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An analysis of the thyroid role in juvenile steelhead (Salmo Gairdneri Richardson) and factors responsible.. 1963

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AN ANALYSIS OF THE THYROID ROLE IN JUVENILE STEELHEAD (SALMO GAJJKDNERI RICHARDSON) AND FACTORS RESPONSIBLE FOR ITS SEASONAL FLUCTUATION IN ACTIVITY by JOHN GEOFFREY EALES B.A., Oxford University, 1959 M.Sc, University of B r i t i s h Columbia, 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s thesis as conforming to the required standard Members of the Department THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1963 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the 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 for. reference and study. I f u r t h e r agr.ee that per- m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives.. I t i s understood that copying, or p u b l i - c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia,. Vancouver 8, Canada. PUBLICATIONS A comparative study of thyroid function i n migrant juvenile salmon. Can. J . Zool 41: ( i n press). 1963. The thyroid gland and low temperature re- sistance of g o l d f i s h . Can. J . Zool., 41: ( i n press), with W.S. Hoar. 1963 The U n i v e r s i t y of B r i t i s h Columbia ' FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of JOHN GEOFFREY EALES B.A., Oxford Un i v e r s i t y , 1959 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1961 TUESDAY, May 7, 1963 at 9:30 A.M. IN ROOM 3332, BIOLOGICAL SCIENCES .BUILDING COMMITTEE,IN CHARGE Chairman: F.H. Soward W.N. Holmes W.D. K i t t s V.J. Okulitch N.J. Wilimovsky. External Examiner: E.J.W. Barrington Department of Zoology, Nottingham U n i v e r s i t y J.R. Adams D.H. C h i t t y I. McT. Cowan W.S. Hoar , AN ANALYSIS OF THE THYROID ROLE IN JUVENILE STEELHEAD (SALMO GAIRDNERI RICHARDSON) AND FACTORS RESPONSIBLE . FOR ITS SEASONAL FLUCTUATION IN ACTIVITY ABSTRACT Investigation of factors c o n t r o l l i n g seasonal changes i n thyroid a c t i v i t y of juvenile steelhead trout (Salmo •gairdneri Richardson) i n fresh water revealed p o s i t i v e c o r r e l a t i o n s between temperature and radioiodine assess- ments of thyroid a c t i v i t y . Temperature and thyroid epi- t h e l i a l height, however, showed negative corr e l a t i o n s . These c o r r e l a t i o n s were v e r i f i e d experimentally. In y e a r l i n g s , the thyroid was refractory to increasing photoperiod (January to July) but two-year-old po t e n t i a l migrants showed a p o s i t i v e response at the same season. Thus, the high smolt thyroid a c t i v i t y i s induced by the combined influence of r i s i n g temperature and increasing spring photoperiod. Body mass (logarithm) was inversely related to various 1^31 parameters (logarithm), so that small f i s h had high- er thyroid a c t i v i t i e s than large f i s h . Precocious sex- ual maturation of two-year-old male parr, increased swim- ming exercise and increased s a l i n i t y were associated with higher thyroid a c t i v i t y . Increase i n ambient 1*27 d e_ pressed thyroid a c t i v i t y and indicated that the a c t i v i t y of the gland (assessed by current methods) i s p a r t l y a compensation for low I•'•27 a v a i l a b i l i t y . The high thyroid a c t i v i t y of the smolt may be due p a r t l y to endemic goitre Since low temperature and 8-hour day length i n h i b i t e d the thyroid a c t i v i t y of potential migrants but did not prevent s i l v e r i n g , the r o l e of thyroxin in guanine depo- s i t i o n under natural conditions i s doubted; Possible radiohormone catab o l i c s i t e s were located i n metaboli- c a l l y a c t i v e tissues including gut, kidney, l i v e r and brain. These findings suggest a general rather than a t i s s u e - s p e c i f i c role of thyroxin i n metabolism. It i s concluded that thyroxin may have no stimulatory role i n s m o l t i f i c a t i o n but r e f l e c t s instead the t o t a l metabolic demands on the t i s s u e s . GRADUATE STUDIES F i e l d of Study: Zoology P.A. Larkin W.S. Hoar W.S. Hoar M.D.F. Udvardy C.V. Finnegan C.C., Lindsey W.N. Holmes P.A. Dehnel Sta f f Other Studies: Quantitative Methods i n Zoology Comparative Physiology Comparative Ethology Comparative Invertebrate Embryology Biology of Fishes Endocrinology Marine F i e l d Course F i s h e r i e s Seminar Biochemistry Faculty of Medicine ABSTRACT I n v e s t i g a t i o n o f f a c t o r s c o n t r o l l i n g s e a s o n a l changes i n t h y r o i d a c t i v i t y o f j u v e n i l e s t e e l h e a d t r o u t (Salmo g a i r d n e r i R i c h a r d s o n ) i n f r e s h w a t e r r e v e a l e d p o s i t i v e c o r r e l a t i o n s b e t w e e n t e m p e r a t u r e and r a d i o i o d i n e a s s e s s m e n t s o f t h y r o i d a c t - i v i t y . T e m p e r a t u r e and t h y r o i d e p i t h e l i a l h e i g h t , h o w e v e r , showed n e g a t i v e c o r r e l a t i o n s . These c o r r e l a t i o n s were v e r i f i e d e x p e r i m e n t a l l y . I n y e a r l i n g s t h e t h y r o i d was r e f r a c t o r y t o i n c r e a s i n g p h o t o p e r i o d ( J a n u a r y t o J u l y ) b u t t w o - y e a r - o l d p o t e n t i a l m i g r a n t s showed a p o s i t i v e r e s p o n s e a t t h e same s e a s o n . T h u s , t h e h i g h s m o l t t h y r o i d a c t i v i t y i s i n d u c e d by t h e c o m b i n e d i n f l u e n c e o f r i s i n g t e m p e r a t u r e and i n c r e a s i n g s p r i n g p h o t o n p e r i o d . Body mass ( l o g a r i t h m ) was i n v e r s e l y r e l a t e d t o v a r i o u s J13>1 p a r a m e t e r s ( l o g a r i t h m ) , so t h a t s m a l l f i s h had h i g h e r t h y r o i d a c t i v i t i e s t h a n l a r g e f i s h . P r e c o c i o u s s e x u a l m a t u r a t i o n o f t w o - y e a r - o l d m a l e p a r r , i n c r e a s e d swimming e x e r c i s e a n d i n c r e a s e d s a l i n i t y were a s s o c i a t e d w i t h h i g h e r t h y r o i d a c t i v i t y . 127 I n c r e a s e i n a m b i e n t I ' d e p r e s s e d t h y r o i d a c t i v i t y and i n d i c a t e d t h a t t h e a c t i v i t y o f t h e g l a n d ( a s s e s s e d by c u r r e n t methods) i s p a r t l y a c o m p e n s a t i o n f o r l o w l 1 2 7 a v a i l a b i l i t y . The h i g h t h y r o i d a c t i v i t y o f t h e s m o l t may be due p a r t l y t o endemic g o i t r e . S i n c e l o w t e m p e r a t u r e a n d 8 - h o u r ( d a y l e n g t h i n h i b i t e d t h e - i i i - t h y r o i d a c t i v i t y o f p o t e n t i a l m i g r a n t s b u t d i d n o t p r e v e n t s i l v e r i n g , t h e r o l e o f t h y r o x i n i n g u a n i n e d e p o s i t i o n u n d e r n a t u r a l c o n d i t i o n s i s d o u b t e d . P o s s i b l e r a d i o h o r m o n e c a t a b o l i c s i t e s were l o c a t e d i n m e t a b o l i c a l l y a c t i v e t i s s u e s i n c l u d i n g g u t , k i d n e y , l i v e r and b r a i n . T h e s e f i n d i n g s s u g g e s t a g e n e r a l r a t h e r t h a n a t i s s u e - s p e c i f i c r o l e o f t h y r o x i n i n m e t a b o l i s m . I t i s c o n c l u d e d t h a t t h y r o x i n may have no s t i m u l a t o r y r o l e i n s m o l t i f i c a t i o n b u t r e f l e c t s i n s t e a d t h e t o t a l m e t a b o l i c demands on t h e t i s s u e s . A C K N O I i V L E D G E M E N T S I wish to express my sincere gratitude to Professor W. S. Hoar, P. R. S. C , Department of Zoology, f o r h i s stimulation, c r i t i c i s m and enthusiastic guidance throughout t h i s project. I am also indebted to Professors J . R. Adams, W. N. Holmes, N. J . Wilimovsky (Department of Zoology) and Professor W. D. K i t t s (Animal Science) f o r t h e i r constructive c r i t i c i s m . I further wish to acknowledge the several graduate students from, whom I gained so much during informal discussions. Dr. K. A. Evelyn of the Strong Laboratory, Vancouver General Hospital, supplied.the radioiodide. Messrs. S. B. Smith and J. G. Terpenning ( B r i t i s h Columbia Pish and Game) arranged f o r the provision of steelhead from the Cultus Lake Hatchery, where the experimental f i s h were tended for several months by Mr. Pat Martin and h i s s t a f f . A great number of h i s t o l o g i c a l preparations were meticulously made by Miss S. Tabata. The s t a t i s t i c a l analyses were f a c i l i t a t e d by use of IBM Computing Centre, University of B r i t i s h Columbia and the co-operation of the s t a f f there was greatly appreciated. To a l l these people I wish to express my deepest appreciation f o r t h e i r various services; without them t h i s study would have been impossible. Pinanc i a l assistance i n the form of a F i s h e r i e s Research Board Studentship enabled me to carry out t h i s study. TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS 4 1. L i v i n g Materials 4 2. Radioiodide Injection 4 3. Blood Sampling and Separation of Protein- bound and Inorganic Radioiodine . . . 6 4. Body and Thyroid . . . . . . . . 8 5. Thyroid Histology 9 6. S t a t i s t i c a l Treatment 11 RESULTS 12 ' I. COMPARISON OF YEARLING STEELHEAD WITH DIFFERENT RATES OF RADIOIODINE METABOLISM . . . . 12 i 1. Extrathyroidal and Thyroidal Clearance of Plasma ±131 13 2. Experimental Measurement of Thyroid l l ? l Accumulation 21 3. Estimation of Radiohormone Output Using the Conversion Ratio 2 7 I I . SEASONAL CHANGES IN RADIOIODINE METABOLISM AND THE HISTOLOGICAL APPEARANCE OF THE THYROID IN JUVENILE STEELHEAD HELD IN FRESH WATER . . 3 0 1. I 1 ? ! Excretion 30 2. Thyroid Uptake 34 3. Conversion Ratio 34 4. H i s t o l o g i c a l Changes 33 V - TABLE OF CONTENTS (Continued) PAGE I I I . EXPERIMENTAL INVESTIGATION OF THE INFLUENCE OF TEMPERATURE ON THE HISTOLOGICAL APPEARANCE OF THE THYROID AND RADIOIODINE METABOLISM IN YEARLING STEELHEAD PARR 36 IV. EXPERDIENTAL INVESTIGATION OF TEMPERATURE AND PHOTOPERIOD INFLUENCE ON THYROID CELL HEIGHT AND RADIOIODINE METABOLISM OF STEELHEAD YEARLINGS (January to July) 44 V. EFFECT OF BODY MASS ON RADIOIODINE METABOLISM 35 VI. EXPERIMENTAL INVESTIGATION OF THE INFLUENCE OF SEASONAL CHANGE IN INCREASING PHOTOPERIOD AND INCREASING TEMPERATURE ON THE RADIOIODINE METABOLISM AND THYROID CELL HEIGHT OF TWO-YEAR- OLD STEELHEAD 6 l 1. l l ? l Excretion 62 2. Thyroid Uptake of I 1 ? ! 64 3 . Conversion Ratio 67 4. C e l l Height 70 VII. INFLUENCE OF PRECOCITY OF MALE PARR ON THYROID ACTIVITY 70 VIII. INFLUENCE OF EXERCISE ON I 1 ? 1 METABOLISM OF STEELHEAD 72 IX. THE INFLUENCE OF CHEMICAL CHANGES (SALINITY AND IODINE LEVELS OF THE MEDIA) ON I 1 ^ ! METABOLISM OF STEELHEAD AND CHUM SALMON (Oncorhynchus keta) 74 - v i - TABLE OF CONTENTS (Continued) PAGE X. THE RELATIONSHIP BETWEEN RADIQIODINE METABO- LISM AND SILVERING 85 XI. PERIPHERAL SITES OF RADIOHORMONE CATABOLISM 89 DISCUSSION 95 I. COMPARISON OF PARAMETERS FOR MEASURING THYROID ACTIVITY AND THE EFFECT OF TEMPERATURE ON THESE PARAMETERS 95 I I . FACTORS CONTRIBUTING TO. SEASONAL CHANGES IN THYROID ACTIVITY IN JUVENILE STEELHEAD . . 102 I I I . THE ROLE OF THE THYROID IN THE STEELHEAD. . 112 SUMMARY AND CONCLUSIONS 116 BIBLIOGRAPHY 119 LIST OP FIGURES FIGURE PAGE 1. Seasonal changes i n water temperature i n the laboratory and i n two streams near Vancouver, B r i t i s h Columbia - 5 2. Plasma clearance following a single J.131 i n - j e c t i o n i n f i s h held at 3° 0 (O) and 10° C (•). Each point represents a mean of 3 to 7 f i s h . (Covariance analysis, Table I) 14 3. T o t a l body clearance (O) and body excluding thyroid clearance (•) following a single 1̂ -31 i n j e c t i o n at 5° C (inactive thyroid) and 10° C active thyroid). Equations calculated separately f o r each phase. Each point represents a mean of 5 to 7 f i s h (Covariance analyses, Tables I I and III) 18 4. (A) Percentage accumulation of 1^51 by the thy- r o i d following a single i l ? 1 i n j e c t i o n at 10° C (active) and 5° C (in a c t i v e ) . Standard deviations shown. Each point represents a mean of 5 to 7 f i s h . (B) TUF values f o r the same f i s h as (A). (C) T/S values f o r the same'fish as (A). The diphasic tendency i s shown i n the active state (Covariance analysis, Table IV) 23 5. Change i n CR following a single 1.131 i n j e c t i o n i n f i s h at 10° C (active) and 5° C ( i n a c t i v e ) . Each point represents a mean of 5 to 7 f i s h . . 28 6. Seasonal change i n plasma T.131, % dose l ! 3 1 i n body, T/S, TUF, CR, mean e p i t h e l i a l cell''height and percentage of f o l l i c l e s containing blue c o l l o i d i n steelhead parr and smolts. Seasonal change i n temperature i s shown. Standard devia- tions are shown for ce r t a i n parameters . . . J>2 7. Correlations between water temperature and four l l 3 1 parameters. A l l values drawn from seasonal data (Fig. 6 ) . A (f. dose i n body); B(CR); C(T/S); D(TUF) . . 33 - v i i i LIST OF FIGURES (Continued) FIGURE 8. Experimental demonstration of the influence of temperature on percentage accumulation of l l 3 1 i n the thyroid 4 and 8 days a f t e r a single l ! 3 1 i n j e c t i o n , on mean c e l l height (O) and on per- centage of f o l l i c l e s containing blue c o l l o i d (•) 9, Experimental demonstration of the influence of temperature on 1"131 l e v e l s i n plasma and t o t a l body 4 days a f t e r a single l"13l i n j e c t i o n . Each point represents 4 to 7 i n d i v i d u a l s (Covariance analysis, Table VI) 10. Experimental demonstration of the influence of temperature on T/S values 4 and 8 days a f t e r a single l ! 3 l i n j e c t i o n . Each point represents a mean of 4 to 7 individuals 11. Experimental demonstration of the influence of temperature on CR values 4 and 8 days a f t e r a single 1131 i n j e c t i o n . Each point represents a mean of 4 to 7 individuals (Covariance analysis, Table VI) 12. Experimental demonstration of the combined i n - fluence of temperature and photoperiod on plasma 1131 l e v e l s and percentage dose i n t o t a l body 4 days a f t e r a single J.131 i n j e c t i o n i n 14 to 18- month steelhead. Each point represents a mean of 8 to 12 f i s h ( S t a t i s t i c s , Table VII I ) . . . . 46 13. Experimental demonstration of the combined i n - fluence of photoperiod and temperature on mean c e l l height and T/S r a t i o s 4 days afte r a single 1131 i n j e c t i o n i n 14 to 18-month steelhead. C e l l height - each point represents a mean of 3 to 4 f i s h ; T/S - each point represents a mean of 8 to 12 f i s h ( S t a t i s t i c s , Table VIII) 48 14. Experimental demonstration of the combined i n - fluence of photoperiod and temperature on plasma clearance following a single l"l31 i n j e c t i o n i n 18-month steelhead. Each point represents a mean of 5 to 7 f i s h (Covariance analysis, Table IX). 51 15. Experimental demonstration of the combined i n - fluence of photoperiod and temperature on T/S values i n 18-month steelhead following a single 1131 i n j e c t i o n . Each point represents a mean of 5 to 7 f i s h (Covariance analysis, Table IX). . 52 PAGE 38 40 42 43 - i x - LIST OF FIGURES (Continued) FIGURE PAGE 16. Experimental demonstration of the combined i n - fluence of photoperiod and temperature on CR values following a single 1131 i n j e c t i o n i n 18- month steelhead. Each point represents a mean of 5 to 7 i n d i v i d u a l s 53 17. Influence of body mass on T/S values and per- centate of l l 3 1 i n the t o t a l body 84 hours a f t e r a single 1131 i n j e c t i o n i n 19-month steelhead.. 56 18. Influence of body mass (gram) on plasma 1.131 l e v e l s and T/S values (4 days a f t e r injection) and CR values (8 days a f t e r i n j e c t i o n ) . Means and standard deviations are shown f o r large, medium and small size groups. No s i g n i f i c a n t difference was noted between medium and large size groups f o r any 1131 parameter, but both d i f f e r e d from the small group f o r a l l parameters (p< . 0 1 ) 59 19. Relationship between the logarithm of body mass and the logarithm of various 1131 parameters. T/S (A), r = - 0 . 5 7 ; CR (•), r = - 0 . 5 3 ; i body (O), r - - 0 . 6 7 60 20. Experimental demonstration of the combined i n - fluence of temperature and photoperiod on plasma 1131 l e v e l s and percentage dose i n t o t a l body 4 days a f t e r J.131 i n j e c t i o n i n 26 to 50-month s t e e l - head (potential migrants). Each point represents a mean of 6 i n d i v i d u a l s ( S t a t i s t i c s , Table XI). 63 21. - Experimental demonstration of the combined i n - fluence of temperature and photoperiod on T/S values 4 and 8 days a f t e r J.131 i n j e c t i o n i n 26 to 30-month steelhead (potential migrants). Each point represents a mean of 6 individuals ( S t a t i s t i c s , TABLE XI) 65 2 2 . Experimental demonstration of the combined i n - fluence of temperature and photoperiod on TUF values 4 and 8 days a f t e r l ! 3 1 i n j e c t i o n i n 26 to 30-month steelhead (potential migrants). Each point represents a mean of 6 i n d i v i d u a l s ( S t a t i s t i c s , Table XI) 66 X - LIST OF FIGURES (Continued) FIGURE PAGE 23. Experimental demonstration of the combined i n - fluence of temperature and photoperiod on mean c e l l height and CR (8 days a f t e r 1.131 injection) i n 26 to 30-month steelhead (potential migrants). C e l l height - means ( A O ) and i n d i v i d u a l values ( A O ) shown; CR - each point represents a mean of 6 i n d i v i d u a l s . . 68 24. TUF (open bars) and CR (shaded bars) f o r preco- cious two-year-old male (U0T and immature 0^ steelhead i n early March under 4 combined condi- tions of temperature and photoperiod . . . . 71 25. Influence of increased swimming a c t i v i t y on various aspects of 1.131 metabolism i n 19-month steelhead. Each point represents a mean of 6 i n d i v i d u a l s (Covariance analysis, Table 2 1 1 ) . . 73 26. E f f e c t of transfer of 20-month steelhead to sea water (25o/oo saline) and iodide-reinforced fresh water on various aspects of 1.131 metabolism mea- sured 4 and 8 days a f t e r 1131 i n j e c t i o n . Mean values represented (SW,X ; FW + J.127,A ; 27. E f f e c t of transfer of 30-month smolts and parr to sea water (25 0/00 saline) on various aspects of 1131 metabolism. Mean values (bar) and i n d i v i - 28. E f f e c t of transfer of underyearling p o t e n t i a l migrant chum salmon to sea water (25 0/00 saline) and iodide-reinforced fresh water. Mean values (bar) and i n d i v i d u a l values shown. X s < 0 . 0 5 ; 29. Demonstration of the lack of a rel a t i o n s h i p be- tween s i l v e r i n g and any aspect of l ! 3 1 metabo- lis m (8 days a f t e r i n j e c t i o n ) . Mean values (bar) and i n d i v i d u a l values shown 88 30. Change i n the t i s s u e : blood r a d i o a c t i v i t y i n a v a r i e t y of tissues from 19-month steelhead. Each point represents a mean of 14 to 16 individuals 91 dual values shown 81 83 - x i - L I S T OF FIGURES ( C o n t i n u e d ) FIGURE PAGE 31 . P e r c e n t a g e change ( r e l a t i v e t o t h e v a l u e 3 days a f t e r i n j e c t i o n ) o f t i s s u e : b l o o d r a t i o s 6 a n d 9 days a f t e r i n j e c t i o n i n 19-month s t e e l h e a d . . 93 32. D i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e h y p o t h e s i s t h a t t h y r o x i n u t i l i z a t i o n and p r o d u c t i o n r e s - pond d i f f e r e n t l y t o t e m p e r a t u r e , x^ and X̂ Q r e p r e s e n t t h e d i f f e r e n c e s b e t w e e n u t i l i z a t i o n and p r o d u c t i o n a t 5 and 10° C . S i n c e x^ i s g r e a t e r t h a n XT_Q i"t w o u l d i m p l y g r e a t e r r e l e a s e o f TSH a t l o w t e m p e r a t u r e s a n d a l s o a g r e a t e r c e l l h e i g h t 99 LIST OF TABLES TABLE - PAGE I. Analysis of covariance f o r plasma l ! 3 1 (plasma 1131 B i o l o g i c a l Concentration Coefficient / 1 0 0 ) and time (hr) f o r active (10° C) and inactive (50 c) thyroid states 15 I I . Analysis of covariance for I 1 ^ 1 clearance from the t o t a l body'and time (hr); and body exclud- ing thyroid and time (hr). S l o p e T t g - Slope^ =' Rate constant for thyroid T!31 uptake (Slope-,). 19 I I I . Analysis of covariance f o r T131 clearance from t o t a l body and time (hr) and body excluding thyroid and time (hr) for active (10° C) and inactive (5° C) thyroid states. The analysis indicates the difference i n excretion rates be- tween the two temperatures i n phase I I . 20 IV. Analysis of covariance f o r T/S and time (hr) f o r active (10© C) and inactive (5° C) thyroid states 24 V. A summary of radioiodine parameters and t h e i r significance 31 VI. Analysis of covariance for the r e l a t i o n s h i p between various 1131 parameters and temperature ( ° 0 ) 41 VII. Summary of the four combined temperature and photoperiod conditions. Symbols shown are used i n figures 45 VTII. Summary of s t a t i s t i c a l l y s i g n i f i c a n t differences for several J.131 parameters between d i f f e r e n t conditions of temperature and photoperiod i n yearlings i n June. White-Wilcoxon non-parametric ranking test used 49 IX. Analysis of covariance performed on plasma 1131 and time (hr), and T/S and time (hr) under four d i f f e r e n t photoperiod and temperature regimes. 54 X. Summary of i n j e c t i o n d e t a i l s f o r size experiment 58 - x i i i - L I S T OF TABLES ( C o n t i n u e d ) TABLE PAGE X I . Summary o f s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r - e n c e s f o r s e v e r a l l ! 3 1 p a r a m e t e r s b e t w e e n d i f f e r e n t c o n d i t i o n s o f t e m p e r a t u r e and p h o t o - p e r i o d i n t w o - y e a r - o l d s ( p o t e n t i a l m i g r a n t s ) i n l a t e May ( W h i t e - W i l c o x o n t e s t ) . . . . 69 X I I . A n a l y s i s o f c o v a r i a n c e f o r t h e r e l a t i o n s h i p b e t w e e n v a r i o u s 1^31 p a r a m e t e r s and t i m e ( h r ) f o r p a r r h e l d i n f a s t and s l o w c u r r e n t s . . 75 X I I I . The e f f e c t o f d i f f e r e n t t e m p e r a t u r e and p h o t o - p e r i o d r e g i m e s on t h e p e r c e n t a g e i n c i d e n c e o f s m o l t i f i c a t i o n i n t w o - y e a r - o l d s t e e l h e a d b e t w e e n 27 A p r i l and 29 May, 1962 87 INTRODUCTION S e a s o n a l changes i n t h y r o i d a c t i v i t y h a v e b e e n r e c o r d e d b y a v a r i e t y o f t e c h n i q u e s i n s e v e r a l t e l e o s t s p e c i e s . T h e s e i n c l u d e M i s g u r n u s f o 3 s i l i s ( L i e b e r , 1936); A n g u i l l a a n g u i l l a ( H a g e n , 1936); P h o x i n u s p h o x i n u s ( B a r r i n g t o n and M a t t y , 1934); E s o x l u c i u s ( Z a i t z e v , 1955); Sa lmo t r u t t a ( S w i f t , 1955, 1959); P l e c o g l o s s u s a l t i v e l i s (Honma, 1959); F u n d u l u s h e t e r o c l i t u s ( B e r g , Gorbman and K o b a y a s h i , 1959)» and P l a t i c h t h y s s t e l l a t u s ( H i c k m a n , 1962). C o m p r e h e n s i v e r e v i e w s r e l a t i n g t o t h e s e changes h a v e b e e n w r i t t e n b y H o a r (1959), M a t t y ( i960) a n d S w i f t ( i 9 6 0 ) . M o s t s t u d i e d o f a l l have b e e n t h e s e a s o n a l changes i n t h y r o i d a c t i v i t y i n anadromous s a l m o n i d s . T h e s e f i s h h a t c h i n f r e s h w a t e r b u t , e x c e p t f o r s p a w n i n g , s p e n d a d u l t l i f e i n t h e s e a . A t a c e r t a i n p e r i o d i n d e v e l o p m e n t , young s a l m o n d e s c e n d t h e r i v e r s t o t h e s e a and a t t h i s t i m e u n d e r g o c h a r a c t e r i s t i c c h a n g e s i n m o r p h o l o g y , b e h a v i o u r a n d m e t a b o l i s m . The most o b v i o u s c h a n g e , s m o l t i f i c a t i o n , i s t h e l o s s o f b l a c k p a r r marks a n d a c q u i s i t i o n o f t h e s i l v e r y g u a n i n e l i v e r y o f t h e s m o l t . One o f t h e most e m p h a s i z e d changes i n t h e u n d e r l y i n g p h y s i o l o g y a t s m o l t i f i c a t i o n i s a m a r k e d t h y r o i d h y p e r a c t i v i t y f i r s t n o t e d h i s t o l o g i c a l l y i n t h e A t l a n t i c s a l m o n (Sa lmo s a l a r ) by H o a r (1939). S i n c e t h i s i n i t i a l d i s c o v e r y , more and more e m p h a s i s h a s been p l a c e d on t h e r o l e o f t h e t h y r o i d d u r i n g s m o l t i f i c a t i o n p r i m a r i l y b y F o n t a i n e and h i s c o - w o r k e r s , ( F o n t a i n e , 1954; L e l o u p a n d F o n t a i n e , i 9 6 0 ) . R o b e r t s o n (1948) o b s e r v e d a s i m i l a r h y p e r t r o p h y and h y p e r p l a s i a i n t h e t h y r o i d o f s m o l t i n g r a i n b o w - 2 - troujt (Salmo gairdnerl) and also claimed that thyroxin stimulated guanine deposition (Robertson, 1949). S i l v e r i n g , however, i s not always dependent upon thyroxin. In P a c i f i c salmon (genus Oncorhvnchus). the thyroids of s i l v e r i n g coho and sockeye are moderately active at migration but those of pink, spring and chum are r e l a t i v e l y i n active (Hoar and B e l l , 1950; Eales, 1961, 1963). The r o l e of the thyroid i n the s m o l t i f i c a t i o n and migration of • salmon i s , therefore, f a r from established. Though c e r t a i n speculations have been made, the control of these seasonal thyroid fluctuations i s almost e n t i r e l y uninvestigated. The matter i s further complicated by the discrepancy between h i s t o - l o g i c a l and radiochemical determinations. With these unresolved points i n mind, a comprehensive h i s t o - l o g i c a l and radiochemical inves t i g a t i o n of thyroid function was attempted i n the steelhead trout (Salmo gairdne r i Richardson). This species i s anadromous and shows an e a s i l y recognizable parr- smolt transformation. The i n v e s t i g a t i o n was divided into four i n t e r r e l a t e d areas of study: (a) A preliminary comparative analysis of radioiodine metabolism i n steelhead with low and high thyroid a c t i v i t y . (b) Seasonal observations on histology and radioiodine metabolism from 17 months p r i o r to migration up to seaward migration i t s e l f to determine seasonal changes. (c) Experimental in v e s t i g a t i o n of environmental factors and i n t e r n a l changes influencing these seasonal fl u c t u a t i o n s . (d) An i n v e s t i g a t i o n of possible s i t e s of radiohormone catabolism i n steelhead and the r e l a t i o n s h i p between radioiodine m e t a b o l i s m a n d s i l v e r i n g . F r o m t h e s e i n v e s t i g a t i o n s i t was hoped t o d e t e r m i n e t h e f a c t o r s c o n t r i b u t i n g t o s e a s o n a l changes i n t h y r o i d a c t i v i t y a n d a l s o t o g a i n k n o w l e d g e c o n c e r n i n g t h e r o l e o f t h e t h y r o i d , p a r t i c u l a r l y a t t h e t i m e o f m i g r a t i o n . MATERIALS AND METHODS 1. L i v i n g M a t e r i a l s S t e e l h e a d were o b t a i n e d as y e a r l i n g s o r t w o - y e a r - o l d s f r o m t h e B r i t i s h C o l u m b i a F i s h and Game H a t c h e r y a t C u l t u s L a k e , B r i t i s h C o l u m b i a , where t h e y w e r e f e d on d r i e d c o m m e r c i a l f o o d a n d k e p t i n l a r g e c o n c r e t e o u t d o o r t a n k s . A t i n t e r v a l s t h e y w e r e t r a n s p o r t e d t o t h e D e p a r t m e n t o f Z o o l o g y , 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 . H e r e t h e y were h e l d i n d o o r s and f e d on c o m m e r c i a l f i s h f o o d ( J . R . C l a r k , S a l t L a k e C i t y , U t a h ) . T h e y r e c e i v e d a n a b u n d a n t f r e s h s u p p l y o f d e c h l o r i n a t e d t a p w a t e r v a r y i n g m a r k e d l y i n s e a s o n a l t e m p e r a t u r e b u t w i t h l i t t l e d i u r n a l c h a n g e . The t e m p e r a t u r e was f o u n d t o a l t e r i n a manner s i m i l a r t o t h a t f o u n d i n c e r t a i n s t r e a m s c l o s e t o V a n c o u v e r known t o c o n t a i n t h e s p e c i e s u n d e r i n v e s t i g a t i o n ( F i g . l ) . I l l u m i n a t i o n was a r t i f i c i a l ( f l u o r e s c e n t l a m p s ) and was c o n t r o l l e d i n l e n g t h b y a n a u t o m a t i c s w i t c h r e g u l a t e d v i a a p h o t o e l e c t r i c c e l l e x p o s e d t o t h e n a t u r a l o u t s i d e i l l u m i n a t i o n . T h u s , a c h a n g i n g s e a s o n a l d a y l e n g t h c o n f o r m i n g t o t h e o u t s i d e p h o t o p e r i o d was i m p o s e d u p o n t h e f i s h . I t d e p a r t e d f r o m t h e n a t u r a l c o n d i t i o n i n n o t b e i n g g r a d u a l i n o n s e t o r o f f s e t ( i . e . t h e r e was no t w i l i g h t p e r i o d ) a n d a l s o i n g e n e r a l i n t e n s i t y . F i s h were removed f r o m t h e s e c o n d i t i o n s f o r a v a r i e t y o f e x p e r i m e n t s i n w h i c h t h e y w e r e s u b j e c t e d t o d i f f e r e n t p h y s i c a l and c h e m i c a l e n v i r o n m e n t s . These c o n d i t i o n s a r e many and v a r i e d and may be c o n s i d e r e d most c o n v e n i e n t l y w i t h t h e r e s u l t s . 2. R a d i o i o d i d e I n j e c t i o n C a r r i e r - f r e e s o d i u m i o d i d e ( N a l ^ l ) w a s d i l u t e d w i t h d i s t i l l e d - 5 - F i g . 1. S e a s o n a l changes i n w a t e r t e m p e r a t u r e i n t h e l a b o r a t o r y and i n two s t r e a m s n e a r V a n c o u v e r , B r i t i s h C o l u m b i a .  - 6 - w a t e r and i n j e c t e d i n t r a p e r i t o n e a l l y v i a t h e d o r s a l m u s c l e i n a v o l u m e b e t w e e n 0.02 and 0.05 m l u s i n g a 0 . 2 5 - m l t u b e r c u l i n s y r i n g e and gauge 30 n e e d l e . D u p l i c a t e s o f t h e i n j e c t e d dose were d i l u t e d t o 100 m l w i t h 3% p o t a s s i u m i o d i d e s o l u t i o n i n 1 0 0 - m l v o l u m e t r i c f l a s k s and u s e d a s s t a n d a r d s ( l ; m l - 1% of i n j e c t e d d o s e ) . The f i s h were n o t a n a e s t h e t i z e d d u r i n g i n - j e c t i o n b u t q u i c k l y l i f t e d o u t o f t h e w a t e r i n a f i n e mesh n e t a n d t h e n i n j e c t e d t h r o u g h t h e n e t t i n g and i m m e d i a t e l y r e t u r n e d t o t h e w a t e r . The e n t i r e o p e r a t i o n t o o k a m a t t e r o f s e c o n d s . U n l e s s o t h e r w i s e s t a t e d , a l l f i s h were k e p t i n r u n n i n g w a t e r a f t e r i n j e c t i o n . A p p r o x i m a t e l y 0.1^uc I 1 ^ ! p e r gram body w e i g h t was a d m i n i s t e r e d t o e a c h f i s h . 3• B l o o d S a m p l i n g a n d S e p a r a t i o n o f P r o t e i n - b o u n d and I n o r g a n i c R a d i o i o d i n e B l o o d s a m p l e s were o b t a i n e d b y c u t t i n g t h r o u g h t h e c a u d a l p e d u n c l e a n d d r a w i n g t h e b l o o d b y c a p i l l a r y a c t i o n i n t o a f i n e h e p a r i n i z e d g l a s s t u b e . V e r y l a r g e f i s h were a n a e s t h e t i z e d f o r t h i s p r o c e d u r e t o f a c i l i t a t e h a n d l i n g ; s m a l l f i s h w e r e n o t a n a e s t h e t i z e d . W i t h i n s e c o n d s o f s a m p l i n g a l l f i s h were k i l l e d b y d e c e r e b r a t i o n . The t e c h n i q u e f o r d e t e r m i n i n g t h e o r g a n i c and i n o r g a n i c r a d i o a c t i v e c o n s t i t u e n t s o f s m a l l s a m p l e s o f f i s h p l a s m a h a s b e e n d e s c r i b e d ( H i c k m a n , 19&0; H o a r and E a l e s , 19&3; E a l e s , 1963). H o w e v e r , i n v i e w o f s l i g h t m o d i f i c a t i o n , t h e t e c h n i q u e i s g i v e n h e r e i n f u l l . On w i t h d r a w a l o f t h e b l o o d s a m p l e , t h e h e p a r i n i z e d c a p i l l a r y t u b e was p l u g g e d a t one end w i t h " p l a s t i c e n e " a n d t h e c o r p u s c l e s separated from the plasma by c e n t r i f u g a t i o n . The plasma was separated from the corpuscles by breaking the tube at t h e i r j u n c t i o n . The tube p l u s plasma contents were weighed and the plasma blown i n t o 4 ml of 12.5f° t r i c h l o r o a c e t i c a c i d i n a 12-ml t h i c k - w a l l e d Tpyrex' c e n t r i f u g e tube. The empty c a p i l l a r y tube was then reweighed t o determine the plasma mass. The plasma p r o t e i n p r e c i p i t a t e was broken up w i t h a g l a s s rod, c e n t r i f u g e d and the supernate decanted o f f and saved. Two ml of 2.5f» t r i c h l o r o a c e t i c a c i d were added and the p r e c i p i t a t e again broken up and c e n t r i f u g e d . T h i s procedure was repeated f o r a second time g i v i n g a t o t a l of 8 ml of supernate c o n s t i t u t i n g the non- p r o t e i n components of the plasma and i n c l u d i n g I 1 ^ 1 . The remaining p r o t e i n s were d i s s o l v e d i n 4 ml of 1.5 N sodium hydroxide (NaOH). This c o n s t i t u t e d the p r o t e i n f r a c t i o n of the plasma and i n c l u d e d the hormonal or protein-bound r a d i o i o d i n e ( P B I ^ l ) . The 4-ml sample of d i s s o l v e d P B I 1 ^ 1 was t r a n s f e r r e d t o a g l a s s counting tube and counted f o r 3000 counts. H a l f of the 1^31 wash d e r i v e d from the same plasma sample was p i p e t t e d t o a s i m i l a r tube and counted f o r 3000 counts or longer depending on i t s a c t i v i t y . By doubling t h i s count the t o t a l I 1 ? ! content o f the sample was found. A l s o counted f o r 30,000 counts were 4 ml (4%) of the standard s o l u t i o n . Thus e i t h e r f r a c t i o n could be expressed as a percentage of the i n j e c t e d dose. A l l counts were made i n a Nuclear Chicago Well Counter (Model DS5 V e r s a t i l e S c i n t i l l a t i o n D e t e c t o r ) . From these data two parameters were determined- ( i ) Conversion r a t i o . OR (Hickman, I960, 1962; E a l e s , l ? 6 l , 1963). - 8 - count/min x 100 OR = p g j l 3 1 count/min + l ! 3 1 count/min _ organic radioiodine(hormonal) t o t a l plasma r a d i o a c t i v i t y ( i i ) B i o l o g i c a l c o n c e n t r a t i o n c o e f f i c i e n t f o r I 1 ^ i n the plasma. This i s d e r i v e d from the b i o l o g i c a l c o n c e n t r a t i o n c o e f f i c i e n t , which expresses plasma 1̂ -31 c o n c e n t r a t i o n as a percentage of the dose i n j e c t e d (Comar, 1955). I t allows f o r the f a c t t h a t the same dose put i n t o a f i s h h a l f the mass of another f i s h w i l l have twice as much r a d i o a c t i v i t y per mass of plasma, and permits comparison of plasma I x 3 1 c o n c e n t r a t i o n between f i s h of d i f f e r e n t s i z e s . b i o l o g i c a l c o n c e n t r a t i o n c o e f f i c i e n t = % of i n j e c t e d dose as l ! 3 1 i n plasma sample x body weight(g) mass of sample(g) The b i o l o g i c a l c o n c e n t r a t i o n c o e f f i c i e n t has always been d i v i d e d by 100. 4. Body and Thyroid Immediately a f t e r death the f i s h was weighed and the b a s i - Tbranchial r e g i o n cut out from the f r o n t of arch I ( a n t e r i o r ) t o the end of arch I I I . This r e g i o n i n c l u d i n g the e n t i r e t h y r o i d t i s s u e was then dropped i n t o a counting tube c o n t a i n i n g B o u i n T s f i x a t i v e and counted f o r 30,000 counts i n a Nuclear Chicago Well Counter. Two 4-ml standards (0.04% of the i n j e c t e d dose) were counted simultaneously and the percentage of the i n j e c t e d dose i n the t h y r o i d area was determined. The t h y r o i d s were counted i n Bouin's f l u i d so t h a t they could be sectioned l a t e r . Some discrepancy may have been introduced by counting e s s e n t i a l l y - 9 - a p o i n t s o u r c e , t h e t h y r o i d r e s t i n g a t t h e b o t t o m o f t h e c o u n t i n g w e l l , a g a i n s t a 4 - m l s t a n d a r d . H o w e v e r , c o m p a r i s o n b e t w e e n u n d i g e s t e d t h y r o i d s and t h e same t h y r o i d d i g e s t e d w i t h . NaOH and t h e n d i l u t e d t o 4 m l r e v e a l e d n e g l i g i b l e d i f f e r e n c e s when m e a s u r e d i n t h e same c o u n t i n g s y s t e m ( W i g g s , 1962). T h i s i s p r o b a b l y due t o t h e l o w a b s o r p t i o n o f t h e h a r d ^ r a d i a t i o n o f j l 3 1 # T h y r o i d u p t a k e was e x p r e s s e d a s t h e p e r c e n t a g e a c c u m u l a - t i o n o f t h e i n j e c t e d d o s e . The body l a c k i n g t h y r o i d was s l i c e d and t h e p i e c e s a r r a n g e d t o f o r m a 5-cm s q u a r e whose t h i c k n e s s v a r i e d w i t h t h e s i z e o f f i s h . By c o u n t i n g t h i s " b o d y " a t a d i s t a n c e o f 10 cm f r o m an e n d - p r o b e s c i n t i l l a t i o n c o u n t e r w i t h a 45 mm d i a m e t e r and 38.5 mm t h i c k N a l ( T l ) c r y s t a l , a n e s t i m a t e o f t h e t o t a l r a d i o a c t i v i t y l e f t i n t h e body c o u l d be made. To c o n v e r t t h i s r a d i o a c t i v i t y t o a p e r c e n t a g e o f t h e i n j e c t e d d o s e , s t a n d a r d s were c o u n t e d a t an i d e n t i c a l d i s t a n c e a n d p o s i t i o n f r o m t h e i n v e r t e d e n d - p r o b e . T h e s e s t a n d a r d s were f o r m e d b y p e r m e a t i n g p i l e s o f a b s o r b e n t p a p e r , c u t t o t h e same s o l i d g e o m e t r y a s t h e s a m p l e s , w i t h 10 m l o f s t a n d a r d s o l u t i o n . E a c h s t a n d a r d t h e n r e p r e s e n t e d 10% o f t h e i n j e c t e d d o s e . Body r e t e n t i o n o f 1.131 was m e a s u r e d , t h e r e f o r e , a s a p e r c e n t a g e o f t h e i n j e c t e d d o s e . T h i s meant t h a t p h y s i c a l d e c a y , c o n s i d e r a b l e i n I 1 ^ 1 w i t h a h a l f - l i f e o f 8.08 d a y s , was e n t i r e l y e l i m i n a t e d a s b o t h s t a n d a r d s and s a m p l e s d e c a y e d a t t h e same r a t e . 5» T h y r o i d H i s t o l o g y T h y r o i d t i s s u e s f i x e d i n B o u i n ' s f l u i d were d e c a l c i f i e d , s e r i a l l y s e c t i o n e d (10 u) i n t h e r e g i o n o f t h e s e c o n d b r a n c h i a l - 10 - a r c h and t h e n s t a i n e d b y t h e A z a n t e c h n i q u e ( G u r r , 1953). T h e o r e t i c a l l y t h i s s t a i n r e n d e r s t h e f o l l i c l e c o l l o i d r e d i n a n i n a c t i v e g l a n d and b l u e i n a n a c t i v e g l a n d ( P i c k f o r d , 1953, 1954; B a r r i n g t o n , 1963). F i f t y u n b r o k e n f o l l i c l e s w e r e e x a m i n e d a n d t h e l o w e s t a n d t h e t a l l e s t c e l l h e i g h t s ( t a l l e s t + l o w e s t / 2 * mean c e l l h e i g h t ) were m e a s u r e d u s i n g a n o c u l a r s c a l e . The c o l l o i d was a s s i g n e d t o one o f f o u r c a t e g o r i e s - b l u e , r e d , m i x t u r e o f r e d and b l u e o r a b s e n t . S i n c e t h e t h y r o i d o f t e l e o s t s i s v a r i a b l e , i t was n e c e s s a r y t o r a n d o m i z e t h e f o l l i c l e s m e a s u r e d . T h i s was done by s e l e c t i n g t i s s u e f r o m a t l e a s t t h r e e d i f f e r e n t a r e a s o f t h e s e c o n d b r a n c h i a l r e g i o n . I f t h r e e s l i d e s were a v a i l a b l e f r o m t h i s a r e a , 17 f o l l i c l e s w o u l d be e x a m i n e d on s l i d e I , 17 on s l i d e I I and 16 on s l i d e I I I . R a n d o m i z a t i o n o f f o l l i c l e s t o be e x a m i n e d on any one s e c t i o n was a c h i e v e d by c e n t r i n g t h e o b j e c t i v e on t h e t h y r o i d t i s s u e u n d e r v e r y l o w power m a g n i f i c a t i o n s u c h t h a t i n d i v i d u a l f o l l i c l e s t r u c t u r e was d i f f i c u l t t o d e t e c t , and t h e n s w i t c h i n g t o h i g h p o w e r . The f o l l i c l e c l o s e s t t o t h e o c u l a r s c a l e u n d e r t h e h i g h power was t h e n t h e f i r s t t o be a s s e s s e d . The n e x t f o l l i c l e t o be e x a m i n e d was t h e one i m m e d i a t e l y t o i t s l o w e r l e f t - h a n d m a r g i n . The f o l l i c l e i m m e d i a t e l y t o t h e l a t t e r ' s l o w e r l e f t was e x a m i n e d t h i r d and so on u n t i l t h e q u o t a f o r t h e s l i d e was e x h a u s t e d . I f t h i s p r o c e d u r e r e s u l t e d i n e x h a u s t i o n o f f o l l i c l e s b e f o r e t h e q u o t a were r e a c h e d , a n o t h e r s e c t i o n was t r e a t e d s i m i l a r l y i n t h e same s l i d e b u t a t l e a s t 150 jx p o s t e r i o r l y . S i n c e f o l l i c l e d i a m e t e r s w e r e u s u a l l y f a r l e s s t h a n 150 u , r e p e t i t i v e measurement o f f o l l i c l e s was a v o i d e d . - 11 - 6. S t a t i s t i c a l Treatment In studies of rates of change of I 1 ^ 1 f r a c t i o n s i n various body compartments, l i n e a r regressions were calculated and either the slopes or adjusted means compared by analysis of covariance (Steel and To r r i e , i 9 6 0 ) . In comparing small samples (frequently below 12), which i n many instances appeared to depart from a normal d i s t r i b u t i o n , the Wilcoxon-White non-parametric ranking t e s t was employed (Snedecor, 1956). - 12 - RESULTS I . COMPARISON OE YEARLING STEELHEAD WITH DIFFERENT RATES OF RADIOIODINE METABOLISM R e l i a b l e r a d i o i o d i d e e s t i m a t e s o f t h y r o i d a c t i v i t y a r e p o s s i b l e o n l y when a t h o r o u g h s t u d y o f r a d i o i o d i n e m e t a b o l i s m h a s been made w i t h t h e a n i m a l i n v a r i o u s s t a t e s o f t h y r o i d a c t i v i t y . D e s p i t e e x t e n s i v e u s e o f r a d i o i o d i n e t o s t u d y t h y r o i d a c t i v i t y i n t e l e o s t s , i n t e n s i v e s t u d i e s o f r a d i o i o d i n e m e t a b o l i s m i n any one s p e c i e s a r e r a r e . L e l o u p and F o n t a i n e ( i960) h a v e p r e s e n t e d a c o m p r e h e n s i v e s u r v e y o f r a d i o i o d i n e m e t a b o l i s m i n l o w e r v e r t e b r a t e s , b u t t h e number o f s p e c i e s c o v e r e d i s so v a s t t h a t i t i s d i f f i c u l t t o r e c o n s t r u c t a c o m p l e t e p i c t u r e o f 1^31 m e t a b o l i s m f o r a n y one s p e c i e s . The w o r k by H i c k m a n (1959, 1962) on P l a t i c h t h y s s t e l l a t u s i s an e x c e p t i o n . S i n c e many e s t i m a t e s o f r a d i o i o d i n e m e t a b o l i s m a t d i f f e r e n t l e v e l s o f a c t i v i t y h a v e been made i n t h e s t u d y , i t i s c o n s i d e r e d n e c e s s a r y t o p r e s e n t a c o m p a r i s o n b e t w e e n r a d i o i o d i n e m e t a b o l i s m i n f i s h w i t h a c t i v e and i n a c t i v e g l a n d s . I n t h i s i n s t a n c e t h e d i f f e r e n c e i n I 1 ^ m e t a b o l i s m was i n d u c e d w i t h a 5° C t e m p e r a t u r e d i f f e r e n c e b y h o l d i n g f i s h a t 5 and 10° C . The o b j e c t o f t h i s s t u d y was t o f o l l o w t h e m e t a b o l i s m o f a s i n g l e dose o f 1^31 f o r a p e r i o d o f s e v e r a l d a y s . T h i s c o u l d b e done i d e a l l y b y m e a s u r i n g t h e d e s i r e d p a r a m e t e r s on t h e same i n d i v i d u a l s . U s e o f t h e same i n d i v i d u a l s f o r t h e e n t i r e p e r i o d p r e s e n t e d t h e p r o b l e m o f k e e p i n g f i s h a l i v e f r o m one d e t e r - m i n a t i o n t o t h e n e x t . B e s i d e s t h e d i f f i c u l t y o f c o u n t i n g t h e d i f f u s e t e l e o s t t h y r o i d i n v i v o i n s m a l l f i s h , t h e r e was t h e - 13 - greater d i f f i c u l t y of extracting adequate blood samples r e p e t i t i v e l y without depleting the blood volume. To obviate these d i f f i c u l t i e s , 55 f i s h (8-25 g, av = 1 2 g) from each temperature regimen (5 and 10° 0) were "simultaneously" injected with 1131 and 5 to 7 f i s h from each group sampled at the required time i n t e r v a l s (12,36,60,84,108,132,156,204 and 276 h r ) . Each f i s h was weighed and plasma I 1 ^ 1 concentration, percentage of I 1 ^ 1 dose i n body, percentage of 1^31 dose i n thyroid and conversion r a t i o recorded. By s e r i a l l y sampling from the popu- l a t i o n s i n t h i s manner and deriving the mean value f o r each sample at a given time under a given condition, i t was possible to follow the changes i n 1^31 concentrations i n several compart- ments as an average of the whole population. 1. Extrathyroidal and Thyroidal Clearance of Plasma I 1 3 1 Radioiodine injected i n t r a p e r i t o n e a l l y i s r a p i d l y absorbed into the blood stream (Hickman, 1959). The plasma l e v e l i s then continually depleted due to loss v i a a va r i e t y of routes (Eig. 2 ) . A small percentage of the dose i s eventually accumulated by the thyroid but the greater part i s excreted. Although plasma I^31 w a s l o s t f a s t e r at the higher temperature, the difference was not s t a t i s t i c a l l y s i g n i f i c a n t (Table I ) . A l l the mean values f o r 5° C f i s h were above those f o r 10° C f i s h , however, the adjusted means were s t a t i s t i c a l l y d i f f e r e n t ( p< 0 . 0 1 ) . This shows convincingly that the temperature eleva- t i o n of 5° 0 had caused an increase i n 1^31 loss from the plasma by one or more routes. Of further i n t e r e s t was the c u r v i l i n e a r tendency at the higher temperature. This i s considered i n greater - 14 - F i g . 2. Plasma clearance following a single I p i n - j e c t i o n i n f i s h held at 5° C (O) and 10° C (•). Each point represents a mean of 5 to 7 f i s h (Covariance analysis, Table I ) . i - 15 - TABLE I Analysis of covariance f o r plasma I - ^ l (plasma I x 3 1 b i o l o g i c a l concentration coefficient / 1 0 0 ) and time (hr) for active (10° C) and inactive (5° C) thyroid states CONDITION SLOPE ?s df P F xm df P INACTIVE -0.0127 1 1 2.08 and 0.05 39.3 and 0.01 ACTIVE -0.0158 101 104 • F g - Variance r a t i o f or slopes. F = Variance r a t i o f o r adjusted means. - 16 - d e t a i l b e l o w . A n e q u a l l y r e l i a b l e and p e r h a p s more d i r e c t method o f d e t e r m i n i n g I 1 ^ ! e x c r e t i o n i s t o measure t h e p e r c e n t a g e o f i n j e c t e d d o s e r e m a i n i n g i n t h e body ( e x c l u d i n g t h y r o i d ) a t v a r i o u s t i m e i n t e r v a l s . T h i s p a r a m e t e r a n d t h e p e r c e n t a g e o f t h e i n j e c t e d dose i n t h e t o t a l body ( i n c l u d i n g t h y r o i d ) a r e shown i n F i g u r e 3« I m m e d i a t e l y e v i d e n t i s t h e c u r v i l i n e a r a s p e c t o f b o t h p a r a m e t e r s . To s i m p l i f y t h e a n a l y s i s , t h e y h a v e b e e n a p p r o x i m a t e d t o d i p h a s i c l i n e a r p l o t s w i t h a n i n f l e x i o n a t 3|- d a y s . T h u s , f o r e a c h p a r a m e t e r u n d e r b o t h c o n d i t i o n s , two r e l a t i o n s h i p s h a v e b e e n c o n s i d e r e d (Day 1-4 a p h a s e I and Day 3-11 • p h a s e I I ) . The s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e s e r e l a t i o n s h i p s a r e g i v e n i n T a b l e s I I and I I I . T a b l e . I I d e m o n s t r a t e s t h e d i p h a s i c ( c u r v i l i n e a r ) n a t u r e o f t h e e x c r e t i o n d a t a . A s y e t t h i s has no c o m p l e t e l y s a t i s - f a c t o r y e x p l a n a t i o n . A c c o r d i n g t o Wiggs (1962) , who o b s e r v e d a s i m i l a r phenomenon i n s t i c k l e b a c k s , i t may be due t o a n i n i t i a l and g e n e r a l r a p i d i o n l o s s i n d u c e d by " l a b o r a t o r y d i u r e s i s " as a r e s u l t o f e x p e r i m e n t a l h a n d l i n g . A p p l i e d t o t h e s e d a t a i t w o u l d s u g g e s t a s i g n i f i c a n t d i u r e s i s f o r 2 t o 3 d a y s a n d t h e n a s t a b i l i z a t i o n . A more p l a u s i b l e e x p l a n a t i o n i s s u g g e s t e d b y Oomar (1955) who d e m o n s t r a t e d m a t h e m a t i c a l l y t h a t a c u r v i l i n e a r r e l a t i o n s h i p i s e x p e c t e d i f a n i s o t o p e i n one compartment i s b e i n g d i l u t e d b y movement t o two o r more c o m p a r t m e n t s a t m a r k e d l y d i f f e r e n t r a t e s . Thus t h e c o m p o s i t e c u r v i l i n e a r r e l a t i o n s h i p may be a c o m b i n a t i o n o f a r a p i d a n d a s l o w T131 l o s s v i a two d i f f e r e n t r o u t e s . A c c o r d i n g t o Comar , t h e r a p i d r e m o v a l w o u l d - 17 - p r e d o m i n a t e i n i t i a l l y b u t w o u l d be r e p l a c e d l a r g e l y b y t h e s l o w e r r e m o v a l r a t e a t l o w I 1 ? 1 l e v e l s . I n c o n s i d e r i n g ' I 1 ? 1 r e m o v a l r o u t e s f r o m t h e s t e e l h e a d b o d y , t h e g i l l c o u l d c o n c e i v a b l y be t h e more r a p i d and t h e k i d n e y t h e s l o w e r r o u t e s i n c e t h e r e i s e v i d e n c e t o s u g g e s t t h a t I - ^ l t , e removed p r e d o m i n a n t l y v i a t h e g i l l s ( H i c k m a n , 1959). The d i p h a s i c n a t u r e o f t h e s e c u r v e s does n o t i n f l u e n c e t h e r a t e o f t h y r o i d l ! 3 1 a c c u m u l a t i o n . R a t e c o n s t a n t s f o r t h y r o i d j.131 a c c u m u l a t i o n h a v e b e e n o b t a i n e d b y s u b t r a c t i n g t h e p e r c e n t - age dose i n body s l o p e f r o m t h e s l o p e d e r i v e d f r o m t h e body and t h y r o i d c o m b i n e d ( T a b l e I I ) . A t 5° C t h e c o n s t a n t i s e s s e n t i a l l y t h e same o v e r b o t h p h a s e s o f t h e c u r v e (6 .0054 a n d 0.0057; a v - 0.00555) and t h e same a p p l i e s a t 10° C (0.0075 and 0.0077; a v = 0 . 0 0 7 6 ) , a l t h o u g h t h e r e a r e v e r y marked d i f f e r e n c e s b e t w e e n mean c o n s t a n t s f r o m t h e two t e m p e r a t u r e s (0.00555 and O.OO76). I t i s c o n c l u d e d t h a t ( i ) t e m p e r a t u r e h a s a s i g n i f i c a n t i n f l u e n c e on t h e t h y r o i d j l 3 1 a c c u m u l a t i o n r a t e c o n s t a n t and ( i i ) t h i s c o n s t a n t i s a r e l i a b l e i n d e x o f t h y r o i d a c t i v i t y u n d e r d i f f e r e n t r a t e s o f l ! 3 1 e x c r e t i o n . O f f u r t h e r s i g n i f i c a n c e was t h e t e m p e r a t u r e i n f l u e n c e on 1.131 e x c r e t i o n r a t e s . E l i m i n a t i o n a p p e a r e d more r a p i d a t t h e h i g h e r t e m p e r a t u r e b u t a g a i n i t was d i f f i c u l t t o d e m o n s t r a t e s t a t i s t i c a l d i f f e r e n c e s between s l o p e s ( T a b l e I I I ) . H o w e v e r , a d j u s t e d mean v a l u e s showed a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two r e g i m e s ( p = 0 . 0 1 ) . T h i s s u p p o r t s t h e e a r l i e r e v i d e n c e t h a t 1131 l o s s f r o m t h e b o d y , l i k e t h y r o i d 1.131 a c c u m u l a t i o n , i s more a c t i v e a t h i g h e r t e m p e r a t u r e s . - 18 - F i g . 3. T o t a l b o d y c l e a r a n c e (O) and b o d y e x c l u d i n g t h y r o i d c l e a r a n c e (•) f o l l o w i n g a s i n g l e 1131 i n j e c t i o n a t 30 c ( i n a c t i v e t h y r o i d ) and 10° C ( a c t i v e t h y r o i d ) . E q u a - t i o n s c a l c u l a t e d s e p a r a t e l y f o r e a c h p h a s e . E a c h p o i n t r e p r e s e n t s a mean o f 5 t o 7 f i s h ( C o v a r i a n c e a n a l y s e s , T a b l e s I I and I I I ) .  TABLE II Analysis of covariance for I ^ l clearance from the t o t a l body and time (hr); and body excluding thyroid and time (hr). Slope™ ~ - Slope = Rate constant f o r thyroid i l j l uptake fSlope-} a % DOSE BODY AND % DOSE THYROID fo DOSE BODY Condition Phase Sloperri + B P df S l o p e B F df Slope T I -0 .0161 1 -0.0215} 18.5** 1. 0.0054 INACTIVE . 9;92** and and II -0.0018 - 56 - 0 . 0 0 7 5 ) 62 0.0057 ACTIVE I -0.0204 ' 1 - 0 . 0 2 7 9 ' 1 0.0075 14.17** and . 9 . 8 0 * * and II - 0 . 0 0 3 5 57 -0.0112 58 0.0077 Denotes s t a t i s t i c a l difference between compared slopes (p = 0 . 0 1 ) . TABLE I I I Analysis of covariance f o r I 1 ? 1 clearance from t o t a l body and time (hr) and body excluding thyroid and time (hr), for active (10° C) and inactive (5 0) thyroid states. The analysis indicates the difference i n excretion rates between the two temperatures i n phase I I f. DOSE BODY f, DOSE BODY + fo DOSE THYROID PHASE CONDITION SLOPE df Fm SLOPE df F s Fm I INACTIVE ACTIVE -0.0161? -0.0204* 1 and 40 0.697 -0 .0215? - 0 . 0 2 7 9 J 1 • and 42 0.80 I I INACTIVE ACTIVE -0 .0018' - 0 . 0 0 3 5 , J 1 . and 72 1.175 58.50** -O.OO75] - 0 . 0 1 1 2j 1 and 68 3 . 8 9 ? 29.87** E s = Variance r a t i o f o r slopes. F m s Variance r a t i o f o r adjusted means. ** denotes highly s i g n i f i c a n t difference (p^O.Ol). ? Almost shows significance between slopes ( p= 0 . 0 5 ) . Other "F" values show no difference between slopes. - 21 - 2. Experimental Measurement of Thyroid I 1 ^ ! Accumulation Ideally, a l l these I 1 ^ ! movements would he represented i n terms of rate constants. In small fish,where s e r i a l sampling f o r determination of regressions f o r rate constants would involve many f i s h , an attempt was made to determine the extent of excretion and thyroid uptake on the basis of single terminal measurements. For comparison these samples were always taken at the same time a f t e r i n j e c t i o n . Due to the influence of temperature on extrathyroidal removal of I - ^ l j mere measurement of the percentage of the dose accumulated i n the thyroid would be unreliable (Fig. 4A). Relative to the standard deviations the differences between the thyroid uptake means are very s l i g h t and are eventually reversed. At a s l i g h t l y f a s t e r rate of extrathyroidal I 1 ^ ! clearance i n f i s h with the active gland, the percentage of the dose accumulated i n the thyroid would have been the same or lower than that i n the inactive gland. Thus thyroid uptake of I x 3 1 measured i n t h i s way i s very dependent on the extrathyroidal clearance rate, and i n any single terminal assessment of thyroid uptake t h i s blood loss of 1^1 m u s t be taken into account. Allowances f o r t h i s have been made i n a va r i e t y of ways. Hoar and Eales (1963) measured the Thyroid Uptake Factor (TUF) where rmT-rr, - f* dose thyroid TUF = • x 100 % dose thyroid + % dose body I t measures at death that percentage of the non-excreted I 1 ^ ! that has been par t i t i o n e d from the body compartment into the - 22 - thyroid. TUT values for the inactive and active thyroids are shown i n Figure 4B. A f a r better separation i s now shown than that obtained from thyroid uptake values. I t i s to be noted that whereas the l i n e a r r e l a t i o n s h i p was maintained u n t i l 12 days a f t e r i n j e c t i o n i n the f i s h with the slower rate of I 1 ^ ! metabolism, i t f e l l markedly a f t e r 6 days i n f i s h with the active thyroid. Further attention i s drawn to t h i s point below. Unlike other 1^21 parameters studied, i t i s a r i t h m e t i c a l l y rather than exponentially related to time a f t e r i n j e c t i o n . A t h e o r e t i c a l explanation for t h i s has not been attempted. It may also be noted at t h i s stage how applicable the TUF measure i s to i n vivo studies providing adequate screening of extra- t h y r o i d a l t i s s u e can be obtained. Leloup and Fontaine ( i960) have used the thyroid/serum or T/S r a t i o /thyroid r a d i o a c t i v i t y / g ^ f o r measuring thyroid uptake, serum r a d i o a c t i v i t y / g In assessing thyroid I - ^ l concentration r e l a t i v e to serum con- centration, t h i s method i s s i m i l a r to thyroid clearance (Hickman, 1959; Baggerman, i 9 6 0 ) . T/S i s a d i r e c t measure, at a c e r t a i n time a f t e r I 1 ^ 1 i n j e c t i o n , of the I 1 ' 5 1 accumulation i n the thyroid r e l a t i v e to that i n the serum. In t h i s study i t was found convenient to use a r a t i o very s i m i l a r to the usual T/S r a t i o ; i t i s r e f e r r e d to as the T/S r a t i o i n t h i s study. It i s expressed as thyroid uptake plasma ll3>l concentration = i of injected I 1 5 1 i n thyroid plasma I x 3 1 b i o l o g i c a l concentration c o e f f i c i e n t 100 - 2 3 - F i g . 4. (A) Percentage accumulation of I by the thyroid following a single i l ^ l i n j e c t i o n at 10° C (active) and 5° 0 ( i n a c t i v e ) . Standard deviations shown. Each point represents a mean of 5 to 7 f i s h . (B) TUF values f o r the same f i s h as (A). (C) T/S values f o r the same f i s h as (A). The diphasic tendency i s shown i n the active state (Covariance analysis, Table IV). ( - 24 - TABLE IV Analysis of covariance f o r T/S and time (hr) f o r active (10° 0) and inactive (5° C) thyroid states CONDITION PHASE SLOPE df P df F I 0.0255 7 1 INACTIVE V and 1.44 II 0.0281 \ 56 ACTIVE I 0.0362 I I 0.0100 and 22 .5** 54 1 J | 1 .and 26.44** }and 50 \ 52 9.06*^ ** Denotes s i g n i f i c a n t difference between slopes - ( p< 0 . 0 1 ) . - 25 - Absolute accumulation r e l a t i v e to the plasma was higher i n the more active group over the entire period of study and there- fore showed a more complete separation of the two thyroid l e v e l s than did the TUF (Fig. 4C). Most information can be gained, however, by a mathematical analysis of the curves since, while the inactive gland showed a s t r i c t l y exponential r i s e with time, the active gland showed a c u r v i l i n e a r or diphasic r e l a t i o n s h i p . In the active gland an i n f l e x i o n of the curve was apparent between 4 and 5 days a f t e r i n j e c t i o n and consequently two regressions were compared separately f o r both the active and inactive states. Thus the exponential relationships between 12 and 108 hours (phase I) and 108 and 276 hours (phase II) were computed fo r each temperature (Table IV). The mathematical analysis revealed ( i ) a s t a t i s t i c a l l y s i g n i f i c a n t reduction i n slope i n the active gland from the f i r s t to the second phase, but no change i n the inactive gland between phases, and ( i i ) that phase I of the active gland had a slope s t a t i s t i c a l l y d i f f e r e n t from phase I of the inactive gland. The difference i n slope during phase I of the two holding conditions i s considered due to greater build-up of I / ^ l l n the thyroid r e l a t i v e to that i n the blood of the f i s h with more active glands. The change i n slope of the active gland i n phase I I could mean either that the a f f i n i t y of the thyroid f o r iodine had been reduced or that there had been a loss of radio- a c t i v i t y from the gland. The former seems un l i k e l y unless the thyroid uptake a c t i v i t y had suddenly changed i n the middle of the experiment. Reference to Figure 4A, however, shows a - 26 - s i g n i f i c a n t drop i n the percentage of the dose i n the thyroid between 8 and 12 days i n the active but not i n the inactive gland. I t i s tempting to i d e n t i f y t h i s l o s s of r a d i o a c t i v i t y with radiohormone output from the active gland. There i s , however, another p o s s i b i l i t y . Although the net r e s u l t i s an accumulation of iodine by the thyroid, there are fluxes of I 1 2*? and I 1 ^ 1 both into and out of the gland. I n i t i a l l y the i n f l u x of I 1 ^ 1 i s high since the r a t i o between plasma I ^ l and plasma l 1 2 7 i s a i s o high. The e f f l u x , however, i s low since there i s l i t t l e I 1 5 1 i n the gland. As the I 1?!;!! 2? r a t i o builds up i n the thyroid and becomes lowered i n the plasma, the s i t u a - t i o n i s reversed and the net tendency f o r 1^31 to come out of the gland i s increased. This could account f o r the change i n slope. The reduced increase i n the T/S r a t i o i s , however, p a r t l y due to an output of radioactive hormone as shown by studies of the plasma P B I ^ l . From these data and t h e o r e t i c a l consideration i t seems evident that a T/S or TUF measurement taken at some f i x e d time a f t e r i n j e c t i o n (4 days or i n some instances 8 days) should give a r e l i a b l e i n d i c a t i o n of the e f f i c i e n c y of the thyroid "iodide pump,, and that these indices are f a r more r e l i a b l e than thyroid uptake alone. In t h i s study most thyroid measurements were done at 4 days a f t e r i n j e c t i o n as shown i n Figure 4C. In t h i s same figure the i n d i v i d u a l values of T/S f o r 84 and 108 hours are shown fo r both the active and inactive states. At both time i n t e r v a l s the active and inactive T/S values were s t a t i s t i c a l l y d i f f e r e n t (p«C0.01, Wilcoxon ranking t e s t ) , and there i s every reason to believe that single terminal assessments - 27 - of T/S or TUF factors at 4 days give an accurate i n d i c a t i o n of the o v e r a l l thyroid I 1 ? ! accumulating e f f i c i e n c y . 3 . Estimation of Radiohormone Output Using the Conversion Ratio As indicated above, i t would be l o g i c a l to expect a s i g n i f i c a n t b u i l d up i n radiohormone ( P B I 1 3 1 ) i n the plasma a f t e r about 5 days i n the f i s h with more active thyroid gland. Figure 5 shows the change i n the conversion r a t i o with time where, „ . 0.. P B I 1 ^ 1 count/min x 100 Conversion r a t i o - P B I 1 ^ 1 count/min + I 1 ^ 1 count/min This r a t i o does indeed ascend steeply at 4 days i n the active gland and a f t e r 4 days the r i s e conforms to an exponential r e l a t i o n s h i p . That the exponential r e l a t i o n s h i p only becomes evident a f t e r 4 days i s probably an a r t i f a c t caused by (i) the very small amounts of radioactive hormone l i b e r a t e d by the gland over the i n i t i a l period and ( i i ) a technical l i m i t a t i o n due to the f a c t that there i s always imperfect washing of from the PBll31 f r a c t i o n such that the conversion r a t i o (CR) i s r a r e l y below 0.5%. Thus at low l e v e l s of radiohormone output the true P B I 1 3 1 l e v e l i s masked by I 1 3 1 contamination of the P B I 1 3 1 f r a c t i o n , the exponential nature of the P B I 1 3 1 build-up i s obscured and a f a l s e picture of a lag phase i s suggested. This exponential r i s e of the CR i s influenced by two factors- output from the gland and PBr̂ "** removal from the blood due to peripheral u t i l i z a t i o n and excretion of hormone. The l a t t e r e f f e c t would cause the CR exponent to f l a t t e n with time. This was not evident during the 12 day observation. Very l i t t l e - 28 - F i g . 5 . Change i n CR f o l l o w i n g a s i n g l e I 1 ^ 1 i n j e c t i o n i n f i s h a t 10° C ( a c t i v e ) and 3° 0 ( i n a c t i v e ) . E a c h p o i n t r e p r e s e n t s a mean o f 3 t o 7 f i s h . / / / 2 0 / x . L O G e Y = L O G r f ) l 6 + O O O I 8 X / • Y = C R X = H R S / CR / y / / / / / / / W A IO / / / / / / / / / /'x / / / / / / - -& • 4 6 8 I O DAYS i s known o f t h e r a t e o f r e m o v a l o f hormone f r o m t h e b l o o d i n f i s h and u n t i l t h i s h a s b e e n d e t e r m i n e d i t s e f f e c t on t h e CR c a n n o t be a s s e s s e d . I t c a n be p o i n t e d o u t , h o w e v e r , t h a t u n t i l r e l a t i v e l y l a r g e q u a n t i t i e s o f P B I 1 ^ 1 h a v e b u i l t up t h e r a t i o o f P B T A ^ P B I 1 2 ' ' i n t h e b l o o d w o u l d be so s m a l l t h a t t h e c h a n c e o f P B I 1 ^ 1 b e i n g d e p l e t e d due t o p e r i p h e r a l u t i l i z a t i o n w o u l d be p r o p o r t i o n a t e l y s m a l l . Thus t h e i n i t i a l p h a s e o f t h e CR c u r v e w o u l d r e p r e s e n t l a r g e l y t h e r a t e o f r a d i o h o r m o n e p r o d u c t i o n . 131 A s w i t h o t h e r a s p e c t s o f I y m e t a b o l i s m , change i n p l a s m a PBI131 l e v e l s o o u l d p r o b a b l y be r e p r e s e n t e d most p r e c i s e l y i n t e r m s o f r a t e c o n s t a n t s . I n t h i s e n t i r e s t u d y , h o w e v e r , t h e e x t e n t o f PB-T131 p r o d u c t i o n was a s s e s s e d by k i l l i n g t h e f i s h 8 d a y s a f t e r i n j e c t i o n and t h e n m e a s u r i n g t h e CR o f a p l a s m a s a m p l e . A t t h i s t i m e PBI131 l e v e l s , a r e b e c o m i n g r e a d i l y d e t e c t a b l e i n t h e p l a s m a and a l s o r e v e a l t h e v e r y marked d i f f e r e n c e b e t w e e n t h e a c t i v e and i n a c t i v e t h y r o i d s ( F i g . 5 ) . I t i s i m p o r t a n t t o n o t e t h a t t h e CR o n l y i n d i c a t e s t h e r a t e o f r a d i o h o r m o n e p r o d u c t i o n and n o t t h e r a t e o f s t a b l e hormone p r o d u c t i o n . To i n d i c a t e t h e l a t t e r , t h e I ^ l must a t t a i n e q u i l i b r i u m w i t h I^ 27 t h r o u g h o u t t h e b o d y . T h i s r e q u i r e s r e p e t i t i v e i n j e c t i o n s t o m a i n t a i n a c o n s t a n t I ^ l l e v e l a n d i s c e r t a i n l y n o t a c h i e v e d i n t h e s e s t u d i e s as a r e s u l t o f a s i n g l e i n j e c t i o n . I n summary, by m a k i n g r o u t i n e measurements o f T I ^ I l e v e l s i n s e v e r a l body c o m p a r t m e n t s a t f i x e d t i m e i n t e r v a l s , a g r e a t d e a l c a n be l e a r n e d a b o u t r a d i o i o d i n e m e t a b o l i s m and t h y r o i d f u n c t i o n . A s a r e s u l t o f t h e s e p r e l i m i n a r y . i n v e s t i g a t i o n s i t - 30 - was d e c i d e d t o d e t e r m i n e r o u t i n e l y t h e p a r a m e t e r s shown i n T a b l e V a t 4 a n d 8 d a y s a f t e r i n j e c t i o n o f I 1 ^ 1 . • I I . SEASONAL CHANGES I N RADIOIODINE METABOLISM AND THE H I S T O - LOGICAL APPEARANCE OF THE THYROID I N J U V E N I L E STEELHEAD HELD I N FRESH WATER F o l l o w i n g t h e p r e l i m i n a r y i n v e s t i g a t i o n o f I 1 ^ ! m e t a b o l i s m c e r t a i n o f t h e p a r a m e t e r s shown i n T a b l e V w e r e d e t e r m i n e d s e a s o n a l l y . Mean c e l l h e i g h t and c o l l o i d c o l o u r were a l s o m e a s u r e d . T h e s e d a t a , t o g e t h e r w i t h s e a s o n a l changes i n w a t e r t e m p e r a t u r e , a r e s u m m a r i z e d i n F i g u r e 6. ! • I 1 ? 1 E x c r e t i o n P l a s m a i l ^ l ( 4 d a y s ) a n d p e r c e n t a g e o f d o s e i n t o t a l body ( 4 d a y s ) , r e v e a l e d d e f i n i t e s e a s o n a l t r e n d s . F r o m May o f t h e p r e - m i g r a n t y e a r u n t i l s m o l t i f i c a t i o n t h e r e was a g e n e r a l n e g a t i v e c o r r e l a t i o n b e t w e e n t h e s e two p a r a m e t e r s and t e m p e r a t u r e , i n d i c a t i n g more r a p i d r e m o v a l o f 1-131 a t h i g h e r t e m p e r a t u r e s . T h i s n e g a t i v e c o r r e l a t i o n i s d e m o n s t r a t e d i n F i g u r e 7 where mean s e a s o n a l v a l u e s f o r t o t a l r e t e n t i o n have been p l o t t e d a g a i n s t t e m p e r a t u r e ( r = - 0 . 7 7 , 26df, p<£ 0.001). I t i s i m - p o r t a n t t o n o t e t h a t t h e s e s e a s o n a l changes i n 1"131 r a t e o f e x c r e t i o n w e r e n o t o n l y d e m o n s t r a t e d by t o t a l p l a s m a c l e a r a n c e b u t a l s o b y e x t r a t h y r o i d a l c l e a r a n c e ( F i g . 7) . T h i s i n d i c a t e s t h a t t h e l o s s o f I 1 ? ! f r o m t h e p l a s m a b o t h t o t h e t h y r o i d and t o t h e o u t s i d e medium was i n c r e a s e d a t h i g h e r t e m p e r a t u r e s . T h e s e o b s e r v a t i o n s s u p p o r t t h e d a t a f r o m t h e p r e l i m i n a r y i n v e s t i g a t i o n s . TABLE V A summary of radioiodine parameters and t h e i r s i g n i f i c a n c e PARAMETER ABBREVIATION USED SIGNIFICANCE I Plasma l!31 b i o l o g i c a l concentration coefficient/100 Plasma 1"131 Measures plasma I 1 ^ 1 concentra- t i o n and allows estimates of t o t a l blood clearance of 1131 II % injected l ! 3 1 dose i n body (excluding thyroid) % body Measures t o t a l clearance of 1131 from the body I I I fo injected 1"131 dose i n ?' whole body ( including thyroid) f» body + fo thyroid Measures the f» of injected dose i n whole body. 100 fo - fo i n whole body = % loss due to extrathyroidal clearance (excretion) IT i injected i W dose i n thyroid Plasma H 3 1 T/S Assesses build-up of 1^31 j _ n the thyroid r e l a t i v e to plasma l l 3 1 . — measures the a f f i n i t y of the thyroid for I x 3 l and allows for excretion rate V % dose thyroid Thyroid uptake Measures that 1 of the % dose body + f» dose thyroid factor TUF unexcreted 1"131 that i s p a r t i - tioned into the thyroid VI Conversion r a t i o Plasma P B I 1 ? 1 x 100 CR Assesses extent of conversion of 1131 to PBli31 Plasma l!31 + plasma PBI X31 - 3 2 - F i g . 6. S e a s o n a l change i n p l a s m a I 1 ? ! , % dose I 1 ? 1 , T / S , T U F , CR, mean e p i t h e l i u m c e l l h e i g h t and p e r c e n t a g e o f f o l l i c l e s c o n t a i n i n g b l u e c o l l o i d i n s t e e l h e a d p a r r and s m o l t s . S e a s o n a l change i n t e m p e r a t u r e i s shown. S t a n d a r d d e v i a t i o n s a r e shown f o r c e r t a i n p a r a m e t e r s .  - 33 - F i g . 7. C o r r e l a t i o n s b e t w e e n w a t e r t e m p e r a t u r e and f o u r 1131 p a r a m e t e r s . A l l v a l u e s drawn f r o m s e a s o n a l d a t a ( F i g . 6). A (% d o s e i n b o d y ) ; B ( C R ) ; C ( T / S ) ; D ( T U F ) . r-=o-77 4 0 h 8 t • 3 2 O B T c O - 4 6 CR 4 0 t - T - O . 7 8 T/S 2 0 t - 6 0 ( - TUF 2 0 U 6 6 5 * • . " • % • • =ae9 —1 L . I O 1 2 8 I O 1 2 °c - 34 - Of f u r t h e r s i g n i f i c a n c e was t h e d e p a r t u r e o f c e r t a i n g r o u p s o f f i s h f r o m t h e g e n e r a l c o r r e l a t i o n . T h e s e f e l l i n t o two c a t e g o r i e s - f i s h b e l o w 20 g i n w e i g h t ( u s u a l l y 18 months o r y o u n g e r ) and s m o l t s (30-31 m o n t h s ) . I n t h e y o u n g e r and s m a l l e r f i s h , t h e l o w r e t e n t i o n o f I 1 ^ 1 i n d i c a t e d d i f f e r e n c e s t h a t c o u l d be a t t r i b u t a b l e t o s i z e o r g r o w t h . These f i s h a r e r e c o n s i d e r e d l a t e r . O v e r t h e p e r i o d o f s m o l t i f i c a t i o n , h o w e v e r , b o t h t h e t o t a l c l e a r a n c e and t h e e x t r a t h y r o i d a l c l e a r a n c e were more r a p i d t h a n a t a n y o t h e r t i m e o f y e a r . A t t h e t i m e o f a n t i c i p a t e d m i g r a t i o n i n May t h e w a t e r t e m p e r a t u r e was 8 t o 9° C and a t t h i s t e m p e r a t u r e t h e p l a s m a a n d body I 1 ^ ! l e v e l s w e r e much l o w e r t h a n t h e p l a s m a and body I 1 ^ ! r e t e n t i o n s a t 12 t o 13° C i n p r e - m i g r a n t s i n S e p t e m b e r o f t h e p r e v i o u s y e a r . T h i s w o u l d i n d i c a t e t h a t some f a c t o r o t h e r t h a n t e m p e r a t u r e m i g h t be i n f l u e n c i n g t h e I x31 e x c r e t i o n r a t e i n s t e e l h e a d a t s m o l t i f i c a t i o n . 2. T h y r o i d U p t a k e T / S and TUF v a l u e s a l s o showed t e m p e r a t u r e c o r r e l a t i o n s b u t w i t h t h e same two e x c e p t i o n s ( s m a l l f i s h a n d s m o l t s ) ( F i g . 6). The t h y r o i d I 1 ^ 1 u p t a k e c o r r e l a t e d p o s i t i v e l y w i t h t e m p e r a t u r e o v e r t h e s e a s o n a l r a n g e o f 4 t o 13° C a t h i g h l y s i g n i f i c a n t s t a t i s t i c a l l e v e l s ( F i g . 7) ( T / S , r = 0.78, 26df, p<0.001; T U F , r = 0.89, 26df, p<0.00l). 3. C o n v e r s i o n R a t i o CR v a l u e s showed a g e n e r a l i n c r e a s e w i t h t e m p e r a t u r e r i s e ( r = 0.46, l8df, p<0.05), b u t t h e e f f e c t was n o t a s m a r k e d as w i t h t h y r o i d u p t a k e ( F i g s . 6 and 7). Of p a r t i c u l a r n o t e w e r e - 35 - the very high CR values of smolts at 8 to 9° C. These values were considerably higher than any recorded during the entire study. No CR estimates were avail a b l e on yearling f i s h below 20 grams. 4. H i s t o l o g i c a l Changes Whereas a l l l!31 parameters were p o s i t i v e l y correlated with temperature and suggested a possible temperature dependence, the c e l l height parameter indicated no such general c o r r e l a t i o n (Fig. 6). I t was conclusively demonstrated that i n yearling steelhead the lowest c e l l heights occurred at the highest and not the lowest temperature, and much of the: seasonal data indicated a tendency f o r higher c e l l height at lower temperatures. In other words, the radioiodine and c e l l height parameters showed opposite trends with respect to temperature. Of some importance, however, was the s i g n i f i c a n t negative c o r r e l a t i o n between mean values f o r c e l l height and mean values f o r percentage of f o l l i c l e s containing blue c o l l o i d (r = -0.66, l l d f , p<0.05). This indicated that the c e l l height data were not i n agreement with the data obtained from c o l l o i d colour, since increasing c e l l height i s associated with an active gland, and absence of blue c o l l o i d indicates decreased thyroid a c t i v i t y . I t i s evident from t h i s b r i e f survey of 'seasonal changes i n 1131 metabolism and h i s t o l o g i c a l change i n steelhead that there i s no absolute pioture of what might be a f f e c t i n g the thyroid. Temperature seems to be correlated with changes i n I 1 ^ ! metabolism. But, i s there a causal r e l a t i o n s h i p between thyroid a c t i v i t y and changing temperature? Furthermore, the - 36 - smolts and small f i s h showed departures from t h i s general trend, while the c e l l height data revealed a c o r r e l a t i o n possibly i n the reverse d i r e c t i o n , and showed a trend opposite to that obtained by the c o l l o i d s t a i ning. There are also other important seasonal variables including photoperiod. C y c l i c a l changes i n radioiodine metabolism independent of environmental influence might also be envisaged. The roles of various factors influencing the thyroid therefore appear uncertain and confused. In the remainder of t h i s study, an attempt has been made to i s o l a t e experimentally the various components that might influence the thyroid and to explain as f u l l y as possible the seasonal changes described above. IT I. EXPER MENTAL INVESTIGATION 0? THE INFLUENCE OF TEMPERATURE ON THE HISTOLOGICAL APPEARANCE OF THE THYROID AND RADIOIODINE METABOLISM IN YEARLING STEELHEAD PARR In the seasonal study of thyroid function correlations between radioiodine metabolism and temperature were established. From such correlations i t might be predicted that temperature causes changes i n radioiodine metabolism. In t h i s aspect of the study i t was hoped to test t h i s point by holding f i s h under conditions which were d i f f e r e n t i n temperature but s i m i l a r i n a l l other respects. To assess experimentally the effects of temperature on thyroid function, 12 f i s h were put into an open system of running water at temperature, regimes of 6, 9, 12, 13 and 18° C, a l l held to +0.05° C. The holding temperature p r i o r to the acclimation - 37 - was 9 + 0.5° G and the acclimation temperature was not imposed suddenly but b u i l t up over a period of several hours. On 18 July, 14 days a f t e r i n i t i a t i o n to the regimes, a l l the f i s h were injected with I - ^ l . S i x were k i l l e d from each condition at four days and d at eight days. The r e s u l t s are shown i n Figures 8 to 11 and i n Table VI. The e f f e c t of temperature on c e l l height and radioiodine metabolism of Salmo gairdneri has been investigated by Olivereau (1955a, b). Thyroid a c t i v i t y was found to be highest between 9 and 12° C using radiochemical techniques. With h i s t o l o g i c a l c r i t e r i a , greatest a c t i v i t y was implied at the lower tempera- tures. At 20° C the thyroid a c t i v i t y was low by both radio- chemical and h i s t o l o g i c a l c r i t e r i a . In the present study, c e l l height showed a trend i d e n t i c a l to that claimed by Olivereau. By t h i s c r i t e r i o n , the gland was s i g n i f i c a n t l y more active at lower temperatures (r = -0.84, 9df, p<0.01) (Fig. 8). However, there was a s t a t i s t i c a l l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between increasing blueness of c o l l o i d and increasing temperature, i n d i c a t i n g a more active thyroid at higher temperatures by t h i s c r i t e r i o n (r - 0.71, 9df, p<0.02) (Fig. 8). In regard to the uptake of radioiodine (Fig. 8), both 4 and 8 day estimates showed a very similar pattern with maximum uptake at 12° C. Olivereau (1955a) measured I ^ l uptake by autoradiography, which i s e s s e n t i a l l y the same technique, and obtained a s i m i l a r r e s u l t . I t has already been pointed out, however, that l A ^ l uptake by the thyroid i s only a v a l i d measure - 38 - F i g . - 8. E x p e r i m e n t a l d e m o n s t r a t i o n o f t h e i n f l u e n c e o f t e m p e r a t u r e on p e r c e n t a g e a c c u m u l a t i o n o f I 1 ? 1 i n t h e t h y r o i d 4 and 8 d a y s a f t e r a s i n g l e 1.131 i n j e c t i o n , on mean c e l l h e i g h t (O) and on p e r c e n t a g e o f f o l l i c l e s c o n t a i n i n g b l u e c o l l o i d ( • ) .  - 39 - o f t h e t h y r o i d a f f i n i t y f o r i o d i n e i f a l l o w a n c e i s made f o r t h e r a t e o f e x t r a t h y r o i d a l I 1 ^ 1 c l e a r a n c e . Thus t h e above c o n c l u s i o n s on m a x i m a l I 1 ^ 1 u p t a k e a t 12° C c o u l d be i n v a l i d a s a n i n d i c a t i o n o f t h y r o i d a c t i v i t y . F i g u r e 9 shows t h e e f f e c t o f t e m p e r a t u r e on i - ^ l r e m o v a l f r o m t h e b o d y as a w h o l e and f r o m t h e p l a s m a a t d i f f e r e n t t e m p - e r a t u r e s (4 a n d 8 d a y s ) . I ^ l r e t e n t i o n f a l l s e x p o n e n t i a l l y w i t h t e m p e r a t u r e . Some a t t e m p t a t a n a n a l y s i s o f t h e s e c u r v e s h a s been p r e s e n t e d ( T a b l e V I ) . I t w o u l d a p p e a r t h a t b o t h t h e 4 and 8 d a y e s t i m a t e s o f I / ^ l r e t e n t i o n i n d i c a t e s l o p e s t h a t do n o t d i f f e r s i g n i f i c a n t l y f r o m e a c h o t h e r . H o w e v e r , t h e h i g h c o r r e l a t i o n c o e f f i c i e n t s e m p h a s i z e a s i g n i f i c a n t i n c r e a s e i n ll31 l o s s w i t h t e m p e r a t u r e . F i g u r e 10 shows t h e a p p r o x i m a t e e x p o n e n t i a l i n c r e a s e i n T / S w i t h t e m p e r a t u r e . The CR v a l u e s f o r t h e same f i s h showed a s i m i l a r e x p o n e n t i a l change ( F i g . 11). H i g h c o r r e l a t i o n c o e f f i c i e n t s were f o u n d f o r t h e r e l a t i o n s h i p and no s t a t i s t i c a l d i f f e r e n c e was e v i d e n t b e t w e e n t h e 4 and 8 day s l o p e s ( T a b l e V I ) . I t i s c o n c l u d e d t h a t t e m p e r a t u r e a c c e l e r a t e s a l l a s p e c t s o f i l ^ l m e t a b o l i s m b e t w e e n 6 a n d 18° C and t h a t r a d i o c h e m i c a l ^ t h e t h y r o i d i s n o t m a x i m a l l y a c t i v e a t 9 t o 12° C a s O l i v e r e a u s u g g e s t e d . Where h i s t o l o g i c a l c r i t e r i a a r e c o n c e r n e d , t h e r e v e r s e seems t o be t r u e s i n c e t h e c e l l h e i g h t i n d i c a t e d g r e a t e r g l a n d a c t i v i t y a t l o w e r t e m p e r a t u r e s . The c o l l o i d c o l o u r , h o w e v e r , c o n f o r m e d w i t h t h e r a d i o c h e m i c a l e s t i m a t e s . A n a t t e m p t h a s been made t o r e s o l v e t h e s e v e r y i m p o r t a n t a n o m o l i e s i n t h e g e n e r a l d i s c u s s i o n . - 40 - F i g . 9. Experimental demonstration of the influence of temperature on l e v e l s i n plasma and t o t a l body 4 days a f t e r a single I x31 i n j e c t i o n . Each point represent 4 to 7 in d i v i d u a l s (Covariance analysis, Table VI) . r PLASMA O Q O O _ — io tj 0\ — M w cn O 4 I L i S 8 % BODY - 41 - TABLE VI Analysis of covariance f o r the relationships between various I 1 * ! parameters and temperature (° C) Time Correlation Parameter (Days) Slope df P c o e f f i c i e n t 4 0.130 1 0.797** CR and 1.14 8 0.172 46 0.710** I ^ l PLASMA 4 -0.226 1 - 0 . 8 1 3 * * and 1.08 8 - 0 . 2 8 5 46 - 0 . 7 9 0 * * i BODY + 4 - 0 . 0 7 2 3 1 -O.665** % THYROID and 0.12 - 0 . 5 8 4 * * 8 - 0 . 0 8 1 8 46 Denotes sig n i f i c a n c e (p^ . 0 . 0 1 ) . - 42 - Ei g . 10. Experimental demonstration of the influence of temperature on T/S values 4 and 8 days a f t e r a single I x31 i n j e c t i o n . Each point represents a mean of 4 to 7 i n d i v i d u a l s .  - 43 - E i g . 11. Experimental demonstration of the influence of temperature on CR values 4 and 8 days a f t e r a single 1131 i n j e c t i o n . Each point represents a mean of 4 to 7 individuals (Covariance analysis, Table VI). r - 44 - IV. EXPERIMENTAL INVESTIGATION OF TEMPERATURE AND PHOTOPERIOD INFLUENCE ON THYROID CELL HEIGHT AND RADIOIODINE METABOLISM OF STEELHEAD YEARLINGS (January to July) The previous seasonal observations and experiments suggest that temperature i s a major environmental fa c t o r influencing the thyroid. This does not exclude, however, the influence of other variables, prominent among which could be the changing photoperiod. Photoperiod i s important i n c o n t r o l l i n g c e r t a i n endocrine changes and the spring increase i n radioiodine metabolism i n yearling and two-year-old steelhead could be influenced by the increasing spring daylight hours. To test t h i s p o s s i b i l i t y , an experiment was conducted from l a t e January to early July. F i s h were held under 4 experimental regimes i n 2 m x 50 cm x 30 cm concrete troughs with a continually replenished supply of dechlorinated water. In two of the troughs the water temperature was not controlled and approximated to the outside conditions. In the other two tanks i t was pre-cooled and main- tained between 5 and 6° G. A l l 4 tanks were covered with l i g h t - proof boxes illuminated by fluorescent bulbs. Two of them were maintained on an 8-hour day and two on a natural daylength using the control mechanism mentioned i n methods (Table VII). F i s h were sampled routinely f o r h i s t o l o g i c a l and radioiodine determinations at approximately monthly i n t e r v a l s . The 1̂ -31 plasma l e v e l s showed a c l e a r separation on the basis of temperature as did the extrathyroidal clearance (Fig. 1 2 ) . There was no noticeable suggestion of a photoperiod influence. - 45 - TABLE YII Summary of the four combined temperature and photoperiod conditions. Symbols shown are as used i n figures TANK SYMBOL PHOTOPERIOD TEMPERATURE 1 • Natural Natural 2 A 8 hours O Natural Controlled 4 A 8 hours (5 - 6° C) - 46 - F i g . 12. Experimental demonstration of the combined influence of temperature and photoperiod on plasma l!31 l e v e l s and percentage dose i n t o t a l body 4 days a f t e r a single 1131 i n j e c t i o n i n 14 to 18-month steelhead. Each point represents a mean of 8 to 12 f i s h ( S t a t i s t i c s , Table VIII).  - 47 - The T/S showed a s i m i l a r increased response to r i s i n g temperature hut showed no response to increasing photoperiod (Fig. 13). By- comparing the four conditions i n June when the greatest diver- gence between the treatment prevailed, i t was not possible to demonstrate a s i g n i f i c a n t e f f e c t of photoperiod, but a very s i g n i f i c a n t influence of temperature on I 1 ^ ! metabolism was shown (Table VIII). In support of the e a r l i e r findings, however, the c e l l height did not agree with the radioiodine parameters (Fig. 13) and i n general the f i s h held at the lower temperature had the higher c e l l height. In f a c t , ignoring photoperiod which did not appear to be influencing the c e l l height s i g n i f i c a n t l y , the means of a l l f i v e seasonal samples of cold-adapted f i s h had a higher c e l l height than those subjected to the increasing temperature. C o l l o i d colour estimates have not been included. They were extremely varied and revealed no apparent trend through the four conditions. Conversion r a t i o s were not available from these experiments since at the time they were conducted i t was not r e a l i z e d how slowly the P B I 1 ^ 1 was produced and no 8-day readings were taken. So f a r , estimates of thyroid a c t i v i t y have been based s o l e l y on;;single terminal measurements and do not include the CR. In order to reach more d e f i n i t e conclusions concerning the e f f e c t s of photoperiod and temperature on yearling f i s h , s e r i a l i n v e s t i - gations were carr i e d out from these same conditions i n early July (high temperature and long photoperiod under the natural conditions). F i f t y to 55 f i s h from each condition were injected with I 1 ^ 1 and 6 from each group k i l l e d at the following i n t e r v a l s : - 48 - Fi g . 13. Experimental demonstration of the combined influence of photoperiod and temperature on mean c e l l height and T/S r a t i o s 4 days a f t e r a single 1"131 i n j e c t i o n i n 14 to 18-month steelhead. C e l l height - each point represents a mean of 3 to 4 f i s h ; T/S - each point represents a mean of 8 to 12 f i s h ( S t a t i s t i c s , Table Y I I I ) .  - 49 - TABLE VIII Summary of s t a t i s t i c a l l y s i g n i f i c a n t differences f o r several i l j l parameters between d i f f e r e n t conditions of temperature and photoperiod i n yearlings i n June. White-Wilcoxon non-parametric ranking test used HIGH TEMPERATURE LOW TEMPERATURE Photoperiod Photoperiod Photoperiod Photoperiod normal 8 hours normal 8 hours I 1 5 1 plasma L ++ fo dose body % dose thyroid ++ T/S ++ - Two groups not s t a t i s t i c a l l y d i f f e r e n t (p-^0.05). ? Two groups almost s t a t i s t i c a l l y d i f f e r e n t (p = 0 . 0 5 ) . + Two groups s i g n i f i c a n t l y d i f f e r e n t (p^.0.05). ++ Two groups s i g n i f i c a n t l y d i f f e r e n t ( p< 0 . 0 1 ) . - 50 - 12, 36, 60, 84, 108, 132, 156, 204 and 276 hr a f t e r i n j e c t i o n . Data are shown i n Figures 14 - 16 i n c l u s i v e ; Table IX sum- marizes the s t a t i s t i c s . The r e s u l t s obtained by the single 4-day measurements were confirmed. Photoperiod at t h i s time of year exerted no influence on plasma I - ^ l clearance nor on thyroid uptake, though temperature had a d e f i n i t e effect (Pigs. 14 and 15) . At the higher temperature, the c h a r a c t e r i s t i c diphasic thyroid uptake curve was evident. A s i m i l a r interpre- t a t i o n i s derived from consideration of the CR (Fig. 1 6 ) . There was a d i s t i n c t separation between the e f f e c t s of the two temperature conditions but no d i f f e r e n t i a l response to photo- period. It i s concluded therefore that i n yearling steelhead (i) increasing temperature stimulates radioiodine metabolism and yet causes a decrease i n c e l l height, ( i i ) there i s no influence of increasing photoperiod on the steelhead thyroid under the conditions investigated and ( i i i ) although the f i s h 131 under the increasing temperature show a higher rate of I ^ metabolism than f i s h at the lower temperature, the increasing temperature regime induces r e l a t i v e l y l i t t l e change. The divergence between the e f f e c t s of the two regimes was not due only to changes i n r ^ l metabolism i n the f i s h under the variable temperature. In f a c t , with I - ^ l excretion data, the greater changes were found i n the f i s h held under constant temperature. This points to some change within the f i s h not governed by temperature and photoperiod. What was common to a l l the f i s h used over this* period was t h e i r r a p i d l y increasing s i z e . One p o s s i b i l i t y i s that t h i s i s a phase of rapid growth requiring - 51 - F i g . 14. E x p e r i m e n t a l d e m o n s t r a t i o n o f t h e c o m b i n e d i n f l u e n c e o f p h o t o p e r i o d and t e m p e r a t u r e on p l a s m a c l e a r - a n c e f o l l o w i n g a s i n g l e I 1 ? ! i n j e c t i o n i n 18-month s t e e l - h e a d . E a c h p o i n t r e p r e s e n t s a mean o f 5 t o 7 f i s h ( C o v a r i a n c e a n a l y s i s , T a b l e I X ) . DAYS - 52 - F i g . 15. E x p e r i m e n t a l d e m o n s t r a t i o n o f t h e combined i n f l u e n c e o f p h o t o p e r i o d and t e m p e r a t u r e on T / S v a l u e s i n 18-month s t e e l h e a d f o l l o w i n g a s i n g l e Il31 i n j e c t i o n . E a c h p o i n t r e p r e s e n t s a mean o f 5 t o 7 f i s h ( C o v a r i a n c e a n a l y s i s , T a b l e E C ) .  - 53 - F i g . 16. Experimental demonstration of the combined influence of photoperiod and temperature on CR values following a single 1^31 i n j e c t i o n i n 18-month steelhead. Each point represents a mean of 5 to 7 i n d i v i d u a l s . 8L ST A TABLE DC Analysis of covariance performed on plasma l x 5 1 and time (hr), and T/S and time (hr) under four d i f f e r e n t photoperiod and temperature regimes PLASMA I 1 ? 1 Temp. Photo- ° C period Phase Slope df P s df E m 8 - -0.013] I ' l l 5 I and 0 .02 \ and 0.44 . 16 - -0.014 J 97 J 99 df F s 1 and 6.67* 105 8 - 0 . 0 1 71 1 n 1 10 \ and 0 .?4 f and I . 6 5 16 - - 0 . 0 1 6 J 102 S 104 1 and - O.63 94 T/S df F s df E s df F s I 0.026 7 1 8 \ and 1.44 K II 0 . 0 2 1 J 56 * I 0.027 7 1 16 I and 3 .08 I I 0 . 0 1 9 ) 48 1 • and 0.07 56 1 .and 4 . 4 8 * 55 I 0.033 7 1 1 8 I and 26 .46** , n • I I 0.014) 38 ± v I O .0367 1 16 > and 26.40** I I O.OIOJ 50 1 - and 0.24 53 , 1 and 2.58 54 n F g = Variance r a t i o f o r slopes. F m » Variance r a t i o f o r adjusted means. * S i g n i f i c a n t difference ( p< 0 . 0 5 ) . * S i g n i f i c a n t difference ( p< 0 . 0 1 ) . - 55 - a g r e a t e r t h y r o x i n demand, b u t a n e q u a l l y p l a u s i b l e h y p o t h e s i s i s t h a t t h e c h a n g e s i n I 1 ^ 1 m e t a b o l i s m u n d e r c o n s t a n t c o n d i t i o n s w e r e s i m p l y a f u n c t i o n o f i n c r e a s i n g s i z e . V . EFFECT OF BODY MASS ON RADIOIODINE METABOLISM To t e s t t h e e f f e c t o f body mass o n I 1 ^ 1 m e t a b o l i s m , two g r o u p s o f y e a r l i n g s t e e l h e a d o f t h e same age were t a k e n i n m i d - J u n e f r o m t h e f o u r h o l d i n g c o n d i t i o n s o f c o m b i n e d t e m p e r a t u r e and p h o t o p e r i o d u s e d a b o v e . One g r o u p f r o m e a c h c o n d i t i o n c o n s i s t e d o f 12 l a r g e f i s h (11-65 g , a v - 19.0 g ) ; t h e s e were i n j e c t e d w i t h 0.02 m l o f I 1 ^ ! (3 JIG). The o t h e r g r o u p c o n s i s t e d o f 12 s m a l l f i s h (3.2-8.0 g , a v = 5.4 g) a n d t h e s e were i n j e c t e d w i t h 0.01 m l ( l ^ i o ) . A l l f i s h were k i l l e d 96 h o u r s a f t e r i n j e c t i o n . The p e r c e n t a g e o f t h e i n j e c t e d dose r e t a i n e d i n t h e e n t i r e b o d y was c o n s i s t e n t l y d i f f e r e n t b e t w e e n t h e s m a l l and l a r g e f i s h , a l t h o u g h b o t h showed t h e e x p e c t e d dependence on t e m p e r a t u r e a n d l a c k o f r e s p o n s e t o p h o t o p e r i o d ( F i g . 17). The f a c t t h a t l a r g e f i s h t e n d e d t o r e t a i n i n j e c t e d I 1 ^ ! l o n g e r t h a n s m a l l f i s h e x p l a i n s why f i s h h e l d u n d e r c o n s t a n t c o n d i t i o n s showed changes i n i o d i n e m e t a b o l i s m as t h e s e a s o n p r o g r e s s e d and t h e a v e r a g e mass i n c r e a s e d . The t e n d e n c y f o r i n c r e a s i n g t e m p e r a t u r e t o i n c r e a s e t h e r a t e o f I x 3 l l o s s was a p p r o x i m a t e l y b a l a n c e d b y t h e t e n d e n c y f o r a g r o w i n g f i s h t o r e t a i n more i o d i n e due t o s l o w e r e x c r e t i o n . Thus t h e a p p a r e n t anomoly i n t h e y e a r l i n g f i s h i s e x p l a i n e d on t h e b a s i s o f a change i n body m a s s . A t a l o w c o n s t a n t t e m p e r a t u r e , i t i n d u c e d a change t h a t w o u l d o t h e r w i s e n o t o c c u r a n d u n d e r t h e i n c r e a s i n g t e m p e r a t u r e t h e s i z e - % - F i g . 17. I n f l u e n c e o f body mass on T/S v a l u e s . a n d p e r c e n t a g e o f I ^ l i n t h e t o t a l body 84 h o u r s a f t e r a s i n g l e 1.131 i n j e c t i o n i n 19-month s t e e l h e a d . I I °C I6HRS a O m 8 0 - 6 0 - 4 0 - 2 0 - 8 0 - o T/S 4 0 - 2 0 - 8 HRS o o • o i j^J S M A L L 5-5 °C 16 HRS L A R G E 8 HRS - 37 - e f f e c t a l m o s t e x a c t l y c a n c e l l e d t h e s t i m u l u s o f i n c r e a s i n g t e m p e r a t u r e . Measurement o f T / S r a t i o s i n g e n e r a l s u p p o r t e d t h e i d e a t h a t l l 3 1 m e t a b o l i s m was more a c t i v e i n a s m a l l e r f i s h . A s w i t h 1.131 e x c r e t i o n , t h e i n d e p e n d e n c e o f p h o t o p e r i o d and dependence on t e m p e r a t u r e were e v i d e n t . W i t h t h e e x c e p t i o n o f one c o n d i - t i o n , s m a l l f i s h had h i g h e r T/S r a t i o s t h a n l a r g e f i s h . S i n c e no f i s h w e r e h e l d t o 8 d a y s , no CR v a l u e s were a v a i l a b l e . To v e r i f y some o f t h e above f i n d i n g s , a n o t h e r e x p e r i m e n t was c o n d u c t e d on 3 g r o u p s o f y e a r l i n g s t e e l h e a d i n S e p t e m b e r a t 1 1 ° C ( T a b l e 2 ) . A l l f i s h were k i l l e d 8 d a y s a f t e r i n j e c t i o n t o o b t a i n CR v a l u e s , l a c k i n g f r o m t h e p r e v i o u s d e t e r m i n a t i o n s . A g a i n t h e r a t e o f e x c r e t i o n and J.131 a c c u m u l a t i o n were a f f e c t e d i n t h e same way b y body mass b u t two f u r t h e r i m p o r t a n t p o i n t s emerged ( F i g . 18); ( i ) 1 t h e CR was a l s o m a r k e d l y s t i m u l a t e d i n t h e s m a l l e r s i z e r a n g e and ( i i ) w i t h e a c h o f t h e 3 p a r a m e t e r s o f J131 m e t a b o l i s m t h e r e was a g r e a t s i z e e f f e c t between 8 and 16 grams and l i t t l e e f f e c t b e t w e e n 16 and 30 g r a m s . The phenomenon may be e x p l a i n e d i n t e r m s o f a l o g body mass - l o g t h y r o i d a c t i v i t y r e l a t i o n s h i p . B o t h T / S a n d CR showed s i g n i f i - c a n t n e g a t i v e c o r r e l a t i o n s when t h e d a t a were t r a n s f o r m e d i n t h i s manner ( F i g . 19). S u c h a t r a n s f o r m a t i o n i s c h a r a c t e r i s t i - c a l l y u s e d t o show dependence o f m e t a b o l i c r a t e on mass and t h e f a c t t h a t r a d i o i o d i n e p a r a m e t e r s o f t h y r o i d a c t i v i t y c o n f o r m t o t h i s r e l a t i o n s h i p s t r o n g l y s u g g e s t s a g e n e r a l m e t a b o l i c r o l e o f t h e t h y r o i d . The e x c r e t i o n r a t e a l s o showed a s i m i l a r r e l a t i o n - s h i p w i t h body w e i g h t ( F i g . 19). S i n c e e x t r a t h y r o i d a l e x c r e t i o n - 58 - TABLE X Summary o f i n j e c t i o n d e t a i l s f o r s i z e e x p e r i m e n t N o . o f V o l . i n j e c t e d f i s h Mass (g) ( c c ) I 1 ^ 1 ijxc) JLC/& 15 8.17(4.40-10.50) .02 1 0.122 4 15.87(12.72-20.00) .04 2 0.126 9 29.28(19.00-41.20) .08 4 0.136 - 59 - F i g . 18. Influence of body mass (gram) on plasma T ' l e v e l s and T/S values (4 days a f t e r i n j e c t i o n) and CR values (8 days aft e r i n j e c t i o n ) . Means and standard deviations are shown f o r large, medium and small size groups. No s i g n i f i - cant difference was noted between medium and large size groups f o r any J.131 parameter, but both d i f f e r e d from the small group f o r a l l parameters (p^.01). to "T o 7} O (0* I'3 1 P L A S M A o o —T— in O o 2 > cn O " O " I — I — I - 60 - F i g . 19. R e l a t i o n s h i p b e t w e e n t h e l o g a r i t h m o f body- mass and t h e l o g a r i t h m o f v a r i o u s l!31 p a r a m e t e r s . T/S ( A ) , r -0.57; CR ( • ) , r -0.53; % b o d y (O), r -O.67.  - 61 - r a t e o f I 1 ' 5 1 was i n d e p e n d e n t o f t h y r o i d a c t i v i t y , i t s r e l a t i o n - s h i p t o body s i z e c o n f i r m s t h a t mass c a u s e s an o v e r a l l change i n m e t a b o l i s m . A s i m i l a r dependence o f e x c r e t i o n on mass h a s b e e n shown i n P l a t i c h t h y s s t e l l a t u s ( H i c k m a n , 1959) and i n G a s t e r o s t e u s a c u l e a t u s ( W i g g s , 1962). The a p p a r e n t s t a b i l i z a t i o n o f t h e mass e f f e c t above a b o u t 16 grams was p r o b a b l y a r e s u l t o f t h e d o u b l e l o g a r i t h m i c r e l a t i o n s h i p w h e r e b y o v e r t h i s r a n g e a r e l a t i v e l y g r e a t change i n mass must be p r e s e n t f o r s i g n i f i c a n t m e t a b o l i c change t o be o b s e r v e d . I n s m a l l e r f i s h t h e r e v e r s e i s t r u e . A s i z e e x c e e d i n g 20 grams i s t h e r e f o r e v e r y d e s i r a b l e f o r e x p e r i m e n t a l w o r k as one i s now w o r k i n g i n a n e s s e n t i a l l y m a s s - s t a b l e r a n g e . V I . SXPERIMBNTAL INVESTIGATION OF THE INFLUENCE OF SEASONAL CHANGE I N INCREASING PHOTOPERIOD AND INCREASING TEMPERATURE ON THE RADIOIODINE METABOLISM AND THYROID C E L L HEIGHT OF TWO-YEAR-OLD STEELHEAD By b o t h h i s t o l o g i c a l a n d r a d i o c h e m i c a l c r i t e r i a t h y r o i d a c t i v i t y i s v e r y h i g h i n s t e e l h e a d a t t h e t i m e o f s m o l t i f i c a t i o n ( F i g . 6) a n d a p p e a r s f a r more a c t i v e t h a n w o u l d be a n t i c i p a t e d on t h e b a s i s o f a t e m p e r a t u r e change a l o n e . I t i s p o s s i b l e t h a t t h i s s u r g e i n t h y r o i d a c t i v i t y i s c a u s e d b y t h e i n f l u e n c e o f i n c r e a s i n g p h o t o p e r i o d . A n a t t e m p t was made t o t e s t t h i s h y p o - t h e s i s b y r e p e a t i n g on t w o - y e a r - o l d s t h e e x p e r i m e n t p r e v i o u s l y c a r r i e d out on y e a r l i n g s u n d e r v a r i o u s p h o t o p e r i o d and t e m p e r a - t u r e r e g i m e s ( T a b l e V I I ) . The e x p e r i m e n t was p e r f o r m e d f r o m l a t e J a n u a r y u n t i l t h e b e g i n n i n g o f J u n e , a . p e r i o d c o v e r i n g t h e m a i n - 62 - extent of increasing photoperiod and also the time of smolti- f i c a t i o n , which normally occurs i n A p r i l and May. The usual 1,131 and h i s t o l o g i c a l parameters were measured. 1. j l 3 1 Excretion This has been assessed by the percentage of the injected dose i n body (4 days) and 1^31 plasma concentration (4 days). From the seasonal data (Fig. 6) i t i s seen that s i g n i f i c a n t changes i n 1^31 metabolism became evident towards the end of March. I f photoperiod were having any e f f e c t on 1^31 metabolism i t might be expected from t h i s time onwards. From the end of March to the end of May the excretion parameters from the 4 conditions did conform to a general scheme (Fig. 20). With but one exception (not s t a t i s t i c a l l y s i g n i f i c a n t ) f i s h subjected 131 to progressively increasing temperature showed a f a s t e r I loss than f i s h at the constant temperature of 5° C. In addition, f i s h subjected to the increasing photoperiod showed a 131 s i g n i f i c a n t l y f a s t e r rate of I removal at any given tempera- ture than those held under the constant 8-hour daylength (Table XI). The influence of the photoperiod on I 131 excretion was not r e a l l y as a t r i g g e r i n the s t r i c t sense since i t did not cause a sudden r i s e i n excretion rate at a p a r t i c u l a r season. From early February onwards, the photoperiods were quite separable with the longer photoperiod causing the more rapid I 1 ^ 1 elimination. The sudden r i s e i n excretion rate i n A p r i l and May was attributed to the influence of temperature which rose quite r a p i d l y at t h i s period and superimposed i t s influence on that of the photoperiod. As f a r as the excretion - 6? - F i g . 2 0 . Experimental demonstration of the combined influence of temperature and photoperiod on plasma T.131 l e v e l s and percentage dose i n t o t a l body 4 days a f t e r 1131 i n j e c t i o n i n 26 to 30-month steelhead (potential migrants). Each point represents a mean of 6 individuals ( S t a t i s t i c s , Table XI).  - 64 - rate i s concerned i t would appear that both temperature and photoperiod combined to induce the high rate of I 1 ^ 1 loss c h a r a c t e r i s t i c of smolting f i s h . There was no i n d i c a t i o n of a synergism between temperature and photoperiod. By l a t e May the effect of increasing photoperiod on f i s h held at 5° C appeared equal to the effect of increasing temperature on f i s h from a constant 8-hour regime. 2. Thyroid Uptake of I 1 ^ 1 This was measured by T/S and TUF parameters (Figs. 21 and 22 r e s p e c t i v e l y ) . For each parameter both 4-day and 8-day values were recorded. Since the f i s h sampled at 4 days were d i f f e r e n t individuals from those sampled at 8 days, t h i s gives a " r e p l i c a t i o n " . As pointed out i n the preliminary study of 1^31 metabolism the 4-day estimate i s more r e l i a b l e , as 131 measurements l a t e r than t h i s may be influenced by I loss from the thyroid as radiohormone. In every instance f o r a p a r t i c u l a r temperature, the increas- ing photoperiod induced a higher thyroid uptake value than the constant 8-hour day. The data also showed that divergence between photoperiod influence became greater as the photoperiod d i f f e r e n t i a l increased; i n May the T/S and TUF indices were high (Table XI). I t may be concluded, therefore, that increasing photoperiod caused a progressive increase i n thyroid uptake of 1^31 and that r i s i n g temperature superimposed a further increase. 131 As with I ^ excretion data, the condition of constant tempera- ture and increasing photoperiod brought about a change i n thyroid I 1 ^ 1 uptake comparable to that i n f i s h held under a - 65 - F i g . 21. E x p e r i m e n t a l d e m o n s t r a t i o n o f t h e combined i n f l u e n c e o f t e m p e r a t u r e and p h o t o p e r i o d on T / S v a l u e s 4 and 8 days a f t e r I 1 ? ! i n j e c t i o n i n 26 t o 30-month s t e e l - h e a d ( p o t e n t i a l m i g r a n t s ) . E a c h p o i n t r e p r e s e n t s a mean o f 6 i n d i v i d u a l s ( S t a t i s t i c s , T a b l e X I ) .  - 66 - F i g . 22. Experimental demonstration of the combined influence of temperature and photoperiod on TUF values 4 and 8 days a f t e r l l 3 1 i n j e c t i o n i n 26 to 30-month s t e e l - head (potential migrants). Each point represents a mean of 6 individuals ( S t a t i s t i c s , Table X I ) .  - 67 - constant 8-hour daylength but with r i s i n g temperature. 3» Conversion Ratio This was measured 8 days a f t e r i n j e c t i o n (Fig. 23). Although there were a few exceptions early i n the year, the f i s h under increasing photoperiod f o r any given temperature showed a higher c e l l height than those under 8 hours. By May the separation was very evident and extremely high CR values were obtained as a r e s u l t of r i s i n g temperature and increasing photoperiod. These differences were highly s i g n i f i c a n t (Table XI). I conclude from the I 1 ^ 1 data on combined temperature and photoperiod influence on two-year-old steelhead, that increasing photoperiod stimulates progressive increase i n rate of I 1 ^ ! excretion, I 1 ^ ! uptake by the thyroid and CR. It does not necessarily have a sudden action but i s influencing the I 1 ^ ! metabolism over a long period. What appears to be a sudden 131 change i n I metabolism i n A p r i l and May i s due to the added stimulation by a temperature r i s e . Radioiodine metabolism i s exponentially dependent upon temperature (Figs. 9 to 11). This r e l a t i o n s h i p would tend to emphasize the response of the thyroid to rap i d l y r i s i n g temperature, and might give the impression of a synergism under the condition of increasing temperature and photoperiod. There i s no d e f i n i t e evidence, however, f o r a synergism between these two important environmental factors. It must be noted that f i s h under constant 8-hour photoperiod and held at 5° C showed no tendency at a l l to change i n any aspect of -rl31 metabolism. It i s f i n a l l y emphasized that the influence of increasing photoperiod on p o t e n t i a l migrants was - 68 - F i g . 23. E x p e r i m e n t a l d e m o n s t r a t i o n o f the combined i n f l u e n c e o f t e m p e r a t u r e and p h o t o p e r i o d on mean c e l l h e i g h t a n d CR (8 days a f t e r I 1 * 1 i n j e c t i o n ) i n 26 t o 30-month s t e e l - h e a d ( p o t e n t i a l m i g r a n t s ) . C e l l h e i g h t - means ( O A ) and i n d i v i d u a l v a l u e s T O A ) shown; CR - e a c h p o i n t r e p r e s e n t s a mean o f 6 i n d i v i d u a l s .  - 69 - TABLE XI Summary of s t a t i s t i c a l l y s i g n i f i c a n t difference f or several i l j l parameters between d i f f e r e n t conditions of temperature and photoperiod i n two-year-olds (potential migrants) i n late May (White-Wilcoxon test) HIGH TEMPERATURE LOW TEMPERATURE Photoperiod Photoperiod Photoperiod Photoperiod increasing 8 hours increasing 8 hours 1^31 plasma (4 days) % dose body t_ % dose thyroid L (4 days) • ++ TUF (4 days) •++ -++ T/S (4 days) •++ \CR (8 days) .++ - Two groups not s t a t i s t i c a l l y d i f f e r e n t (p-0.05). ? Two groups almost s t a t i s t i c a l l y d i f f e r e n t (p*0.0j?) + Two groups s i g n i f i c a n t l y d i f f e r e n t (p 4.0.05). + Two groups s i g n i f i c a n t l y d i f f e r e n t (p<0.01). + - 70 - c o m p l e t e l y a b s e n t i n y e a r l i n g n o n - m i g r a n t : f i s h . 4. C e l l H e i g h t Measurement o f c e l l h e i g h t , t h o u g h s a m p l e s were s m a l l , showed a d e f i n i t e s e p a r a t i o n due t o p h o t o p e r i o d b e t w e e n A p r i l a n d May ( F i g . 2 3 ) . No c l e a r - c u t e f f e c t o f t e m p e r a t u r e on c e l l h e i g h t c o u l d be d e m o n s t r a t e d a l t h o u g h i n 8 - h o u r p h o t o p e r i o d f i s h t h e l o w e r t e m p e r a t u r e was g e n e r a l l y a s s o c i a t e d w i t h a t a l l e r c e l l h e i g h t t h a n t h e h i g h e r t e m p e r a t u r e . T h i s e f f e c t o f l o w t e m p e r a t u r e on c e l l h e i g h t c o n f i r m s e a r l i e r f i n d i n g s . I n t e r m s o f c o l l o i d c o l o u r , i t was d i f f i c u l t t o r e c o g n i s e any d i f f e r e n c e s a t a l l b e t w e e n f i s h f r o m t h e c o n d i t i o n s . V I I . INFLUENCE OF PRECOCITY OF MALE PARR ON THYROID A C T I V I T Y I t was n o t e d i n e a r l y s p r i n g t h a t up t o 13% o f t h e t w o - y e a r - o l d s t e e l h e a d s a m p l e d w e r e p r e c o c i o u s s e x u a l l y m a t u r e m a l e s . T h e s e n e v e r showed t h e t e n d e n c y t o w a r d s s i l v e r i n g and p r e s e r v e d a m o d i f i e d p a r r c o l o r a t i o n c h a r a c t e r i s t i c o f s p a w n i n g members o f t h e s p e c i e s . H i s t o l o g i c a l s t u d i e s o f t h y r o i d s o f t h e s e p a r r r e v e a l e d t h a t c e l l h e i g h t s w e r e g e n e r a l l y h i g h e r t h a n i n n o n - m a t u r e i n d i v i d u a l s ( F i g . 2 3 ) . T h i s was p a r t i c u l a r l y n o t i c e a b l e i n e a r l y M a r c h and s u g g e s t e d t h a t s e x u a l m a t u r a t i o n c o u l d be a n o t h e r v a r i a b l e a f f e c t i n g t h e t h y r o i d a c t i v i t y . T h e r e a r e many i n s t a n c e s i n t h e l i t e r a t u r e a s s o c i a t i n g i n c r e a s e d t h y r o i d a c t i v i t y w i t h s e x u a l m a t u r a t i o n i n t e l e o s t s . T h e s e h a v e been s u m m a r i z e d by P i c k f o r d and A t z (1957). To i n v e s t i g a t e t h i s h y p o t h e s i s f u r t h e r , a n e x a m i n a t i o n was made o f t h e TUF a n d CR d a t a a v a i l a b l e f r o m t h e s e same s e x u a l l y - 71 - F i g . 24. TUF (open bars) and CR (shaded bars) f o r precocious two-year-old male 09ff and immature (0#) steelhead i n early March under 4 combined conditions of temperature and photoperiod.  mature indivi d u a l s (Fig. 24). Although no d e f i n i t e conclusions could he drawn, cert a i n sexually mature male parr did have thyroid a c t i v i t i e s considerably higher than the other members of t h e i r group. It i s t e n t a t i v e l y suggested that precocious sexual maturation i n male parr i s correlated with increasing thyroid a c t i v i t y . The causal aspects of t h i s c o r r e l a t i o n were not investigated. VIII. INFLUENCE OF EXERCISE ON I 1 ? 1 METABOLISM OF STEELHEAD Mass, increasing photoperiod, temperature and possibly state of sexual maturation have been shown to influence annual cycles i n thyroid a c t i v i t y . One other f a c t o r that could be contributing to c y c l i c a l changes i n the natural environment i s the water current. F l u v i a t i l e f i s h , i n general, maintain t h e i r p o s i t i o n by rheotaxis. In a f a s t r i v e r the energy expenditure fo r maintaining p o s i t i o n i s presumably greater i n a sluggish r i v e r and extra^metabolic demands could be made on the animal and r e f l e c t e d by increase i n thyroid a c t i v i t y . Such an increase i n thyroid a c t i v i t y i n f a s t currents has been claimed for Salmo gairdneri (Fontaine, 1959). In t h i s experiment 48 f i s h were injected with I 1 ^ ! and immediately 24 of these were placed i n a f a s t current and 24 i n a slow current; the slow current was approximately 20f» of the f a s t current. In both tanks the temperature was 10- to 10.5° C. At 2, 4, 6 and 8 days, 6 f i s h were k i l l e d from each condition (Fig. 25). Three parameters were measured - percentage of the dose i n - 73 - F i g . 25. I n f l u e n c e o f i n c r e a s e d swimming a c t i v i t y on v a r i o u s a s p e c t s o f I l p l m e t a b o l i s m i n 19-month s t e e l h e a d . E a c h p o i n t r e p r e s e n t s a mean o f 6 i n d i v i d u a l s ( C o v a r i a n c e a n a l y s i s , T a b l e X I I ) .  - 74 - t o t a l body, percentage of the dose i n the thyroid and CR. They a l l showed s i g n i f i c a n t differences between treatments (Table XII). The increased swimming caused an increased rate of extrathyroidal jl31 excretion, a decrease i n the t o t a l l!31 uptake by the thyroid and an increase i n CR. The increased CR indicates that radiohormone production i s augmented, despite a decreased 1^31 uptake by the thyroid. The reduced accumulation of 1^31 i n the thyroid i s probably due to loss of r a d i o a c t i v i t y as hormone and would add support to the contention that muscular a c t i v i t y induces an increased demand f o r thyroxin. These findings agree with the general conclusions of Fontaine (1959h IX. THE INFLUENCE OF CHEMICAL CHANGES (SALINITY AND IODINE LEVELS OF THE MEDIA) ON I 1 ? 1 METABOLISM OF STEELHEAD AND CHUM SALMON (Oncorhynchus keta) Apart from the effects of physical aspects of the environ- ment on thyroid a c t i v i t y and the influence of body mass and possibly sexual maturation, there i s also the chemical environ- ment to consider i n these anadromous te l e o s t s . Hoar (1939) surveyed the chemical factors that possibly influence the thyroid and concluded that the s a l i n i t y and iodine concentration were most important. The influence of s a l i n i t y was p a r t i c u l a r l y pertinent here, i n view of the various theories that have been put forward to explain the r o l e of the thyroid i n a p o t e n t i a l migrant. The previous observations suggested the influence that various factors have on the thyroid, but i t i s now necessary to consider what t h i s increased thyroid a c t i v i t y means i n terms of - 75 - TABLE XII Analysis of covariance for the r e l a t i o n s h i p between various 1.131 parameters and time (hr) f o r parr held i n f a s t and slow currents Parameter Condition Slope df P F m xm % dose Calm - . 0 0 3 1 7 1 7.5** 4 .1* t o t a l , and body Torrent -.0084 j 48 CR Calm Torrent .0064 ] .0083 J 1 , and 1 48 1.4 12.8** F g = Variance r a t i o f o r slopes. F m = Variance r a t i o f o r adjusted means. * S i g n i f i c a n t difference between slopes (p*C . 0 5 ) . ** S i g n i f i c a n t difference between slopes ( p< . 0 1 ) . - 76 - metabolism. This involves consideration of the various thyroid roles suggested for a migrating f i s h : ( i ) Thyroxin stimulates c e r t a i n changes i n metabolism, such as s i l v e r i n g and s a l i n i t y preference that occur at s m o l t i f i c a t i o n (Fontaine, 1954; Baggerman, i 9 6 0 , 1963). ( i i ) A f i s h p r i o r to seaward migration tends to become demineralized. Such demineralization has been described f o r several species (reviewed by Hoar, 1959) and may be due to a v a r i e t y of factors including the increased production of metabolic water or the functioning of c e r t a i n s a l t elimination mechanisms p r i o r to the actual entrance to the sea. Olivereau ( i960) has shown a great increase i n adrenocortical volume at t h i s time of s m o l t i f i c a t i o n i n Salmo s a l a r . Hoar (1952, 1959) suggested that such changes i n the i n t e r n a l environment could promote increased metabolic demands r e s u l t i n g i n increased thyroid a c t i v i t y . ( i i i ) The f i n a l p o s s i b i l i t y stems from the demineralization described above. Namely, that under conditions of elimination of body s a l t s the a v a i l a b i l i t y of iodine to the thyroid could become reduced, e s p e c i a l l y i f the surrounding medium were d e f i c i e n t i n iodine. Under conditions such as these, the f i s h would not be i n iodine equilibrium. The thyroxin supply f o r the body would have to be produced despite progressively lower l e v e l s of available , Active transport mechanisms would be r e l i e d upon even more for building up the iodine gradient between thyroid and plasma. I f the gland were compensating f o r 127 low plasma I ' l e v e l s i n t h i s way then i t could appear active - 77 - by b o t h h i s t o l o g i c a l and r a d i o c h e m i c a l c r i t e r i a d e s p i t e t h e f a c t t h a t t h e a c t u a l o u t p u t o f hormone i n t o t h e p l a s m a had n o t r e a l l y i n c r e a s e d . I n o t h e r w o r d s , t h e h i g h t h y r o i d a c t i v i t y o f t h e s m o l t may be i n p a r t a n a r t i f a o t and m e r e l y a r e f l e c t i o n o f l o w a v a i l a b l e I 1 2 7 i n d u c e d by a r a p i d r a t e o f I 1 2 ^ e x c r e t i o n . T h i s 131 w o u l d be s u p p o r t e d by t h e d a t a s h o w i n g v e r y r a p i d I e l i m i n a - t i o n i n s t e e l h e a d s m o l t s and o t h e r s p e c i e s a t m i g r a t i o n ( L e l o u p a n d F o n t a i n e , i 9 6 0 ) . I n a n a t t e m p t t o e v a l u a t e t h e s e p o s s i b i l i t i e s f i s h were k e p t i n ( i ) f r e s h w a t e r , ( i i ) s e a w a t e r (250/00 s a l i n e ) a n d ( i i i ) f r e s h w a t e r c o n t a i n i n g t h e same amount o f r*-27 a s 25 0/00 s e a w a t e r (36 j i g / l ) . By c o m p a r i n g t h e f r e s h - w a t e r and s e a w a t e r , f i s h t h e n e t e f f e c t o f s e a w a t e r on t h e t h e t h y r o i d c o u l d be a s s e s s e d . By c o m p a r i n g f r e s h w a t e r w i t h I 1 2 ? a g a i n s t s e a w a t e r , t h e o s m o t i c i n f l u e n c e on t h e t h y r o i d c o u l d be i s o l a t e d s i n c e t h e i o d i n e c o n c e n t r a t i o n w o u l d be t h e same i n b o t h m e d i a . A s H i c k m a n (1959) h a s p o i n t e d o u t , t h e l e v e l s have t o be made 127 t h e same i n b o t h m e d i a s i n c e t h e l e v e l o f I ' c a n a l t e r 131 c o n s i d e r a b l y t h e m e t a b o l i s m o f I . F i n a l l y , c o m p a r i s o n o f f i s h i n f r e s h w a t e r w i t h f i s h i n f r e s h w a t e r c o n t a i n i n g i o d i n e m i g h t i n d i c a t e , by t h e r e s p o n s e o f t h e t h y r o i d , w h e t h e r t h e f i s h was l i v i n g i n i o d i n e d e f i c i e n c y . These t e s t s were c a r r i e d o u t on b o t h y e a r l i n g s ( p a r r ) and f i s h o f s m o l t i n g age ( s m o l t s and p a r r ) . Y e a r l i n g f i s h , a l l w i t h v e r y p r o n o u n c e d p a r r marks a n d no s i l v e r i n g , were p l a c e d i n t h e t h r e e m e d i a i n S e p t e m b e r a t a w a t e r t e m p e r a t u r e o f 1 3 . 6 ° C. T h e y s u r v i v e d t h e t r a n s i t i o n w h e t h e r - 78 - the introduction was d i r e c t or v i a intermediate s a l i n i t i e s . A f t e r 10 days they were injected with I 1 ^ 1 . Half of the f i s h were k i l l e d at 4 days; h a l f were k i l l e d at 8 days. A c u r v i l i n e a r r e l a t i o n s h i p f o r I 1 ^ 1 clearance was apparent i n f i s h from a l l three media. The i n i t i a l rates of loss of plasma I 1 ^ ! from f i s h held i n iodine deplete and iodine replete fresh water did not d i f f e r . In sea water, on the other hand, t h i s rate of loss was greater. In the second phase, the rates of loss were again s i m i l a r i n both fresh-water media but the sea water adapted f i s h showed a pronounced decrease i n the rate of plasma I ^ l removal (Fig. 26). A s i m i l a r difference between 131 fresh water and sea water was noted with I J elimination from the whole body. The more diphasic curves i n sea water suggest that there i s more than one mechanism of elimination i n the steelhead. Furthermore, since the c u r v i l i n e a r form was more pronounced i n sea water, i t i s possible that the extrarenal excretory pathway i s better developed i n t h i s medium. Possibly transfer to sea water increased general s a l t loss through the g i l l s and reduced further the r o l e of the kidney mechanism. The 127 addition of I ' into the water i t s e l f does not a f f e c t the loss of 1131. Addition of l l 2 7 appeared to reduce the thyroid I 1 3 1 uptake (TUF) by a small amount and r e l a t i v e to t h i s reduced a c t i v i t y i n iodine reinforced fresh water, the enhanced TUF values i n the sea water suggested that increased osmotic pressure may have stimulated the "iodine pump". The conversion r a t i o , however, showed no marked difference between the three conditions. - 19 - F i g . 26. E f f e c t of transfer of 20-month steelhead to sea water (25 o/oo saline) and iodide-reinforced fresh water on various aspects of I ^ ? 1 metabolism measured 4 and 8 days a f t e r I 1 * ! i n j e c t i o n . Mean values represented (SW,X ; FW + I 1 2 ' , A ; FW,0 ).  - 80 - In l a t e May the experiment was repeated at 8 . 5 ° 0 on po t e n t i a l migrants kept under the increasing photoperiod and increasing temperature regime. Unfortunately the temperature f e l l to a very low l e v e l (1° C) i n the iodine control tank (F.W. + I 1 2 7 ) and i n view of the temperature influence on I 1 ^ ! metabolism these data were discarded. Comparison could only be made, therefore, between fresh dechlorinated water and sea water (25 o/oo saline) (Fig. 27) . For comparison, smolts have been treated separtely from parr, though t h i s breakdown con- siderably reduced the sample size and made a f i n a l c r i t i c a l examination d i f f i c u l t . Certain important trends appear from the data. F i r s t , the extent of I 1 ? 1 l o s s , both t o t a l and extrathyroidal, was reduced i n sea water while the parameters measuring the a c t i v i t y of gland, T/S and CR were lower than i n fresh water. No difference between si l v e r e d or s i l v e r i n g f i s h and parr was noted. I t was concluded that introduction into sea water of p o t e n t i a l l y migrating f i s h (parr or smolt) possessing a high thyroid a c t i v i t y considerably lowered thyroid hyperfunction and also reduced 131 the former rapid rate of I excretion. It i s inferred either that s t a b i l i z a t i o n of plasma iodine at a higher l e v e l has inhi b i t e d the thyroid I 1 ^ 1 accumulating e f f i c i e n c y , or that sea water i t s e l f lowered thyroid a c t i v i t y . A t h i r d p o s s i b i l i t y i s that higher iodine l e v e l s i n themselves reduced thyroid a c t i v i t y but that t h i s trend was masked by the tendency f o r the increased osmotic pressure to stimulate a r e a l increase i n hormone output. Without controls i n iodine reinforced water - 81 - F i g . 27. E f f e c t o f t r a n s f e r o f 30-month s m o l t s a n d p a r r t o s e a w a t e r (23 0 / 0 0 s a l i n e ) on v a r i o u s a s p e c t s o f l l 3 1 m e t a b o l i s m . Mean v a l u e s ( b a r ) and i n d i v i d u a l v a l u e s shown. 1 2 «/> < _l Q. 5 - I 4 0 T / S 2 C f S M O L T S f 1 E 3 f f l P A R R • • 3 - 3 t9 o o o o T O T A L • • 3 - 3 t 8 ft o 8 8 sw O F W - 82 - T h i s i s s u e c a n n o t be r e s o l v e d . A t t h i s p o i n t i n t h e p r o c e e d i n g s t h e s t o c k s o f s t e e l h e a d o f m i g r a n t age were e x h a u s t e d so t h e e x p e r i m e n t c o u l d n o t be r e p e a t e d . Chum s a l m o n were a v a i l a b l e , h o w e v e r , i n a more o r l e s s i d e n t i c a l p h y s i o l o g i c a l c o n d i t i o n . By mid-summer u n d e r - y e a r l i n g chum s a l m o n ( w h i c h s h o u l d have m i g r a t e d i n t h e s p r i n g ) show v e r y h i g h r a t e s o f I * ^ 1 m e t a b o l i s m when r e t a i n e d i n f r e s h w a t e r . The b i o l o g i c a l h a l f - l i f e f o r t h e l o s s o f serum I 1 ^ ! d r o p s as l o w as 23 h o u r s ( E a l e s , 1961), w h i l e h i g h t h y r o i d u p - t a k e and t h e r a p i d p r o d u c t i o n o f v e r y h i g h CR v a l u e s (60% o r o v e r i n some c a s e s ) a l s o i n d i c a t e s a v e r y r a p i d I 1 ^ 1 m e t a b o l i s m v i a t h y r o i d r o u t e s . I t h a s been s u g g e s t e d b y H o a r (1932, 1959) and E a l e s (1961, 1963) t h a t t h e s e f i s h may show i n an e x a g g e r a t e d f o r m t h e t h y r o i d h y p e r a c t i v i t y u s u a l l y e x h i b i t e d i n a s m o l t i n g f i s h . I t was d e c i d e d t h e r e f o r e t o i n v e s t i g a t e t h e i n f l u e n c e o f s e a w a t e r and change i n i o d i n e c o n c e n t r a t i o n on t h e chum s a l m o n t h y r o i d . T w e l v e f i s h p o s s e s s i n g t h y r o i d a c t i v i t i e s o f t h i s k i n d were p u t i n t o e a c h o f t h e f o l l o w i n g : ( i ) s e a w a t e r (25 0 / 0 0 •. s a l i n e ) ( i i ) f r e s h w a t e r w i t h t h e same I 1 2 ^ c o n t e n t as 25 0 / 0 0 s e a w a t e r and ( i i i ) f r e s h w a t e r i t s e l f . The w a t e r t e m p e r a t u r e was 18° G . The f i s h were h e l d u n d e r t h e s e c o n d i t i o n s f o r 6 d a y s and t h e n i n j e c t e d w i t h I 1 ^ 1 and k i l l e d 108 h o u r s a f t e r i n j e c t i o n . A t t h i s t i m e t h e e x t r a t h y r o i d a l e x c r e t i o n and CR w e r e measured ( F i g . 2 8 ) . S t a b l e i o d i n e r e i n f o r c e m e n t , as w i t h y e a r l i n g s t e e l h e a d , d i d n o t a l t e r t h e I 1 ? ! r a t e o f e x c r e t i o n b u t l o w e r e d t h e CR - 83 - F i g . 28. E f f e c t o f t r a n s f e r o f u n d e r y e a r l i n g p o t e n t i a l m i g r a n t chum s a l m o n t o s e a w a t e r (25 o/oo s a l i n e ) and i o d i d e - r e i n f o r c e d f r e s h w a t e r . Mean v a l u e s ( b a r ) and i n d i v i d u a l v a l u e s shown Xs<.0.05; XX«=<0.01.  - 84 - greatly compared to the s t r i c t l y fresh-water s i t u a t i o n . In addition, s a l i n i t y depressed 1^31 loss r e l a t i v e to either fresh water or iodine reinforced controls. This suggests that the reduced 1^31 excretion observed i n sea water was due to the ov e r a l l osmotic e f f e c t and not due to high ambient I 3 - 2 7 . The conversion r a t i o i n sea water was less than i n fresh water although the influence was not as pronounced as with the migrant steelhead. In these data on chum, the eff e c t s of iodine and osmotic change become much clearer. I t appears that increased iodine l e v e l s caused a reduction i n the CR. The hyperactivity of the thyroid as measured i n fresh water by radiochemical methods 127 could be abolished by a high ambient I l e v e l , even though the rate of iodine elimination from the body had not been altered. In sea water, however, thyroid a c t i v i t y was increased over the iodine controls and t h i s revealed response to the osmotic change. As with steelhead yearlings, there was d e f i n i t e stimulation of the thyroid i n a medium of high s a l t content. I f the migrant steelhead responded s i m i l a r l y to the chum i n the iodine reinforced medium, i t would be concluded that (i ) the high l e v e l of 1^31 loss i s reduced when the steelhead moves into the sea ( i i ) the high thyroid a c t i v i t y i s p a r t l y due to a state of iodine deficiency and ( i i i ) when the f i s h enters the sea i t s thyroid may again be activated due to the increased s a l i n i t y . Whether the change from fresh to sea water produces an o v e r a l l increase or decrease i n thyroid a c t i v i t y would be due to several factors prominent among which would be the blood - 85 - l e v e l o f I 1 2 * ? a t t h e t i m e o f t r a n s i t i o n and a l s o t h e r e s p o n s e t o t h e s a l t c o n c e n t r a t i o n . T h u s , a l t h o u g h t h e n e t r e s u l t o f t r a n s f e r f r o m f r e s h w a t e r t o s e a w a t e r may he a d r o p i n t h y r o i d a c t i v i t y , a n a l y s i s o f t h e i o d i n e a n d s a l i n i t y i n f l u e n c e s g e n e r a l l y show a d e p r e s s i o n i n t h y r o i d a c t i v i t y due t o t h e i n c r e a s e i n I ^ 2 ^ i e v e i D U t a s t i m u - l a t i o n due t o t h e s a l i n i t y e l e v a t i o n . I f t h e t r a n s i t i o n i n v o l v e s a n e t i n c r e a s e i n t h y r o i d a c t i v i t y t h e n i t c a n he a r g u e d t h a t t h e s t i m u l a t o r y e f f e c t o f s a l t w a t e r o u t w e i g h s t h e r e s p o n s e due t o t h e i n c r e a s e d i o d i n e l e v e l . The two g r o u p s o f s t e e l h e a d w o u l d show, t h e r e f o r e , some d i f f e r e n c e i n t h i s r e s p e c t . I n t h e y e a r l i n g s , t h e r e was v e r y l i t t l e r e s p o n s e t o e i t h e r medium h u t t h e i o d i n e s l i g h t l y d e - p r e s s e d T / S r a t i o s and t h e s e a w a t e r c a u s e d s l i g h t s t i m u l a t i o n . I n t h e s m o l t s , h o w e v e r , t h e n e t r e s u l t was a d e p r e s s i o n o f t h y r o i d a c t i v i t y by a l l p a r a m e t e r s and a m a r k e d t h y r o i d i n h i b i t i o n b y h i g h i o d i n e l e v e l s i s s u g g e s t e d . T h i s i m p l i e s t h a t t h e h i g h t h y r o i d a c t i v i t y i n t h e s m o l t i s p a r t l y due t o a n i o d i n e d e f i c i e n c y . X . THE RELATIONSHIP BETWEEN RADIOIODINE METABOLISM AND SILVERING T h y r o i d h y p e r f u n c t i o n i n j u v e n i l e s a l m o n i s c o r r e l a t e d w i t h s i l v e r i n g and s m o l t i f i c a t i o n . Though c e r t a i n e x p e r i m e n t s c o n f i r m t h i s b e l i e f , t h e r e i s c o n s i d e r a b l e d i s a g r e e m e n t r e g a r d i n g t h e r o l e o f t h y r o x i n . A s n o t e d p r e v i o u s l y , w h i l e t h y r o x i n may s t i m u l a t e s u b e p i d e r m a l g u a n i n e d e p o s i t i o n i n t h e l a b o r a t o r y i t may n o t be r e q u i r e d u n d e r n a t u r a l c o n d i t i o n s . T h i s seemed t o be i n d i c a t e d by c e r t a i n d a t a o b t a i n e d i n t h i s i n v e s t i g a t i o n . I t - 86 - was noted that f i s h kept under an 8-hour daylength and at a temperature of 5 "to 6° C showed no seasonal increase i n thyroid function either with radioiodine or h i s t o l o g i c a l methods and yet certain of them s i l v e r e d . Between the period 27 A p r i l to 29 May when investigations of thyroid function were being made of smolting f i s h from the four photoperiod and temperature regimes outlined i n Table 711, a record was made of the state of smolti- f i c a t i o n of each f i s h (Table X I I I ) . Of the 4 categories (smolts, parr-smolts, parr and sexually mature males) considered, s m o l t i f i c a t i o n was not favoured by any one environmental regime. It i s s t i l l possible that the smolts under any one condition might tend to have a higher thyroid a c t i v i t y than the parr under the same condition. To investigate t h i s more f u l l y , two-year-olds from the increasing photoperiod and low temperature regime were considered i n greater d e t a i l as these constituted a larger sample of examined f i s h . Each aspect of 1^31 metabolism was treated separately i n r e l a t i o n to the state of s m o l t i f i c a t i o n (Pig. 29). I t i s concluded that there 131 was no consistent trend r e l a t i n g any aspect of I metabolism to s i l v e r i n g , and the causal r e l a t i o n s h i p between thyroxin and subepidermal guanine deposition i s therefore questioned. Of further i n t e r e s t was the constancy between the 4 holding conditions i n the t o t a l number of steelhead showing any tendency to s i l v e r (smolts + parr-smolts). This was always between 65 and 75% of the t o t a l number examined i n any one condition (Table X I I I ) . According to Maher and Larkin (1954), approximately 60% of young steelhead trout from the Chilliwack River, B r i t i s h Columbia, - 87 - TABLE XIII The e f f e c t of d i f f e r e n t temperature and photoperiod regimes on the percentage incidence of s m o l t i f i c a t i o n i n two-year-old steelhead between 27 A p r i l and 29 May, 1°62 HIGH TEMPERATURE LOW TEMPERATURE Photoperiod Photoperiod Photoperiod Photoperiod increasing 8 hours increasing 8 hours No. i No. % No. % No. % Smolts (no parr marks) 24 40 7 28 39 58 7 30 Parr-smolts Silver, but with parr marks) 18 30 9 36 13 19 11 48 Parr (no s i l v e r 10 17 7 28 10 15 3 13 Sexually mature male parr 8 13 2 8 5 8 2 9 TOTALS 6o 100 25 100 67 100 23 100 - 88 - F i g . 29. D e m o n s t r a t i o n o f t h e l a c k o f a r e l a t i o n s h i p between s i l v e r i n g and any a s p e c t o f 1131 m e t a b o l i s m (8 d a y s a f t e r i n j e c t i o n ) . Mean v a l u e s ( b a r ) . a n d i n d i v i d u a l v a l u e s shown.  - 89 - migrate as two-year-olds at an average length of 16.49 cm. Both the length of the migrating f i s h and the percentage showing s m o l t i f i c a t i o n agreed favourably with the data from the laboratory-held f i s h considered above. XI. PERIPHERAL SITES OF RADIOHORMONE CATABOLISM Any attempt to c l a r i f y the r o l e of thyroxin i n teleosts w i l l depend on knowledge of i t s s i t e of action. So f a r the target organs of thyroxin i n f i s h are unknown. One method would be to in j e c t a sample of radioactive hormone and then follow i t s route into the various tissues. No pure radioactive thyroxin was avai l a b l e i n the present study and so a les s d i r e c t technique was employed. When I - ^ l i s injected, the inorganic ion ( I ^ l ) very quickly reaches an equilibrium between the various body compartments. Thus as f a r as inorganic r*"^ i s concerned, the r a t i o of tissue count/min/g s h o u l d r e m a i n constant f o r as long as there blood count/min/g i s I 1 ^ ! i n the body. Owing to the r e l a t i v e l y rapid d i f f u s i o n of the ion, departure from t h i s r a t i o would only be noted i n those tissues such as the thyroid which a c t i v e l y concentrate iodide. It may also be argued that since 1^31 i s the predominant radio- active constituent i n the blood that the same r a t i o should hold between the t o t a l r a d i o a c t i v i t y i n the tissues and blood, i . e . tissue I ^ 1 count/min/g t o t a l tissue r a d i o a c t i v i t y count/min/g blood I 131 count/min/g " t o t a l blood r a d i o a c t i v i t y count/min/g I f t h i s r a t i o based on t o t a l r a d i o a c t i v i t y i n tissue and blood should change, i t would imply a change between blood and tissue - 90 - of an organic 1^1 f r a c t i o n . A r i s e i n the r a t i o would mean accumulation of organically bound i n the tissues - pre- sumably an accumulation of radiohormone. Thus by measuring the r a t i o of r a d i o a c t i v i t y of tissue to blood at various times 131 a f t e r I ' i n j e c t i o n some ind i c a t i o n might be gained of s i t e s of peripheral metabolism of radiohormone. This was the t h e o r e t i c a l basis for the experiment carried out below i n which 48 f i s h (10-40 g) were injected with 4 uc l i 3 1 and one-third of t h i s t o t a l k i l l e d at 3, 6 and 9 days. From each f i s h a small whole blood sample was removed i n the usual manner and blown into a tared planchet. The planchet was reweighed and the whole blood digested f o r 2 days with one ml of 2N NaOH and counted at a distance of 4 cm from the end-probe s c i n t i l l a t i o n counter f o r 3,000 counts. Weighed pieces of g i l l , kidney, skin, l i v e r , gut, brain and muscle were digested and counted i n a s i m i l a r manner. In each f i s h the r a d i o a c t i v i t y per gram tissue was expressed r e l a t i v e to the r a d i o a c t i v i t y per gram blood. At each time period, 16 values fo r each r a t i o were obtained and means calculated (IPig. 30). With the exception of the g i l l t i ssue, there was l i t t l e change i n the r a t i o between 3 and 6 days. No significance i s placed on the absolute l e v e l s of r a d i o a c t i v i t y i n the various tissues as t h i s was undoubtedly due to greater va s c u l a r i t y of the tissue - i f the tissue were completely vascular i t would possess a r a t i o of exactly 1. Any increase i n r a d i o a c t i v i t y above 1 means an active accumulation of iodine from the blood. This i s found only i n the g i l l t i s s u e . For other tissues, the constancy of the r a t i o i n general between 3 and 6 days would bear out the i n i t i a l - 91 - F i g . 30. Change i n the ti s s u e : blood r a d i o a c t i v i t y i n a variety of tissues from 19-month steelhead. Each point represents a mean of 14- to 16 individuals.  - 92 - assumption that iA^l moves ra p i d l y between body compartments and provides support f o r the suggestion that any l a t e r change i n the r a t i o would be due to change i n d i s t r i b u t i o n of organic f r a c t i o n s , which even at 6 days are only present i n small quantities i n the blood (Figs. 5 and 16). The change i n the r a t i o i n the g i l l t i s s u e i s i n t e r e s t i n g and, i n view of the small amount of P B I131 usually encountered i n blood at 6 days, presumably involves an accumulation of This was also found by Hickman (1959) i n the starry flounder and i t was concluded by Hickman that the high I - ^ l concentration there could be reconciled with the known function of the g i l l s i n s a l t excretion. However, while the data of Hickman showed a steady r a t i o between g i l l and blood 24 hours a f t e r i n j e c t i o n , these data on steelhead indicate a progressive accumulation of r a d i o a c t i v i t y i n the g i l l with time. No s a t i s f a c t o r y explanation for t h i s phenomenon can be given, unless the l a t e r high values were due to accumulation of i n a f o m other than the f r e e l y d i f f u s i b l e ion. The tissue:blood r ^ l r a t i o changed between 6 and 9 days i n a l l tissues, but was consistently most pronounced i n the very metabolically active tissues, i . e . brain, gut, l i v e r and kidney. Skin showed only a s l i g h t percentage increase and muscle only a moderate percentage change from 6 to 9 days (Figs. 30 and 31). That the r i s e i n the tissue:blood r a t i o between 6 and 9 days i s due to an accumulation of radiothyroxin i s borne out by the observation that over t h i s period, detectable amounts of are released from the gland (Figs. 5 and 16). - 93 - F i g . 31. P e r c e n t a g e change ( r e l a t i v e t o t h e v a l u e 3 d a y s a f t e r i n j e c t i o n ) o f t i s s u e : b l o o d r a t i o s 6 and 9 d a y s a f t e r i n j e c t i o n i n 19-month s t e e l h e a d . 6 DAYS - 94 - It i s therefore concluded that i f change i n t o t a l tissue count/min/g blood count/min/g i s i n d i c a t i v e of build up of radioactive hormone i n the t i s s u e , then the highest accumulation was observed i n brain, l i v e r , kidney and gut. In a l l these tissues energy demands are probably high. In less active tissues such as muscle and skin, the P B I 1 ^ accumulation was l e s s . This suggests that the thyroid hormone i s not u t i l i z e d i n s p e c i f i c areas but probably required by any metabolically active system. It would argue against a speci- f i c a l l y l o c a l i z e d r o l e of the thyroid i n teleosts and implies a general metabolic r o l e . - 95 - DISCUSSION I . COMPARISON OF PARAMETERS FOR MEASURING THYROID A C T I V I T Y AMD THE EFFECT OF TEMPERATURE ON THESE PARAMETERS S e v e r a l d i s c r e p a n c i e s between t e c h n i q u e s f o r m e a s u r i n g t h y r o i d a c t i v i t y may be c i t e d . S w i f t (1959), s i m u l t a n e o u s l y u s i n g h i s t o l o g y and r a d i o c h e m i s t r y t o measure s e a s o n a l change i n t h y r o i d a c t i v i t y i n brown t r o u t , d e m o n s t r a t e d t h a t t h e two t e c h n i q u e s were n o t n e c e s s a r i l y c o r r e l a t e d . O l i v e r e a u (1955) showed t h a t h i g h t e m p e r a t u r e c a u s e d no h i s t o l o g i c a l change i n t h e e e l , b u t L e l o u p and F o n t a i n e ( i960) h a v e shown a v e r y s i g n i f i c a n t s t i m u l a t i o n o f t h e t h y r o i d by t e m p e r a t u r e w i t h I131 t e c h n i q u e s . E a l e s (1963) o b t a i n e d some c o n f o r m i t y between h i s t o l o g i c a l and r a d i o c h e m i c a l methods i n two s p e c i e s o f j u v e n i l e O n c o r h y n c h u s . coho and s o c k e y e , b u t i n two o t h e r s p e c i e s , p i n k and chum t h e r e was a c o m p l e t e d i s a g r e e m e n t . O t h e r i n s t a n c e s o f d i s a g r e e m e n t have b e e n c i t e d by M a t t y ( i 9 6 0 ) . I t i s e m p h a s i z e d t h a t i n g e n e r a l t h e s e d i s c r e p a n c i e s r e l a t e e i t h e r t o c o m p a r i s o n s o f t h y r o i d a c t i v i t y a t d i f f e r e n t t e m p e r a t u r e s o r t o s e a s o n a l changes p r o b a b l y i n d u c e d by t e m p e r a t u r e d i f f e r e n c e s . T h a t d i s a g r e e m e n t between h i s t o l o g i c a l and r a d i o i o d i n e measurements may o n l y o c c u r when t e m p e r a t u r e i s t h e m a i n v a r i a b l e i s v e r y i m p o r t a n t f o r t h e e x p l a n a t i o n t h a t f o l l o w s . I n t h e p r e s e n t s t u d y , e s t i m a t i o n o f t h y r o i d a c t i v i t y b a s e d on p e r c e n t a g e l ! 3 1 a c c u m u l a t i o n , c a n be e n t i r e l y e l i m i n a t e d f r o m f u r t h e r c o n s i d e r a t i o n as i t does n o t a l l o w f o r change i n e x t r a - t h y r o i d a l I 1 3 1 l o s s . T h i s d i s a d v a n t a g e i s e l i m i n a t e d by TUF and T/S w h i c h show c l o s e c o r r e s p o n d e n c e b o t h i n s e a s o n a l d a t a - 96 - a n d i n e x p e r i m e n t a l t e m p e r a t u r e t r e a t m e n t s . The same i s t r u e o f t h e CR w h i c h c o n f o r m s w i t h t h e TUF and T/S i n d e x . Thus t h e b e h a v i o u r o f r a d i o i o d i n e a t v a r i o u s l e v e l s o f t h y r o i d a c t i v i t y seems c o n s i s t e n t b u t i t does n o t n e c e s s a r i l y c o r r e l a t e w i t h t h e h i s t o l o g i c a l o b s e r v a t i o n s . T h i s i s s t r i k i n g l y e v i d e n t where t h e e x p e r i m e n t on t e m p e r a t u r e i s c o n c e r n e d ( F i g s . 8 t o 11) . The CR was c o r r e l a t e d p o s i t i v e l y w i t h t e m p e r a t u r e w h i l e t h e c e l l h e i g h t a s s e s s m e n t s showed a n e g a t i v e c o r r e l a t i o n . The c o l l o i d c o l o u r showed a p o s i t i v e c o r r e l a t i o n s i m i l a r t o t h e 1^31 d a t a . C o m p a r a b l e d i s c r e p a n c i e s were d e m o n s t r a t e d i n t h e s e a s o n a l d a t a , e s p e c i a l l y i n l a t e summer. The m a i n q u e s t i o n t o be r e s o l v e d i s why t h e r a d i o c h e m i c a l d a t a a n d c o l l o i d d a t a s h o u l d show i n c r e a s e i n t h y r o i d a c t i v i t y w i t h r i s i n g t e m p e r a t u r e and t h e c e l l h e i g h t d e c r e a s i n g a c t i v i t y w i t h r i s i n g t e m p e r a t u r e . I s i t p o s s i b l e t o r e c o n c i l e t h e s e two c o n f l i c t i n g o b s e r v a t i o n s , b e a r i n g i n m i n d t h a t w i t h r e s p e c t t o o t h e r e n v i r o n m e n t a l i n f l u e n c e s , c e l l h e i g h t a n d r a d i o i o d i n e a s s e s s m e n t s g e n e r a l l y a g r e e ? The f o l l o w i n g a t t e m p t t o do t h i s , r e s t s on 3 a s s u m p t i o n s - ( i ) c e l l h e i g h t i s c o n t r o l l e d s o l e l y b y TSH ( i i ) a s s u m i n g no c o n t r o l f r o m h i g h e r n e r v o u s c e n t r e s , t h e r e l e a s e o f TSH i s c o n t r o l l e d by a f e e d b a c k mechans im t h a t i s d e p e n d e n t upon t h e b l o o d hormone ( t h y r o x i n and p r o b a b l y t r i i o d o t h y r o n i n e l e v e l s ) a n d ( i i i ) t h a t w i t h i n c r e a s i n g t e m p e r a t u r e t h e r e i s a g r e a t e r need by t h e t i s s u e s f o r t h y r o x i n . The l a t t e r i s n o t a n u n - r e a s o n a b l e a s s u m p t i o n i f t h y r o x i n i s i n v o l v e d i n some b a s i c a s p e c t o f c e l l u l a r m e t a b o l i s m whose r a t e i n c r e a s e s w i t h t e m p e r a t u r e . - 97 - C o n s i d e r a f i s h a c c l i m a t e d t o a l o w t e m p e r a t u r e . I t i s a p o i k i l o t h e r m i c a n i m a l and t h e r a t e c o n s t a n t s f o r many b i o c h e m i c a l r e a c t i o n s w o u l d t e n d t o be l o w . A s t h e t e m p e r a t u r e i s r a i s e d s o - i s t h e t h y r o x i n r e q u i r e m e n t . Due t o t h e p o s t u l a t e d i n c r e a s e d p e r i p h e r a l m e t a b o l i s m o f h o r m o n e , t h e b l o o d l e v e l s w o u l d be d e p l e t e d and t h e o u t p u t o f TSH augmented v i a t h e f e e d b a c k . The c e l l h e i g h t w o u l d become g r e a t e r a n d so w o u l d t h e a c t i v i t y o f t h e g l a n d as m e a s u r e d r a d i o c h e m i c a l l y . H o w e v e r , i n a p o i k i - l o t h e r m o n e must assume t h a t r a t e c o n s t a n t s f o r a l l t h e body r e a c t i o n s w i l l be i n c r e a s e d by r i s i n g t e m p e r a t u r e a n d , r a t e c o n s t a n t s d e t e r m i n i n g t h e r a t e o f o p e r a t i o n o f t h e t h y r o i d w i l l a l s o be s t i m u l a t e d . I f t h e s t i m u l a t o r y e f f e c t on t h e p e r i p h e r a l c a t a b o l i s m o f t h y r o x i n were s i m u l t a n e o u s l y b a l a n c e d b y a n i d e n t i c a l s t i m u l a t i o n o f s y n t h e s i s , t h e n t h e r e w o u l d be no f a l l i n b l o o d l e v e l s and no i n c r e a s e d r e l e a s e o f TSH f r o m t h e p i t u i t a r y . I f t h e same r e l a t i o n s h i p w e r e t o h o l d o v e r t h e e n t i r e t e m p e r a t u r e r a n g e t h e n t h e i n c r e a s i n g t i s s u e demands f o r t h y r o x i n w o u l d be c o m p l e t e l y compensated by t h e s t i m u l a t i o n o f i t s s y n t h e s i s . Thus one w o u l d e x p e c t no change i n h i s t o l o g y w i t h t e m p e r a t u r e b u t a marked s t i m u l a t i o n where r a d i o c h e m i c a l t e c h n i q u e s w e r e c o n c e r n e d . A s O l i v e r e a u (1955a,b,c) h a s shown t h e f o l l o w i n g s p e c i e s r e v e a l no h i s t o l o g i c a l change t o t e m p e r a - t u r e - C y p r i n u s c a r p i o , T i n e a v u l g a r i s , A n g u i l l a a n g u i l l a , M u g i l a u r a t u s and S c y I l i u m c a n i c u l a . I n a t l e a s t one o f t h e s e s p e c i e s , A n g u i l l a a n g u i l l a , h o w e v e r , L e l o u p and F o n t a i n e (i960) h a v e shown b y I - ^ l methods a n e x t r e m e l y m a r k e d p o s i t i v e r e s p o n s e t o r i s i n g t e m p e r a t u r e . A l l t h e s e e x a m p l e s w o u l d c o n f o r m , t h e r e f o r e , - 98 - w i t h t h e above t h e o r y t h a t t h y r o x i n p r o d u c t i o n and c o n s u m p t i o n r e s p o n d t o t e m p e r a t u r e t o t h e same d e g r e e . Though s u c h a n i d e a l and e c o n o m i c a l t e m p e r a t u r e c o n t r o l o f i o d i n e m e t a b o l i s m may o p e r a t e , i t need n o t h o l d o v e r t h e e n t i r e t e m p e r a t u r e r a n g e , n o r f o r a l l s p e c i e s . F o r i n s t a n c e , t h e r e i s e v i d e n c e t o s u g g e s t t h a t t h y r o x i n demands may be g r e a t e r t h a n t h y r o x i n o u t p u t a t l o w t e m p e r a t u r e i n g o l d f i s h ( H o a r , 1958, 1959; H o a r and S a l e s , 1965). A l s o t h e d a t a on S a l m o g a i r d n e r i i m p l y more TSH p r o d u c t i o n a t l o w t e m p e r a t u r e s when t h e c e l l h e i g h t i s g r e a t e s t . T h i s i n d i c a t e s t h a t t h e enzymes c o n t r o l l i n g t h e t h y r o x i n p r o d u c t i o n and s e c r e t i o n a r e i n h i b i t e d more b y t h e t e m p e r a t u r e r e d u c t i o n t h a n t h o s e g o v e r n i n g i t s p e r i p h e r a l c a t a - b o l i s m . U n d e r t h e s e c o n d i t i o n s t h e serum l e v e l o f t h y r o x i n w o u l d d r o p , TSH w o u l d be r e l e a s e d and c e l l h e i g h t s s t i m u l a t e d . T h i s h a s b e e n h y p o t h e t i c a l l y r e p r e s e n t e d ( F i g . 5 2 ) . F r o m t h i s f i g u r e , a t 5° C, a h i g h c e l l h e i g h t ( p r o p o r t i o n a l t o x^) m i g h t be e x p e c t e d s i n c e , due t o t h e e f f e c t o f t e m p e r a t u r e a l o n e , t h e l e v e l o f hormone p r o d u c t i o n i s c o n s i d e r a b l y l e s s t h a n i t s u s e . A t 10° C, h o w e v e r , b o t h o u t p u t and p e r i p h e r a l u t i l i z a t i o n have i n c r e a s e d , b u t t o d i f f e r e n t d e g r e e s . U n d e r t h e s e c o n d i t i o n s t h y r o x i n w o u l d b u i l d up i n t h e p l a s m a , and TSH p r o d u c t i o n w o u l d be i n h i b i t e d w i t h a r e s u l t i n g r e d u c t i o n i n c e l l h e i g h t ( p r o p o r t i o n a l t o XIQ). Thus one w o u l d e x p e c t a g r e a t e r c e l l h e i g h t a t l o w e r t e m p e r a t u r e s , b e c a u s e t h e t h y r o x i n p r o d u c i n g s y s t e m i s i n h i b i t e d more t h a n t h e t h y r o x i n u t i l i z a t i o n s y s t e m . The e n s u i n g l o w b l o o d t h y r o x i n l e v e l s w o u l d c a u s e a p r o d u c t i o n o f TSH and i n c r e a s e d c e l l h e i g h t . I n o t h e r s p e c i e s i t i s p o s s i b l e t h a t b l o o d t h y r o x i n l e v e l s m i g h t - 99 - F i g . 32. Diagrammatic representation of the hypothesis that thyroxin u t i l i z a t i o n and production respond d i f f e r e n t - i a l l y to temperature. X5 and X I Q represent the differences between u t i l i z a t i o n and production at 5 and 10° C. Since Xcj i s greater than X ± Q i t would imply greater release of TSH at low temperatures and also a greater c e l l height.  - 100 - d r o p a t h i g h e r t e m p e r a t u r e s . T h e s e s p e c i e s w o u l d show i n c r e a s i n g c e l l h e i g h t w i t h i n c r e a s i n g t e m p e r a t u r e a s d e m o n s t r a t e d by P h o x i n u s p h o x i n u s ( B a r r i n g t o n a n d M a t t y , 1954). The above h y p o t h e s i s may a l s o a c c o u n t f o r t h e a n o m o l y r e p o r t e d by H o a r and E a l e s (1965). They showed t h a t a l t h o u g h t h y r o i d a c t i v i t y measured r a d i o c h e m i c a l l y was much l o w e r a t l o w t e m p e r a t u r e s , TSH was s t i l l o f i m p o r t a n c e i n p r e v e n t i n g c o l d d e a t h i n g o l d f i s h . T h i s c o u l d be e x p l a i n e d by a s s u m i n g t h a t a t l o w t e m p e r a t u r e s ( i ) t h y r o x i n i s needed i n a d e q u a t e amounts a n d ( i i ) due t o a d i f f e r e n t e f f e c t o f c o l d on t h y r o x i n p r o - d u c t i o n and p e r i p h e r a l m e t a b o l i c s i t e s , t h y r o x i n was b e i n g removed f r o m t h e b l o o d a t a g r e a t e r r a t e t h a n i t was b e i n g r e p l e n i s h e d . I n a d d i t i o n , H o c h a c h k a and H a y e s (19&2) and H o c h a c h k a (1962) have shown t h a t t h y r o x i n t r e a t m e n t d i r e c t s c a r b o h y d r a t e m e t a b o l i s m v i a t h e p e n t o s e s h u n t i n S a l v e l i n u s f o n t i n a l i s . T h e y have shown a l s o t h a t , a t l o w t e m p e r a t u r e s , t h e p e n t o s e p h o s p h a t e s h u n t becomes more p r o m i n e n t . T h i s s u g g e s t s t h e r e may a l s o be e x t r a t h y r o x i n demands a t l o w t e m p e r a - t u r e s i n g o l d f i s h (Hoar and E a l e s , 1963). U n d e r t h e s e c o n d i t i o n s a n y t h i n g t h a t s t i m u l a t e d t h e a c t i v i t y o f t h e t h y r o i d w o u l d enhance s u r v i v a l and t h e a c t i o n o f TSH i s i m m e d i a t e l y u n d e r - s t a n d a b l e . B o t h c e l l h e i g h t and r a d i o i o d i n e a r e i m p o r t a n t i n e s t i m a t i n g t h y r o i d a c t i v i t y u n d e r d i f f e r e n t t e m p e r a t u r e r e g i m e s . W h i l e t h e j l 3 1 c o n s i s t e n t l y a l l o w s a dynamic a n a l y s i s o f t h y r o i d a c t i v i t y , t h e c e l l h e i g h t i s d e p e n d e n t on TSH s e c r e t i o n . T h i s TSH s e c r e t i o n may be s t i m u l a t e d v i a t h e h y p o t h a l a m i c o - h y p o p h y s e a l a x i s o r by t h e p l a s m a f e e d b a c k m e c h a n i s m . I n t h e l a t t e r c a s e t h i s c a n g i v e - 101 - r i s e t o d i s c r e p a n c y b e t w e e n h i s t o l o g i c a l a n d r a d i o i o d i n e c r i t e r i a . A l t h o u g h t h e h i s t o l o g i c a l change may measure r e l a t i v e d i f f e r e n c e s b e t w e e n hormone p r o d u c t i o n and c o n s u m p t i o n i t c a n be u n r e l i a b l e f o r a s s e s s i n g t h e g e n e r a l a c t i v i t y o f t h e g l a n d . Why t h e c o l l o i d s h o u l d show changes p a r a l l e l i n g t h e r a d i o - c h e m i c a l a s s e s s m e n t s r a t h e r t h a n c e l l h e i g h i s n o t c l e a r . P r o m what i s now known o f t h y r o x i n b i o s y n t h e s i s , i t a p p e a r s t h a t a p a r t f r o m t h e i n i t i a l u p t a k e o f i o d i n e t h e r e m a i n d e r o f t h e m e t a b o l i c p a t h w a y i s p u r s u e d i n t h e c o l l o i d i t s e l f (Gorbman and B e r n , 1962). The r o l e o f t h e c e l l s i s u n c e r t a i n b u t t h e y p r o b a b l y p r o d u c e t h e c o l l o i d t h y r o g l o b u l i n s . S i n c e t h e c o l l o i d a p p e a r s t o be t h e m a i n c e n t r e f o r i o d i n a t i o n o f t y r o s i n e and r e l e a s e o f t h e h o r m o n e , i t i s p e r h a p s n o t s u r p r i s i n g t h a t i t s s t a t e s h o u l d c o n f o r m more c l o s e l y w i t h t h e r a d i o c h e m i c a l e s t i m a t e s . F i n a l l y , I e m p h a s i z e t h a t t h e e f f e c t o f t e m p e r a t u r e on i o d i n e m e t a b o l i s m i s p r o b a b l y u n i q u e among a l l t h e f a c t o r s c o n s i d e r e d . I t may be t h e o n l y e n v i r o n m e n t a l f a c t o r t h a t d i r e c t l y a f f e c t s t h e t h y r o i d t i s s u e . A l l t h e r e m a i n i n g f a c t o r s - s e x u a l m a t u r i t y , p h o t o p e r i o d and s a l i n i t y m i g h t be e x p e c t e d t o a c t more i n d i r e c t l y , p r e s u m a b l y by c h a n g i n g TSH l e v e l s e i t h e r v i a t h e f e e d b a c k o r v i a t h e h y p o t h a l a m i c o - h y p o p h y s e a l a x i s . T h u s , i n a l l i n s t a n c e s e x c e p t t e m p e r a t u r e s t i m u l a t i o n , one w o u l d e x p e c t agreement between h i s t o l o g i c a l and I ^ l c r i t e r i a . I n g e n e r a l t h e l i t e r a t u r e r e v e a l s t h i s t o be t r u e . B e f o r e c o n c l u d i n g t h e d i s c u s s i o n o n t e m p e r a t u r e , r e f e r e n c e must be made t o t h e w o r k o f S w i f t (1955, 1959) who c o n c l u d e d t h a t i n brown t r o u t (Salmo t r u t t a ) ( i ) r a d i o i o d i n e a s s e s s m e n t s - 102 - of thyroid a c t i v i t y p a r a l l e l e d the c e l l height measurements and ( i i ) thyroid a c t i v i t y was inversely related to temperature. These conclusions d i r e c t l y oppose my own and since they are derived from-:a species very cl o s e l y r e l a t e d to the steelhead they require closer examination. Swift's conclusions based on c e l l height could be unreliable indications of thyroid a c t i v i t y i f the thesis developed here on temperature influence i s correct. Furthermore, Swift based his 131 131 I ' estimates on the rate of loss of I ' from the head region between 6 and 72 hours. Yet, from my studies on the steelhead thyroid, maximal thyroid uptake of I ^ ^ l ± 3 only achieved at 6 days. This i s also the time at which detectable amount of p B I 1 3 1 f i r s t appear i n the blood. It i s possible that Swift 131 was measuring predominantly rate of extrathyroidal I ' l o s s , from the vascular head region and f o r t h i s reason not measuring thyroid a c t i v i t y at a l l . I I . FACTORS CONTRIBUTING TO SEASONAL CHANGES IH THYROID ACTIVITY IN JUVENILE STEELHEAD Seasonal change i n thyroid a c t i v i t y has been interpreted s o l e l y i n terms of radiochemical data. The important influence of temperature has been discussed at considerable length, but s i z e , sexual maturation, photoperiod, iodine l e v e l of the environ- ment and s a l i n i t y also modify thyroid a c t i v i t y . S ize: The effect of s i z e on I - ^ l metabolism agrees with the work of Hickman (1959) and Wiggs (1°62) and indicates a more rapid rate of radioiodine metabolism i n smaller f i s h . That - 103 - t h e i n c r e a s e d m e t a b o l i s m i s due t o t h e s m a l l s i z e i t s e l f a n d n o t due t o a p h a s e o f i n c r e a s e d g r o w t h i s i n d i c a t e d by s e v e r a l f a c t o r s . P r o m i n e n t among t h e s e i s t h e q u a l i t a t i v e c o n f o r m i t y o f t h e body mass a n d t h y r o i d d a t a t o t h e d o u b l e l o g a r i t h m i c r e l a - t i o n s h i p o f body mass and t i s s u e m e t a b o l i s m . T h i s w o u l d s u g g e s t t h a t t h e t h y r o i d may have a v e r y g e n e r a l r o l e i n m e t a b o l i s m a n d be r e l a t e d t o t h e h i g h e r 0£ demands o f s m a l l e r f i s h . T h i s h a s b e e n d e m o n s t r a t e d i n P l a t i c h t h y s s t e l l a t u s b y H i c k m a n (1959). F u r t h e r e v i d e n c e a g a i n s t t h e h i g h e r t h y r o i d a c t i v i t y o f s m a l l f i s h b e i n g a s s o c i a t e d w i t h p e r i o d s o f more a c t i v e g r o w t h i s t h e s i m i l a r age o f s m a l l a n d l a r g e f i s h u s e d i n t h e s e s t u d i e s . T h i s w o u l d mean t h a t t h e s m a l l f i s h were s t u n t e d r e l a t i v e t o t h e l a r g e ones and h a d a s l o w e r g r o w t h r a t e . I f s t u n t e d f i s h had a h i g h e r t h y r o i d a c t i v i t y t h a n more r a p i d l y g r o w i n g f i s h , t h i s w o u l d c o n t r a d i c t t h e f i n d i n g s o f B a r r i n g t o n (19&3) t h a t t h y r o x i n s t i m u l a t e s g r o w t h i n Sa lmo g a i r d n e r i . I t l e n d s f u r t h e r s u p p o r t t o t h e i d e a t h a t i t i s a change i n b o d y mass and n o t g r o w t h a l o n e t h a t i s i n f l u e n c i n g t h y r o i d a c t i v i t y i n t h i s i n v e s t i g a t i o n . The i n f l u e n c e o f body m a s s , p a r t i c u l a r l y on s m a l l f i s h , c o u l d e x p l a i n t h e s e a s o n a l changes o b s e r v e d i n c e r t a i n s p e c i e s o f j u v e n i l e O n c o r h y n c h u s where t h e r e was c o n s i d e r a b l e d e p a r t u r e f r o m a t e m p e r a t u r e c o r r e l a t i o n ( E a l e s , 1963). I t c o u l d a l s o h a v e some b e a r i n g on t h e d i s c r e p a n c y n o t e d by S w i f t (1955, 1959) between brown t r o u t o f d i f f e r e n t age c l a s s e s . S e x u a l P r e c o c i t y i n M a l e P a r r : The r e l a t i o n s h i p between s e x u a l m a t u r a t i o n and t h y r o i d a c t i v i t y i s one o f t h e l e a s t c o n t r o v e r s i a l a s p e c t s o f t h y r o i d f u n c t i o n . T h y r o i d a c t i v i t y , - 104 - w i t h few e x c e p t i o n s , i n c r e a s e s e i t h e r b y h i s t o l o g i c a l o r r a d i o - c h e m i c a l c r i t e r i a o v e r t h e p e r i o d o f s e x u a l m a t u r a t i o n . The most r e c e n t o f many a p p r a i s a l s o f t h i s a s p e c t o f t h y r o i d f u n c t i o n i s b y M a t t y ( i 9 6 0 ) . T h i s s t u d y s u p p o r t s t h e c o n c l u s i o n t h a t t h e t h y r o i d and g o n a d a l a c t i v i t y a r e r e l a t e d . I n t w o - y e a r - o l d s t e e l h e a d , i r - r e s p e c t i v e o f t h e i m m e d i a t e t e m p e r a t u r e o r p h o t o p e r i o d c o n d i t i o n , c e r t a i n e x t r e m e l y h i g h h i s t o l o g i c a l a n d r a d i o c h e m i c a l a s s e s s m e n t s o f t h y r o i d a c t i v i t y were n o t e d i n s e x u a l l y m a t u r e m a l e i n d i v i d u a l s . T h i s a d d i t i o n a l i n f l u e n c e on t h e t h y r o i d a c t i v i t y must be r e c o g n i s e d , b u t o n l y a t c e r t a i n t i m e s o f t h e y e a r . M e t a b o l i c demands d u r i n g s e x u a l m a t u r a t i o n w o u l d t e n d t o be h i g h e r , a n d i t i s p o s s i b l e t h a t t h e demands f o r t h y r o x i n w o u l d a l s o be i n - c r e a s e d . T h e r e i s no e v i d e n c e f a v o u r i n g t h e t h e o r y t h a t t h y r o x i n s t i m u l a t e s t h e o n s e t o f s e x u a l m a t u r i t y i n p r e c o c i o u s m a l e s . P h o t p e r i o d : I t seems c o n c l u s i v e l y d e m o n s t r a t e d i n t h i s s t u d y t h a t i n c r e a s i n g p h o t o p e r i o d ( J a n u a r y t o Jiune) i n d u c e s no change i n t h e t h y r o i d p h y s i o l o g y o f y e a r l i n g s t e e l h e a d , b u t h a s a v e r y d e f i n i t e e f f e c t on l a r g e r members o f t h e same s p e c i e s p r e c i s e l y one y e a r o l d e r ( p o t e n t i a l m i g r a n t s ) . W h e t h e r s i z e o r age i s i m p o r t a n t i n p o t e n t i a t i n g r e s p o n s e t o p h o t o p e r i o d h a s n o t been r e v e a l e d by t h i s s t u d y . T h i s w o u l d i n d i c a t e t h a t t h e i n c r e a s i n g p h o t o p e r i o d i s o f i m p o r t a n c e i n c a u s i n g m e t a b o l i c c h a n g e s w i t h i n t h e f i s h p r i o r t o m i g r a t i o n . H o w e v e r , t h e e x a c t s t a t u s o f t h e t h y r o i d i n t h e m e t a b o l i c sequence has n o t been r e s o l v e d . One p o s s i b i l i t y i s t h a t t h e h y p o t h a l a m i c o - h y p o p h y s e a l - 105 - s y s t e m s r e c e i v i n g i m p u l s e s f r o m p i n e a l o r o p t i c c e n t r e s r e l e a s e TSH w h i c h s t i m u l a t e s t h y r o x i n p r o d u c t i o n w h i c h i n t u r n c a u s e s changes i n t h e s m o l t . E v i d e n c e f o r t h i s i s n e g l i g i b l e and an e q u a l l y r e a l i s t i c e x p l a n a t i o n i s t h a t i n c r e a s i n g p h o t o p e r i o d i n d u c e s i n c r e a s e d m e t a b o l i c demands ( p e r h a p s due t o i n c r e a s e d g e n e r a l a c t i v i t y ) t h a t s e c o n d a r i l y b r i n g a b o u t i n c r e a s e i n t h y r o i d a c t i v i t y v i a t h e f e e d b a c k . A t h i r d p o s s i b i l i t y i s t h a t p h o t o p e r i o d a c t i n g v i a t h e p i t u i t a r y and a d r e n a l c o r t e x (shown by O l i v e r e a u i n 1?60: t o be a c t i v e i n m i g r a t i n g s m o l t s ) a f f e c t s m i n e r a l w a t e r m e t a b o l i s m t o t h e e x t e n t t h a t i o d i d e s a r e l o s t f a s t e r t h a n t h e y a r e r e p l a c e d , so t h a t t h e b l o o d i o d i n e l e v e l a v a i l a b l e t o t h e t h y r o i d i s r e d u c e d . T h i s p o s s i b i l i t y i s c o n s i d e r e d i n d e t a i l b e l o w . I o d i n e A v a i l a b i l i t y : A s H i c k m a n (1962) h a s e m p h a s i z e d , b e f o r e v a l i d c o m p a r i s o n s c a n be made b e t w e e n I-*-31 d e t e r m i n a t i o n s , t h e f i s h must be i n i o d i n e b a l a n c e w i t h i t s e n v i r o n m e n t , i . e . t h e amount o f I ^ 7 e n t e r i n g t h e body and t h y r o i d i s e q u a l t o t h a t l e a v i n g t h e body and t h y r o i d . One ; s i t u a t i o n i n w h i c h t h i s c o n - d i t i o n may n o t be s a t i s f i e d i s i n an i o d i n e d e f i c i e n t e n v i r o n - m e n t , where p l a s m a I-*- 2 7 l e v e l s c o u l d be c o n s i d e r a b l y r e d u c e d . A s L e l o u p and F o n t a i n e (i960) have shown, m a r i n e t e l e o s t s g e n e r a l l y have a h i g h e r p l a s m a 1^27 t h a n f r e s h - w a t e r f o r m s . T h i s i s a t t r i b u t e d t o t h e d i f f e r e n t amounts o f i o d i n e p r e s e n t i n t h e two m e d i a . R o b e r t s o n and Chaney (1953) d e m o n s t r a t e d i n S a l m o g a i r d n e r i t h a t t h e amount o f a m b i e n t i o d i n e d i r e c t l y d e - t e r m i n e d t h e p l a s m a i o d i n e l e v e l s . S i n c e t h e t h y r o i d u p t a k e o f I 1 2 7 i s d i r e c t l y d e p e n d e n t on - 106 - 127 the plasma supply of I , any c o n d i t i o n tending t o lower plasma l e v e l s becomes l i m i t i n g . I f a c e r t a i n minimal uptake of l 1 2 7 i s r e q u i r e d by the t h y r o i d t o produce an adequate hormone supply, 127 i n order to maintain t h i s l e v e l i n the gland, the I ' gradient between t h y r o i d and plasma becomes gr e a t e r and more demands are placed on a c t i v e t r a n s p o r t mechanisms. This i s the t h e o r e t i c a l b a s i s of the i o d i n e d e f i c i e n c y g o i t r e described f o r S a l v e l i n u s f o n t i n a l i s (Marine and Lenhart, 1910), Salmo g a i r d n e r i (Robertson and Ghaney, 1953) and f o r Salmo s a l a r (La Roche, 1952). Under these c o n d i t i o n s of low plasma i o d i n e the gland increases i t s i o d i n e concentrating e f f i c i e n c y by hypertrophy and hyper- p l a s i a . As the above authors demonstrated, t h i s h y p e r p l a s i a and hypertrophy can be completely removed by p r o v i d i n g the f i s h w i t h adequate i o d i n e . The same response to changing i o d i n e l e v e l s could con- c e i v a b l y occur i n l e s s s t r i n g e n t circumstances. The higher the l e v e l of a v a i l a b l e i o d i n e the l e s s e f f i c i e n t the " i o d i n e pump" has to be. None of the methods c u r r e n t l y used i n determining t h y r o i d a c t i v i t y w i l l d i s t i n g u i s h true and apparent increases i n t h y r o i d a c t i v i t y . I n the former c o n d i t i o n , the net output of hormone i s increased w h i l e under c o n d i t i o n s of low environ- mental i o d i n e , the i o d i n e accumulation i s more e f f i c i e n t but the net hormone production i s not augmented. There i s evidence to suggest that many fresh-water f i s h are compensating f o r some measure of i o d i n e d e f i c i e n c y . This can e a s i l y be demonstrated by observing the h i s t o l o g i c a l or r a d i o - chemical response by the t h y r o i d when the a v a i l a b l e s t a b l e - 107 - i o d i n e i s i n c r e a s e d . I f t h e s e p a r a m e t e r s do n o t change a t a l l t h e n a d e q u a t e i o d i n e i s a v a i l a b l e ; i f t h e y a r e r e d u c e d t h e n t h i s i s i n d i c a t i v e t h a t p a r t o f what was o r i g i n a l l y b e i n g m e a s u r e d as t h y r o i d a c t i v i t y was r e a l l y o n l y a n a d a p t a t i o n t o l o w i o d i n e l e v e l s . T h e r e a r e many i n s t a n c e s i n t h e l i t e r a t u r e o f i o d i n e l e v e l s r e d u c i n g t h y r o i d a c t i v i t y i n c o n d i t i o n s n o t g e n e r a l l y c o n s i d e r e d as g o i t r o u s . H i c k m a n (1959) o b s e r v e d , i n t h e s t a r r y f l o u n d e r , t h a t i n c r e a s e i n t h e i o d i n e l e v e l o f t h e w a t e r d e c r e a s e d t h y r o i d u p t a k e and B e r g , G-orbman a n d K o b a y a s h i (1959) have d e m o n s t r a t e d t h i s f o r s e v e r a l f r e s h - w a t e r s p e c i e s . I n no i n s t a n c e has a d d i t i o n o f i o d i n e i n c r e a s e d e i t h e r t h e h i s t o l o g i c a l o r r a d i o c h e m i c a l a c t i v i t y o f t h e g l a n d . I n t h e p r e s e n t s t u d y , u n d e r y e a r l i n g s t e e l h e a d showed o n l y a m i l d r e d u c t i o n i n t h y r o i d u p t a k e o n t h e a d d i t i o n o f h i g h l e v e l s o f i o d i n e t o t h e medium. I t i s t h e r e f o r e c o n c l u d e d t h a t a t t h i s s t a t e o f d e v e l o p m e n t t h e a m b i e n t i o d i n e l e v e l was more o r l e s s a d e q u a t e . The same s p e c i e s t e s t e d a t t h e t i m e o f s m o l t i f i c a t i o n showed a v e r y s i g n i f i c a n t d e p r e s s i o n o f t h y r o i d a c t i v i t y when i n t r o d u c e d i n t o s e a w a t e r d e s p i t e t h e f a c t t h a t t h i s t r e a t m e n t g e n e r a l l y makes h i g h e r demands on t h e t h y r o i d . I t i s c o n c l u d e d , t h e r e f o r e , t h a t t h e s m o l t has v e r y d e f i n i t e p r o b l e m i n o b t a i n i n g a d e q u a t e i o d i n e . The e x p e r i m e n t a l w o r k on t h e chum s a l m o n w o u l d s u p p o r t t h i s . S i n c e t h e w a t e r a v a i l - a b l e t o b o t h n o n - m i g r a n t and s m o l t i n g f i s h came f r o m t h e same r e s e r v o i r ( C l e v e l a n d Dam) and t h e e x t e r n a l a v a i l a b i l i t y o f i o d i n e was p r o b a b l y unchanged one must t h e r e f o r e i n v e s t i g a t e endogenous p h y s i o l o g i c a l c h a n g e s . A change i n e f f i c i e n c y o f - 108 - 1127 a s s i m i l a t i o n from the food could also have some e f f e c t , hut t h i s has not been considered further. A s i g n i f i c a n t point to emerge was the extremely rapid elimination of injected 1.131 i n steelhead at the time of migration. This was higher than at any other time of the year. Such a rapid loss of I x31 W a s also found i n A t l a n t i c salmon smolts by Leloup and Fontaine (l°60) and i n several species of P a c i f i c salmon (Eales, l Q 6 l ) . It i s considered that t h i s loss of I - ^ l i s a consequence of the general demineralization that has been observed i n many diadro- mous species immediately p r i o r to descent to the ocean (reviewed by Hoar, 1939). A f i s h l i v i n g i n waters already low i n iodine could show a greater loss from the body than uptake from the medium i . e . plasma iodine l e v e l s would be low and thyroid compensation fo r t h i s would occur as outlined above. Leloup and Fontaine ( i960) contend that the rapid loss i s merely a r e f l e c t i o n of a more active 1^31 metabolism v i a the thyroid. This study shows that t h i s i s not the case and that the bulk of the I 1 ^ 1 i o s t from the body i s v i a extrathyroidal routes. On t h i s basis one would anticipate low l e v e l s of l l 2 7 both i n the thyroid and i n the blood of a migrating salmon. Leloup and Fontaine ( i960) have made a comprehensive study of the d i s t r i b u t i o n of stable iodine i n the various body compartments of the A t l a n t i c salmon smolt and parr. While the thyroid iodine l e v e l s are much lower i n smolts than i n parr, the blood iodine <, i n the smolt i s a c t u a l l y higher. At f i r s t sight t h i s argues against the above hypothesis, but Leloup and Fontaine have also shown that most of t h i s I 1 2 7 i s loosely bound to protein. (This - 109 - i s a "binding d i f f e r e n t from that formed i n P B I 1 ^ sinoe i t i s s p l i t by t r i c h l o r o a c e t i c acid). This e f f e c t i v e l y makes the I 1 2 7 molecule much larger and less d i f f u s i b l e and would explain why although the injected non-bound r ^ l ±3 r a p i d l y l o s t , the smolt 127 s t i l l has high plasma I ' l e v e l s . According to Leloup and Fontaine t h i s binding mechanism, also present i n steelhead, permits a store of iodine to be b u i l t up i n the blood at smolti- f i c a t i o n . This i s claimed to be of selective advantage due to the high thyroid a c t i v i t y at t h i s time when the demands for iodine would be considerable. It i s argued here, however, that a binding of I-*-27 to plasma proteins does not necessarily increase 127 the I ' plasma l e v e l s available f o r t h y r o i d uptake. The very mechanism that renders I^ 27 bound to proteins and therefore reduces i t s rate of extrathyroidal elimination may also render i t unavailable f o r uptake by the thyroid. In other words the binding of proteins to iodide may decrease the free iodide a v a i l a b l e f o r thyroid uptake and may accentuate any tendency towards go i t r e . One experiment to tes t t h i s hypothesis would be to i n v e s t i - gate the uptake of J.131 from a pool of protein-bound j!31 i n the blood. I f the hypothesis of Leloup and Fontaine were correct then ( i ) there would be a reduced rate of extrathyroidal loss of the protein-tagged I 131 a n d because t h i s l 1 3 1 w a s being l o s t so slowly from the blood i t would be taken up i n increased amounts by the thyroid. Such a experiment was performed by Chavin (1956) on Carassius auratus. He injected one group of gol d f i s h with I 1 5 1 and one group with I 1 3 1 bound to albumin. The excretion rate of the protein-tagged I 131 was very slow but - 110 - the thyroid accumulation of r a d i o a c t i v i t y was no higher than with free I 1 ^ 1 . This supports the present theory that binding of I 1 2 7 to proteins does not necessarily augment the pool of iodine available to the thyroid. One can therefore conclude that the smolt may be i n an iodine d e f i c i e n t condition despite the apparent abundance of 1"127 i n i t s body. The very low l e v e l s of I-1-27} either organically bound or free, i n the thyroid i t s e l f would support t h i s . The binding of iodine to protein i s possibly a fortuitous change occasioned by an increase at s m o l t i f i c a t i o n of a blood protein f r a c t i o n that has an exceptional a f f i n i t y f o r iodides. It i s concluded, therefore, that the fresh-water te l e o s t thyroid a c t i v i t y can be influenced markedly by the plasma iodine l e v e l , and that t h i s l e v e l i n turn i s dependent on the net r e s u l t of both the uptake of I 1 2 7 from the medium v i a the food or the water and on i t s t o t a l rate of loss from the body. It i s suggested that the altered physiology of the smolt renders less iodine available to the thyroid and that this, to some yet unascertained degree, contributes to the high thyroid " a c t i v i t y " at s m o l t i f i c a t i o n . S a l i n i t y : The p r i n c i p l e object of the entire investigation was an analysis of factors contributing to seasonal change i n ll31 metabolism and thyroid function during the fresh-water phase of development. On t h i s basis i n v e s t i g a t i o n of the e f f e c t of s a l i n i t y appears inconsistent, as f i s h i n fresh water (by d e f i n i t i o n ) would never be exposed to such a s i t u a t i o n . A study of response to s a l i n i t y , however, could help to elucidate the - I l l - change i n i o d i n e m e t a b o l i s m a t s m o l t i f i c a t i o n . I t has a l r e a d y - b e e n i n d i c a t e d t h a t t h i s c a n be e x p l a i n e d on t h e b a s i s o f i o d i n e d e f i c i e n c y b u t t h e . e x t e n t o f t h i s e f f e c t i s n o t known a n d o t h e r t h e o r i e s p u t f o r w a r d t o e x p l a i n t h i s i n c r e a s e c a n n o t be e x c l u d e d i m m e d i a t e l y . One s u g g e s t i o n , r e f e r r e d t o e a r l i e r , i s t h a t a s m o l t i n g f i s h p o s s i b l y due t o i n c r e a s e d amounts o f m e t a b o l i c w a t e r and t o changes i n i t s i o n r e t a i n i n g mechanisms p r i o r t o movement t o t h e s e a , has p r o b l e m s a s s o c i a t e d w i t h s a l t and w a t e r b a l a n c e ( H o a r , 1952, 1959; E a l e s , 1961). C o n s e q u e n t l y , t r a n s f e r t o s e a w a t e r s h o u l d a l l e v i a t e t h e o s m o t i c p r o b l e m s e n c o u n t e r e d by t h e f r e s h - w a t e r s m o l t . Thus t h y r o i d a c t i v i t y w o u l d be h i g h i n t h e s m o l t i n f r e s h w a t e r where t h e m e t a b o l i c demands a r e p r e s u m a b l y g r e a t , b u t r e d u c e d i n s e a w a t e r . The d a t a f r o m b o t h y e a r l i n g s t e e l h e a d and p o s t - m i g r a n t chum where m e t a b o l i c demands m i g h t be h i g h i n f r e s h w a t e r , h o w e v e r , show i n c r e a s e d t h y r o i d a c t i v i t y i n s e a w a t e r . T h i s a g r e e s w i t h t h e f i n d i n g s o f H i c k m a n (1959) on t h e e u r y h a l i n e f l o u n d e r , P l a t i c h t h y s s t e l l a t u s . where s a l i n i t y i n c r e a s e d b o t h o x y g e n c o n s u m p t i o n and t h y r o i d a c t i v i t y . I t does n o t s u p p o r t t h e t h e o r y t h a t t h y r o x i n demands o f t h e s m o l t a r e g r e a t e r i n f r e s h w a t e r due t o o s m o t i c s t r e s s . I n c o n c l u s i o n , many f a c t o r s a f f e c t t h y r o i d a c t i v i t y and c o n t r i b u t e t o t h e s e a s o n a l c y c l e o f t h e s t e e l h e a d . U n i v e r s a l among t h e s e i s t e m p e r a t u r e a n d a t a l l s e a s o n s t h e t h y r o i d r e s p o n d s t o t h i s i m p o r t a n t v a r i a b l e . S u p e r i m p o s e d on t h e b a s i c r e s p o n s e t o t e m p e r a t u r e a r e t h e e f f e c t s o f o t h e r f a c t o r s . Of t h e s e , b o d y s i z e i s p r o b a b l y a l w a y s e x e r t i n g some e f f e c t . T h y r o i d a c t i v i t y - 112 - i s l o garithmically related to body mass. As a re s u l t of t h i s relationship, appreciable e f f e c t s on the thyroid are noted "only over the lower size range and become ne g l i g i b l e above 20 grams. At a r e s t r i c t e d time of year, the state of sexual maturation (as shown here i n precocious male parr) can place an extra demand on the thyroid. In general, the thyroid appears refractory to changes i n photoperiod but p o t e n t i a l migrants show a very d e f i n i t e response that i s superimposed on a positive response, to temperature i n the spring. The ph y s i o l o g i c a l route by which the increasing photoperiod progressively exerts i t s effect i s unknown. F i n a l l y , c e r t a i n of these changes i n thyroid may be apparent rather than r e a l . Due to the e f f e c t s of d i f f e r e n t factors on plasma I l 2 7 l e v e l s , the a v a i l a b i l i t y of iodine to the thyroid may be altered. It i s considered that t h i s e f f e c t may be p a r t i c u l a r l y important i n the smolt but n e g l i g i b l e i n the parr. I I I . THE ROLE OF THE THYROID IN THE STEELHEAD Any comprehensive theory concerning the thyroid r o l e i n steelhead must take i n t o account that r i s i n g temperature, small size, sexual maturation and exposure to higher s a l i n i t y a l l exert greater demands on the thyroid. Without exception a l l these factors have been associated with higher rates of metabolism i n poikilotherms. This suggests that the thyroid i s involved i n some basic metabolic r o l e . There i s no evidence from t h i s study to implicate a s p e c i f i c r ole of the thyroid i n a diadromous teleost although several such roles have been suggested i n the - 113 - l i t e r a t u r e . These re l a t e to the r o l e of thyroxin i n inducing s a l i n i t y preference (Baggerman, i 9 6 0 , 1963), s i l v e r i n g (Robertson, 1949) and changes i n glycogen metabolism (Fontaine and Hatey, 1950) . Data presented e a r l i e r showed the independence of s i l v e r i n g on increased thyroxin output and I concluded that the stimulation of guanine deposition i s a pharmacological effect of thyroxin, since increased thyroxin i s not required i n nature. Furthermore, thyroxin administration does not always produce s i l v e r i n g and thyroid extracts containing mainly iodine and thyroglobulin are often more potent. The same i s true where experiments with thyroxin and thyroid extracts have been used to stimulate mobilization of glycogen c h a r a c t e r i s t i c of the smolt. In addition, though Baggerman (1963) obtained p o s i t i v e r e s u l t s with the e f f e c t of TSH on s a l i n i t y preference and an i n h i b i t i o n of s a l i n i t y preference with thyroid i n h i b i t o r s i n juvenile Oncorhvnchus, she could obtain no change i n s a l i n i t y response with thyroxin alone (Baggerman, i 9 6 0 ) . Although such s p e c i f i c r o l e s of thyroxin cannot be e n t i r e l y eliminated, consideration of the better established general metabolic roles of thyroxin might be more p r o f i t a b l e . Such a general metabolic r o l e i s i n fact suggested by the data on s i t e s of peripheral catabolism of radioactive hormone. A l l the metabolically active tissues showed a s i m i l a r r i s e i n hormone accumulation suggesting a systemic rather than an organ or t i s s u e - s p e c i f i c action. Muscle with a lower metabolism than kidney, l i v e r , brain or probably gut had a much lower accumu- l a t i o n of presumed hormonal r a d i o a c t i v i t y , while skin the most - 114 - m e t a b o l i c a l l y i n a c t i v e o f a l l showed t h e l e a s t r i s e . I t i s c o n c l u d e d t h e r e f o r e t h a t t h y r o x i n i s p r o b a b l y i m p o r t a n t t o a l l c e l l s o f t h e body b u t t h a t q u a n t i t a t i v e r e q u i r e m e n t s o f e a c h w i l l be g e a r e d t o t h e i r r a t e o f m e t a b o l i s m . One o f t h e p r i n c i p l e r e a s o n s f o r n o t a c c e p t i n g a g e n e r a l r o l e o f t h y r o x i n i n m e t a b o l i s m , s i m i l a r t o t h a t d e s c r i b e d i n mammals, i s t h e a l m o s t c o m p l e t e i n a b i l i t y o f t h y r o x i n t o s t i m u l a t e o x y g e n c o n s u m p t i o n i n p o i k i l o t h e r m s . T h e r e a r e a few n o t a b l e e x c e p t i o n s and t h e p o s i t i v e r e s u l t s o b t a i n e d by M u l l e r (1953) i n t h e g o l d f i s h a r e b e s t known. H o w e v e r , m e r e l y b e c a u s e t h y r o x i n w i l l n o t s t i m u l a t e a change i n m e t a b o l i c r a t e does n o t mean t h a t i t h a s no m e t a b o l i c * r o l e . I t i s p o s s i b l e t h a t t h y r o x i n p l a y s a s i m i l a r r o l e a t t h e b i o c h e m i c a l l e v e l i n b o t h warm and c o l d - b l o o d e d v e r t e b r a t e s . I n t h e f o r m e r n o t o n l y does t h y r o x i n t a k e p a r t i n t h e r e a c t i o n b u t i t i s p r e s u m a b l y a l i m i t i n g f a c t o r t o c e r t a i n r e a c t i o n s and f o r t h i s r e a s o n c o n t r o l s t h e m . I n t h e l a t t e r , h o w e v e r , t h o u g h t h y r o x i n may s e r v e e x a c t l y t h e same s u b c e l l u l a r r o l e , i t may be r a r e l y t h e s o l e l i m i t i n g f a c t o r . I t i s s p e c u l a t e d t h a t i n s u c h a s i t u a t i o n i n c r e a s e i n t h y r o x i n l e v e l m i g h t n o t be a b l e t o c a u s e a n i n c r e a s e i n m e t a b o l i s m b e c a u s e o t h e r v i t a l s u b s t a n c e s were a l s o l a c k i n g . H o w e v e r , u n d e r c e r t a i n r a t h e r r a r e e x p e r i m e n t a l c o n d i t i o n s t h y r o x i n m i g h t be t h e s o l e l i m i t i n g f a c t o r . I f t h i s were t h e c a s e (as c o u l d c o n c e i v a b l y be t r u e w i t h M u l l e r ' s w o r k ) t h e n a d m i n i s t r a t i o n o f t h y r o x i n w o u l d a l l o w m e t a b o l i s m t o p r o c e e d a t a f a s t e r r a t e and o x y g e n c o n s u m p t i o n w o u l d i n c r e a s e . S u c h a p a s s i v e r o l e o f t h y r o x i n w o u l d p r o b a b l y be a c c o m p a n i e d - 115 - by an equally passive form of control i . e . v i a the plasma feed- back method postulated e a r l i e r . It i s proposed that as a r e s u l t of a l l the thyroxin-demanding processes ( r i s e i n body temperature, small s i z e , sexual maturation, increased s a l i n i t y and possibly increased exercise) plasma l e v e l s of hormone would be depleted and then reinstated v i a the hypophyseal feedback system. Where the e f f e c t of photoperiod i s concerned i t i s tempting to suggest that t h i s stimulates the thyroid d i r e c t l y v i a the hypothalamico-hypophyseal system. But there i s no d i r e c t evidence f o r t h i s and i t i s equally l i k e l y that i t could either induce demineralization with secondary effects on the thyroid due to iodine deficiency. A t h i r d p o s s i b i l i t y i s that lengthening photoperiod stimulates the smolt to higher l e v e l s of general a c t i v i t y that secondarily cause increased thyroxin production. The general speculative conclusion i s that thyroxin may subserve a very s i m i l a r biochemical role i n both poikilotherms and homiotherms. While i n the l a t t e r , however, there are d i s t i n c t advantages to i t s c o n t r o l l i n g metabolism by being a l i m i t i n g factor i n c e r t a i n reactions, i n poikilotherms t h i s i s not the case. Instead, the production of thyroxin may be governed s o l e l y by the demands of the tissues and i t s production controlled to a large extent, or e n t i r e l y by the hypophyseal feedback mechanism. - 116 - SUMMARY AND CONCLUSIONS I. Reliable estimates of thyroid a c t i v i t y can be made using single terminal assessments of TUF, T/S or CR at 4 or 8 days a f t e r i n j e c t i o n i n juvenile steelhead. II. Seasonal changes i n radioiodine metabolism revealed some c o r r e l a t i o n with temperature but smolts departed from t h i s rule i n having higher than anticipated values. Smolts had a h i s t o l o g i c a l l y active gland but, i n general, c e l l height showed a negative c o r r e l a t i o n with temperature. I I I . Experimental investigation of temperature on thyroid a c t i v i t y revealed a d i r e c t dependence of a l l aspects of J.131 metabolism on temperature from 4 to 18° C. H i s t o l o g i c a l l y , however, the gland was more active at low temperatures. IV. Photoperiod exerted no eff e c t on c e l l height or radio- iodine metabolism of steelhead yearlings from January to July. V. Body mass had an important ef f e c t on radioiodine meta- bolism with small f i s h having the higher a c t i v i t y . The logarithmic dependence of thyroid a c t i v i t y on mass permitted neglect of t h i s factor i n f i s h over 20 grams. VI. P o t e n t i a l migrant steelhead examined from January to June showed a marked p o s i t i v e response to both i n - creasing temperature and increasing photoperiod. The high smolt value was due to summation of the photo- period and temperature response. There was no evidence - 117 - f o r a synergism between temperature and photoperiod influences. VII. Precocious sexually mature male parr i n March frequently had a higher thyroid a c t i v i t y both by h i s t o l o g i c a l and radiochemical techniques (than immature forms). VIII. Exercise s l i g h t l y increased the conversion r a t i o . IX. S a l i n i t y generally caused an increase i n thyroid a c t i v i t y . Parr i n mid-summer were only s l i g h t l y sensitive to increased iodine l e v e l s i n the medium but smolts and chum salmon showed a great reduction i n thyroid a c t i v i t y v/hen the iodine concentration of the medium was raised. This indicates that part of the high a c t i v i t y of the smolt i s due to an iodine deficiency. X. Potential migrant steelhead held under conditions of low temperature and 8-hour daylength had unchanging thyroid a c t i v i t y . A normal percentage of these f i s h s i l v e r e d and the role of thyroxin i n s m o l t i f i c a t i o n i s therefore questioned. XI. "Radiohormone" was shown to b u i l d up i n several tissues but e s p e c i a l l y i n those that were metabolically most active. G i l l showed an accumulation of !-131 that could be i n d i c a t i v e of a role i n ion exchange with the surrounding medium. Integration of these findings suggests that environmental temperature and mass of f i s h always play a r o l e i n determining thyroid a c t i v i t y . However, i n fish- over 20 grams the l a t t e r can be discounted as an important variable. It i s believed that - 118 - t e m p e r a t u r e i s u n i q u e amongst t h e v a r i a b l e s a f f e c t i n g t h e t h y r o i d i n t h a t i t may a l t e r t h y r o i d f u n c t i o n w i t h o u t t h e m e d i a t i o n o f T S H . T h i s c o u l d e x p l a i n d i s c r e p a n c i e s between h i s t o l o g i c a l and r a d i o c h e m i c a l d a t a , o b s e r v e d i n t h i s s t u d y and i n t h e l i t e r a t u r e . S u p e r i m p o s e d on t h e s i z e and t e m p e r a t u r e i n f l u e n c e s may be t h y r o i d r e s p o n s e s t o gonad m a t u r a t i o n , i n c r e a s e d e x e r c i s e , s a l i n i t y , p h o t o p e r i o d and i o d i n e c o n c e n t r a t i o n o f t h e w a t e r . I t i s s u g g e s t e d t h a t gonad m a t u r a t i o n , e x e r c i s e and i n c r e a s e d o s m o t i c p r e s s u r e a l l r e q u i r e m e t a b o l i c w o r k and a n i n c r e a s e i n t h y r o i d a c t i v i t y w o u l d be a n t i c i p a t e d . The r e s p o n s e t o i n c r e a s e d i o d i n e c o n c e n t r a t i o n i s , h o w e v e r , a n i n d i c a t i o n t h a t p a r t o f what i s o f t e n c o n s i d e r e d t o be t h e a c t i v i t y o f t h e g l a n d i s r e a l l y a c o m p e n s a t i o n whereby t h e t h y r o i d " i o d i n e pump" becomes more e f f i c i e n t a t l o w i o d i n e l e v e l s . I n t h e s m o l t s u c h a c o m p e n s a t i o n seems v e r y a p p a r e n t . I t i s p o s t u l a t e d t h a t p h o t o - p e r i o d c o u l d cause i o n i c i m b a l a n c e i n t h e f i s h and t h a t l o w i o d i n e l e v e l s d e v e l o p a t s m o l t i f i c a t i o n w h i c h l e a d t o an a p p a r e n t i n c r e a s e i n t h y r o i d a c t i v i t y . I n v i e w o f t h e l a c k o f a s p e c i f i c r o l e i n s i l v e r i n g and i t s g e n e r a l a c c u m u l a t i o n i n a c t i v e l y m e t a b o l i z i n g t i s s u e s , i t i s c o n c l u d e d t h a t t h y r o x i n s u b s e r v e s a v e r y f u n d a m e n t a l b i o c h e m i c a l r o l e a n d t h a t i t s p r o d u c t i o n i s p r o b a b l y l a r g e l y g o v e r n e d by t h e demands o f t h e t i s s u e s a c t i n g v i a a f e e d b a c k m e c h a n i s m . - 119 - BIBLIOGRAPHY Baggerman, B. i 9 6 0 . S a l i n i t y preference, thyroid a c t i v i t y and the seaward migration of four species of P a c i f i c salmon (Oncorhvnohus). J. F i s h . Res. Bd. Canada, 17: 295-322. Baggerman, B. 1963. The effect of TSH and antithyroid sub- stances on s a l i n i t y preference and thyroid a c t i v i t y i n juvenile P a c i f i c salmon. Can. J . Zool., 41: 307-319. Barrington, E. J . W. 1961. Metamorphic processes i n fishes and lampreys. Am. 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