complete oxygenation at the g i l l s . Thus at physiological dorsal a o r t i c tensions of carbon dioxide (Holeton and Randall, 1967; Stevens and Randall, 1967) the Root s h i f t calculated by Eddy (1971) would indicate that only 90% oxygen saturation would be acheived during pas-sage of the blood through the g i l l s . U n t i l information is a v a i l -able r e l a t i n g PC^, PC02 a^d oxygen content of the blood and tissues at the c a p i l l a r y l e v e l , the function of the Root ef f e c t in salmonids must remain i n question. It has been concluded that the increased demand for oxygen during exercise i n trout i s met by increased cardiac output and that only minor increases (about 9%) i n the amount of oxygen released from each hemoglobin molecule occur when the f i s h are exercised (Stevens and Randall, 1967). Although PCO^ of ventral a o r t i c blood increased from 5.7 to about 8 mm Hg during exercise the dorsal a o r t i c PC02 remained unchanged at 2.7 mm Hg (ibid.) . Only 60% of the hemoglobin bound oxygen was delivered to the tissues i n a re s t i n g f i s h and this value increased to 70% during exercise (ibid.). Holeton and Randall (1967) demonstrated that only when environmental P02 was re-duced to about 30 mm Hg did the ventral a o r t i c P02 decrease to about 0 mm Hg. Under these conditions the dorsal aortic blood was only 37% saturated with oxygen. In both the lungfish - 126 -Neoeeratodus f o r s t e r i (Lenfant, Johansen and Grigg, 1966) and rainbow trout (Cameron, 1971) ventral a o r t i c oxygen saturations exceed 50! i n non-exercised f i s h . Although the int e r p r e t a t i o n of the foregoing ob-servations i s complicated by the fact that the blood of the ventral aorta represents the mixed venous return from the various organs and muscles which could vary s u b s t a n t i a l l y i n t h e i r extraction of oxygen from the blood, i t i s clear that a portion of the hemoglobin-bound oxygen i s not released during i t s c i r c u i t through the body. Furthermore, increased a c t i v i t y does not re s u l t i n a s i g n i f i c a n t reduction in this "stored oxygen". This suggests that the in vivo blood oxygenation c h a r a c t e r i s t i c s of active f i s h serve to maintain a r e l a t i v e l y constant blood-to-tissue oxygen gradient. It also i l l u s t r a t e s the possible error i n a r b i t r a r i l y designating Pr-Q as an in d i c a t i o n of oxygen a f f i n i t y since this may be more of a biochemical than a phy s i o l o g i c a l parameter. It i s also evident that variations in heme-heme int e r a c t i o n at a constant P m a y be more p h y s i o l o g i c a l l y important than pre-viously supposed since such variations would s u b s t a n t i a l l y a l t e r the release of oxygen i n the upper portions of the equilibrium curve. Interpreting the oxygen equilibrium character-i s t i c s of adult coho blood in r e l a t i o n to" the foregoing ob-servations i t is evident that they would serve to maintain a r e l a t i v e l y constant blood-to-tissue Q2 gradient under a wide variety of environmental conditions which these f i s h would encounter during the extensive and frequently quite strenuous - 127 -migrations i n the sea and i n freshwater when returning to their spawning grounds. Since these f i s h seldom encounter hypoxic conditions i t i s probable that the dorsal a o r t i c i s maintained above the 60 mm Hg required to achieve com-plete oxygen saturation of the blood. Salmon do not normally reside in water containing high carbon dioxide tensions and the reduced Bohr e f f e c t i s probably not an adaptation to continual high environmental PC02 as has been suggested for the blood of ce r t a i n t r o p i c a l f i s h which i n h i b i t sluggist back-waters (Willmer, 1934). Over a 10 to 20 hour period, PC02 of .surface water i n coastal areas can vary from 0.1 mm Hg during intense phytoplankton blooms to 0.5 mm Hg during intense upwelling i n the northern P a c i f i c Ocean (L. S. Gordon, personal communication). A d d i t i o n a l l y up to 0.9 mm Hg carbon dioxide have been observed near the ocean bottom (depth of 50 m) i n areas of 10 to 20 km offshore during the upwelling season (ibid.). Such variations i n PC02 could re s u l t i n blood pH changes of 0.04 to 0.09 units i f the blood pH versus PC02 relationships published for rainbow trout blood (Cameron and Randall, 1972) are applied. It i s probable that coho post-smolts would encounter these fluctuations i n carbon dioxide tension. Since the Bohr e f f e c t i s not very great these f i s h would not experience aberrations in the oxygen carrying c h a r a c t e r i s t i c s of the blood. The absence of a Root s h i f t would also prevent the reduction in oxygen capacity which could occur under such conditions. Although d a i l y and seasonal fluctuations i n Ocean water temperature are r e l a t i v e l y small adult coho often encounter - 128 large d i e ! and seasonal temperature fluctuations during the freshwater spawning migration. This i s also a period when the metabolic demands of migratory and spawning a c t i v i t y are severe. Under such conditions a large temperature -dependence of the hemoglobin oxygen a f f i n i t y would be d i s -advantageous to the maintenance of e f f i c i e n t oxygen trans-port. Although some f i s h are capable of adjusting the oxygen a f f i n i t y of t h e i r blood i n response to environmental tempera-ture changes (Grigg, 1969) such adaptations probably require some time to accomplish and would not be responsive to d i e l temperature fluctuations. It would seem probable, therefore, that the small influence of temperature upon the oxygen equilibrium of adult coho blood would serve to maintain a r e l a t i v e l y uniform pattern of gas transport during large and rapid changes i n environmental temperature. It i s much more d i f f i c u l t to account for the oxygenation c h a r a c t e r i s t i c s of f r y blood. Blood with a high oxygen a f f i n i t y i s normally found in f i s h residing i n hypoxic environments and the Bohr s h i f t functions to oxygen-unloading tensions of hemoglobin when f i s h are exposed to low environ-mental temperatures (Krough and L e i t c h , 1919; Black, 1940; Black, Kirkpatrick and Tucker, 1966 a,b; Black Tucker and Kirk p a t r i c k , 1966a). It is obvious that the oxygenation c h a r a c t e r i s t i c s of the blood must s a t i s f y two sets of requirements. F i r s t , the oxygen a f f i n i t y at the g i l l s must be s u f f i c i e n t l y high to a t t a i n near saturation of the hemoglobin at ambient oxygen tensions. If environmental 129 -PC02 is high then the presence of a large Bohr s h i f t would int e r f e r e with the saturation of the hemoglobin with oxygen. Secondly, oxygen should be released to the tissues at a s u f f i c i e n t l y high P02 to maintain an adequate oxygen gradient between the blood and the t i s s u e s . The tissue P02 i s not known for f i s h but i s probably less than 15 mm Hg (Randall, 1969). At the tissue l e v e l the presence of a Bohr ef f e c t in the blood would increase the oxygen tensions at which oxygen i s released from the hemoglobin. Considering the high oxygen a f f i n i t y of f r y blood at 0.2 mm PC02 and the sharp increase i n a f f i n i t y associated with decreases i n e q u i l i b r a t i o n temperature i t i s probable that the large Bohr s h i f t functions i n part at l e a s t , to elevate blood:tissue oxygen gradients during the near-freezing temperatures encountered during the winter. This int e r p r e t a t i o n i s somewhat complicated, however, since the Bohr s h i f t occurring in f r y hemoglobin solutions between pH 7.4 and 7.9 i s less at 9.8 C than at 15.2 C. Although part of this c o n f l i c t i s re-solved by the small difference in the lower pH value at 9.8 and 15.2 C (the exact pH values were 7.44 and 7.38, respectively) a f u l l explanation must await further i n v e s t i g a t i o n . Consider-in g , however, the high metabolic rate of fry and i t s dependence upon environmental temperature the increase i n the magnitude of the Bohr s h i f t at elevated temperature would tend to increase the tension and thereby the rate at which oxygen i s delivered i n the c a p i l l a r y beds. Although the Bohr s h i f t is available to decrease the oxygen a f f i n i t y of fry hemoglobin the question arises as - 130 -to why this hemoglobin exhibits such a high a f f i n i t y for oxygen. High oxygen a f f i n i t y i n f i s h blood has been associa-ted with hypoxic environments (Krough and L e i t c h , 1919; Black, 1940) whereas active coho f r y generally occupy well aerated streams and riv e r s in which the PCG^ seldom exceeds 0.2 mm Hg. During extreme winter conditions, however, these f i s h often occupy crevasses i n the stream bed probably to conserve energy when their food sources have been eliminated. Under such conditions i t is l i k e l y that the l o c a l PG^ of the water would be reduced e s p e c i a l l y i n areas containing large amounts of organic material. Under such conditions the high a f f i n i t y of the hemoglobin would insure adequate saturation of the blood with oxygen. Although the environ-mental PCC^ would also increase under such conditions the magnitude of the Bohr s h i f t may be decreased and interference with oxygenation of the blood would be minimized. Although the foregoing functional interpretations of the oxygen equilibrium c h a r a c t e r i s t i c s would seem to s a t i s f y the requirements of oxygen transport i n coho salmon during d i f f e r e n t portions of the l i f e cycle the in vivo pattern of gas transport may be altogether d i f f e r e n t . Although ATP does not appear to modify the oxygen equilibrium of coho hemoglobin and of a wide variety of metabolites present i n f i s h erythrocytes could perform a regulatory function s i m i l a r to that of 2,3-DPG i n human red c e l l s . Since environmental factors may exert some sel e c t i v e influence upon erythrocyte metabolism the p o s s i b i l i t y of a l l o s t e r i c effector substances modifying oxygenation of the hemoglobin molecule i n response - 131 -to cert a i n environmental changes cannot be eliminated. These p o s s i b i l i t i e s emphasize the need for in vivo p h y s i o l o g i c a l experimentation in conjunction with in v i t r o biochemical i n -vestigations into questions of this kind although i n most cases there exist considerable overlap between the two approaches. S p e c i f i c a l l y measurements of tissue PG^ and P C O 2 , rates of carbonic anhydrase a c t i v i t y and patterns of blood flow under d i f f e r e n t environmental and physio l o g i c a l conditions are required i n addition to present knowledge in order to understand the f i n e r d e t a i l s of this v i t a l process i n f i s h . SUMMARY III 1. The hemoglobin concentration of the blood of coho salmon i n freshwater was d i r e c t l y related to the hematocrit by the least squares regression Hb = 0.514 + 0.255 Hct. The blood of seawater g r i l s e also exhibited this r e l a t i o n s h i p . 2. The concentration of ATP i n the erythrocytes of adult coho decreased from 1.0 to 1.2 ymoles/mole hemoglobin i n f i s h which had just recently returned to freshwater, to about 0.8 ymoles/mole Hb near the end of the spawning period. 3. At physiol o g i c a l concentrations ATP did not influence the oxygen equilibrium curve of hemoglobin solutions from adult coho. The PJ-Q only decreased by 1 or 2 mm at ATP concentrations which were about 5 times those occurring i n intact erythrocytes. 4. Hemoglobin components A6-8, prepared by ion-exchange chromatography from hemolyzates of adult coho blood, exhi-bited a high oxygen a f f i n i t y at a pH above 7.5 ( P^Q < 6.2 mm Hg), a large temperature dependence which varied with pH and a non-linear Bohr e f f e c t which was highest i n the pH range of 7.08 to 7.50. Since these hemoglobin components account for v i r t u a l l y a l l the hemoglobin of the blood i n coho f r y , these oxygen equilibrium c h a r a c t e r i s t i c s were con-sidered to be representative of fry hemoglobin. 5. The hemolyzates of freshwater adult coho blood had a lower oxygen a f f i n i t y ( P 5 0 = 17.9 at pH 7.43 and 9.8 C) and a small, almost l i n e a r , Bohr ef f e c t (0 = 0.172). the oxygen a f f i n i t y of the whole blood of adult coho exhibited a small - 133 -temperature dependence. 6. The oxygen e q u i l i b r i a of the pooled whole blood of 113 coho fry and of freshwater adult coho were determined at a PC02 of 0.37 mm Hg (air e q u i l i b r a t i o n ) , and at a, PC02 of 3.4 mm Hg. The oxygen a f f i n i t y of the fr y blood was higher than that of adult blood and a larger Bohr e f f e c t was observed in the juvenile f i s h confirming the observations made on the hemoglobin solutions. The ef f e c t of elevated C02 tensions was upon the decrease i n oxygen a f f i n i t y was greater i n adult blood than that expected from the observed Bohr eff e c t i n adult hemoiyzates which indicated that only q u a l i -tative comparisons could be made between the oxygen equi-l i b r i a observed in whole blood and hemoglobin so l u t i o n s . 7. The implications of the d i f f e r e n t oxygen equilibrium c h a r a c t e r i s t i c s of the hemoglobin of fry and adult coho salmon are discussed. - 134 -MAJOR FINDINGS OF THIS THESIS As stated i n the introduction, the purpose of this thesis was to determine in.some d e t a i l , the ontogeny of the multiple hemoglobins of coho salmon, discover whether or not environmental factors influenced the expression of the hemoglobin patterns and to provide some information as to whether the changes i n the multiple hemoglobin pattern re-sulted in changes i n the oxygenation c h a r a c t e r i s t i c s of the blood. From these observations i t might then be possible to draw certai n conclusions regarding the functional significance of the multiple hemoglobins of the blood of coho salmon. I n i t i a l l y i t was determined that the expression of twelve to thirteen anodic and fi v e to six cathodic hemo-globin components can be observed in the blood of coho salmon when the hemoiyzates were subjected to electrophoresis on starch-gel i n borate buffer at pH 8.5. A l l of these components did not occur at any one developmental stage but various com-binations of the polymorphs were associated with d i s t i n c t ontogenetic stages of the f i s h . Thus coho embryos and alevins exhibited an electrophoretic hemoglobin pattern of thirteen anodic and one cathodic components while f r y hemoglobin con-tained only three anodic components a l l of which were also present in alevin blood. Postsmolt coho exhibited a hemo-globin pattern comprised of f i v e anodic and f i v e cathodic components four to five of which were not present i n ale v i n or f r y blood or, i f present, only i n trace amounts. Very l i t t l e change occurred in the multiple hemoglobin pattern - 135 -during subsequent growth, and sexual development after the f i s h had been i n seawater for more than two months. Since the t r a n s i t i o n from one hemoglobin pattern to another was associated with a change i n behaviour and habitat of the f i s h i t appeared reasonable to suspect that the environment may have been d i r e c t l y or i n d i r e c t l y respon-s i b l e for the p a r t i c u l a r composition of the multiple hemo-globins observed in coho at various developmental stages. Experimental exposure of very young f r y and older presmolt coho to water of various temperatures, oxygen concentrations and s a l i n i t i e s for periods of forty-nine to s i x t y days did not re s u l t i n any changes i n the hemoglobin composition of the blood. These observations e f f e c t i v e l y eliminated the p o s s i b i l i t y that the ontogenetic variations i n hemoglobin pattern were the r e s u l t of variations i n environmental f a c t o r s . In addition maintaining postsmolts i n freshwater during the normal period of early marine residence did not i n h i b i t the expression of the postsmolt hemoglobin pattern. These observations are i n agreement with the observation that both landlocked and sea-run A t l a n t i c salmon exhibit simi-l a r ontogenetic changes i n hemoglobin pattern (Westmann, 1971). Although ontogenetic changes i n the hemoglobin pattern have been correlated with the size rather than age of c e r t a i n salmonids (Kock et al., 1964; Wilkins and l i e s , 1966; Westman, 1970) this does not appear to occur in coho salmon. Presmolts which had been exposed to elevated temperatures i n freshwater or at 10 C in d i l u t e seawater for 60 days were larger than either normal smolts or postsmolts i n - 136 -seawater for t h i r t y days, but s t i l l retained the "presmolt" hemoglobin pattern. In this connection, i t i s interesting to note that * intercept of the weight-length rela t i o n s h i p only decreased s l i g h t l y i n these accelerated presmolts suggesting that the increase i n growth was not accompanied by the normal streamlining of the f i s h as observed i n smolts. A l l of the foregoing observations suggest that i n salmon the genetic control of the expression of the multi-ple hemoglobins i s i t s e l f t i g h t l y controlled by some factor or factors associated with the ontogenetic development of the f i s h . Such a control mechanism could operate by regu-l a t i n g d i f f e r e n t erythropoietic organs which could produce erythrocytes capable of synthetizing only certain hemoglobin polymorphs. Such a s u b s t i t u t i o n of one erythroid c e l l l i n e f o r another which synthetizes a d i f f e r e n t hemoglobin molecule has been observed i n mice (Fantoni et al., 1969). There i s also evidence that erythropoietic factors i n the plasma may s e l e c t i v e l y stimulate the synthesis of one or another of the hemoglobin components of c a l f erythrocytes (Jonxis and Nijhof, 1969). In this connection the s i m i l a r i t y i n timing of the switch over from the blood islands to the kidney as the major erythropoietic organ i n A t l a n t i c salmon alevins (Vernidub, 1966) and the change in hemoglobin pattern i n coho alevins may support the contention that the transformation from the alevin to the f r y hemoglobin pattern involves the substitution of a new erythroid c e l l l i n e . At the present time, however, the control mechanism regulating the trans-formation of one hemoglobin pattern to another remains obscure. - 137 -Whatever the control mechanism i t i s evident that environmental factors play an i n s i g n i f i c a n t role i n the regulation of the select i v e synthesis of the multiple hemoglobins of coho salmon. The functional s i g n i f i c a n c e of the various combinations of hemoglobin polymorphs observed i n coho s a l -mon was investigated by determining some of the oxygenation c h a r a c t e r i s t i c s of hemoglobin solutions containing the hemo-globin components c h a r a c t e r i s t i c of the f r y and adult stages and r e l a t i n g these observations to the oxygen e q u i l i b r i a of fry and adult whole blood measured at two tensions of carbon dioxide. It was observed that f r y hemoglobin had a higher oxygen a f f i n i t y at pH greater then 7.3, a larger Bohr s h i f t and a larger decrease i n oxygen a f f i n i t y with increased tem-perature than adult hemoglobin. In addition, the Bohr ef f e c t was non-linear i n fry hemoglobin and rose sharply i n the pH range of 7.1 to 7.5, whereas the Bohr ef f e c t of adult hemo-globin was approximately l i n e a r over the pH range of 6.95 to 8.20. In general then the oxygenation c h a r a c t e r i s t i c s of adult hemoglobin were r e l a t i v e l y i n s e n s i t i v e to variations in pH and temperature, while the oxygen equilibrium of fry hemoglobin was greatly influenced by such f a c t o r s . When the oxygen e q u i l i b r i a of whole blood of fry and adult coho salmon were measured at a PCC^ of 0.2 and 3.4 mm Hg. the q u a l i t a t i v e relationships observed i n the hemoglobin solutions were evident although quantitative com-parisons of the observations on whole blood and hemolyzates - 138 -were not poss i b l e . The s i g n i f i c a n t observation, however, was that the oxygen carrying c h a r a c t e r i s t i c s of the blood of coho fry and adults are d i f f e r e n t and that i t i s the di f f e r e n t hemoglobin components present i n the erythrocytes of these two age groups that account for these oxygenation c h a r a c t e r i s t i c s . The foregoing studies demonstrate the necessity of confirming the i d e n t i t y of the multiple hemoglobin pattern during comparisons of the in vivo patterns of gas transport i n f i s h e s p e c i a l l y when such comparisons are made on a seasonal basis. It i s also evident that the use of blood equilibrium curves of a species of f i s h from one area may not represent those of the same species i n another l o c a l i t y , e s p e c i a l l y i f the rates of development are d i f f e r e n t i n the two areas. 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(Tokyo) 63: 70-76. 152 APPENDIX A C a l c u l a t i o n of the r e l a t i o n s h i p between the r e l a t i v e amount o f hemoglobin a p p l i e d t o m i c r o - s t a r c h - g e l s and the e s t i m a t i o n of the r e l a t i v e concentrations of the hemoglobin components of pre-smolt coho salmon. 153 TABLE XVIII Spearman Rank Correlation Coefficient test of significance of the relationship between the relative amount of hemoglobin (density index) applied to the starch-gel and the estimation of the combined relative concentration of hemoglobin components A 6-8 of pre-smolt coho salmon. RELATIVE CONCENTRATION SOURCE DENSITY INDEX COMPONENTS A6-8 (date) Observed Rank Observed Rank Creek 1 3 1 . 3 2 7 9 . 3 1 ( 1 6 / 2 / 7 1 ) 1 2 3 . 1 1 8 8 . 4 6 145.8 4 8 7 . 2 5 184.6 6 85.0 3 149.8 5 8 6 . 3 4 . 1 3 3 . 5 3 8 3 . 0 2 Creek 3 £ 4 . 2 6 8 6 . 5 3 ( 2 / 3 / 7 1 ) 2 9 2 . 1 4 91.3 6 2 8 3 . 7 3 8 8 . 0 5 3 3 4 . 1 5 8 7 . 6 4 2 2 9 . 1 2 80.7 1 1 7 0 . 2 1 8 5 . 9 2 Creek 3 3 8 . 2 6 7 6 . 1 1 ( 1 6 / 3 / 7 D 3 2 7 . 5 5 84.1 6 2 5 4 . 0 4 8 3 . 0 5 2 3 5 . 8 2 7 7 . 3 2 2 3 7 . 4 3 7 9 . 0 3 2 0 6 . 7 1 8 2 . 6 4 Combined 1 2 9 . 8 1 8 8 . 5 3 Controls and 1 5 9 . 0 • 2 9 0 . 3 4 1 0 . 2 C treat- . 1 5 9 . 5 3 8 5 . 5 1 ment 1 6 3 . 9 4 9 1 . 2 5 (16/2/71) 178.7 5 8 8 . 0 2 ( 2 / 3 / 7 D 187.0 1 8 8 . 1 4 2 2 2 . 7 2 8 6 . 9 3 2 8 3 . 3 3 8 8 . 7 6 3 3 2 . 9 4 8 8 . 6 5 3 6 8 . 6 5 8 6 . 8 2 404.1 6 8 3 . 5 1 ( 1 6 / 3 / 7 D 1 9 6 . 3 1 8 2 . 1 1 2 2 4 . 0 2 8 7 . 1 6 2 3 8 . 2 3 8 3 . 6 2 2 5 6 . 0 4 8 3 . 9 3 3 0 3 . 1 5 8 5 . 1 4 3 1 3 . 0 6 8 6 . 5 5 CORRELATION COEFFICIENT - 0 . 0 8 6 n.s, + 0 . 4 2 9 n. s, - 0 . 0 2 9 n.s. - 0 . 0 5 6 n.s, - 0 . 5 4 3 n.s, - 0 . 9 0 5 (.01>p>.05) 

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