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A comparative study of iodine metabolism in juvenile Oncorhynchus 1961

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A COMPARATIVE STUDY OF IODINE METABOLISM IN JUVENILE ONCORHYNCHUS by JOHN GEOFFREY EALES ' B.A., Oxford University, 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard from candidates for the degree of MASTER OF SCIENCE Members of the Department THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1961 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for e:cfcensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of B r i t i s h Columbia, Vancouver 8, Canada. •» i i - ABSTRACT Comparative histological and radiochemical studies of iodine meta- bolism in juvenile Oncorhynchus revealed good agreement between thyroid epithelial height and a b i l i t y to convert il31 i n t o protein-bound I^^PBI*"^) • The ratio of I131 to PBll31 in plasma samples (Conversion Ratio) was con- sidered superior to other thyroid assays reviewed. Peaks in thyroid a c t i v i t y and loss of I * 3 1 from the body occurred in sockeye and coho at the time of downstream migration, but i n chum and pink only when postmigrants were retained in fresh water* In coho and sockeye these changes were transitory, i n chum irreversible and i n pink prolonged. On the above basis, thyroxine was assigned no specific role but a theory of smolt evolution was proposed and related to a phylogeny within the genus Oncorhynchus. - i i i - TABLE OF CONTENTS PAGE I. INTRODUCTION 1 II . SURVEY OP METHODS FOR DETERMINING THYROID ACTIVITY. 4 A. CHEMICAL METHODS 4 B. RADIOIODINE METHODS 4 C. HISTOLOGICAL METHODS 8 1. Fol l i c u l a r and Extra-follicular changes • 8 2. Changes i n the Colloid • • • • • 10 3* Cytological Changes • 11 III . MATERIALS AND METHODS 12 A. LIVING MATERIALS 12 B. RADIOIODINE TECHNIQUE 13 1. Injection . . . . . . . . 13 2. Blood Sampling and Conversion Ratio Technique . . . . . . . . 13 3. Thyroid Sampling . . . . . . 14 4. Body Sampling . . • • e . . 14 C. HISTOLOGICAL TECHNIQUE 15 IV. RESULTS 16 A. RADIOIODINE 16 1. Data on General l!31 Metabolism . . . 16 2. Comparative Data . . . . . . 17 3. Data on Technique • a • • • • 18 a. Dose Size . . . . . . . 18 - i v - TABLE OF CONTENTS (Continued) PAGE b. Plasma Sample ' • • • • • . 1 9 c. Diurnal Variation . . . . . 19 B. HISTOLOGICAL . » 20 V. DISCUSSION 22: A. IODINE METABOLISM AND EVALUATION OF THYROID ASSAY METHODS . . . . . . . . . 22 B. THE ROLE OF THE THYROID IN AN ADROIT . . . 31 C. SPECULATION ON SMOLTIFICATION AND THE PHYLOGENY WITHIN GENUS ONCORHYNCHUS . . . 36 VI. SUMMARY AND CONCLUSIONS 39 VII. BIBLIOGRAPHY 41 VIII. APPENDIX 44 - V - LIST OF FIGURES FIGURE FOLLOWING PAGE 1» Loss of injected 1131 f r o m blood plasma in underyearling coho and chum salmon . . . » 16 2. Loss of injected il31 from body (less thyroid) in underyearling coho and chum salmon . . . 16 3. Thyroid uptake of injected I131 in juvenile Oncorhynchus • 16 4. Changes in FBI-'-5'"- concentrations in plasma samples of juvenile Oncorhynchus . . . . . . 17 5. Juvenile Oncorhynchus conversion curves. . . 17 6. il31 metabolism in underyearling coho salmon (inactive thyroid) . . . . . . . . 17 7. jl31 metabolism in underyearling chum salmon (inactive thyroid) . . . . . . . . 17 8. i l S l metabolism i n underyearling chum salmon (active thyroid) 18 9. I131 metabolism in sockeye smolts (very active thyroid) 18 10. Relationship between Conversion Ratio and thyroid uptake of injected I131 ±a under- yearling chum salmon 18 11. Seasonal variation in Conversion Ratio values in juvenile Oncorhynchus held in fresh water . 19 12. Seasonal variation in the per cent retention (after 108 hrs) of injected l!31 in the bodies (less thyroid) of juvenile Oncorhynchus held in fresh water ( 1 9 13. Relationship between extent of vacuolation of colloid and lowest c e l l height in sockeye smolts . . . . . . . . . . . 19 14. Relationship between per cent of f o l l i c l e s containing no colloid and lowest c e l l height i n sockeye smolts . . . . . . . . . 20 - v i - LIST OF FIGURES (Continued) FIGURE FOLLOWING PAGE 15. Relationship between mean f o l l i c l e diameter and lowest c e l l height in sockeye smolts « . • 20 16o Relationship between mass and mean f o l l i c l e diameter in sockeye smolts • • • • • • 20 17. Relationship between depth of staining and lowest c e l l height in sockeye smolts . . . • . 20 18. Regressions of Conversion Ratio and lowest, ta l l e s t and mean epithelial c e l l height in sockeye smolts . . . . . . . . . 21 19. Seasonal changes in Conversion Ratio and mean c e l l height in two-year-old coho smolts . . . 21 20. Seasonal changes in Conversion Ratio, mean c e l l height and thyroid uptake in underyearling chum salmon 21 21. Seasonal changes in Conversion Ratio and mean c e l l height in sockeye smolts . • • • • 21 - v i i - LIST OF TABLES TABLE PAGE I. Effect of I 1 3 * dose on Conversion Ratio in underyearling chum salmon • • • • • • 45 II . Decrease in Conversion Ratio in second plasma sample in sookeye smolts • • • • • • 46 III . Diurnal variation in Conversion Ratio i n chum salmon • • • • • • • • • • • 47 IV. Seasonal change in Conversion Ratio in juvenile Onoorhynohus held i n fresh water • • • • 48 V. Seasonal change in the per oent retention (after 108 hrs) of injected I131 in plasma and body (less thyroid) of juvenile Oncorhynchus held in fresh water • • • • . • • • 4 9 VI. 1131 metabolism in underyearling coho salmon (thyroid inactive) • • • • • • • • 5 0 VII. I131 metabolism in underyearling chum salmon (thyroid inactive) . o e » o . . o 5 1 VIII. l!31 metabolism i n underyearling chum salmon (thyroid act ive) • • • • • • • • 52 IX. il31 metabolism in three-year-old sockeye smolts (thyroid active) • • • • • • • 5 3 X. Quant a l and semi-quantal determinations of histological characters i»:sockeye smolts . . 5 4 XI. Cell heights and Conversion Ratio in sockeye smolts . . . . . . . . . . . 5 5 XII. Seasonal changes in Conversion Ratio and mean c e l l height in two-year-old coho smolts . . 5 6 XIII. Seasonal changes in Conversion Ratio, mean ce l l height and thyroid uptake in underyearling chum salmon . . . 5 7 XIV. Seasonal changes in Conversion Ratio and mean c e l l height in two-year-old sockeye smolts . . 5 8 - v i i i - ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Professor W. S. Hoar, F. R. S; C , Department of Zoology, for suggesting and supervising this study and to Professors J. R. Briggs, W. N. Holmes and C. C. Lindsey for their c r i t i c a l advice. The author i s indebted to Dr. Evelyn and Mr. Tang of the Strong Laboratory, Vancouver General Hospital, for supplying radioiodine and to Loyd Royal and Harold Harvey, both of the International Salmon Commission, and to Mr. S. B. Smith of the B r i t i s h Columbia Game Commission, for providing and also maintaining most of the live material. Mr. R. W. McLaren of the Department of Fisheries was responsible for supplying live coho and steelhead downstream migrants. The histological preparations were made by Miss F. van Eerten and Miss S. Tabata. To his fellow graduate students, Mr. J. E. Mclnerney and Mr. A. J. Wiggs, the author extends his thanks for stimulating discussion and constructive criticism, and to the former for his conscientious maintenance of the hatchery f i s h . Financial assistance in the form of a Fisheries Research Board Studentship enabled the author to carry out this study. INTRODUCTION Life cycles of anadromous salmonids follow a generally consistent pattern. Spawning and juvenile development occur in fresh water# while the usually longer phase of adult growth takes place in the sea. Such a l i f e history involves precise timing of both upstream and downstream migration. At the c r i t i c a l period i n juvenile l i f e when the downstream migration commences, the parr may undergo a marked metamorphosis termed smoltification, which i s also accompanied by behavioural and physiological changes . Morphological changes include a subepidermal guanine deposition and melanophore disintegration (Robertson, 1948), a thickening of the epidermis (Pickford & Atz, 1957, p. 172), an increase i n adrenocortical volume (Olivereau, I960), while chloride secreting cells appear i n the g i l l s (Nishida, 1953)• These changes are associated both with increased s a l i n i t y tolerance (Fontaine and Baraduc, 1954) and with a marked preference for salt as opposed to fresh water (Baggerman, 1960). At the metabolic level, the smolting parr has been observed to have increased oxygen consumption, a f a l l i n hepatic glycogen (Fontaine & Hatey, 1950) and also changes in l i p i d metabolism (Lovern, 1934). Of equal significance are certain be- havioural changes which favour downstream displacement (Fontaine, 1954; i Hoar, 1958). Closely correlated i n time with a l l these changes is a marked thyroid hyperfunction as determined by the histological state of the f o l l i c l e s (Pickford & Atz, 1957, p. 133; Hoar, 1939, 1950). Certain experiments involving TSH, thyroxine or iodinated casein administration seem to i n - dicate thyroid hyperfunction as causal to many features of smoltification (summarized by Pickford & Atz, 1957). However, the parr induced to smolt by such means does not show exactly the same complex of physiological change found i n nature. There are two alternative explanations. Either the thyroid has a compensatory function allowing a f i s h preadapted to salt water to continue to live in fresh water, i . e . alleviates osmotic stress or, histological picture i s an artif a c t and is merely the result of continued existence in the iodine deficient fresh water habitat (Hoar, 1950, 1952). Hoar and B e l l (1950) refer especially to the confusing picture in Pacifio salmon. Histologically they show low thyroid activity at the time of migration, only becoming hyperactive i f detained in fresh water. This is in marked contrast to Atlantic salmon (Hoar, 1939; Leloup & Fontaine, 1960) where, by various methods, heightened thyroid activity has been associated with smoltification and downstream migration. However, even in this species, i t has been claimed that the thyroid of the smolt has a re- duced a b i l i t y to f i x iodine and that the ratio of thyroxine iodine to t o t a l iodine in the plasma i s reduced (Pickford & Atz, 1957, p. 133). I t i s evident from the above that confusion exists concerning not only the role of the thyroid, but also concerning i t s state of a c t i v i t y . This stems partly from lack of a precise definition of thyroid activity - namely, tot a l rate of hormone output, as opposed to either an increase i n efficiency of any iodine trapping mechanism or to the level of hormone in the blood, which can i t s e l f be influenced by peripheral u t i l i z a t i o n . Different methods of determining thyroid activity only take certain of these aspects into account and are not necessarily equivalent. There is also an obvious need for a study of radioiodine metabolism in juvenile Oncorhynchus. On this basis, a study of iodine metabolism has been made on four species of the genus Oncorhynohus in an attempt to follow seasonal changes i n thyroid a c t i v i t y . Ah improved radioiodine technique (Conversion Ratio method) has been employed and f u l l y described. In the course of the i n - vestigation, data obtained by this method has been compared to histological and other radioiodine techniques i n order to evaluate the various methods of thyroid assay. This was necessary to assess earlier work and to f a c i l i t a t e later study. I I . SURVEY OP METHODS FOR DETERMINING THYROID ACTIVITY Methods for determining thyroid activity f a l l into three main cate- gories* a l l of which have been applied to salmonids* They are ( i ) CHEMICAL ( i i ) RADIOIODINE and ( i i i ) HISTOLOGICAL. In the following account emphasis has been l a i d on histological and radioiodine techniques. No observations were made using chemical techniques and only a brief summary has been given. A. CHEMICAL^ METHODS By such methods, i t i s possible to determine the t o t a l 1127 i n the thyroid or blood plasma and also to discriminate between free iodide ( l ~ ) and that bound to protein (hormonal). Reference to application of such methods to salmonids has been made by Leloup & Fontaine (1960). It is important to remember in evaluating such data that the blood concentration of thyroxine i s not necessarily indicative of the gland's output. One possible way to increase hormone output owuld be to increase peripheral u t i l i z a t i o n . Under suoh conditions, the blood thyroxine level would temporarily drop and acting via the feed-back mechanism to the hypo- thalamus-pituitary axis (Brown-Grant, 1957) could stimulate increased hormone output so that the blood level would eventually be reinstated. Thus although there might be l i t t l e change in blood thyroxine level, the hormone output could be greatly increased. Determination of total thyroxine level does not take into account the dynamic aspects of hormonal production, and since i t s value is always prone to changes in peripheral u t i l i z a t i o n , i t should not necessarily be considered a reliable indication of thyroid a c t i v i t y . B. RADIOIODINE METHODS Several methods have been used to determine thyroid activity in f i s h . They can be considered in two categories. There are those determinations In whioh the a f f i n i t y of the gland for iodine i s considered indicative of the thyroid state while others implicate the rate of hormone output. Since under the action of certain thyroid inhibitors, the thyroid may accumulate vast quantities of I ^ l convert none to thyroxine^3"'-, i t is evident that the two process are independent to some extent and not necessa- r i l y comparable measures. The most fundamental measure of thyroid activity would be to calculate the rate of output of thyroxine from the gland and any method whioh i s not concerned with this aspect of thyroid activity i s therefore suspect. In the former category, the most common method i s to measure the per- 131 centage of an injected dose of I taken up by the gland over a fixed time period (% thyroid uptake). Setting aside the above criticism, the method has the following disadvantages. (i) It assumes a constant loss of radioiodine from the body between successive determinations. Thyroid uptake i s dependent on the reservoir of available I ^ 3 ^ . Where loss of i l s l i s great, there i s reduced chance of any molecule being taken up and thyroid uptake would appear less even though the activity of the gland was unaltered. Such depletion of the i ^ l pool could be brought about either by excretion or by the a f f i n i t y of extra- thyroidal tissues for I * 3 * . Where excretion i s concerned, marked variation has been shown in the Atlantic salmon (Leloup & Fontaine, I960). Also significant i s the a f f i n i t y in some f i s h of certain tissues such as noto- chord, ovary and musole for 1*31 (Leloup & Fontaine, 1960). Variation in the rate of l!31 uptake by the blood from the ooelom i s another source of 131 variation capable of effecting the i J - W J - blood level and hence thyroid uptake. ( i i ) Hickman (1959) has shown that the i ^ 3 * - accumulating potential of the thyroid i s significantly deoreased by the addition of iodine to natural fresh watero This i s in accordance with a homoiostatic mechanism i n whioh the gland's a f f i n i t y for iodine i s altered relative to the i!27 concentration i n the water. It i s generally considered to be greater in the lower concentration. ( i i i ) Mere thyroid uptake measurement after a fixed time period gives no idea of the loss of I f r o m the gland as thyroxine or protein - bound i131 (= ps i * 3 * ) . In some cases this loss i s appreciable and occurs quite early. Working from this principle, some workers (Framm & Reineke, 1956; Leloup & Fontaine, 1960) have measured the biological h a l f - l i f e for the loss of l 1 3 l (presumably as PBI 1 3 1) from the thyroid. This has been claimed as a measure of the loss of hormone. (iv) Finally, the method involves accurate dissection of the thyroid gland which in teleosts is notoriously diffuse and in certain f i s h such as goldfish i s p a r t i a l l y located in the head kidney region (Chavin, 1956)• However careful the dissection, an inevitable source of error arises from the blood trapped in the thyroid region i t s e l f . In counts taken soon after injeotion this source of error w i l l be high and then exponentially decrease. In this respect, a l l counts on thyroid uptake w i l l be falsely high and the source of error i s always l i k e l y to be greatest after injection< The main criticism of thyroid uptake concerns lack of appreciation of the rate of extrathyroidal I i 3 l clearance. This has been taken into account by the thyroid clearance method employed on f i s h by Hickman (1959) and Baggerman (i960) where the method has been f u l l y explained. Essentially, this i s a ratio between two rates of l l 3 l clearance, (a) the general clear- ance from the blood and (b) the thyroid uptake which indicates what per cent of the loss has gone to the thyroid, i . e . 131 Thyroid clearance s rate of I A " A uptake during *t' minutes 131 mean blood concentration of I during 't* minutes - 7 - - # thyroid uptake I 1 5 1 i n time 't' mean blood concentration I * 3 * in time ! t ' 131 This method is s t i l l susceptible to certain hazards of estimating I concentrating in fish by thyroid uptake. As employed by Baggerman (1960), the method involves measurement of 1131 i 9 v e i at 5 - 10 hours after injec- t i o n . At such a time i t i s considered that essentially only l!31 w i l l be in the blood, but Baggerman herself has emphasized the need for investiga- tion on this point. Secondly, such a choice of time means that i!31 ioss is being measured at i t s most rapid and variable phase (Hickman, 1959). A far superior method would not involve direct measurement of the per cent of the dose taken up by the thyroid and also take into account the rate of secretion of PBI into the blood stream. An approach to such an ideal i s provided by the C. R.:(Conversion Ratio) method. This has been mathe- matically derived by Riggs (1952) and adapted to the present microtechnique by Hickman (personal communication-). After a fixed interval of time the following ratio i s determined i n a given sample.of plasma. PBI131 hormonal I 1 3 1 X 100 = X 100 I 1 3 1 / P B I 1 3 1 total l " l Superficially i t i s a percentage representation of the a b i l i t y of the thyroid to remove iodine from the blood, convert i t to thyroxine and secrete i t to the blood where i t i s found as the protein-bound form. It i s thus representative of iodine uptake, thyroxine synthesis and hormone output, the three main phases of thyroid function. The v a l i d i t y of the method has been more f u l l y c r i t i c i s e d in the following study of iodine metabolism in the juvenile Oncorhynchus. The account i s based partly on personal data, partly on results from the literature and partly on speculation* C. HISTOLOGICAL METHODS Many workers are of the opinion that the histological appearance of the teleost thyroid i s indicative of the gland's activity (Pickford & Atz, 1957. p. 129). However, Swift (1955) and Fontaine (1953) have disagreed with this generalization. Swift (1955) refers to the following quotation from Carter (1933) "neither the size nor the histological appearance of a gland i s necessarily correlated with the amount of secretion which is pouring into the circulation"• However, in a later paper he does demonstrate a good general agreement between radioiodine and histological technique (Swift, 1958). Despite such contradictions almost a l l aspects of thyroid histology have been related to the activity of the gland. In the following treatment a general survey has been made of the reliance of the c r i t e r i a used. Histological changes may be considered as follows: 1. F o l l i c u l a r and extra-follicular changes 2. Changes in the colloid 3. Cytological changes. 1. Fo l l i c u l a r and Extra-follicular Changes Total f o l l i c u l a r mass i s claimed to increase with sustained thyroid hyperfunction in both Atlantic and Pacific salmon (Hoar, 1939, 1952). To estimate thyroid activity in this manner, detailed examination of the entire thyroid must be made. In theory a l l f o l l i c l e s should be accounted for. In the very variable f i s h thyroid, this presents a definite problem as i t i s d i f f i c u l t to make allowance for duplicate counting or omission of same f o l l i c l e s . In view of i t s dependence on prolonged growth processes, total f o l l i c u l a r area i s probably one of the slowest changing features of the gland. i n addition to being perhaps the most tedious to measure. According to Hoar & B e l l (1950) the quiescent thyroid f o l l i c l e i s of spherical shape in Onoorhynchus. In hyperthyroid chum salmon they become tufted and irregular in shape. This i s probably a direct result of the increased mass of cells per f o l l i c l e which causes mechanical buckling and irregular extrusion into extra-follicular spaces, while frequent budding of the active thyroid would also contribute to irregularity. Gaylord & Marsh (1912) and Hoar (1952) mention in addition a "pathological condition" diagnosed by the disorganised appearance of clumps of thyroid epithelial cells • Such a character i s d i f f i c u l t and tedious to quantify, but various workers have based conclusions on observations partly dependent on f o l l i c l e diameter. Hoar & B e l l (1950) were of the hesitant opinion that more active thyroid f o l l i c l e s had a larger diameter, but pointed out that the trend was not inconsistent with the growth of the f i s h . On the other hand, Stolk (1951) has claimed that in Lebistes the f o l l i c l e diameter i s smaller in the hyperthyroid gland. This could be due to the fact that i n an active thyroid more smaller f o l l i c l e s have just budded and i s indicative of the lack of reliance that might be placed on f o l l i c l e diameter as a measure of the glands a c t i v i t y . The principle extra-follicular change associated with hyperthyroidism i s a general increase in vascularity. The erythrocytes may even come to l i e in the f o l l i c l e (Gudernatsch, 1911). The erythrocytes are capable of intruding between the f o l l i c l e cells (Rasquin, 1949) and may eventually cause rupture of the f o l l i c l e . Aocording to Bargmann (1939) this i s not an uncommon means of f o l l i c l e discharge. However, vascularization would be d i f f i c u l t to quantify. A possible method might be to estimate the percentage of f o l l i c l e s containing erythrocytes but this might only provide estimates - 10 - above the level of activity at which the phenomenon occurs. 2. Changes in the Colloid Peripheral vacuolation i s evident in histological preparations as secretion occurs. This i s an artifact but i s s t i l l indicative of hyper- activity (De Robertis, 1949). Vacuolation or amount of colloid present i s d i f f i c u l t to quantify but a method has been employed by Fortune (1955). By projection of outlines on to paper, then cutting out and weighing these outlines, the ratio of colloid mass to epithelial mass has been determined. This ratio though not precisely definable i n physiological terms, takes into acoount both the change in colloid content and the increase in epithelial mass (hyperplasia and hypertrophy). It has many advantages and no major criticism apart from i t s lengthiness. A comparable mathematical derivation has been used by Lever (1949) and Stoik (1951) relating the ratio of f o l l i c u l a r internal diameter to the number of cells in the same f o l l i c l e . It appears in this instance that internal diameter represents the amount of colloid present, but f a i l s to take into account the appreciable effect of vacuolation. Other disadvantages include restriction of observation to spherical f o l l i c l e s , which in some instances may be rare and may even be spherical as a result of reduced activity (Hoar & B e l l , 1950). Staining has also been shown to vary (Pickford & Atz, 1957, p. 129j Hoar, 1952). In the inactive gland the colloid i s very visoous, acidophilic and homogeneous. The change in staining reaction to acid dyes (eosin) i s brought about by a change in pH i n the colloid as the result of enzymatic hydrolysis (De Robertis, 1949). This i s associated with hormone release and i s stimulated by TSH. As such i t is d i f f i c u l t to quantify. According to Pickford (Pickford & Atz, 1957, p. 129), the staining reaction i n Fundulus - 11 - may not always be i n agreement with other histological c r i t e r i a . 3• Cytological Changes Since primary tyrosine iodination and also hormonal release are currently considered simultaneous properties of each and every epithelial c e l l , and since both phases of thyroxine metabolism are indicative of gland activity, i t i s not surprising that many workers have concentrated on measuring thyroid function in terms of epithelial changes. Hoar & B e l l (1950) and Vivien (1958) have described various cytological states appropriate to different phases of a c t i v i t y . Changes occur in nuclear position, vacuola- tion of cytoplasm, staining reaction of cytoplasm and presence of colloid i n the c e l l . Most noticeable, however, i s a change in c e l l height (Pickford & Atz, 1957, p. 129j Hoar, 1939, 1950, 1952). These have been the most widely used of a l l histological c r i t e r i a . However, as pointed out earlier, i t s applicability i s controversial. The aim of current observations in sockeye smolts was therefore twofold. In the f i r s t place evaluation of c e l l height against other easily quantified histological c r i t e r i a was made. Cell height was then assessed as an index of thyroid level against more modern radioiodine techniques. - 12 I I I . MATERIALS AND METHODS A. LIVING MATERIALS PINK SALMON (Oncorhynchus gorbuscha) and CHUM SALMON (Oncorhynchus keta) These were obtained on May 7th as recently hatched fry from Cultus Lake Hatchery* The pinks were kept indoors throughout and a few f i s h survived u n t i l December* They ranged in size from an average of 0.2 gram in June to a maximum of 10 grams in September. I n i t i a l l y the chum were also kept indoors but early in July were transferred to a large out- door concrete pool where they survived well u n t i l Ootober when fungus k i l l e d most of them. They ranged i n size from an average of 0.3 gram in June to 17 grams in November. COHO SALMON (Oncorhynchus kisutch) These were obtained by seining from Salmon River. Hauls were made in November and January (1959) and August (i960). The size range was 2 to 6 grams depending on time of capture. SOCKEYE SALMON (Oncorhynchus nerka) A l l sockeye came from Cultus Lake and were trapped as yearling downstream migrants ( 4 - 6 grams in June). During June and early July, heavy mortality was suffered partly from fungus, partly from wearing away of the snout on the rough concrete surface of their tank and partly from unknown causes. Some survived to November of the same year and attained a weight of 30 grams. Sockeye smolts were also available whioh had been retained i n fresh water under hatchery conditions (University of Bri t i s h Columbia) three and four years, and by November of their fourth year weighed up to 150 grams. It i s interesting to note that none of these sockeye ever developed red or pink flesh and that some of the males showed partly developed gonads. A l l f i s h were kept (unless otherwise stated) under hatchery - 13 - conditions (University of B r i t i s h Columbia). They were held in freely running dechlorinated water which ranged i n temperature from 4 to 15° C, reaching i t s peak value in September. No sudden fluctuations in temperature occurred. Pood consisted of Clark's Commercial Trout Food which was administered i n one of four grades appropriate to the size of the f i s h * Most f i s h were fed twice a day and fry at more frequent intervals. B. RADI01OPINE TECHNIQUE 1. Injection Radioiodine in the form of carrier-free iodine, diluted with d i s t i l l e d water, was injected intraperitoneally using a 30-gauge needle and 0.25-ml tuberculin syringe. The injeoted dose was usually of the order of one microcurie per gram of f i s h , and was injeoted in a volume appropriate to the size of f i s h (0.005 to 0.05 ml). To prevent leakage, the method advocated by Hickman (1959) was employed, i n which the dose was injected through dorsal musculature which acted as a seal to the wound. Aliquots of each dose were reserved as standards (for thyroid uptake and excretion estimates) and were diluted with potassium iodide. 2. Blood Sampling and Conversion Ratio Technique At fixed periods after injection, the f i s h was k i l l e d , weighed, measured and a sample of blood taken by cleanly cutting the t a i l in the region of the caudal peduncle and drawing up the blood into a fine heparinised capillary tube as i t welled from the haemal artery. Care was taken to with- draw as pure a sample as possible, avoiding contamination either from body slime or especially from the cerebrospinal f l u i d . One end of the capillary tube was plugged with 'plasticine* and the sample centrifuged. When separation of corpuscles and plasma was complete. 14 - the tube was broken at their junction and the length of the plasma in the capillary tube measured. This was later used as a measure of the amount of the amount of plasma. Direct weighings were also taken in many cases. The plasma was then blown into 12.5$ trichloracetic acid (TCA) in a thick-walled 12-ml centrifuge tube where precipitation of proteins immediately occurred. The precipitate was centrifuged and the supernatant decanted o f f . The precipitate was then washed two or three times with 2.5$ TCA to ensure that a l l I 1 3 1 was washed free from the protein-bound I 1 3 1 ( F B I 1 3 1 ) . The P B I 1 3 1 1 31 was then dissolved by adding IH NaOH. Aliquots of the PBI solution and jl51 solution were then counted i n a well s c i n t i l l a t i o n counter. The volumes of reagents used varied with the size of the blood sample. In large f i s h 4 ml of 1Z»5% TCA were used and with smaller fish 2 ml. Similar amounts of 2.5$ TCA were used for washing the precipitate. 3. Thyroid Sampling The standard method of thyroid dissection was to remove the basi- branchial region of the f i r s t three g i l l arches, trimming away a l l hyoidal musculature, g i l l filaments and most of the g i l l arches. In sockeye smolts i t was found that the dissected thyroid constituted 0.23$ of the body weight with a standard deviation for ninety-six f i s h of 0.045. A similar relation- ship held for large as well as small f i s h . The trimming technique was there- fore considered consistent. The thyroid was then dropped into a "clearsite" counting tube containing Bouin's fixative (many thyroids were histologically examined after counting) and then counted against a standard of comparable volume (4 ml) i n a well s c i n t i l l a t i o n counter. Thyroid uptake was expressed as a percentage in terms of the injected dose. 4« Body Sampling The bodies, lacking throat thyroid, were oounted against a standard - 15 - to determine the percentage of the dose s t i l l retained. The standard consisted of piles of f i l t e r paper cut to the shape and thickness of the fi s h and evenly permeated hy a known proportion of the dose diluted with potassium iodide. Both bodies and their standards were counted under an end-probe s c i n t i l l a t i o n counter with a 45 mm diameter and 58.5 mm thick Nal (Tl) orystal shielded in a lead castle. C. HISTOLOGICAL TECHNIQUE Thyroids were dissected from the basibranchial region in accordance with the distribution of thyroid tissue described by Hoar & Bell (1950). Tissues were fixed i n Bouin's formol-picric-acetic acid fixative and pre- pared for histological study by routine methods. Tissues from the region of the seoond basibranchial region were s e r i a l l y sectioned (lOu). Harris* haematoxylin and eosin stains were used throughout. - 16 - 17. RESULTS A. RADIOIODINE 1. Data on General I * 2 * Metabolism Studies of il31 metabolism were made on underyearling coho in early September with low thyroid activity(Table VI; Fig. 6), in underyearling chum in July (Table VIII; Fig. 8) and August (Table VII; F i g . 7) when the thyroid a c t i v i t y was low and high respectively, and in three year sockeye smolts with an extremely active thyroid (Table IX; Fig. 9). In order to follow the fate of I 1 3 * in these small f i s h , up to 100 individuals were injected and then k i l l e d i n groups of 3 to 12 at intervals from 8 to 24 hours. Means were calculated for each group and considered representative of the i l ^ l metabolic state i n the population as a whole. Where possible the following were measured at each time interval: ( i ) I 1 3 1 Biological Coefficient 1 (Fig. 1) ( i i ) $£. Retention of dose in body (Fig. 2) ( i i i ) % Uptake of dose by thyroid (Fig. 3) (iv) P B l l 5 ! Biological Coefficient (Fig. 4) (v) Conversion Ratio (Fig. 5) The data have also been presented collectively for eaoh separate con- version curve in an attempt to show how the Conversion Ratio for each species can be interpreted i n terms of the measurements made (Table VI - IX; F i g . 6 - '9)T 1 The n i l 3 1 Biological Coefficient n = % of the dose represented as il31 i n the sample x body weight mass of the sample This particular measurement was chosen as i t makes allowance for variations in specimen size. I f the same dose i s injected into different sized f i s h , then the absolute concentration i s inversely related to i t s body weight. Thus allowance for size i s merely made by multiplying "concentration" by "body weight" (Camar, 1955). To follow page 16. Fi g . 1. Loss of injected 1131 from blood plasma in underyearling coho and chum salmon. DAYS DAYS To follow page 16 F i g . 2. Loss of in j e c t e d i l ' l from body (less thyroid) i n underyearling coho and chum salmon. D A Y S To follow papje 16. Underyearling COHO (thy r o i d i n a c t i v e ) Underyearling CHUM (thyroid i n a c t i v e ) COHO smolts (thyroid active) Underyearling CHUM (thy r o i d active) SOCKEYE smolts (thyroid active) F i g . 3. Thyroid uptake of i n j e c t e d I 1 3 1 i n juvenile Oncorhynchus. % THYROID J I I I L _ J 1 I I I I I I I - 17 - 2. Comparative Data Conversion Ratio (C. R.) determinations were made on juvenile Oncorhynchus maintained in fresh water (Table IV; Fig. 11). These tests were checked over certain phases of the season by histological observations and in certain cases by determinations of per cent uptake by the thyroid (Tables XII - XV; Fig. 19 - 21). CHUM SALMON had a high C. R. when f i r s t examined in July. This value declined i n August and then rose to a steady value of 25 - Z0% which was maintained u n t i l November. In addition, histological measurement revealed that the f i r s t peak in thyroid a c t i v i t y as measured by the C. R. method had only recently been attained (Fig. 20). This implies that at the time of migration, their thyroid activity was probably lower. COHO and SOCKEYE SALMON showed definite seasonal trends i n their f i r s t year. There was low thyroid activity in winter followed by a marked peak value in spring coinciding with the period of downstream migration. A very similar pattern seemed evident in underyearling coho, except that thyroid decline appeared slower and even in August showed relatively high activity (Fig. 11). PINK SALMON showed a decline i n thyroid activity from July to October. However, survival was poor and i t i s possible that data were obtained from a highly select group of fish.(Fig. 11). That the state of thyroid activity i n these hatchery maintained fi s h was representative of changes in nature was indicated by histological observations made on coho downstream migrants from the Capilano River in early June. Four migrants examined had mean c e l l heights of 4.58yj (individual values were 4.71, 5.00, 3.99, 4.59 )• This showed very good agreement with the laboratory-reared fi s h ( f i g . 19). The retention of I 1 3 1 in the body (less thyroid) after 108 hours was _ To follow page 17. Underyearling COHO (thyroid i n a c t i v e ) Underyearling CHUM (thyroid i n a c t i v e ) Underyearling CHUM (thyroid a c t i v e ) SOCKEYE smolts (thyroid active) F i g . 4. Changes in PBI1*5-1 concentrations i n plasma samples of juve n i l e Oncorhynchus. DAYS To follow page 17 COHO CHUM SOCKEYE F i g . 5. Juvenile Oncorhynchus conversion curves. 30: 13 To follow page 17 Fig. 6. i l o l metabolism in underyearling coho salmon (inactive thyroid).  To follow page 17. Fig. 7. j metabolism in underyearling chum salmon (inactive thyroid). 2 3 4 5 6 2 3 4 5 6 7 DAYS - 18 - also measured (Table Vj Fig. 12), and consistent seasonal trends demonstrated. At migration, the per cent retention in the body was always low but during the summer and winter climbed to a very high value in coho and sockeye smolts especially, implying a very slow loss of iodine at this season. A similar more restricted trend may also have been present in pink. In chum, however, the retention was very low throughout the entire season u n t i l the f i n a l determination was made in mid-November. At this point, two completely aber- rant f i s h had very high retention of approximately 20% at 108 hours. Plasma levels of 1131 were also measured at certain seasons and the rate of loss represented as a biological h a l f - l i f e (Table V). In contrast to the great extremes noted for biological half-lives for loss of I131 f o r the body as a whole, calculations of half-lives for the plasma f e l l between olosely defined limits, usually 20 - 30 hours. In chum salmon, there was l i t t l e difference between the h a l f - l i f e for loss of i!31 from the body and that from the plasma. In other species, the body values were usually con- siderably longer than those for the plasma. 3. Data on Technique a. Do3e Size Excessive amounts of i!31 o a n injure the thyroid (La Roche and Leblond, 1954j Harris, 1959). Relatively large doses of 1131 ( yie/gm) were used to obtain appreciable PBI131 levels, and i t was therefore con- sidered necessary to investigate the effect of i!31 dose on thyroid activity. Varying doses were administered to underyearling chum s t i l l l i v i n g in fresh water. The results were as shown in Table I . It can be seen that there was relatively l i t t l e variation in Conversion R a t i o for both sockeye and chum over a considerable dose range. In the chum salmon, the thyroid activity was rising sharply at the time when determinations were made and this could account for the low intermediate value (dose 8LLc)» That the C. R. remained To follow page 18. F i g . 8. metabolism i n underyearling chum salmon (active t h y r o i d ) .  To follow page 18. F i g . 9. I 1 5 1 metabolism i n sockeye smolts (very active t h y r o i d ) .  To follow page 18. F i g . 10 Relationship between Conversion Ratio and thyroid uptake of i n j e c t e d I131 i n underyearling chum salmon. THYROID UPTAKE j i i i i constant implied that during the 72 hour period after injection ( i ) the radioactivity had had no detrimental effeot on thyroxine synthesis and ( i i ) the amount of carrier-free I 1 3 1 (0.79 X 10~5 jxga/jio) was within the realms of a 'tracer dose* and had not flooded the system with iodine to the extent that i t s metabolism was altered. In chum salmon, at least seven miorocuries oould be given per gram of f i s h without detriment. This compared favourably with the dose-level used (1 - 2yuc/gm). D • Plasma Sample When several consecutive blood samples were removed from the same fi s h , i t was noted that the second C. R. was usually lower than the f i r s t (Table I I ) . This was due to both a rise in I ^ l concentration and a 1 5 1 drastic lowering of FBI concentration. The following factors could have contributed to t h i s . ( i ) Rapid d o t t i n g (as is known to occur in fish) at the wound, such that thyroxine^ 3 1 with the attached proteins could not escape through the clot. On the other hand, the inorganic i A , a x in the free state could i n i t i a l l y pass through unimpeded. ( i i ) There was almost oertainly a local reduction i n blood volume on sampling. This would be supplemented by extracellular f l u i d , which, for the 131 1^1 aame reasons of permeability, would be rich in I and low i n FBI*" • Consequently, only the i n i t i a l gush of blood was sampled and great care taken not to squeeze the f i s h unduly in the hope of obtaining a largeri sample. I f an adequate dose was injected (2juc/gm), i t was possible to obtain consistent C. R. values with fi s h of 0.6 gram providing plasma samples of 0.001 gram. c. Diurnal Variation Subsequent investigations on chum (Table III) revealed a possible diurnal variation in C. R., reflecting different relative output Fig.11. Seasonal variation in Conversion Ratio values in juvenile Onoorhynehus held in fresh water. COHO smolts H3 O SOCKEYE smolts ^ o H3 (B to Underyearling PINK Underyearling CHUM 40 - 20 - Fig. 12. Seasonal variation in the per cent retention (after 108 hrs) of injected 11*1 in the bodies (less thyroid) of juvenile Oncorhynchus held in fresh water. COHO smolts 4 >-3 O S O C K E Y E smolts {L % o (-• Underyearling P I N K Underyearling CHUM A0O9 % To follow page 19. Fig. I S . Relationship between extent of vacuolation of colloid and lowest c e l l height in sockeye smolts.  - 20 - levels of P B I 1 3 1 at various times of day (high i n the evening, low i n the morning). In view of this, a l l f i s h were k i l l e d between 8:30 A.M. and 10:30 A.M., unless otherwise stated. B. HISTOLOGICAL The following were quantified or semi-quantified in sockeye smolts ranging from 2 to 80 grams. (i ) Lowest c e l l height j > ( i i i ) Mean c e l l height (= Average of 1 & 2) ( i i ) Tallest c e l l height) (iv) Greatest external f o l l i c l e diameter ] / (vi) Mean external f o l l i o l e (v) Greatest external f o l l i c l e diameter I f diameter (=Average of 4 & 5) at 90° to the f i r s t diameter j ( v i i ) Percentage of f o l l i c l e s containing no colloid ( v i i i ) Extent of vacuolation (ix) Depth of staining By arbitrary assignment to one of 8 classes Determination were made on 100 f o l l i c l e s (per individual f i s h ) , selected from the second basibranchial region and means were calculated (Table X). Since, i n the literature, c e l l height has been the most widely used c r i - terion, i t s relationship was tested against the other thyroid characters (Fig. 13, 14, 15 and 17). ^owest c e l l height was used as this minimized the chance of the plane of section going through the base and not the height of the c e l l , thereby giving a falsely high value. It may be noted that extent of "vacuolation"' and "per cent of f o l l i c l e s with no colloid" showed remarkably good agreement with the c e l l height measurement. In view of possible variations i n teohnique and the arbitrary nature of the estimation, i t was not surprising that the point scatter, was wide in the "depth of stain" regression. This negative regression line did however reveal that as the thyroid became more active ( c e l l height), then the staining reaction was l o s t . Cell height and mean f o l l i c l e diameter also To follow page 20. Fig. 14. Relationship between per cent of f o l l i c l e s containing colloid and lowest c e l l height in sockeye smolts. % N O 6 C O L L O I D t\j o i i i i 1 ro O o \ — C E  o ^ o - H E 1  ° Vo I G H T  o \ o \ o | - 1 1 1 1 — To follow page 20. Fig. 15. Relationship between mean f o l l i c l e diameter and lowest c e l l height in sockeye smolts. O o 5 s LU 50h LLI < U J _J (J o LL 30[ o O o o o o —I l__ 3 4 C E L L H E I G H T C / O To follow page 2 0 . Fig. 16. Relationship between mass and mean fol l ic le diameter in sockeye smolts.  To follow page 20. Fig. 17. Relationship between depth of staining and lowest cell height in sockeye smolts. S T A I N I N G t o in T" 'O o O m w r r~ x m o x H O o o o - 21 - showed negative regression and also with a very wide scatter. However, there was an unfortunate bias in the sample, whereby generally smaller f i s h had the highest oell height. It was therefore deoided to test the relation- - ship between mean f o l l i c l e diameter and mass of f i s h (Fig* 16). A much tighter regression resulted implying that changes i n f o l l i c l e diameter, as a result of increased thyroid activity* could be masked by dependence on the mass of the speoimen. To te.st the exact nature of possible interaction between thyroid activity, f o l l i c l e diameter and mass of f i s h , individuals of the same size but with different thyroid activities would have to be used. Many of the above measurements were taken on thyroids in which radio- iodine determinations (C. R. method) had already been made. In sockeye smolts, relationships were tested between lowest, mean, and tallest c e l l height and C. R. Regression coefficients were not calculated, but i t can be seen that there was generally good agreement between c e l l height and C. R. with a sli g h t l y greater scatter where largest c e l l height was measured (Fig. 18). This could be due to the sectioning artifact mentioned e a r l i e r . The agreement between mean c e l l height and C. R. was further substan- tiated by seasonal observations on sockeye, coho and chum salmon (Tables XII - XV; F i g . 19 - 2 l ) • It w i l l be noticed i n chum underyearlings (Fig. 20) that c e l l height also showed a consistent trend with the per cent uptake of jl31 by the thyroid (another estimate of thyroid a c t i v i t y ) . To follow page 21• Fig* 18. Regression of Conversion Ratio and lowest, t a l l e s t and mean epithelial c e l l height in sockeye smolts.  To follow page 21. Pig* 19. Seasonal changes i n Conversion Ratio and mean c e l l height in two-year-old ooho smolts.  F i g . 20. Seasonal changes i n Conversion Ratio, mean c e l l height and t h y r o i d uptake i n underyearling chum salmon. 1-3 O *T> O t-> o £ P C M CD r o u C E L L HEIGHT o- 1 r o 1 1 l± o 1 CD O 1 l 1 1 1 1 r~ THYROID UPTAKE F i g . 21. Seasonal changes i n Conversion Ratio and mean c e l l height in sockeye smolts. t-3 O o M I—*  - 22 - V, DISCUSSION A. IODINE METABOLISM AND EVALUATION OF THYROID ASSAY METHODS When 1*31 ^ s injected into the coelom i t rapidly enters the blood stream. Maximum blood concentration occurs in flounder and Pacific salmon -within 3 or 4 hours after injection (Hickman, 1959; Baggerman, I960). Current studies on juvenile Oncorhynchus suggest that maximum blood concentration occurs before ten hours, but no time period shorter than this was investigated in detail. 151 After uptake into the serum the I*-** becomes uniformly dispersed and can leave by a variety of routes. (i ) It may enter red blood c e l l s . As Leloup and Fontaine (1960) have pointed out the extent to which this occurs i s quite variable and i n the Atlantic salmon and other amphibiotio forms may be prevented by a widespread but loose binding of 1*31 p r o t e i n s in the slow albumin or theO^-1-globulin zone. ( i i ) It may be taken up by the thyroid which concentrates iodine against a steep gradient. This i s dealt with l a t e r . ( i i i ) It may be selectively withdrawn into certain organs of the body. In some salmonids, a very marked concentration can occur in the ovary (Leloup & Fontaine, 1960). (iv) The I131 may be eliminated permanently via the gut, the g i l l s or the kidneys. The relative regies of these potential sites of 1*31 excretion have not been investigated in any detail, but in flounder a very considerable percentage i s thought to be lost v i a the g i l l s (Hickman, 1959). Assuming that each of these various sites of l!31 elimination i s operat- ing at a consistent level, then the depletion of i!31 ±a the serum w i l l show a regular pattern, and i n terms of measured amounts of l!31 i n the blood w i l l - 23 - show an exponential relationship with time (Fig. 1). Consequently, the rate of i l s l removal i s best determined in terms of a "biological h a l f - l i f e ( i t ) , which may vary with species and season. In the accompanying figures, each point represents the n l l 3 1 Biological Coefficient/100" at a particular time for a mean of 10 to 12 f i s h . 131 Indication of excretion rate i s also given by comparing the r A Biological Coefficients after a particular time period. Such values depict relative differences in excretion but are less meaningful. Excretion from the entire body (less thyroid) can also be measured (Fig. 2). This i s extremely variable from species to species and in juvenile Oncorhynchus depends largely on the season. It w i l l be noted in under- yearling chum and coho that i n terms of the per cent dose retained in the body, there are marked differences even at the same time of year (|t ohum z 29 hrsj gt coho - 90 hrs). This i s explained by a greater a f f i n i t y of the peripheral tissues for i!31 or iodine i n general and i s discussed later at some length. It i s from this continually diminishing level of ll31 i n the plasma 1S1 and the body as a whole that the thyroid I concentration occurs. I f the sole ro*le of the thyroid were to accumulate iodide, then the hypothetical curve would show logarithmic form, the exponential f a l l off in rate of I131 accumulation being a direct function of the fact that i!31 blood concentra- tion i s also f a l l i n g i n an exponential manner. However, i t i s the function of the thyroid to synthesize and secrete thyroxine and triiodothyronine which combine in the blood with a thyroid- binding protein (TBP). This i s believed to be an cx.-l-globulin in mammals (Barker, 1955), although in some teleosts i t appears that the binding i s not complete (Leloup & Fontaine, I960). This loss of il31 a a the protein- bound form ( P B l l 3 1 ) w i l l exert a continual influence on the thyroid uptake - 24 - curve. The hormone synthesized w i l l progressively oonfcain a greater and greater relative proportion of P B I x 3 i as more and more i s taken up by the gland. Therefore, assuming total thyroxine output (PBI A^ 7 & PBI13'*') i s occurring at a constant rate, there w i l l be an ever increasing amount of F B I 1 3 1 lost from the,gland. At a c r i t i c a l time period the loss of pgjlSl w i l l eventually exceed the ever diminishing uptake of I 1 3 1 . Under these conditions the total level of 1*31 (protein-bound and inorganic) w i l l show a peak. Leloup & Fontaine (1960) have demonstrated that this peak turnover of iodine occurs earlier in the active thyroid. This i s supported by data presented below (Fig. 3). Therefore, ignoring any discrepency in excretion rate, i t seems that (i) a f f i n i t y of thyroidal tissue for iodine, ( i i ) rate of hormone synthesis and ( i i i ) output of P B I 1 3 1 can a l l influence the thyroid uptake curve. However, merely by measuring i!31 uptake by the thyroid i t i s impossible to analyse separately these various simultaneously operating phases of l!31 metabolism, but different states of activity may be associated with different shaped curves. In F i g . 3A and 3B, the thyroid i s inactive and the uptake curve shows a gradual climb implying a steady increase i n I131 uptake and a l i t t l e loss of P B I 1 3 1 . The latter was borne out by observation (Fig. 4A and 4B). In a thyroid with heightened activity the curve shows a peak at approximately 120 hours (Fig. 3C) and the concentration of I 1 3 1 in the gland may rise up to 10% of the injected dose (Fig. 3D). In F i g . 3D and 3E, extremely active thyroids are being considered. These show a distinct peak which in the case of chum salmon occurs as early as 96 hours• In the very active sockeye this may be as early as 72 hours. This rapid turnover of iodine i s possibly reflected by (i) the relatively low maximum concentration of i!31 i n the gland and ( i i ) the very drastic rise of P B I 1 3 1 i n the plasma with which i t coincides (Fig. 4D). Thus data on Oncorhynchus show that the level of l!31 25 in the thyroid can increase in a very characteristic manner which i s depend- ent on three phases of intra-thyroidal metabolism a l l operating synchronously. Most meaningful appears to be the shape of the curve. A comparison of the sockeye and chum (Fig. 3C and 3D) shows that peak values may vary even in active glands. The slopes of thyroid uptake curves are probably more mean- ingful and as mentioned earlier, the biological h a l f - l i f e for loss of radioactivity from the thyroid has been used as an indication of activity in the trout by Fromm & Reineke (1956) and for other speoies (Leloup & Fontaine, 1960). The closest approximation to absolute thyroid activity would be to measure the rate at which thyroxine i s produced by the thyroid. As yet there i s no direct measure of this quantity. By chemical methods i t has been possible to determine the concentration of thyroxine and triiodothyronine in the blood. However, this cannot be used to measure thyroxine output as peripheral u t i l i z a t i o n i s continually depleting this concentration. On the other hand, one could measure the rate of production of radio- active hormone from the gland (Fig. 4). This quantity i s just as prone to peripheral u t i l i z a t i o n as the non-radioiodine form. However, in the early stages of i ^ S l metabolism, the relative amount of PBI^ 3! added to the plasma from the thyroid i s greater than that catabolised and i s therefore not as susceptible to the above effect. This can be illustrated by the following hypothetical situation i n which the hormone level i n the blood i s constant, but i s being both synthesized and peripherally catabolised at a uniform instantaneous rate. Shortly after injection, a very small percentage of the total hormone output w i l l be radioactive. Owing to the considerable dilution occurring when this FBI* 3! becomes mixed with the stable hormone, only a small fraction w i l l be lost due to peripheral catabolism. Thus the plasma level of P B I 1 3 1 - 26 - w i l l rise and w i l l be further augmented b y the ever increasing ratio of PBilSl to P B I 1 2 7 free for liberation by the gland. After the peak in thyroid uptake, however, this ratio w i l l become progressively smaller and less P B I I 3 I w i l l be released per unit time. At the same time, owing to the increase in PBI A31 concentration i n the plasma, more w i l l be lost peri- pherally. Eventually, the rate of loss of P B I a 3 a from the plasma exoeeds i t s addition and this accentuates the f a l l i n F B I 1 3 1 l e v e l . These aspects of P B I 1 3 1 metabolism cause the N P B I 1 3 1 Biological Coefficient/time" curve to show a distinct peak, which i s evident for a l l species. The graphs are representative of both iodine uptake and hormone syn- thesis and secretion. As such the following points may be noted. (i ) In the inactive thyroid both the climb and f a l l are very slow (Pig. 4A). This i s indicative of slow peripheral catabolism and low pro- duction. The maximum P B I i 3 l concentration in the plasma i s extremely low. Figure 4B shows the plasma P B l l ^ l concentrations in what is considered to be a relatively inactive chum thyroid. In general, the level of radio- active hormone is low but the early minor rise and f a l l i s baffling. Relatively high values of P B I 1 3 x have been noticed within one or two days of injection in other instances and the only explanation is that i t i s an after effect of injeotion treatment. It i s therefore an artifact, dis- rupting the normal smooth continuity of thyroid metabolism. ( i i ) In the active thyroid (Fig. 4C and 4D), the absolute height of the peak is considerable and rise and f a l l i s extremely sharp. This is indicative of an active thyroid gland, i n which both thyroxine output and catabolism are proceeding very rapidly. ( i i i ) The height of the peak represents the concentration of P B I 1 3 1 in the plasma when the output of radioactive hormone by the thyroid i s equal to the rate of i t s catabolism. This peak value is one which w i l l depend on the maximum extent to which PBI131 replaces P B I 1 ^ in the thyroxine pro- duced and also on the rate at which thyroxine i s produced. Thus this peak value w i l l depend on output of hormone, uptake of 1131 extent of syn- thesis incorporating l!31. This peak value i s therefore representative of the several phases of iodine metabolism and may be considered a good indication of the rate of thyroxine output. However, comparisons between peak PBI1^1 values, only have significance 131 i f to each thyroid there was the same ava i l a b i l i t y of I • This d i f f i c u l t y is partly removed by calculating PBI131 plasma concentration in terms of 131 per cent of the injected dose. However, the I x pool i s continually being depleted as the result of renal and extrarenal loss. Consequently, the most 131 logical prooedure would be to relate the PBI concentration peak to the rate of il31 loss from the blood. This could be estimated most precisely in terms of the h a l f - l i f e or rate constant. Suoh determinations involve many measurements. A quicker and less precise, but at the same time quite meaningful method, would be to measure the relative concentrations of PBI 1 31 and I131 at a time corresponding to peak PBI 1 31 concentration in the plasma. This may be expressed as follows: PBll31 Biological Coefficient I131 Biological Coefficient for a single blood sample from the same f i s h this may be represented as P B I 1 3 1 (c.p.m.) 1131 (c.p.m.) or, expressed i n terms of a percentage of the total iodine i n the blood. % COFVERSION RATIO. = PBI (o.p.m.) ^ ^ PBll31 (c.p.m.) / I 1 3 1 (c.p.m.) - 228 - Such a ratio may be determined at any time after injection. Examples of Conversion Ratio curves for several species are given (Fig* 5). The Conversion Ratio curves can be easily interpreted in terms of i A O > l meta- bolism by consideration of Fig* 6, 7, 8.and 9 (Tables VI - IX). In active thyroids, there i s a peak in the C. R. occasioned by high p B I131 a t a time of low il 3 1 concentration. In very active thyroids the peak is very sharp (Fig. 8). In inactive thyroids, though the PBI concentrations peaks, no peak occurs in the C R. and the ratio gradually r i s e s . This i s because the rate i n f a l l of P B I i 3 i concentration in the plasma i s lower than the loss of I 1 3 1 (Fig. 6 and 7). In practice most observations were made at 108 hours after injection as this corresponded quite closely to most of the peaks i n the active thyroid. In the very active thyroids, examination should be made up to 24 hours earlier otherwise a falsely low thyroid level w i l l be recorded (Fig. 8). It i s also equally important not to use C. R. measurements made too long after injection. This w i l l give a ratio which is falsely high as the rate of loss of P B I x 3 i never approaches that of il31 loss (Fig. 6) and in theory the ultimate C. R. after a sufficient length of time would be 100%. Thus the success of the Conversion Ratio i s dependent on a certain knowledge of the overall i 1 3 x metabolism of the individual. This i s a criticism whioh can be levelled at any radioiodine determination and does not detract from i t s applicability. There are, however, certain possible disadvantages associated with the method (i) It assumes the binding of thyroxine and triiodothyronine with plasma globulins to be complete and assumes that no organio binding of 1131 W i t h plasma proteins oocurs. As Leloup & Fontaine (i960) have pointed out v a r i a b i l i t y in the former i s a possib i l i t y , while the latter combination - 29 i s a certainty i n amphibiotic f i s h . But, i t is also shown that under the experimental conditions the latter combination i s s p l i t by trichloracetic acid. ( i i ) There may also be v a r i a b i l i t y i n extent of I penetration into erythrocytes. This has. been shown to vary with species and season (Leloup & Fontaine, I960). One way to eliminate such discrepency i n the inorganic count would be to count the red blood cells with this fraction or to haemo- lyse them prior to precipitation . Despite these possible anomolies (which could be allowed for), i t i s considered that the C. R. method takes into account most aspects of 1*31 metabolism and is free from disadvantages associated with measurement of 1*31 Uptake by the thyroid. It is a quick and sensitive method and can be used even to measure diurnal variation in thyroid activity, i f such does exist. It requires only small blood samples and in large specimens does not necessitate k i l l i n g the f i s h i f sampling i s done by lateral puncture of the haemal artery. In almost every instance i t agrees with data obtained by histological techniques (Fig. 18 - 21). In the case of underyearling chum salmon re- tained in fresh water, remarkable agreement (on the basis of means of 10-- 12 fish) i s also shown with per cent il31 uptake by the thyroid (Fig. 19). Also significant in these f i s h i s the consistent loss of il31 from the body throughout the season (Table V; F i g . 12). Most important i s the good agreement between determinations of thyroid activity by radioiodine and by histological techniques, especially c e l l height. There is also good agreement between c e l l height and other histo- logical characters. Certain anomolies have been observed by other workers. In particular, Pickford (Pickford & At/z, 1957, p. 129) mentions the dis- crepency between c e l l height and staining reaction in Fundulus. - 30 - A less c r i t i c a l but far quicker estimate would be i n terms of the extent of Mvacuolation r t. In particular, estimation of the n % of f o l l i c l e s with no colloid" could be used to very great advantage for quick determinations and seems a generally reliable and precisely quantified measure. Far less reliance should be placed on estimates of depth of stain, f o l l i c l e shape, and especially mean f o l l i c l e diameter* The latter i s almost certainly dependent in part on size in sockeye smolts. The best method i s that of Fortune (1955). As described earlier, this accounts for c e l l height and extent of colloid, both of which are reliable and measurable characteristics. However, the method appears tedious. Taking into account the above data and the central role played by the epithelial c e l l in iodine uptake, i n i t i a l iodination and thyroxine release, i t i s suggested that the simplest and most straightforward method i s to measure the epithelial height. Such a method has often been used but has rarely been compared to more reliable radioiodine techniques and thereby never substantiated before. In conclusion, a l l methods for determining thyroid activity oan be usefully employed in poikilothe-rms. Data presented reveals good agreement between histological and radioiodine determinations. Chemical methods may also be helpful in completing the picture of iodine metabolism, but could possibly be misleading in determinations of level of hormone output, though this has not been borne out by any personal observations. The value of reliance on histology i s particularly evident for f i e l d determinations. The C» R. method is chosen where an accurate assessment of thyroid overall activity i s required and i s the generally preferred measure. However, i t would be inapplicable for small f i s h where the i n - dividuals have to be kept alive. In this case, thyroid uptake of iodine, though not so reliable, could be adapted to an in vivo£ technique. - 31 - Despite above criticism, sudden increase in thyroxine output appears in many cases to cause an increased plasma thyroxine level (Leloup & Fontaine, 1960), while stable inorganic iodine determinations are of value i n assess- ing general iodine depletion of the tissues or the environment. A l l methods have their use, but must be used with discrimination and inter- preted as much as possible in terms of the complete iodine metabolic pathway. B. THE ROLE OF THE THYROID IS ANADROMY Seasonal fluctuation of thyroid activity in f i s h has been confirmed by many workers - Hoar (1939) and Leloup & Fontaine (1960) in the Atlantic salmonj -Hoar & Bell (1950) i n the Pacific salmon; Hoar (1952) in the alewife and smelt; Buchmann (1940) in the herring; Barrington & Matty (1954) in the minnow and Swift (1955, 1958) in the brown trout. In certain species, especially amphibiotic salmonids, peaks in thyroid activity have been correlated with migration disposition. In the Atlantic salmon and rainbow trout, there is evidence to suggest that thyroxine i s causal to many aspects of smoltification. It i s significant that the effects of thyroxine on the "pseudosmolt" are often less marked than those in nature, despite a frequent hormone dosage above normal physiological l e v e l . The stimulatory role of thyroxine has therefore not been f u l l y established and i t i s certainly not stimulatory to a l l aspects of smolti- f i c a t i o n . One line of evidence not supporting such a role was supplied by Hoar & Be l l (1950) in Oncorhynchus on the basis of histology. It was found that although sockeye and coho might have more active glands at the time of migration, pink and chum had inactive glands, but would become extremely hyperplastic in postmigrants retained in fresh water. Late pink and chum migrants also showed increased thyroid a c t i v i t y . 32 Such observations suggested that the thyroid was acting i n a compen- satory role. The thyroid was considered to offset osmotic unbalance occasioned by increased salt loss, induced by retaining a f i s h already adapted to marine l i f e for an extended period i n fresh water. However, doubt was cast on the va l i d i t y of the histological observations. Never- theless, data presented here suggest a very good agreement in juvenile Oncorhynchus between histological and radioiodine determinations. On that basis, the earlier work by Hoar & Bell has been accepted as representative of the thyroid state and has been combined with current histological and radioiodine data to give a more complete comparative picture for Oncorhynchus, which i s as follows. In a l l fry, the thyroid i s quiescent. During this stage, sockeye may move from stream to lake and chum and pink move to the sea. Some of the late sea-running pink and chum may become slightly more active and on reten- tion in fresh water become extremely hyperplastic. In chum salmon, this hyperplasticity temporarily drops in mid-August but i s then maintained at a high level u n t i l November. In pink the thyroid activity i s high i n June, but moderately low in October. It may be noted that chum and pink in their f i r s t year of marine l i f e have quiescent or mildly active glands. On the other hand, sockeye and coho normally remain in fresh water and migrate as yearlings. They both have an extended parr l i f e and during this period thyroid activity i s low, though in underyearling coho a moderate fluctuation in activity may be observed (Fig. 11). At smoltification, the gland shows a very marked increase in ac t i v i t y . However, unlike chum and pink, retention in fresh water induces not an active but an extremely i n - active thyroid state, which occurs very suddenly in June. It i s also interesting to note that sockeye can survive in fresh water for at least four years and that i n their t h i r d year they show a cyole of thyroid change - 33 - comparable to that in the younger f i s h . Such observations could suggest a compensatory rather than a stimu- latory thyroid role i n alleviation of osmotic stress (Hoar & B e l l , 1950; Hoar, 1952). It should be remembered, however, that the thyroid a f f i n i t y for iodine i s well adjusted to the concentration of iodine in the environ- ment . In a f i s h destined to move into iodine-rich marine conditions, retention in iodine deficient fresh water could lead to a goitrogenic conditions ..offset by greater hyperplasticity• Thus the hyperactivity of the chum thyroid could be apparent rather than real* But both hypotheses would suggest thyroid hyperactivity as being secondary to other changes* There i s certain evidence to support the osmotic theory* In part, many of the changes observed in smoltification are superficially either adaptations enabling the young salmon to be displaced downstream or a form of "preadaptation" for "anticipated" marine l i f e . Most evident is the loss of parr marks and acquisition of the "mirrored" sides of the pelagic f i s h . Preadaptation for an environment of very high osmotic pressure i s of greater importance. In general, there i s adaptation towards water reten- tion and ion loss. This i s reflected by a thickened epidermis, the develop- ment of chloride secreting cells in the g i l l s , increased s a l i n i t y tolerance and a marked s a l i n i t y preference. Should these aspects be functioning while the animal is in fresh water, then osmotic stress in the form of deminerali- zation might be expected. Such demineralization has been shown to occur in the e e l . As Hoar (1959) has pointed out, a loss in the inorganic constituents also precedes the seaward migration of Salmo salar (Fage & Fontaine, 1958), Oncorhynchus masou (Kubo, 1955) and Salmo gairdneri (Houston , 1960) and may be a characteristic feature of a l l such migrations. Data presented here on the variation i n retention of i l s l in the body also suggest demineralization, i f one assumes that i!31 ioss ±s ±n part a - 34 - 131 reflection of overall ion loss. Coho and sockeye had a very low i A U J - retention at the time of high thyroid activity, but later when thyroid activity dropped, this changed to low retention. In chum, there was a persistent very rapid loss of any I 1 3 1 injected and this was coincident with a very high thyroid activity. Pink salmon showed an intermediate condition. Like chum salmon, they had a low retention in September but i n October i t 131 was much higher. Again i x c , x retention paralleled thyroid a c t i v i t y . In such instances i t might be argued that since i!31 retention i s generally high when thyroid activity i s low, the low body retention could be due to a rapid i l ^ l turnover by the thyroid. But when one considers that the per cent uptake in chum salmon by the thyroid at 108 hours may vary from 0.6% to 10%, with no perceptible change in iodine loss, and also that the P B l l 3 * level in the blood i s but a minute fraction of the original dose, then such an explanation seems unlikely. If i t i s assumed that loss of i X O J - reflects general ion loss, then the "demineralization theory" i s partly substantiated. But, chum salmon in August (Fig. 11 and 12) have both an inactive thyroid and a very low body retention and this alone would tend to suggest that the two phenomena are not directly related. However, from the beginning of September to mid- November, the C R. in these f i s h did not vary. It i s possible that the i n i t i a l rise and f a l l in C. R. value prior to this stabilization reflects a period of homoiostatic adjustment. Indirect evidence negating such a role of thyroxine was presented by Hickman (1959) who showed in the starry flounder that thyroxine was asso- ciated with increased loss of ions and not their retention. In addition, Baggerman (I960) claimed that the high thyroid activity in chum salmon retained in fresh water was not reduced by immersion in salt water. Baggerman1 s work was done on the basis of thyroid uptake. In an exploratory - 35 - study, her experiment was repeated using the C. R. method. The results were far from conclusive hut did suggest that not only may salt water have no inhibiting action on the thyroid, but could occasion an appreciable r i s e . These experiments are to be repeated. The relationship between thyroid activity and osmoregulation i s far from settled. There are many experiments demonstrating changes in thyroid activity when f i s h are transferred from fresh to salt water and also with the reverse procedure. Such data would suggest a compensatory and prob- ably transitory rise i n thyroid activity whenever the osmotic pressure of the surrounding medium i s drastically altered. However, this does not preclude the possible stimulatory role implied by Baggerman's work. This i s a view frequently expressed by Fontaine (1950) whereby thyro- xine stimulates or i s closely associated with ion loss. This loss then brings about more profound neuroendocrinological changes. These changes in conjunction with appropriate external conditions give rise to changes in behaviour favouring displacement of the parr to the sea. In many ways this i s an attractive theory as i t combines both aspects of smoltification, "marine preadaptation" and "downstream displacement", into a single inte- grated physiological and ethological change. Although Fontaine's idea lack direct support, they pose no serious criticism when related to Atlantic salmon. As applied to Pacific salmon, certain anomolies are evident when chum and pink are considered which have histologically low thyroid activity at migration. The survival value of very high thyroid activity in a stimulatory capacity at a time well past their normal time of migration i s d i f f i c u l t to interpret and one wonders i f i t has any biological meaning in the normal l i f e cycle. Concerning the role of thyroxine, l i t t l e oan be said other than i t i s part of the neuroendocrinological transition whioh must occur when the 36 physiologically drastic process of smoltification occurs. It i s closely associated with smoltification hut i t s specific role is s t i l l unknown. Changes at the neuroendocrinological level rarely occur singly but as a complex and integrated association. It could be that in terms of such ohanges, the thyroid plays a very subordinate role and that early investi- gations of smoltification in salmonids, by placing great weight on this relatively easily quantified gland, have inadvertently led science away from the main governing factors in the neuroendocrine system. Irrespective of i t s function, however, changes i n thyroid activity can be used as indications of migration disposition in the genus Oncorhynchus. C. SPECULATION OW SMOLTIFICATION AND THE PHYLOGENY WITHIN THE GENUS ONCORHYNCHUS Certain differences exist between species of Onoorhynohus in both thyroid activity and body retention of I a 3 a , which, as a possible reflec- tion of t o t a l ion loss, might be indicative of marine preadaptation. In both respects chum are extreme. Although thyroid activity i s low at the time of downstream migration, retention in fresh water causes a rise main- tained u n t i l death. This i s accompanied by drastic demineralization. These features suggest an irreversible adaptation to marine l i f e compatible with their obligatory seaward migration. Accepting Neave's theory (Neave, 1958) concerning the role of pleisto- cene glaoiation i n the origin to speciation of the genus Oncorhynchus, i t is not d i f f i c u l t to envisage possible conditions under which chum could have evolved. Severe glaciation, restricting p a r t i a l l y diadromous f i s h to lo c a l i t i e s of reduced food and fresh water, would favour selection for development of an extended phase of marine l i f e . The advantages afforded by faster growth in a medium of higher osmotic pressure (Canagaratnam, 1959) - 37 would only be offset by the physiological need for reproduction in fresh water (Tchernavin, 1939). The high thyroid activity and extreme ion loss resulting from prolonged retention of the fry i n fresh water suggests in a b i l i t y to readapt to fresh water. I f one accepts the Oncorhynchus ancestor as being only weakly amphibiotic, then i t i s quite conceivable that more complete smoltification as evidenced in sockeye and coho was never attained in chum salmon. In sookeye and coho, smoltification i s transitory with respect to l ^ l thyroid hyperfunction and iJ-<iX loss from the body. The change i s precisely timed and normally occurs after a prolonged period of juvenile l i f e in fresh water. It probably develops as the result of external influence on an appropriately primed neuroendocrine system. In many respects some of the changes parallel those in chum salmon and i t i s speculated that smolti- fication now seen i n sockeye and coho i s a reflection of genetically con- troll e d marine preadaptation, whioh has more recently been incorporated as a mechanism for displacing the parr to the sea. Expression of marine behaviour in the fresh water habitat could i n i t s e l f provide the basis on which selection might act. Thus though sockeye and coho have a more elab- orate smoltification, i t s transitory nature might suggest a closer a f f i n i t y of' these fish towards a possible fresh water or p a r t i a l l y diadromous ancestor. Pink are intermediate between sockeye and chum. The low thyroid activity at migration and elevation on prolonged retention in fresh water i s characteristic of chum. But the decline in thyroid activity and increase 131 in. r 4 retention in the body i s reminiscent of the transitory hyperfunction of sockeye. It i s postulated that a sockeye ancestor isolated under glacial conditions, as severe as these considered to govern the evolution of chum salmon, could have led to a seoondary obligatory migration now witnessed in - 38 - pink* In conclusion, i t might be stated that coho, sockeye and chum could a l l have evolved independently from an Oncorhynchus ancestor under a variety of glacial conditions* However, pink may have been secondarily evolved from sockeye* In many respects, this conclusion supports the phylogeny suggested by Hoar (1958), who, on the basis of a study of juvenile behaviour, aooepted the fresh water ancestry proposed by Tchernavin (1939)* Coho were claimed to represent the primitive stream-dwelling ancestor most closely, while from a sockeye-like ancestor, probably l i v i n g in lakes, more extreme migrants as chum and ultimately pink were evolved* Both physiological and ethological evidence suggest that coho and sockeye are more primitive with respect to tendency to stay in fresh water* However, on physiological evidence alone, chum could be independently evolved from the ancestral form while pink seem much closer to sockeye than Hoar indicated. It i s l i k e l y that adequate f o s s i l evidence w i l l f i n a l l y resolve the problem of the phylogeny of the genus Oncorhynchus. - 39 - VI. SUMMARY AND CONCLUSIONS From radioiodine metabolism and histological studies on juvenile Oncorhynchus, the following conclusions are drawn. (i) That histological and radioiodine determinations show good agreement • ( i i ) That the Conversion Ratio method i s a valid one and i s repre- sentative of the metabolic pathway of iodine, but should not entirely replace other methods. ( i i i ) Comparative studies (in conjunction with the data of Hoar & Be l l , 1950) reveal marked seasonal change in thyroid activity associated with downstream migration. (iv) In coho and sockeye this oocurs simultaneously with smoltification, a process which both "preadapts" the f i s h for marine l i f e and also brings about i t s downstream displacement. Evidenoe for drastic physiological change at this time i s the rapid loss of I1^1, probably indicative of greatly increased deficiency of a l l ions. Sockeye and coho can readily revert to the fresh water physiological state of low thyroid and high ion retention and enjoy a relatively high survival in fresh water. (v) In chum, the mechanism operates differently. These young f i s h are never really adapted to fresh water, but undergo their f i n a l transition in the sea. Evidence that such change has become inherent, i s the irrevers- ible thyroid hyperplasticity and ion loss induced by retaining them in fresh water. (vi) Pink are superficially similar to chum i n a l l respects but do show readaptation to fresh water in terms of low thyroid a c t i v i t y and reduced i!31 loss when fresh water retention i s prolonged. ( v i i ) Thyroxine has been assigned no specific role. It appears to be - 40 - closely correlated with i l 3 l i o a s may have an osmoregulatory role but there is no direct evidence to support this in Oncorhynchus. In the present treatment, i t has been considered as a physiological indication of migration disposition. It may even be a reflection of continued existence in a iodine deficient fresh water environment. ( v i i i ) In view of the lack of available evidence, detailed speculation on the evolution within the genus would be f u t i l e . However, combining physiological data with the geological presentation by Neave, general state- ments can be made. It i s suggested that ohum, ooho and sockeye could have evolved independently from the ancestral Oncorhynchus species and derived their varied l i f e histories during glacial isolation. Pink, however, may have been secondarily derived from sockeye. (ix) Smoltification has been considered a specialized process com- bining marine preadaptation with a mechanism for downstream displacement. It could have been derived by delaying the type of marine preadaptation shown to occur i n postmigrant chum salmon held in fresh water. - 41 - VII. BIBLIOGRAPHY Baggerman, B. Salinity preference, thyroid activity and the seaward migration of four species of Pacific salmon (Oncorhynchus)o J . Fish. Res. Bd. Canada, 17: 295-322 (i960). Bargmann, W. Uber intrafollikulare Blutungen i n der Sohilddruse der Selachien. Anat. Anz., 88: 41-45 (1939). Barker, S. B. The circulating thyroid hormone. Brookhaven Symp. B i o l . , 7: 74-89 (1955). Barrington, E. J . W. and Matty, A. J . Seasonal variation in the thyroid gland of the minnow, Phoxinus phoxinus L>, with some observations on the effect of temperature. Proc. Zool. Soc. London, 124: 89-95 (1954). Brown-Grant, K. The "feed back" hypothesis of the control of thyroid function. In Ciba Foundation Colloquia on Endocrinology, 10: 97-116 (1957). Buchmann, H. Hypophyse und Thyroidea im Individualzyklus des Herings. Zool. Jb. Abt. 2 Anat. Ontog., 66: 191-262 (1940). Canagaratnam, P. Growth of fishes in different s a l i n i t i e s . J . Fish. Res. Bd. Canada, 16: 121-130 (1959). Carter, G. S. On the control of the level of activity of the animal body. 1* The endocrine control of seasonal variations of activity in the frog. J . Exptl. B i o l . , 10: 256-273 (1933). Chavin, W. Thyroid distribution and function i n the goldfish, Carassius auratus L. J . Exptl. Zool., 133: 259-279 (1956). Comar, C. L. Radioisotopes in biology and agriculture. McGraw-Hill Book Co., Inc., Toronto (1955). De Robertis, E. Cytological and cytochemical bases of thyroid function. Ann. N. Y. Acad. Sc i . , 50: 317-335 (1949). Fage, L. and Fontaine, M. Migrations. _In Traite de Zoologie, 13: 1850- 1884, edited by P. Grasse, Masson, Paris (1958). Fontaine, M. Faoteurs externes et internes regissant les migrations des poissons. Ann. B i o l . , 27: 569-580 (1951). Fontaine, M. Du determinisme physiologique des migrations. B i o l . Rev., 29: 390-418 (1954). Fontaine, M. and Hatey, J. Variations de l a teneur du foie en glycogene chez le jeune saumon (Salmo salar L.) au cours de l a "smoltification". C. R. Soc. Bio l . , Paris, 144: 953-955 (1950). Fortune, P. Y. Comparative studies of the thyroid function i n teleosts of tropical and temperate habits. J . Exptl. B i o l . , 32: 504-513 (1955). - 42 - Fromm, P. 0. and Reineke, E. P. Some aspects of thyroid physiology in rainbow trout. J . C e l l . Comp. Physiol., 48: 393-404 (1956). Gaylord, H. R. and Marsh, M« C. Carcinoma of the thyroid i n salmonid fishes. B u l l . U. S. Bur. Fish., 32: 367-524 (1912). Gudernatsch, J . F. The thyroid gland of the teleosts. J . Morphol., 21: 709-782 (1911). Harris, P. J. A study of thyroid function in Fundulus heteroclitus. B i o l . B u l l . , 117: 88-99 (1959). Hickman, C. P. The osmoregulatory role of the thyroid gland in the starry flounder, Platichthys stellatus. Can. J . Zool., 37: 997-1060 (1959). Hoar, W. S. The thyroid gland of the Atlantic salmon. J . Fish. Res. Bd. Canada, 4: 442-460 (1939). Hoar, W. S. Thyroid function 1 i n some anadromous and landlocked teleosts. Trans. Roy. Soc. Canada, 46: 39-53 (1952). Hoar, W. S. The evolution of migratory behaviour among juvenile salmon of the genus Oncorhynchus. J . Fish. Res. Bd. Canada, 15: 391-428 (1958). Hoar, W. S. Endocrine factors in the ecological adaptation of fishes. Symposium on Comparative Endocrinology, edited by A. Gorbman, John Wiley & Sons, New York, pp. 1-23 (1959). Hoar, W. S. and B e l l , G. M. The thyroid gland in relation to the seaward migration of Pac i f i c salmon. Can. J . Res., 28: 126-136 (1950). Houston, A. H. Variations in the plasma-level of chloride i n hatchery- reared yearling Atlantic salmon during parr-smolt transformation and following transfer into sea water. Nature, 185: 632-633 (1960). Kubo, T. Changes of some characteristics of blood of smolt of Oncorhynchus masou during seaward migration. B u l l . Fac. Fish. (Hakodate), Hokkaido Univ., 6: 201-207 (1955). La Roche, G. and Leblond, C. P. Destruction of thyroid gland of Atlantic salmon (Salmo salar L.) by means of radioiodine. Proc. Soc. Exptl. B i o l . Med., N. Y., 87: 273-276 (1954). Leloup, J . and Fontaine, M. Iodine metabolism in lower vertebrates. Ann. N. Y. Acad. Sci., 86: 311-676 (1960). Lever, J . A mathematical method for the determination of the state of activity of the thyroid gland. Proc. Kon.. Ned. Akad. v. Wetenseh., Amsterdam, 51: 1302-1309 (1948). Lovern, J . A. Fat metabolism in fishes. V. The fat of the salmon in i t s young fresh water stages. Biochem. J., 28: 1961-1963 (1934). Neave, F. The origin and speciation of Oncorhynchus. Trans. Roy. Soc. Canada, 52: 25-39 (1958). - 43 - Nishida, H. Cyto-histolagical observations on the gland c e l l of the branchial epidermis, with the comparison of two types of Oncorhynchus masou, landlocked and sea-run form. S c i . Rep. Hokkaido Fish.Hatch., 8; 33- 37 (1953). Olivereau, M. Etude volumetrique de l'interrenal anterieur au cours de l a smoltification de Salmo salar L. Aota Endocrinologica, 33: 142-156 (1960). Pickford, G. E. and Atz, J . IT. The physiology of the pituitary gland of fishes. N. Y. Zool. S o c , N. Y. (1957). Rasquin, P. The influence of light and darkness on thyroid and pituitary activity of the oharacin, Astyanax mexicanus, and i t s cave derivatives. B u l l . Amer. Mus. Hat. Hist., 93: 497-537 (1949). Riggs, D. S. Quantitative aspects of iodine metabolism in man. Pharm. Rev., 4: 284-370 (1952). Robertson, 0. H. The occurrence of increased activity of the thyroid gland in rainbow trout at the time of transformation from parr to silvery smolt. Physiol. Zool., 21: 282-294 (1948). Stolk, A. Histo-endocrinological analysis of gestation phenomena in the cyprinodont, Lebistes reticulatus Peters. I. Thyroid activity during pregnancy. Proc. Akad. Sci., Amst., C. 54: 550-557 (1951). Swifb, D. R. Seasonal variations in the growth rate, thyroid gland aotivity and food reserves of brown trout (Salmo trutta L.). J . Exptl. B i o l . , 32: 751-764 (1955). ~~~ Swift, D. R. Seasonal variation i n the activity of the thyroid gland of yearling brown trout, Salmo trutta L. J . Exptl. B i o l . , 36: 120-125 (1959). Tchernavin, V. The origin of salmon. Salmon and Trout Mag., 95: 1-21 (1939). Vivien, J . Les glandes endocrines. In Traite de Zoologie, 13: 1470-1544, edited by P. Grasse, Masson, Paris (1958). - 44 - T i l l APPENDIX 45 - TABLE I Effect of I13! dose on Conversion Ratio in underyearling chum salmon Mean mass Mean C.R. Dose No* of (S.D.) (S.D.) injected (tic) f i s h 3.8 13.0 2 11 (1.47) (3.56) 5.0 12.0 4 11 (2.18) (5.60) 4.8 7.60 8 12 (2.39) (5.81) 5.7 15.80 16 8 (1.29) (8.15) 5.7 15.35 40 9 (2.52) (5.06) - 46 - TABLE II Decrease in Conversion Ratio in second plasma sample in sockeye smolts Body Plasma % change % change % mass mass I131/mgm PBI 1 3 1/agm change No. C.R. (m) (mgm) C.R. A 1 73.0 25.00 41.3 3.53 6.9 3.01 2 70.8 29 «2 increase decrease decrease 1 57.7 26.6 66.30 16.8 29.65 B 2 40.6 23.35 21.6 increase decrease decrease 1 49.7 38.65 0.379 14.2 5.84 C 2 45.8 18.80 30.00 increase decrease decrease 1 18.06 13.50 16.5 0.234 89.0 86.80 D 2 2.37 35.6 increase deorease decrease E 1 36.9 16.80 27.0 10.80 86.2 77.90 2 8.2 40.0 decrease decrease decrease 1 4.15 57.0 4.12 21.6 10.60 F 2 3.71 33.00 61.0 decrease deorease decrease 1 40.70 23.80 45.0 215.5 93.8 96.6 G- 2 1.33; 50.0 increase deorease decrease 1 1.97 22.20 64.0 0.90 3.4 5.3 H 2 1.87 48.0 increase deorease decrease 47 - TABLE III Diurnal variation in Conversion Ratio in chum salmon K i l l e d August 15th. Kil l e d July 25th. Conversion Ratio lOtOO a.m. 10:00 p.m. 11:00 a.m. 11:00 p.m. 5.2 10.6 1.0 12.2 7.1 9.2 3.2 51.8 9.2 11.6 7.2 9.1 8.0 11.2 7.1 9.3 11.0 7.1 3.6 8.1 13.2 9.8 25.6 24.5 19.6 12.1 7.0 14.2 3.2 13.4 37.5 12.5 6.6 - 3.5 9.6 2.2 - 1.3 17.4 1.5 21.4 30.9 14.2 3.0 10.0 - 8.9 Mean - 7.7 11.6 11.8 16.0 Significantly different by Significantly differnt by ranking at 0.01$ le v e l . ranking at 0.05^ le v e l . TABLE IT Seasonal change in Conversion Ratio in juvenile Onoorhynohus held i n fresh water Aug* Sept* Oct* Jan* May May June July Date COHO 15 7 1 1 20 30 13 11 underyear lings and smolts 15.3 7.9 6.0 4.0 2.4 0.9 1.7 0.6 34.0 12.2 19.3 11.3 12.2 3.9 1.9 1.6 C.R. S.D. May June June July July Aug. Sept. Oct. Date 29 6 15 2 18 9 7 12 Yearling 3 9 # 4 2 3 < 6 3 1 # 6 3 < 4 6 # 1 3 # 4 2 # 5 8 . 3 C . R . SOCKEYE 1 2 < 2 1 5 < 6 3 0 # 1 1 # 9 2 # 5 2 . 4 0 # 9 3 # 0 s.D. Jan. May* May* June June July July Sept. Oct. Date Three and four- 10 20 28 4 16 2 18 7 12 ? A ^ S d 3*3 75#4 6 2 , 9 3 , 2 3 , 8 1 , 6 1 2 , 5 6 , 0 5*2 C*E# SOCKEYE Q # 9 1 3 # 2 1 5 # 8 2 # 2 2 < 1 0 # 2 4 # 6 0 # 1 1 < 9 S > D # July Aug. Aug. Oct. Date 18 10 50 12 Underyearling 4 2 < 0 1 8 # ? 2 3 # 0 6 # 9 C # R > P I N K 18.9 6.7 2.2 1.3 S.D. July July July Aug. Aug. Aug. Aug. Sept. Oot. Nov. Date 7 18 25 6 15 22 30 7 12 20 Underyearling 4 g # g 4 ? < 0 n < 6 2 Q > 5 ? # 5 1 Q # 8 l g # 0 2 ? # 6 2 6 # g 3 0 # Q Q ^ m M 29.5 9.6 14.5 5.9 5.4 8.8 3.6 19.5 19.5 f s.D. * Examined 84 hours - a l l other determinations at 108 hours, •j- Estimated from per cent uptake by thyroid. - 49 - TABLE V Seasonal change in the per cent retention (after 108 hrs) of injected I 1 3 1 in plasma and body (less thyroid) of juvenile Oncorhynchus held in fresh water A. Underyearling CHUM stserum Ijtbody % in body Date (hr) (hr) at 108 hrs July 7 32 mm mm Aug.' 6 24* 2.8 Aug. 22 24 23 2.2 Aug.-30 19* 1.3 Sept. 7 19* 1.4 Oct. 12 28* 4.4 Nov. 20 26* 4.2** B. Underyearling COHO Sept. 3 30 89 24.0 Oct. 1 50* 10.8 Nov. 20 69* 13.9 C. COHO smolts June 15 24 25 2.7 Nov. 20 (very long)* 34.7 D. Underyearling PINK salmon Aug. 6 > - 47* 8.9 Aug. 29 21.5* 1.7 Oct. 12 72* 15.6 E. Yearling SOCKEYE smolts June 14 25 23 2.5 Sept. 7 (very long)* 24.8 Oct. 12 74 16.1 Nov. 20 (very long)* 35.0 F. Three-year-old SOCKEYE smolts May 16 23 _ „ Sept. 7 (very long)* 25.0 Oct. 12 (very long)* 18.5 Nov. 20 (very long)* 26.3 •Calculated from per cent in body at 108 hrs. **2 f i s h in this group had values of 22.7 and 20.0$. TABLE YI ll31 metabolism in underyearling coho salmon (thyroid inactive) I 1 3 1 B i o l . F B I 1 3 1 PBIt C. R. % thyroid Coeff./lOO B i o l . Coeff. % body (hr) Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. 24 0.7 0.15 1.6 0.33 9.9 4.5 0.0 0.0 35.8 12.4 48 1.2 1.11 2.2 1.07 8.8 4.4 1.6 1.3 36.8 7.6 72 2.1 0.35 2.1 0.92 2.1* , 1.3 2.4 1.6 27.5 10.9 96 3.0 1.34 3.8 0.83 1.3 0.7 2.5 2.0 27.6 7.6 120 6.0 4.00 3.7 1.23 1.5 0.6 5.2 2.12 22.1 3.6 144 3.4 1.38 3.6 1.30 0.6 0.3 2.2 2.3 14.8 7.3 312 8.7 2.89 5.0 1.95 0.024 0.007 1.3 1.2 3.8 2.9 Sampled September 2 - 15, 1960. Each mean comprises 10 f i s h . Size range 0.85 - 2.6 gm; 4.1 - 7.2 am TABLE VII ll31 metabolism in underyearling chum salmon (thyroid inactive) 1131 B i o l e P B I 1 3 1 PBU C R. % thyroid Coeff./lOO B i o l . Coeff. % body (hr; Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. 12 2.2 0.69 0.93 0.41 16.10 5.31 1.80 2.40 30.5 8.15 36 7.7 2.89 1.38 0.53 9.50 4.99 33.60 5.80 15.1 2.33 60 9.4 8.46 1.68 0.49 3.45 2.43 14.50 3.90 8.0 4.00 84 5.2 2.82 1.70 0.34 1.90 1.18 3.30 2.80 4.1 1.90 108 10.0 8.66 1.67 0.55 0.83 0.45 3.60 0.60 2.2 1.33 132 12.1 6.48 - - 0.53 0.33 4.8 0.50 1.8 0.9& 156 17.9 11.10 2.07 0.62: 0.37 0.30 2.8 0 . 80 1.2 0.63 Sampled August 18 - 24, 1960. Each mean comprises 11 - IS fis h Size range 1.3 - 4.3 gm. TABLE VIII 1131 metabolism i n underyearling chum salmon (thyroid active) i l S l B i o l . PBI131 PBI-£ C R. % thyroid Coeff./lOO B i o l . Coeff. (hr) Mean S.D. Mean S.D. Mean • S.D. Mean S.D. 20 7.6 2.45 48 17.6 8.90 72 9.8 5.94 96 44.7 12.30 108 49.6 21.50 120 40.2 13.10 150 24.8 23.40 3.72 1.23 8.39 4.93 1.42 1.56 7.07 2.69 1.28 10.33 1.43 0.73 9.20 3.84 0.52 8.40 2.24 0.25 5.80 3.00 0.28 9.10 3.1 1.25 0.80 20.5 1.28 0.39 4.3 2.00 0.21 65.2 31.50 0.80 55.5 22.60 0.29 18.4 15.60 0.12 22.0 8.70 Sampled July 2 - 8 , 1960. Each mean comprises 3 - 8 f i s h . Size range 0.4 - 1.2 gmj 3.5 - 6.0 cm TABLE IX l!31 metabolism, in three-year-old sockeye smolts (thyroid active) j l S l B i o l . PBI 131 C R. % thyroid Coeff./100 B i o l . Coeff. (hr) Mean S.D. Mean S.D. Mean S.D. Mean S.D. 11.5 2.5 2.36 0.52 0.35 8.15 7.91 3.9 2.9 19.0 0.5 0.12 0.77 0.53 10.06 2.75 4.3 4.9 35.0 0.7 0.10 1.44 0.12 11.67 5.84 6.7 2.5 43.0 1.1 0.40 1.61 0.20 8.93 2.74 7.6 2.4 59.5 1.1 0.66 2.04 0.34 7.28 3.59 5.7 4.6 67.0 1.1 0.35 3.10 0.93 7.84 3.42 7.0 4.0 83.5 47.6 50.20 2.23 1.10 2.60 1.86 223.5 175.0 92.0 45.0 53.40 2.08 1.13 1.92 1.90 128.7 90.9 107.0 5.3 17.50 1.64 0.56 1.42 2.00 17.3 12.9 118.0 4.3 1.80 1.81 0.44 1.40 1.03 21.3 11.6 131.5 1.4 0.94 1.28 0.63 1.62 0.90 13.5 10.0 143.0 7.2 3.38 1.76 0.77 1.62 1.20 35.0 48.5 Sampled May 17 - 28, 1960. Each mean comprises 3 - 6 f i s h . Size range 10 - 30 gmj 10 - 18 cm. TABLE X Quantal and semi-quantal determinations of histological characters in sockeye smolts Lowest c e l l % f o l l i c l e s F o l l i c l e Mass f i s h height(u) Vacuolation with no colloid Staining diameter^u) (gm) Mean Range Mean Range Mean Range Mean Range Mean Range Mean Range 1.95 1.03 3 2.87 44.7 14.5 2.24 2.55 2.61 1.30 1.50 1.29 4.57 6 8 3.12 3.46 2.06 61.0 76.7 59.6 19.4 23.0 8.3 3.20 3.82 2.66 1.86 2.18 1.54 17.40 25 13 2.51 3.25 1.69 62.5 65.4 63.7 14.5 21.3 16.8 3.27 3.83 4.01 1.78 2.02 1.47 19.00 23 12 2.21 2.73 1.61 66.4 71.2 28.9 17.8 18.8 V3.2 4.63 5.17 1.98 2.37 19.25 26 2.17 2.89 33.2 36.8 5.4 8.4 3.23 1.79 1.51 14.00 10 2.94 2.58 39.9 37.1 4.0 3.0 3.56 3.89 2.08 18 3.31 42.8 5.0 1.90 1.20 6 one one 26.1 21.7 2.20 2.30 1.23 1.27 6.00 6 2.98 fi s h 77.8 fi s h 30.4 2.70 1.03 0.94 13.25 8 3.05 35.3 29.9 3.8 3.22 3.64 1.11 20 3.33 3.62 38.2 4.3 4.7 3.75 2.92 - 17.00 15 4.5 4.0 4.58 2.36 - 19 - — 5.0 2.40 2.43 1.24 — 8.00 - 2.98 - 77.8 — 45.7 _ 1.96 1.44 _ 6 21.3 2.40 - - 9.80 12 — - - 37.2 50.1 2.64 - 24.30 20 - - 4.3 2.90 3.24 - — 31 — - — 6.4 7.8 2.28 2.12 2.45 - 9.00 • 8 10 - - 5.6 5.4 5.8 2.40 9.50 6 47.0 2.68 2.96 — - 13 — — _ 60.6 74.0 2.30 _ 7.00 6 _ 10.5 2.50 2.73 — - 8 - - - 11.2 12.0 Notes In each thyroid, 100 fo l l i o l e s were measured and means i calculated. The mean of these mean values is given above for groups of 1 - 7 f i s h . TABLE XI Cell heights and Conversion Ratio in sockeye smolts HISTOLOGY' DATA RADIOIODINE DATA Mean oell heights (100 f o l l i c l e s / f i s h ) pu) No. of Mean . . PBIt No. of Lowest Range Highest Range Mean Range f i s h CR. S.D. (hr) f i s h 2.24 1.95 2.55 5.07 4.33 5.62: 3.65 3.13 4.03 7 3.3 0.94 108 7 4.63 4.01 5.17 6.54 6.02 6.96 5.58 5.00 5.81 4 39.4 12.20 108 9 3.56 3.23 3.89 6.36 5.94 6.76 4.97 4.59 5.53 3 23.6 15.60 112 3 2.20 1.90 2.50 4.52 3.70 5.34 3.36 2.80 3.93 2 3.2 2.17 108 4 3.22 2.70 3.64 5.59 4.97 5.85 4.41 3.84 4.76 4 31.6 30.10 108 8 2.40 2.36 2.43 5.13 4.62 5.71 3.78 3.49 4.07 3 3.8 2.11 108 3 1.96 1.44 2.40 4.68 3.84 5.27 3.32 2.64 3.83 4 1.6 0.24 108 6 2.90 2.64 3.24 5.43 5.21 5.66 4.16 4.14 4.20 3 3.4 1.89 108 9 2.28 2.12 2.45 4.81 4.79 4.83 3.93 3.64 4.23 2 6.1 2.55 108 7 2.68 1.96 2.40 6.56 6.33 6.79 4.62 4.36 4.88 2 12.5 4.60 108 8 2.50 2.30 2.73 5.59 5.55 5.62 4.16 3.96 4.14 2 3.4 2.41 108 2 Note: See note to Table X concerning the mean c e l l height. TABLE'XII Seasonal changes i n Conversion Ratio and mean c e l l height in two-year-old ooho smolts Mean c e l l No. of No. of height(yu) Range f i s h C.R. S.D. f i s h Date 3 . 9 2 4 . 4 3 2 . 8 9 2 . 7 8 3 . 2 5 * Underyearling f i s h . Note: See note to Table X concerning the mean c e l l height. 2 . 8 9 May 5 # 1 8 4 3 3 . 0 1 2 . 2 6 2 0 3 . 7 4 May 5 #j_2. 2 1 9 . 3 1 1 . 3 2 JJQ 2 . 1 3 „ , „ „ „ „„ . June 3 > 4 7 3 1 2 . 2 3 . 9 8 4 13 2 . 2 2 July 3 . 3 4 2 1 , 9 1 , 6 2 2 1 1 3 . 3 3 Aug. 3 . 4 1 3 1 5 ' 3 7 « 9 0 9 1 5 * TABLE XIII Seasonal changes in Conversion Ratio, mean ce l l height and thyroid uptake in underyearling chum salmon HISTOLOGY RADIOIODINE Mean c e l l height(u) No. of C.R. % thyroid No. of PBIt Mean Range fish Mean S.D. Mean S.D. f i s h (hr) Date 4.82 - 1 - - - - - - June 8 6.08 5.72 6.44 2 - - - - - - June 30 - - - 49.9 29.50 10.2 3.85 6 108 July 7 - - - 47.0 9.61 5.3 1.63 11 108 July 18 3.65 3.58 3.89 4 11.6 14.50 0.7 0.19 12 108 July 25 - - - 20.5 5.89 1.5 1.39 12 108 Aug. 6 - - - 7.5 5.36 0.6 0.16 12 108 Aug. 15 3.45 2.97 3.87 3 10.8 8.85 1.7 0.62 12 108 Aug. 22 - - - 20.6 13.30 2.0 0.94 12 108 Aug. 30 - - - 27.6 19.60 6.1 2.57 12 108 Sept. 7 _ — 26.6 19.50 4.5 0.51 7 108 Oct. 12 Note: See note to Table X concerning the mean c e l l height. 58 TABLE XIV Seasonal changes in Conversion Ratio and mean c e l l height in two-year-old sockeye smolts Mean c e l l Ho* of No. of height(u) Range f i s h c.R. S.D* f i s h Date 5.00 May 5 , 6 8 5.81 4 3 9 , 4 12*20 9 2 9 4.59 June 4 » 9 6 5.3S 3 2 3 ° 6 15.60 8 6 3.84 June 4.42 4 . 7 6 4 31.6 30.10 8 1 3 . 4.05 June 5 * 0 0 5.83 3 " 19 4.14 July 4.16 4.20 3 3 , 4 1.89 9 2 3.64 July 3.93 4.23 2 2.55 7 3.96 Aug. 4 , 0 5 4.14 2 3 , 4 2 , 4 1 3 9 3.13 Jan. 3.66 4 #o3 7 3 , 3 0 , 9 4 7 10 Note: See note to Table X concerning mean c e l l height


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