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The osmoregulatory metabolism of the starry flounder, Platichthys stellatus Hickman, Cleveland Pendleton Jr. 1958

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THE OSMOREGULATORY METABOLISM OP THE STARRY FLOUNDER, PLATICHTHYS STELLATUS by CLEVELAND PENDLETON HICKMAN, JR. B.A., DePauw University, 1950 M.S., University of New Hampshire, 1953 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA June, 1958 Faculty of Graduate Studies PROGRAMME OF THE F I N A L O R A L E X A M I N A T I O N F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y of C L E V E L A N D P E N D L E T O N H I C K M A N JR. B.A. DePauw University, 1950 M.S. University of New Hampshire, 1953 IN R O O M 187A, B I O L O G I C A L SCIENCES B U I L D I N G M O N D A Y , J U N E 30, 1958 at 10:30 a.m. C O M M I T T E E IN C H A R G E D E A N F. H . S O W A R D , Chairman H . A D A S K I N W. S. H O A R W. A . C L E M E N S W. N . H O L M E S I. McT. C O W A N C. C. L I N D S E Y P. A . D E H N E L H . M c L E N N A N R. F . S C A G E L External Examiner: F. E . J. F R Y University of Toronto T H E O S M O R E G U L A T O R Y M E T A B O L I S M O F T H E S T A R R Y F L O U N D E R , PLATICHTYS STELLATUS A B S T R A C T Energy demands for osmotic regulation and the possible osmoregulatory role of the thyroid gland were investigated in the euryhaline starry flounder, Platichthys stellatus. Using a melt-ing-point technique, it was established that flounder could regulate body fluid concentration independent of widely divergent environ-mental salinities. Small flounder experienced more rapid disturb-ances of body fluid concentration than large flounder after abrupt salinity alterations. The standard metabolic rate of flounder adapted to fresh water was consistently and significantly less than that of marine flounder. In supernormal salinities standard metabolic rate was significantly greater than in normal sea water. These findings agree with the theory that energy demands for active electrolyte transport are greater in sea water than fresh water. Thyroid activity was studied in flounder adapted to fresh water and salt water. Correlative with the higher metabolic rate of small flounder was the more rapid turnover and excretion of radioiodine and greater thyroid uptake of small than large flounder. Percentage uptake of radioiodine by the thyroid was shown to be an insensitive and inaccurate criterion for evaluating thyroid activity in different salinities because removal rates of radioiodine from the body and blood differed between fresh water and marine flounder.. Using thyroid clearance of radioiodine from the blood as a measure of activity, salt water flounder were shown to have much greater thyroid clearance rates and, hence, more active thy-roid glands than flounder adapted to fresh water. The greater activity of the thyroid of marine flounder correlates with greater oxygen demands in sea water and suggests a direct or adjunctive osmoregulatory role of the thyroid gland of fish. G R A D U A T E S T U D I E S Field of Study: Comparative Animal Physiology Environmental Physiology W . S. Hoar Comparative Physiology W . S. Hoar Other Studies: Biochemistry Biochemistry Staff Mammalian Physiology D . H . Copp and E. C. Black Marine Invertebrate Zoology.. D . L. Ray (Friday Harbor Laboratory) i ABSTRACT Energy demands for osmotic regulation and the possible osmoregulatory role of the thyroid gland were investigated i n the euryhaline starry flounder, Platichthys stellatus. Using a melting-point technique, i t was established that flounder could regulate adequately body f l u i d concentration independent of widely divergent environmental s a l i n i t i e s . Small flounder experienced more rapid d i s -turbances of body f l u i d concentration than large flounder after abrupt s a l i n i t y alterations. The standard metabolic rate of flounder adapted to fresh water was consistently and significantly less than that of marine flounder. In supernormal s a l i n i t i e s standard metabolic rate was significantly greater than i n normal sea water. These findings agree with the theory that energy demands for active electrolyte transport are greater i n sea water than fresh water. Thyroid a c t i v i t y was studied i n flounder adapted to fresh water and salt water. Correlative with the higher metabolic rate of small flounder was the more rapid turnover and excretion of radioiodine and greater thyroid uptake of small than large flounder. Percentage uptake of radioiodine by the thyroid was shown to be an insensitive and inaccurate c r i t e r i o n for evaluating thyroid a c t i v i t y i n different s a l i n i t i e s because removal rates of radioiodine from the s body and blood differed between fresh water and marine flounder. Using thyroid clearance of radioiodine from the blood as a measure of a c t i v i t y , s a l t water flounder were shown to have much greater thyroid clearance rates and, hence, more active thyroid glands than flounder adapted to fresh water. The greater a c t i v i t y of the thyroid of marine flounder correlates with greater oxygen demands i n sea water and suggests a direct or adjunctive osmoregulatory role of the thyroid gland of f i s h . In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada. Department i i TABLE OP CONTENTS I INTRODUCTION A* Environmental relations of starry flounder, lemon sole and speckled sand dab • ••••eo«...o«eo.»......<>oo 3 Be Collection and care of f i s h ••o.eeo»<>.«» I I I THE OSMOTIC RESPONSE OF STARRY FLOUNDER TO CHANGES IN ENVIRONMENTAL SALINITY „.„».. 9 A. Determination of body f l u i d concentration »•••••••••• 10 1. Melting-point determination «..••.••••••••••••« 10 2© Experimental procedure M « M « » « « « « » » « « t « « t M « « 11 B. ReSUltS ..eo........•<,•••••»..«oo...o...........°..c. 13 1« Starry flounder i n normal sea water 13 2. Starry flounder i n hypotonic media: regulation against overhydration and salt depletion „ „ „ 17 a, concentration disturbances •»•••••••••»••• 17 b, volume disturbances • • » « « i«««n« M M t M M 18 3. Starry flounder i n supernormal hypertonic media: regulation against dehydration and s a l t excess 20 4* The effect of body size on alterations i n body f l u i d osmolarity after abrupt changes i n envir-onmental s a l i n i t y ••<«•>••«««•<•••••«««« 20 C. Comment «••••<••»••••«•««•••••«<••»•••«•«••••<•(••«< 23 IV THE EFFECT OF ENVIRONMENTAL SALINITY ON THE OXYGEN CONSUMPTION OF FIATFISH 27 A. Determination of standard metabolic rate ........e.o. 32 i i i 1. Apparatus •••••<,«••••••« <>«••»•••••••••••••••••• 32 2, Chemical analysis o» 35 3.o Experimental procedure •<»«<«•<«•«•••«•<«••••» 35 4« S t a t i s t i c a l procedures „ „ ( „.,•«« 36 B. Results ............... o • 37 1» Standard metabolic rates of starry flounder, lemon sole and speckled sand dab •••••••••••••• 37 a. interspecific comparison ••••••••••••<,•••<, 37 b. significance of the slope of regression of metabolic rate ••••••.••••»•••*•« 40 2. Diurnal rhythm i n the metabolic rate of Starry flounder ••oa«oo*ooo*«ooe««o«««*«*o*9eaa 43 3» Effect of starvation on the standard metabolic rate of starry flounder •••••»....•<>••<>••.0oa«* 48 4* Effect of s a l i n i t y on the standard metabolic rate of starry flounder O » M . O 55 5« The effect of s a l i n i t y on the standard metabolic rate of lemon sole and speckled sand dab 9 » M M « . < » M M < c t « M i » » 68 C. Comment •••.»««..*.«.o<>.e....*«..0.....* »«• 72 V THE EFFECT OF ENVIRONMENTAL SALINITY ON THYROID ACTIVITY AND RADIOIODIDE METABOLISM OF THE STARRY FLOUNDER , 80 A, Methods: determination of iodide movement and thyroid a c t i v i t y with radioiodine •••••••••••••«••••• 86 1. Injections •«•««••••«•• •««•••••«• 86 2. Collection, treatment and counting of samples 87 a. thyroid 90 b. blood .,. 90 c. urine («<o«*Mi«ti««» g M i » « « i « i • 91 d. body ............. 91 3. Expression of results e...•...•.•«•••<>•«•.••••c 92 B. Results .... o 9 5 1. Factors influencing the excretion of radioiodide 9 5 a. effect of radioisotope reentry ••••••••o.. 9 5 b. effect of s a l i n i t y »•.••••...••• 9 7 c. relative importance of renal and extrarenal excretion of I 102 d. effect of size • •»...e.o««.e.o* 107 2. Factors influencing uptake of radioiodide by the thyroid gland •••• •«•«••• ... 107 a. effect of iodide content of the water «... 107 b. effect of s a l i n i t y .........<>•. I l l I. the behavior of radioiodide i n the blood 112 i i . the thyroidal clearance of radio-iodide from the blood 116 c. effect of body size on thyroid a c t i v i t y of starry flounder and speckled sand dab 118 VI DISCUSSION • » 123 VII SUMMARY AND CONCLUSIONS .. 134 APPENDIX 136 LITERATURE CITED 140 V LIST OF FIGURES" l a Average melting points of serum and urine of Pl a t i c h t /oo stellatus transferred abruptly from sea water of 25 to dilute sea water and fresh water ......<>».......o...*•••• 14 l b Average melting points of serum and urine of Platichthys  stellatus transferred abruptly fgom sea water of 25 "/oo to concentrated sea water of 46 /oo ••••••••••••«•••••••«• 14 2 Melting-points of serum and urine of Platichthys stellatus transferred abruptly to concentrated sea water of 46 v/oo 21 3 The effect of body size on the rapidity of change ofsenum melting-point of PlatichthyB stellatus ...».<>•..•..<,•<> 22 4 A diagrammatic representation of f l u i d s h i f t s attending concentration and volume disturbances of flounder transferred to fresh water. « 25 5 A view of part of the appartus for standard metabolism measurements . M . O M > O M » M < 34 6a Standard metabolic rate of Platichthys stellatus. Parophrys vetulus and Citharichthys stigmaeus .....es. ••• 38 6b Standard metabolic rate of winter Platichthys stellatus: showing the departure from the linear log weight-log rate relationship seen i n summer flounder ....e...<>».•••»•<> 38 7 Metabolic rates of 14 Platichthys stellatus measured at 3-hour intervals over one 24 hour period ••«••<•••«•«••««••• 44 8 Diurnal variations i n metabolic rate of Platichthys stellatus 4 7 9 Standard metabolic rate of spring,Platichthys stellatus starved 4, 7 and 20 days ••••• .••..••••<>•.....<><...o*o« 49 10a Standard metabolic rate of Platichthys stellatus starved 2 and 11 days ••••• 50 10b Decrease i n standard metabolic rate of Platichthys stellatus due to starvation •••• 50 11a Comparisons of standard metabolic rate of Platichthys stellatus i n 20 °/oo and 8 /oo sea water ••••«..«••*••<>••• 56 l i b Comparison of standard metabolic rate of Platichthys stellatus i n 20 °/oo sea water and after 20 hours i n fresh water 56 12a Effect of adaptation time i n fresh water on the standard metabolic rate of Platichthys stellatus ..........<,•......«• 60 v i 12b Comparison of standard metabolic rate of Platich-foys stellatus i n 25 /oo sea water and i n 49 /oo sea water <>.........•.<.. 60 13a Comparison of standard metabolic rate of Platichthys stellatus i n 22.8 /oo sea water and i n fresh water, 20 hour adaptation 63 13b Comparison of standard metabolic rate of Platichthys'stellatus i n 25 /oo sea water and i n fresh water, 5 day adaptation .. 63 14 Comparison of standard metabolic rate of Platichthys stellatus i n 25 /oo sea water and i n 43*2 /oo sea water ••••••o.««.* 67 15a Comparison of standard metabolic rate of Citharachthys stigmaeus i n 23 /oo sea water and i n 43.4 V 0 0 s e a water 69 15b Comparison of standard metabolic rate of Parophrys vetulus i n 24.3 %x> sea water and i n 5*8 /oo sea water •••••••••• 69 16 Diagrammatic representation of relative energy demands of starry flounder for osmotic regulation i n hypotonic and hypertonic media .......o.................<>.............».o. 73 17 The effect of self-absorption on the observed counting rate of I X 3 1 i n thyroid samples as measured with an end-window Geiger-Mueller counter , „ > M . H < • > « 89 18 Effect of reentry into the body of excreted isotope on the I, excretion curves „«••••••• « . « • » « « < > • . • . 9 6 131 19 Effect of s a l i n i t y on the cumulative tota l excretion of I by Platichthys stellatus ................................... 98 131 20 Rate of removal of I from the bodies of Platichthys  stellatus ............. .................... ......... • 100 131 21a Effect of body size on the exgretion of I from Platichthys stellatus i n sea water of 25 /oo s......................... 108 131 21b Effect of body size on the excretion of I from Platichthys  stellatus i n fresh water, normal sea wa ter and concentrated sea water ,„ ,„.i <>.••»••.•••• 108 22 Effect of elemental iodine content of the water on thyroid I uptake of Platichthys stellatus 109 23 Effect of s a l i n i t y on thyroid I 1 3 1 uptake and blood I 1 5 1 disappearance rate of Platichthys stellatus ................ 113 24 Behavior of radioiodine i n the blood of 3 large (102 to 213 gm.) Platichthys stellatus i n sea water ......... 114 25 Effect of body size on thyroid a c t i v i t y of Platichthys  stellatus • ....«......*... 119 v i i 26 Effect of body size on thyroid a c t i v i t y of Citharichthys 27 Effect of body size on thyroid a c t i v i t y of Citharichthys  stigmaeus and Platichthys stellatus ............».•*....»••• 122 28 Diagrammatic representation of the weight dependency and interrelationships of thyroid a c t i v i t y , excretion and metabolic rate of starry flounder „•••...«.. 129 v i i i LIST OP TABLES I Melting-points of serum and urine and urine:serum ratios of Platichthys stellatus i n the control s a l i n i t y of 25 ' /oo and after transfer to 46 /oo, 5#45 /oo and fresh water ..<> 15 I I The influence of s a l i n i t y on oxygen consumption of teleost f i s h as reported i n the literature 28 I I I Standard metabolic rates of Platichthys stellatus. Parophrys vetulus and Citharichthys stigmaeua i n sea water ........... 39 IV Diurnal variation of metabolic rate i n Platichthys stellatus 45 V Effect of starvation on the standard metabolic rate of summer Platichthys stellatus .................. .«•••• 51 VI Effect of starvation on the standard metabolic rate of winter Platichthys stellatus ....................... 52 VII The standard metabolic rate of Platichthys stellatus i n 20 /oo sea water and fresh water . . . . . . . . 5 7 VIII Standard metabolic rate of Platichthys stellatus i n 25 °/oo and 49 /oo sea water ..................... • ••• 61 IX Standard metabolic rate of Platichthys stellatus i n fresh water, normal sea water and concentrated sea water 64 X Standard metabolic rate of Citharichthys stigmaeus i n 24.4 °/oo and 43.4 /oo sea water .................................... 70 XI Standard metabolic rate of Parophrys vetulus i n 24.35 °/oo and 5.8 /oo sea water ..•..................•....•*.•.....•. 71 XII Excretory clearance of radioiodine from the blood of flounder i n s a l t and fresh water ••••« 103 XIII Calculated urine flows of individual fresh and j a j t water flounder, assuming no extrarenal excretion of I ......... 105 131 XIV The concentration of I i n the g i l l lamellae of starry flounder expressed as percentage of the concentration of I i n the blood 106 XV Thyroid clearance of radioiodide from the blood of Platichthys stellatus 117 i x ACKNO\fLEIXJilENTS I t i s a pleasure to acknowledge my indebtedness and gratitude to Dr. W.S. Hoar, Department of Zoology who suggested the problem and under whose direction and encouragement this study was made. I wish to thank Dr. P.A. Dehnel, Department of Zoology for advice and c r i t i c i s m during the study. Other members of my doctoral committee, Drs. I. McTaggart-Cowan and C.C. Lindsey, Department of Zoology were generous i n their assistance. Dr. P.A. Larkin and Mr. R.R. Parker, Department of Zoology gave valuable help with s t a t i s t i c a l treatment of the data. I am also indebted to Drs. D.H. Copp, E.C. Black and CP. Cramer, Department of Physiology and to Dr. B. Baggerman, University of Groningan for generous advice i n several aspects of the work. Dr. A. Gorbman, Department of Zoology, Barnard College, Columbia University gave valuable advice during the i n i t i a l work on thyroidal uptake of radioactive iodine. The use of the end-probe s c i n t i l l a t i o n counter was generously supplied by Dr. D.H. Copp, Department of Physiology. Dr. P.P. Solvonuk, B r i t i s h Columbia Medical Research Institute, kindly granted the use of the well-crystal scin-t i l l a t i o n counter. This study would not have been practical without the help of those who made available a continual supply of l i v e f l a t f i s h . I refer to Mr. S.U. Quadri, Institute of Fisheries, Mr. Joseph Bauer of Steveston and Mr. Norman Pelkey of Vancouver. Their kindness i s gratefully acknowledged. I am obligated to my fellow graduate students, Dr. M.A. A l i , Mr. H.H. Harvey and Mr. A.H. Houston for helpful advice and many stimulating discussions during the course of the study. The Vancouver Public Aquarium Association kindly provided research f a c i l i t i e s . Financial support for this research was received from National Research Council of Canada through grants-in-aid of research to W.S. Hoar and a studentship to CP. Hickman. I INTRODUCTION In recent years the problem of body f l u i d regulation i n the lower vertebrates, i n particular the fishes, has become of considerable interest. Perusal of a recent review (Black, 1957) shows that considerable research has been directed toward the elucidation of detailed mechanisms by which adult teleosts achieve osmotic inde-pendence from their environment. With the demonstration of basic regulatory mechanisms for both fresh and salt water f i s h , i t became apparent that fundamental to their operation was the movement of charged particles from a lower to a higher potential - either primarily from the external environment to the internal milieu, i n the case of fresh water teleosts, or primarily i n the opposite direction i n the case of marine teleosts. The certain involvement of active u p h i l l particle move-ment means that a significant portion of c e l l u l a r metabolism must be directed to the maintainance of this work. Thus, osmotic independence from the environment demands an expenditure of energy. More recently, the role of the endocrines i n s a l t and water metabolism of fishes has come under surveillance of an increasing number of workers. The subject i s reviewed by Pickford and Atz (1957). No precise osmoregulatory function has been demonstrated conclusively for the endocrine glands, although the evidence i s insufficient at present to preclude such a function for any of them. Often implicated i n an osmoregulatory role i s the enigmatic thyroid gland of teleosts. However, i t s precise action i n this regard has remained obscure. Even more puzzling has been the i n a b i l i t y of numerous workers to demonstrate i n teleosts the fundamental calorigenic action of the thyroid of higher vertebrates. The present investigation i s a study of the osmoregulatory metabolism of a euryhaline teleost, the starry flounder, (Platichthys stellatus). subjected to varied environmental s a l i n i t i e s , with particular reference to the role of the thyroid gland i n osmotic regulation. The design of this investigation embodies 2 two assumptions, both hypothetical at the outset, that (a) using a euryhaline teleost, consistent quantitative differences i n metabolic rate should be measurable i n individuals adapted to different osmotic concentrations accordant with the osmotic gradient imposed and that (b) elucidation of an osmoregulatory role for the thyroid could be approached most successfully by looking for a general metabolic effect of the thyroid hormone rather than a specific action on one or more target organs* Thus, the hypothesis was proposed that i f energy demands for osmotic regulation are mitigated or intensified by changing the ambient s a l i n i t y , such changes should be reflected i n a concomitant increase or decrease i n both tota l metabolic rate and thyroid a c t i v i t y . In this thesis, experimental evidence i s presented which demonstrate that energy demands of flounder are consistently and significantly greater i n sea water than fresh water, and that the a c t i v i t y of thyroid gland as evaluated by thyroidal clearance rates of radioiodine from the blood i s greater i n sea water than fresh water adapted flounder. The starry flounder was chosen as the experimental animal because i n addition to possessing the prime attribute of euryhalinity, i t i s a hardy, rugged f i s h that lends i t s e l f exceptionally well to experimental treatment, i t i s r e l a t i v e l y unexcitable, and could be readily collected from i t s marine environment during most of the year. Occasionally, other species of f l a t f i s h were collected, and isolated comparative experiments were performed when possible. I t i s convenient to divide the presentation of this work into three sections: establishment of the osmotic capacity of the species i n terms of i t s a b i l i t y to regulate body f l u i d concentration i n widely divergent s a l i n i t i e s , the relative energy demands for regulation i n these s a l i n i t i e s and f i n a l l y the part played by the thyroid gland. 3 I I EXPERIMENTAL ANIMALS A. ENVIRONMENTAL RELATIONS OF STARRY FLOUNDER.  LEMON SOLE AND SPECKLED SAND DAB The f l a t f i s h e s (Order Heterosomata) occupy a unique and interesting position among the Teleostei because of their remarkable departure from b i l a t e r a l symmetry characteristic of the rest of the vertebrates. Except during the symmetri-ca l l a r v a l stages prior to metamorphosis, f l a t f i s h are distinguished by having both eyes on the same side of the head. In the definitive adult body form, the eyed side of the body i s pigmented and the blind side white. Flatfishes are t y p i c a l l y demersal, both lying and swimming i n a horizontal position. Norman (1934) distinguishes fi v e families of the Heterosomata, of which two are represented on the Pacific Coast of North America: Bothidae and Pleuronectidae. Only one genus of the Bothidae (left-eyed flounders) i s found here, the genus Citharichthys. A l l . of the rest of the f l a t f i s h e s (14 genera i n B r i t i s h Columbia waters) belong to the family Pleuronectidae, the right-eyed flounders. This investigation i s concerned primarily with the starry flounder, Platichthys stellatus, a euryhaline pleuronectid. In addition, an incomplete series of experiments were carried out on two p a r t i a l l y euryhaline f l a t f i s h e s , a pleuronectid, Parophrys vetulus, and a bothid, Citharichthys stigmaeus. The starry flounder, Platichthys stellatus, i s one of the most widely distributed of the Pacific Coast f l a t f i s h e s , ranging from central California northward along the Canadian and Alaskan coasts and west along the Aleutian Islands to Tokyo Bay. I t i s also found i n the Bering Sea and Arctic Ocean (Orcutt, 1950). I t i s a true coastal f i s h , inhabiting i n l e t s , bays and estuaries of the North Pacific as well as the adjacent oceanic waters, to 300 meters. 4 Juvenile flounders (1-6 cm.) axe found abundantly i n t e r t i d a l l y i n the spring and summer. Adults frequent somewhat deeper water, but are also common i n t e r t i d a l l y at certain seasons. Food i s chiefly Crustacea, polychaetes and molluscs. The spawning season of starry flounder i s during the winter, from November through February i n California (Orcutt, 1950), while perhaps a month later i n B r i t i s h Columbia. Males reach maturity during their second year with a length of about 225 mm., females a year lat e r with a length of about 250 mm. The starry flounder's habit of entering brackish and fresh water a l l along the Pacific coast i s well documented (e.g. Carl, 1937; Gunter, 1942; Hubbs, 1947; Clemens and Wilby, 1949; Orcutt, 1950; Roedel, 1953; Westrheim, 1955). Orcutt reports that a large number of small flounder 19 to 109 mm. i n length (less than 30 grams) were collected i n two California r i v e r s . In B r i t i s h Columbia, several juvenile flounder, a l l less than 15 grams, were collected i n a tributary of the Fraser River more than 20 miles above the riv e r mouth. These specimens are deposited i n the museum of the Institute of Fisheries, University of B r i t i s h Columbia. Presumably, large flounder also penetrate fresh water, for Westrheim (1955) reports that a number of tagged flounder, 6 to 27 inches i n length (roughly 100 to 4000 grams) were recaptured inside the Columbia River, some as far as 20 miles upstream. In many cases of "fresh water" penetration, the f i s h may actually have been i n brackish water; frequently a tongue of saline water underlies water of coastal rivers for some distance upstream. There i s no question, however, that Platichthys stellatus voluntarily enters entirely fresh water on occasion. The lemon sole, Parophrys vetulus, (known i n American waters as the English sole) i s limited i n distribution to the Northeastern P a c i f i c from the Gulf of Alaska to southern California (Norman, 1934). Juvenile forms (2-5 cm.) are commonly collected i n t e r t i d a l l y along with juvenile starry flounder. Older stages move into progressively deeper water. The adults most frequently are found on soft 5 sand or mud bottoms at depths of 60-100 meters (Ketchen, 1956). Food i s mostly bottom-living invertebrates. Females reach maturity i n the i r t h i r d or fourth year with a length of about 295 mm., the males i n their second year at 250 mm. (Ketchen, 1947). As with the starry flounder, spawning occurs i n the winter months of January through March. There i s no record of lemon sole entering fresh water. The speckled sand dab, Citharichthys stigmaeus. i s an active f l a t f i s h ranging from southern California to southeastern Alaska (Clemens and Wilby, 1949). I t i s found i n shallow water and to a depth of 80 meters. Because of i t s small size (reaching less than 17 cm. or 60 grams), i t i s of no commercial importance and has received v i r t u a l l y no attention from fishery biologists. Nothing i s known of i t s early l i f e history or food habits. The closely related, though larger Citharichthys sordidus (reaching 1000 grams) i s a summer spawner (Arora, 1951) and observations made during the present investigation indicate that C. stigmaeus. also, spawns during the summer months. The speckled sand dab i s a s t r i c t l y marine f i s h , never entering fresh water, though i t i s not infrequently found i n the i n t e r t i d a l zone. I t was often taken during the spring and summer on Vancouver beaches by the author when the spring run-off from the nearby Eraser River had lowered the s a l i n i t y to near isotonicity with the fishes' body f l u i d s . Platichthys stellatus i s not the only euryhaline f l a t f i s h . The European flounder Pleuronectes flesus i s well known for i t s habit of entering fresh water. Both this species and Pleuronectes platessa (plaice) penetrate extensively into the low-salinity B a l t i c Sea, but the l a t t e r i s unable to l i v e i n fresh water. Henschel (1936) established that plaice could not inde f i n i t e l y withstand a s a l i n i t y below 8°/oo (<4.43). Norman (1934) l i s t s two additional pleuronectids that enter fresh water: Liopsetta g l a c i a l i s . the Arctic flounder which i s distributed along the Arctic shores of Russia, Alaska and Canada, and Rhombosolea r e t i a r i a . the black flounder of New Zealand. 6 Euryhaline bothids are known: Citharichthys stamflu of West Africa and Citharichthys g i l b e r t i of the Paci f i c coast of tropical America from Lower California to Peru (Norman, 1934). Both of these species have been recorded i n fresh water. B. COLLECTION AND CARE OF PISH Experimental work was carried out oyer a two year period from summer, 1956 to spring, 1958. During most of the work, the laboratory f a c i l i t i e s of the Vancouver Public Aquarium were u t i l i z e d . A constant supply of both dechlorinated fresh water and f i l t e r e d sea water was available. The s a l i n i t y of the sea water i n the Aquarium varied seasonally. During the winter the s a l i n i t y usually remained high - 25-27°/oo, - but i n the early summer i t dropped to as low as 16°/oo because of the heavy discharge of the nearby Fraser River at that time of year. The s a l i n i t y rarely rose above 20°/oo u n t i l f a l l . During 1956, flounder were collected at frequent intervals by means of a beach seine at Locarno Beach, Vancouver. The following year, the service of a small otter trawl was successfully employed, and thereafter, a l l flounder were caught by this method on the North'Bank of the Fraser River, Steveston, B r i t i s h Columbia. Both of these areas are characterized by low s a l i n i t i e s i n the summer-time - the Locarno Beach area as low as 10°/oo and the North Bank of the Fraser as low as 6°/oo. The l a t t e r i s largely protected from the f u l l influence of the river by a j e t t y which extends several miles offshore from the r i v e r mouth. Flounder were transported to the laboratory i n 10 gallon containers with aeration. Fish were kept i n tanks containing about 130 l i t e r s of running sea water at a temperature within 1°C. of the environmental temperature i n which the f i s h were captured. After 2 or 3 days i n running water, the flounder were transferred to tanks containing 130 l i t e r s of aerated water adjusted to the desired experimental s a l i n i t y . Temperature was maintained within ±1.0 C. of the environmental temperature by means of running fresh water cooling tubes. Water was changed at intervals when any sign of fouling appeared, but t h i s was rarely any problem i f the f i s h were given the i n i t i a l two-day period i n sea water for the elimination of metabolic wastes. The tanks were p a r t i a l l y covered, but daylight was not excluded. The f i s h appeared to thrive under these conditions and v i r t u a l l y no mortality occurred between the time of capture and the end of the experiment (two weeks or l e s s ) . Sand dab and lemon sole were captured at Locarno Beach, occasionally with flounder i n a beach seine, but these species usually remain i n somewhat deeper water and were not readily taken by this method. To capture these f i s h i n deeper water i n the same area, a small beam trawl was used. However, the capture of f i s h of sufficient numbers and of a suitable size range was never easy and the supply proved to be unpredictable. Sand dab and lemon sole were transported to the laboratory and treated i n similar fashion to the flounder. Since the metabolism of foodstuffs forms a large portion of the t o t a l c a l o r i f i c output of animals, i t i s usually found advantageous to fast experimental animals before measuring standard"'' metabolism. Accordingly, none of the f l a t f i s h were fed i n the laboratory. There i s no evidence that the drop i n metabolic rate, caused by the cessation of food assimilation and protein storage for growth, would i n any way interfere with the fish's capacity to perform osmotic work. In the present experiments, although starved flounder li v e d for l£ months i n the summer 1. Basal metabolism, a term i n common usage by medical physiologists and c l i n i c i a n s , refers to the metabolic rate of a resting (but not sleeping) and fasted animal, removed from discomfort and distracting influences. Because the metabolism of vertebrates can be lowered from the "basal" rate by drugs or sleep, Krogh (1914) suggested the term standard metabolism for the energy requirements under normal resting conditions. Standard metabolism would seem to be a more accurate term and has been adopted by most comparative physiologists studying energy metabolism i n animals. 8 P 1 • • and about 3 months i n the winter before appreciable mortality began, a l l exper-imental work was begun at least within 10 days (usually within 4 days) and terminated within 15 days after collection of the f i s h . Thermal acclimation i s one of the most important factors i n oxygen con-sumption comparisons of f i s h measured at different times. If the ambient environ-mental acclimation temperature i s changed, there w i l l occur a measurable systematic change i n the metabolic rate to a new l e v e l . In an effort to eliminate any s h i f t due to changes i n thermal acclimation, the f i s h were held i n temperature controlled tanks within ±1.0°C. of the surface temperature measured at the collection locale. However, surface temperatures are not always representative of bottom temperatures (though nearly so i n the marine i n t e r t i d a l zone) and may fluctuate considerably with t i d a l movements. In addition, there i s the p o s s i b i l i t y that the f l a t f i s h which are known to carry on diurnal migrations, may actually be acclimated to a colder deep water temperature. Thus the thermal history of the f l a t f i s h was, within certain l i m i t s , unknown. Changes i n temperature acclimation, with con-comitant shi f t s i n the fishes' metabolic response to the experimental temperature was then always a possible variable. At the very extreme, however, the d i s -crepancy between environmental and laboratory temperature did not exceed 1.5°C., hence the resulting metabolic shi f t s due to any departure between these temperatures must be small. 9 I I I THE OSMOTIC RESPONSE OP STARR! FLOUNDER TO CHANGES IN ENVIRONMENTAL SALINITY In fishes, the principle organs for osmotic exchange with the environment are the kidneys and the g i l l s . Fresh water f i s h depend primarily for the prevention of overhydration on the excretion of a copious and dilute urine, whereas the kidney i s more of a l i a b i l i t y than an asset to the marine teleost. In the l a t t e r there have developed i n the g i l l adaptive c e l l s capable of concentrating and excreting ions to the exterior. In spite of the fundamental opposition of direction of active s a l t and water exchange i n fresh and s a l t water fishes, (overhydration vs. dehydration) both regulate the osmolar concentration of their body fluids'at comparable levels: fresh water fishes at.AO.5 to 0.7°C. and marine fishes at about A 0,7 to 0.9°C. (Black, 1957). Reported values for body f l u i d osmolarity of fishes vary greatly between species and often within a species. Interspecific variations are due to several contributing factors such as error i n various methods of measurement and abnormal changes i n plasma osmolarity due to "laboratory diuresis" (Forster, 1953). In addition, body f l u i d osmolarity i s never maintained absolutely constant even under r e l a t i v e l y stable environmental conditions, and a certain amount of l a b i l i t y i s to be expected, parti c u l a r l y i f the osmotic gradient changes. The capacity of fishes to adapt to vicissitudes i n the t o n i c i t y of the environment varies markedly. I t i s generally recognized that the terms stenohaline. referring to animals that can tolerate only a narrow environmental concentration range, and euryhaline, referring to animals tolerating a wide s a l i n i t y range, are inadequate for categorizing a l l fishes. Most oceanic marine fishes are more or less stenohaline, but toward coastal areas there occur i n -creasing numbers of fishes that tolerate dilute sea water. In the i n t e r t i d a l region, i n estuaries and at ri v e r mouths marine fishes are found l i v i n g 10 comfortably i n near-fresh or even fresh water. Thus, the above terminology w i l l not cover the r e l a t i v e l y frequent case of the marine f i s h that enters brackish water but cannot survive i n fresh water. The degree of euryhalinity of a species may depend to some extent on the degree of di l u t i o n or concentration of to t a l body electrolyte that can be tolerated, but ultimately i t i s contingent upon the capacity of the organs of exchange to adapt to changes or reversal of the concentration gradient. Evaluation of euryhalinity usually i s based on the a b i l i t y of f i s h to survive a wide range of s a l i n i t i e s . Such a c r i t e r i o n i s crude, for i t cannot reveal the extent of disturbance of body f l u i d composition and concentration, hence the degree of perfection of homeostatic mechanisms. Presented i n this section i s a precise method for describing the a b i l i t y of an aquatic animal to regulate under a wide range of concentration gradients by a technique hitherto unused by f i s h physiologists. A. DETERMINATION OF BODY FLUID CONCENTRATION 1. Melting-point Determination. Total osmotic pressure of serum and urine was determined by a melting-point method similar to that used by Gross (1954) for the measurement of osmotic pressure changes i n the body f l u i d s of a sipunculid. This method involves com-parison of the time of melting of frozen solutions of unknown melting point with solutions of known melting point, when allowed to warm very slowly and l i n e a r l y i n a cold brine bath. Because only enough solution (.01 - .001 ml.) i s needed to f i l l a small portion of a capillary tube, the method i s particularly desirable where small quantities of f l u i d are available. For this study, t h i s method offered the following advantages: (a) i t i s applicable for osmocentration measure-ments of body f l u i d s of small f i s h (less than 10 grams) as well as large, (b) urinary catheterization i s unnecessary for urine collection since only a drop of urine i s needed, an amount easily expressed by gentle pressure over the urinary bladder, and (c) many of the problems encountered i n freezing-point determinations are eliminated, e.g. coagulation and supercooling* The brine bath consisted of a 2 - l i t e r capacity plastic box insulated with cork and rock wool* Windows were provided on the top and bottom of the chamber for illumination and observation of the tubes. Standard sodium chloride solutions with melting points of about -2°. -1°, -0.5° and 0°C. were prepared and the exact osmotic concentrations of each determined to an accuracy of ,005°C. by the freezing-point method. In an experimental run, capillary tubes containing approximately equal quantities of the unknowns were quick-frozen on dry ice and placed side by side on a rack i n the brine bath at an i n i t i a l temperature of -9.0°C. Beside these were placed capillary tubes containing equal quantities of the four standard solutions, similarly frozen on dry ice . The brine bath was then covered and sti r r e d gently during the period of warming. Warming was rapid i n i t i a l l y , but soon slowed. During the c r i t i c a l period (-2.0 to 0°C.) the temperature rose about 1°C. every 35 minutes i n a nearly linear fashion. The tubes were watched with a long-arm binocular microscope and the time of melting of both standard and unknowns recorded by marking a kymograph drum with signal magnets arranged for the purpose. Then, by plotting and f i t t i n g a l i n e through the points, the melting-points of the unknowns could be interpreted relative to those of the standards. The method used here had an accuracy to within .01°C, a l i t t l e less than the accuracy possible to achieve by careful freezing point determinations (.005°C.). 2. Experimental Procedure. I t has been shown frequently that marine f i s h held i n overcrowded con-ditions i n the laboratory or subjected to handling develop a "laboratory" or "osmotic diuresis" that may persist for a long period of time (Grafflin, 1931; P i t t s , 1934; Clarke, 1934; Meyer, 1948; Forster, 1953; Porster and Berglund, 12 1956). Evidence indicates that a spontaneous increase i n urine flow occurs immediately after capture whether or not subsequent hand!ing or treatment i s imposed. However, cannulation of the urinary bladder for renal clearance studies seems to augment diuresis considerably. Most apparent physiological changes are the rise i n osmoconcentration of both blood and urine and f i n a l l y isotonicity between blood and urine. The selective electrolyte reabsorption by the kidney tubules breaks down so that i n addition to other changes, an excessive renal loss of chloride occurs. The reason for the onset of laboratory diuresis i s not known other than that i t i s the result of the trauma imposed during capturing, holding and handling. In this investigation, single urine samples were collected from each f i s h , thus eliminating two important contributing factors to severe laboratory diuresis: bladder catheterization and the continual handling necessary for s e r i a l sampling of individual f i s h . After collection, f i s h were held for three days i n flowing sea water at a temperature of 14-15°C. At the beginning of the experiment, a group of flounder were transferred rapidly from the holding tank to the desired s a l i n i t y . Controls (flounder maintained i n normal, 25°/oo sea water) also were transferred to a similar aquarium so that effects due to handling i n transfer would be the same i n both control and experimental f i s h . At selected intervals of time following transfer, f i s h were removed, rinsed i n fresh water and blotted dry. A urine sample was collected by touching the t i p of a capillary tube to the urinary papillae and pressing gently over the urinary bladder. The drop of urine was tapped to the center of the tube, the tube sealed at both ends with a heavy inert grease (Nevastane, Heavy X, Keystone Co.) and immediately placed on dry ice. Blood was collected by puncturing the dorsal aorta above B"^ s l i g h t l y posterior to the center of the coelom with a narrow, sharp-pointed scalpel blade. Because of the l a t e r a l compression of f l a t f i s h , the dorsal aorta l i e s r e l a t i v e l y near the surface and may be easily punctured from the side of the animal without entering either the coelom or spinal cord. By 113 inverting the animal, blood was allowed to flow onto a clean glass slide and l e f t undisturbed u n t i l i t had clotted (20-50 seconds). Serum was drawn into the capillary tube and the tube sealed and frozen. Melting-points were determined either immediately or the tubes were stored b r i e f l y i n a brine solution at -10°C. u n t i l such time as the measurement could be made. B. RESULTS 1. Starry Flounder i n Normal Sea Water. At the outset of the experiment the osmoconcentration of the serum of flounder i n 25°'/oo sea water averages A 0.69°C. (Fig. l a , Table I ) . At 76 hours the average serum osmolarity has increased to A 0.702°C. and to A 0.706°C. at 14 days. The urine melting-point was invariably lower than the serum melting-point of the same f i s h , as has been repeatedly demonstrated for other marine teleosts (Dekhuyzen, 1905; Smith, 1932; Martret, 1939; Forster, 1953; B r u l l and Cuypers, 1954; Forster and Berglund, 1956). I t i s notable that with the large increase i n v a r i a b i l i t y of serum and urine concentration at 14 days, urine always remains hypotonic to the serum when values are compared on an individual basis. In comparing relative degrees of hypotonicity of urine to serum, the urine: serum melting-point ra t i o i s useful; these values have been calculated and are included i n Table I . The average U/S ratios of the i n i t i a l and 76 hour samples are essentially the same (.834 and .835), but the average 14 day U/S ratio has increased to .91. Forster (1953) and Forster and Berglund (1956) have shown that the onset of laboratory diuresis i s accompanied by a s h i f t i n t o t a l electrolyte composition of plasma and urine and an increase i n urine flow and loss of chloride, a l l of which con-tribute to a marked increase i n toni c i t y of both urine and extracellular f l u i d . They noted a progressive r i s e i n the urine:plasma ratio during laboratory 14 Figure l a . Average melting points of serum (solid lines) and urine (broken lines) of Platichthys stellatus. transferred abruptly from sea water of 25 °/oo (ZM.35°C.) to dilute sea water of 5.45 °/oo (^0.29°C) and fresh water. Individual values summarized i n Table I. Figure l b . Average melting points of surum (solid lines) and urine (broken lines) of Platichthys tt e l l a t u s transferred abruptly from sea water of 25 °/oo (/4l.35°C.) to concentrated sea water of 46 °/oo (^l2.49 0C.). Individual values summarized i n Table I. 5 - \ O \ S2 4 h ^ w 3 r ^ r \ ^ ^ ""^ .Q fresh water \ ^ ^ ° ~ ~ 2 ~ " » — — — 8 / -I 1 1 1 1 I I L_ 10 20 30 40 50 60 70 80 H O U R S A F T E R T R A N S F E R Table I. Melting points of serum and urine and urine:serum ratios of Platichthys stellatus i n the control s a l i n i t y of 25°/oo, and after transfer to 46/oo sea water, 5.45%o sea water and fresh water. Sa l i n i t y 25%o Melting Point Time Weight Serum Urine U/S hrso gm. -°C. -°c. 0 13.8 .68 .58 .852 17.3 .705 22.5 .687 28.6 .705 .58 .823 35.1 .672 .555 .826 Average .689 .571 • 834 10.1 .71 .62 .873 10.4 .70 .59 .833 18.2 .71 .60 .845 48.2 .72 .58 .805 57.5 .76 .622 .818 Average .702 .602 .835 14 days 7.2 .645 .602 .933 12.3 .748 .715 .955 .515 .70 21.0 .763 .672 .854 45.6 .675 .608 .90 Average .706 .644 .91 Sali n i t y 46%o Melting Point Time Weight Serum Urine U/S hrs. gm. -°c. -°c. 4i 8.5 .778 .66 .848 12.2 .89 .68 .775 15.9 .73 .66 .905 39.5 .715 139.2 .68 Average .759 .666 .843 12 9.1 .78 .745 .955 12.7 .80 .697 .871 25.9 .73 .696 .953 33.5 .752 .707 .94 130.7. .73 Average .758 .711 .930 24 8.7 .792 10.8 .825 13.1 .845 31.0 .792 .685 .865 210.1 .765 .685 .895 Average .803 .685 .88 74 10.5 .912 .89 .976 12.5 .845 .835 .99 25.4 .835 .765 .915 34.7 .87 .837 .962 77.3 .81 .765 .945 Average .854 .818 .958 Table I (Continued) Sal i n i t y 5.45%>o Melting Point Time hrs. 41 Weight gm. 6.9 13.0 15.3 48.1 93.2 Average Serum -°C .630 .662 .68 .668 .66 Urine -°C .60 .125 .575 .433 U/S .883 .86 .871 Fresh Water Time hrs. Weight gm. Melting Point Ser t im - ° C . Urine -°C. ~u7s 5 7.4 18.5 19.4 45.3 68.5 Average .58 .642 .612 .642 .63 .621 .515 .085 .435 .545 .515 .419 .888 .132 .71 .849 .817 .679 6.7 .695 .147 .212 12 10.0 .635 .264 .417 17.7 .646 15.6 .644 .288 .447 23.1 .672 .155 .231 19.5 .61 57.9 .662 .115 .174 21.7 .65 .304 .458 58.0 .68 .105 .154 76.5 .66 .134 .203 Average .671 .131 .193 Average .64 .248 .381 9.2 .645 .30 .455 24 9.3 .66 .254 .384 11.5 .642 .20 .311 9.7 .622 .192 .308 18.7 .65 .19 .292 20.2 .665 .335 .504 67.7 .662 .08 .121 29.4 .615 .142 .23 170.0 .612 .11 .18 74.3 .615 .15 .244 Average .642 .176 .271 Average .635 .214 .334 11.8 .688 .311 .451 75 12.7 .505 .295 .584 14.7 .59 14.1 .597 .265 .444 16.3 .634 .616 .971 19.9 .535 .107 .20 25.4 .679 .14 .206 35.5 .575 .107 .186 77.3 .69 .088 .127 64.6 .587 .107 .199 Average .559 .176 .322 14 3.6 .544 .47 .865 11.0 .583 .105 .18 17.3 .105 50.0 .58 .145 .25 Average .569 .206 .431 17 diuresis and eventually blood and urine become isotonic. Inasmuch as the U/S ratio of flounder i n this study increased not at a l l between 0 and 76 hours and only s l i g h t l y at 14 days, i t i s unlikely a laboratory diuresis developed during the experiment. I t i s conceivable, nevertheless, that a change had occurred i n the U/S ratio i n the 3-day interval between capture and i n i t i a l sampling. Forster, (1953), working with three marine species, two aglomerular and the other glomerular, found urine:plasma freezing point ratios of samples collected immediately after capture to vary between 0.81 and 0.87. These values compare closely with urine:serum ratios obtained for the flounder. I f we assume Forster*s findings are representative of the normal condition, the U/S ratios obtained for flounder i n the present investigation f a l l i n the normal range for marine teleosts. 2. Starry Flounder i n Hypotonic Media: Regulation Against Overhydration and Salt Depletion. a. Concentration disturbances Abrupt transfer to hypotonic media results i n an immediate d i l u t i o n of the blood of flounder (Fig. l a ) . Since water freely traverses c e l l u l a r membranes the net result i s a d i l u t i o n of both c e l l u l a r and extracellular f l u i d s . The average serum osmolarities of flounder i n both fresh water and hypotonic sea water are lower at 5 than at 12 hours, suggesting that the i n i t i a l i n f l u x of water into the extracellular space overwhelms the capacity of the kidney to excrete the excess f l u i d . A brief recovery occurred at 12 hours and was followed by a slower drop i n the body f l u i d concentration u n t i l a balance was struck between water influx and output. This occurred within 24 hours for flounder i n 5.45o/oo sea water, but the concentration continued to drop i n fresh water flounder u n t i l equilibrium was reached, sometime between the f i r s t and t h i r d days following transfer. The concentration of the urine decreased markedly during the f i r s t day (Fig. l a ) . Surprisingly, the drop i n urine concentration of flounder i n 18 dilute sea water i s i n i t i a l l y greater than that of fresh water flounder, a phenomenon that must be presented without an attempt at explanation. As would be expected, urine concentration reached equilibrium at about the same time as did the blood concentration, i.e., 24 hours for flounder i n dilute sea water and about 3 days for fresh water flounder. The slight increase i n osmolarity of the serum and urine of the flounder i n fresh water between the t h i r d and fourteenth days i s probably the result of a net retention of solutes with inanition as demonstrated with goldfish by Meyer, Westfall and Flatner (1956) and Jorgensen and Rosenkilde (1956). b. Volume disturbances Starry flounder tolerate abrupt transfer from sea to fresh water with absolutely no outward appearance of distress or asthenia. In fresh water they remain alert and vigorous under the added imposition of starvation for many weeks. Some changes i n body volume and, as has already been discussed, concentration, do occur which have significance i n assessing the actions of the osmotic stress imposed. Measurements of weight changes were not carried out i n this study. Henschel (1936) has, however, followed weight changes of the euryhaline Pleuronectes flesus (very similar to Platichthys stellatus) after transfer from a hypertonic to hypotonic medium. No change i n weight occurred when transferred to 8°/oo (Zl0.42°C.) from 16°/oo ( A 0.95°C.) but some increase i n weight occurred at 4°/oo ( A 0.21°C.), due to water loading. Henschel also made an interesting comparison between the flounder and less adaptable plaice, P. platessa. In 8°/oo sea water the plaice gained weight over the 16°/oo controls and i n 4°/oo they gained as much as 20$ i n body weight before death within 3 days. In other experiments he found that plaice l o s t sal t as rapidly as they gained water, resulting i n a rapid f a l l i n serum osmolarity and death with l i t t l e weight change. Whereas flounder were able to excrete the excess 19 water by increasing urine flow, urine production of plaice i n the 4 /oo sea water actually decreased or even stopped, thus vastly increasing the disparity between water influx and excretion. In addition, Henschel found that flounder transferred to hypotonic solutions stopped drinking water, while plaice continued. Thus Pj_ flesus i s many times better suited to l i f e i n low s a l i n i t i e s than P.  platessa. Starry flounder i n the present study were observed to increase body volume somewhat after transfer to fresh water, but no excessive hydration was apparent u n t i l after several weeks of starvation at 15°C. Inanition became severe after 5 to 6 weeks i n fresh water, when mortality began. The immediate and direct cause of death appeared almost invariably to be the result of a breakdown i n osmotic regulation, caused by acute caloric starvation. A notice-able edema occurred and was accompanied by a stiffening of the body and a marked decrease i n swimming a c t i v i t y when disturbed. Moribund f i s h were occasionally so s t i f f that they could hardly be forcefully bent when picked up. Also characteristic of osmotic f a i l u r e were cardiovascular disturb-ances. Collection of blood samples became increasingly d i f f i c u l t , suggesting a marked decrease i n blood volume and/or cardiac output, producing a near circulatory stasis. Such c r i t i c a l changes i n circulation occurred during the terminal stages of osmotic f a i l u r e , but some small decrease i n the effective blood flow was occasionally witnessed i n healthy fresh-water flounder and appeared to be a normal consequence of the relative over-hydration of these animals. 20 3. Starry Flounder i n Supernormal Hypertonic Media: Regulation Against Dehydration and Salt Excess* The blood and urine osmolarities of flounder abruptly transferred to concentrated sea water of 46°/oo ( A 2.49°C.) from the control s a l i n i t y of 25%>o ( A 1.35°C.) quickly r i s e (Fig. l b , Table I ) . The average serum melting-point drops from A 0.69 to A0.76°C. during the f i r s t 4-J hours, a decrease of about 10$ (10$ increase i n t o t a l osmotic pressure). Between 4% and 12 hours the concentration changes very l i t t l e , but i s then followed by a gradual con-tinuous r i s e to 74 hours, when the experiment was terminated. Whether the brief pause between A\ and 12 hours has special significance i s not known, though a similar "recovery" period was noted i n the progressive decrease i n blood concentration of flounder transferred to fresh water. 4. The Effect of Body Size on Alterations i n Body F l u i d  Osmolarity After Abrupt Changes i n Environmental S a l i n i t y . To assess the effect.of size on the rapidity of concentration changes affected by abrupt s a l i n i t y alterations, the individual melting points of serum were plotted on a linear axis against body weight on a logarithmic axis. An example i s shown i n Figure 2. The progressive increase i n serum melting-points of flounder abruptly transferred to concentrated sea water are plotted for each sampling period following the transfer. Included are the urine melting-points (open c i r c l e s ) . From each eye-fitted l i n e through the values, 2 points were graphically derived representing the serum melting points of a 10 gram i and a 60 gram starry flounder and plotted against time after transfer (Fig. 3). I t i s evident from Figure 3 that the osmotic disturbance following a s a l i n i t y change i s considerably greater i n smaller flounder. The body f l u i d concentration of smaller flounder i s shifted more rapidly and to greater degree than large flounder, i n the direction of the osmotic concentration of the external media, i.e., serum osmolarities are greater i n concentrated sea water and less i n fresh water i n smaller individuals. 21. Figure 2. Melting points of serum (•) and urine (o) of Platichthys stellatus transferred abruptly to concentrated sea water of 46 °/oo (A 2.49°C.). .9r 4 1/2 H O U R S .8 .7 .6 i i i i i i • • • I •9i-.8 oY .71 I -O Q. 10 100 12 H O U R S J L J l l—J. 1 5 -81 U J 71 JO 0L_l i i i 100 2 4 H O U R S J i L J I L .9-.8-.7-i i i i 1 100 7 4 H O U R S J L J 1 I—L 10 100 BODY WEIGHT IN GRAMS 22 Figure 3. The effect of body size of the rapidity of change of serum melting point of Platichthys stellatus transferred abruptly from sea water of 25 °/oo to concentrated sea water of 46 °/oo, to dilute sea water of 5.45 °/°° and to fresh water. The points representing 10 gram (•) and 60 gram (o) starry flounder were graphically derived from eye-fitted lines through individual serum melting points for each sampling period of each experimental s a l i n i t y (see Figure 2 for example). Individual values are summarized i n Table I. HOURS AFTER TRANSFER 23 C. COMMENT It has been suggested (Gordon, 1957) that euryhaline teleosts may be divided into two groups with respect to thei r a b i l i t y to regulate the osmo-concentration of "the body f l u i d s at the same or at different levels when trans-ferred from hypo- to hypertonic media or the reverse. Certain teleosts apparently experience only transitory changes i n body f l u i d osmolarity when subjected to s a l i n i t y changes. The stickleback Gasterosteus (Gueylard, 1924; Koch and Heuts, 1943) and the k i l l i f i s h Fundulus heteroclitus (Burden, 1956) maintain essentially the same plasma osmolalities or chloride concentration i n either hypo- or hypertonic media. For nearly a l l of the Salmonoids examined however, rather large sh i f t s (12 to 25$) occur i n plasma concentration levels of f i s h acclimated to hypo- and hypertonic media, the s h i f t tending to decrease the concentration gradient between the internal and external milieu (Greene, 1904, 1926; Benditt et. a l . 1941; Fontaine, 1943, 1948; Fontaine and Koch, 1950; Fontaine, Callamand and Vibert, 1950; Kubo, 1953; Gordon, 1957). However, i n this laboratory, Houston (1958) has demonstrated that the plasma chloride concentration of steelhead trout trans-ferred abruptly to sea water from fresh water may return to pre-transfer levels. Several authors have found appreciable l a b i l i t y i n serum and tissue concentration of the European eel, Anguilla vulgaris (Portier and Duval, 1922; Duval, 1925; Boucher-Firly, 1935) and the American eel, A. rostrata (Smith, 1932) exposed to fresh and salt water. The a b i l i t y of some f i s h to control body f l u i d osmolarity at one level regardless of external variations does not necessarily imply that these f i s h osmoregulate more e f f i c i e n t l y than those that allow substantial internal osmotic fluctuations i n the direction of the external s h i f t . On the contrary, whereas the former possess very effective and capable homeostatic mechanisms, such r i g i d homeostasis i s considerably less efficient i n terms of thermodynamic work than the 24 less advanced method of reducing the osmotic gradient* As Potts (1954) has pointed out, the most important mechanism for reducing the osmotic strain imposed on a marine animal entering brackish water i s to decrease the concentration of the blood* I t should be emphasized that a l l euryhaline teleosts are s t i l l homoiosmotic regulators and that the osmotic l a b i l i t y i l l u s t r a t e d by some i s only relative with fluctuations always held within certain s t r i c t l i m i t s * In t h i s investigation . blood melting-points dropped from -0.70°C. i n salt water flounder to -0*55°C* i n fresh water flounder adapted 75 hours - a 20$ decrease. Henschel (1936) transferred the euryhaline European flounder, Pleuronectes flesus from dilute sea water of 1 6 % o ( A 0.95°C.) to a hypotonic s a l i n i t y of 5-6%o (about A 0.3°C), and measured a 10$ decrease i n the freezing point depression of the blood, A 0.58 to 0.52°C. Under the same conditions the plaice, Pleuronectes platessa, a hypo-osmotic regulator, rapidly l o s t salt and soon died at a body f l u i d t o n i c i t y approaching that of the external milieu. The division of body f l u i d compartments and the chemical and physical forces that determine movements and distribution of f l u i d s and solutes are essentially the same throughout the vertebrates. Hence, reference to the vast mammalian literature i s valuable to an interpretation of f l u i d balance i n fishes. Excellent comprehensive treatments on body f l u i d dynamics are found i n the writings of Peters (1953), Elkinton and Danowski (1955), and Gamble (1958). The disturbances of the body f l u i d s seen i n the flounder transferred to fresh water can be i l l u s t r a t e d with Darrow-Tannet diagrams (Pig. 4) adapted from Elkinton and Danowski (1955). Osmotic water gained by the flounder enters v i a the g i l l s and oral membranes into the plasma and i n t e r s t i t i a l f l u i d , together known as the extracellular space. I t i s this compartment, "le milieu interieur" of Claude Bernard, that acts as a buffer between the vicissitudes of the external environment and the c e l l s . Normally the osmotic pressures between the i n t e r -s t i t i a l f l u i d and int r a c e l l u l a r f l u i d are equal though the relative composition 25 Figure 4. A diagrammatic representation of f l u i d s h i f t s attending concen-tration and volume disturbances of flounder transferred to fresh water. The extracellular and intracellular spaces are separated by the heavy v e r t i c a l l i n e . 4a. Overhydration: A pure influx of water without s a l t depletion decreases the concentration of the extracellular f l u i d , and water shifts into the c e l l s . The volume of the extracellular space i s increased. 4b. Salt depletion: Loss of electrolytes with no change i n t o t a l f l u i d volume results i n s h i f t of extracellular f l u i d into the c e l l s . The volume of the extracellular space i s decreased. ECS INCS E C S INCS 1 •" " 7 j j * volume >• E C S INCS volume E C S INCS volume 26) of solutes — electrolytes and proteins - that produce this pressure i s different i n the two phases. The influx of water lowers the osmotic pressure i n the extra-c e l l u l a r space. Water then freely passes the ce l l u l a r membranes to the region of higher osmotic pressure, the intr a c e l l u l a r space, thereby causing a swelling of the l a t t e r . The result i s an increase i n volume and weight and a decrease i n concentration of both f l u i d phases (Fig. 4a). The second major osmotic problem of fresh water flounder i s the loss of s a l t s . Pure salt depletion reduces the concentration of the extracellular phase and induces a movement of extracellular water into the c e l l s (Fig. 4b). In this case the volume of the extracellular space decreases while the intracellular volume i s increased. Thus, either water excess or salt depletion causes a swelling of the c e l l s , though alterations of the extracellular f l u i d volume di f f e r s i n each case. I t i s evident that concentration disturbance cannot be dissociated from volume disturbances, since a change i n one affects the other. A fresh water flounder i s regulating against both disturbances simultaneously -a superimposition of over-hydration on salt depletion. In osmotic f a i l u r e i n fresh water, the breakdown appears to affect the active transport of solutes — both the uptake of ions by the g i l l s and the reabsorption of electrolytes from the glomerular f i l t r a t e by the renal tubules. With the drop i n osmolarity of the plasma and i n t e r s t i t i a l f l u i d s , water moves into the c e l l s , causing the marked edema noted i n flounder starved for long periods. The extracellular volume, however, decreases as water s h i f t s to the c e l l s and produces a drop i n plasma volume with a decline i n cardiac output and blood pressure. These cardiovascular changes were readily apparent i n moribund fresh water flounder and at times i n otherwise healthy flounder. With circulatory f a i l u r e , renal plasma flow and glomerular f i l t r a t i o n diminish so that even osmotic water which continues to enter the body cannot be eliminated. This vastly intensifies the problem and total osmotic collapse comes quickly. 271 rv , THE EFFECT OF ENVIBONMENTAL SALINITY ON THE OXYGEN CONSUMPTION OF FLATFISH Osmotic independence of l i v i n g organisms appears to nave originated a number of times during nri-imal evolution and radiated along several lines resulting i n the appearance of rather diverse regulatory mechanisms to solve osmotic problems encountered i n nature. Because of such diversity, i t i s not surprising that investigations of energy output associated with osmotic work i n different groups of aquatic animals have resulted i n a seemingly irreconcilable array of experimental findings. Tablen presents a summary of the literature on the subject of the influence, of s a l i n i t y on oxygen consumption of fishes. A study of the table w i l l convince the reader that i t i s not possible to f i t a l l of these findings, some of which seem contradictory, together into one picture. In addition, the variety of techniques employed contribute to the inadequacy of the results for comparison with one another. In one case (Keys) standard metabolism was measured by the constant flow technique, but the usual method was simply to place the f i s h i n a closed container for a measured period of time then remove i t and determine the amount of dissolved oxygen consumed from -the water (e.g. Busnel, Cordier and Leblanc. Cordier and Maurice, Graetz, Henschel, Raffy). Wohlschlag measured active metabolism i n a closed circ u l a r plastic con-tainer. Leiner used the Warburg apparatus for his measurements of sea horse respiration. Deviations i n metabolic rate as affected by s a l i n i t y have not always been accepted as indicative of altered energy demands for osmotic work. Schlieper (1929, 1935) i n particular, has proposed alternate "theories to account for respiratory changes i n marine animals, primarily invertebrates, exposed to variations i n environmental s a l i n i t y . In general, the theories are attempts to explain the frequently observed increase i n metabolic rate of marine invertebrates Table I I . The influence of s a l i n i t y on oxygen consumpt Species Salmo iridaeus & Salmo salar (eggs & alevins) Habitat fresh water Regulatory capacity byper-osmotic regulator Tinea vulgaris fresh water stenohaline hyper-osmotic regulator Tinea tinea fresh water stenohaline hyper-osmotic regulator Carassius carassius freshwater stenohaline hyper-osmotic regulator Anguilla vulgaris (juvenile) migrating euryhaline sea to fresh water u i l l a vulgaris adult) migrating euryhaline fresh to sea water Fundulus parvipinnis marine euryhaline of teleost f i s h as reported i n the literature. S a l i n i t y effect Author Salinity change has no effect. During growth 0^ consumption increases more rapidly i n fresh water. Busnel, e t . a l . , 1946 Transfer to s a l i n i t i e s of 10 to 15°/oo results i n gradual fa.ll i n 0 consumption to death, more rapid i n higher concentrations. Death apparently by asphyxiation. Cordier and Maurice, 1957 Increased s a l i n i t y caused f a l l i n i n 0^ consumption followed by death. Raffy, 1932, 1933. In s a l i n i t i e s up to Isotonicity, 0^ consumption increased. In hypertonic media, 0£ consumption decreased to 60$ of normal. Veselov, 1949 0^ consumption less.in fresh water. Raffy and Fontaine, 1930. 0^ consumption less i n fresh water. Raffy, 1933 Abrupt transfer to fresh water causes Keys, 1931 decrease i n 0^ consumption. Returns to normal after fresh water acclimation. Table I I . (continued). G-asterosteus fresh water euryhaline Coregonus sardinella fresh water euryhaline and marine migratory Scorpaena porcus marine stenohaline hyper-osmotic regulator Scyllium catulus marine stenohaline hyper-osmotic regulator Sargus rondeletei marine stenohaline hyper-osmotic regulator Pleuronectes platessa marine (juvenile) hypo-osmotic regulator Pleuronectes platessa marine (adult) hypo-osmotic regulator Hippocampus marine stenohaline hypo-osmotic regulator O2 consumption 20 - 30$ higher i n Graetz, 1931 fresh water than isotonic sea water. No further decrease with increase i n s a l i n i t y . Marine (migratory) forms have higher Wohlschlag, metabolic rates than fresh water forms 1957 Transfer to fresh water causes immediate drop i n respiration to half normal sea water rate, then followed decline to death. Cordier and Leblanc, 1955 Transfer to dilute sea water or fresh water causes 0^ consumption drop followed by death. Transferred to dilute sea water, 0^ consumption at f i r s t rises, then drops suddenly followed by death. Raffy, 1932, 1933 Raffy, 1933 ro wo Transfer to dilute sea water and fresh water had no significant effect on oxygen consumption. Raffy, 1955 Low s a l i n i t i e s cause increased 0, consumption. Transfer to dilute sea water causes transitory increase i n respiration. Large dil u t i o n causes continual f a l l . In concentrated sea water, 0^ con-sumption rises b r i e f l y then returns to normal. If to saline, respiration f a l l s continually. Henschel, 1936 Leiner, 1938 30 moved into brackish or fresh water. His f i r s t theory (1929), which he himself subsequently refuted, assumed that oxygen demands were less i n high s a l i n i t i e s because carbon dioxide could be eliminated more e f f i c i e n t l y i n s a l t than fresh water, thus reducing the carbon dioxide content of the blood. In the second theory (1935) Schlieper proposed that the oxygen content of tissues was d i r e c t l y related to their water content. In low salt concentrations water would flood and swell the c e l l s , thereby increasing their surface area and f a c i l i t a t i n g the exchange of respiratory gases. Arguments have been raised against the theory (e.g. Kuenen, 1939) but i t does offer a convenient explanation for the o f t -observed increase i n respiration of marine poikilosmotic animals introduced into low s a l i n i t i e s . I t i s paramount i n any study of the energetics of osmotic regulation that a f u l l appreciation i s given the osmotic capabilities of the species studied. Measurements of oxygen consumption of a f i s h subjected to a s a l i n i t y beyond that which i t can indefinitely tolerate have doubtful significance, because the observed changes may be of a pathological nature. Even i n euryhaline forms, a change i n "the energy requirements concomitant with s a l i n i t y changes must be interpreted with caution. Transitory increases i n oxygen consumption usually are measured when a f i s h i s introduced into a new s a l i n i t y and are the result of the stress imposed. It i s important, therefore, that the animal be adapted to the experimental s a l i n i t i e s before definite determinations of oxygen consumption are made. In the published literature on the effect of s a l i n i t y on "the oxygen consumption of fishes, i t would appear that inadequate attention has been paid to either proper s a l i n i t y adaptation or the osmotic capacity of the species. The indirect determination of metabolic rate i n fishes as measured by the consumption of dissolved oxygen needs to be carried out with certain pre-cautions i n mind. Fortunately the problems of such measurement have recently become appreciated and the unrecognized p i t f a l l s encountered by the ea r l i e r 331 workers can now be largely avoided. The most important considerations, as discussed i n a recent review by Pry (1957) are: 1) Body size. 2) Experimental temperature and thermal history. 3) Oxygen and carbon dioxide tensions. 4) Seasonal influences. 5) Diurnal variations. 6) A c t i v i t y . 7) Nutritional state. 8) Sex and sexual maturity. 9) Environmental s a l i n i t y . In this investigation the attempt has been made to control, eliminate, or take into account a l l of these influences, leaving one variable, s a l i n i t y , to be experimentally altered. Because the effect of size on the metabolic rate-thyroid a c t i v i t y interrelationship formed an important part of the investigation, a f a i r l y large size range of flounder (about 4 to 300 grams) was used. This was as wide a range as the sensit i v i t y of the method and the capacity of the apparatus would allow. Among the important advantages of using a wide range of sizes are that the data lend themselves well to s t a t i s t i c a l treatment and y i e l d a great deal more information regarding the influences of the experimental variable, i n this case s a l i n i t y . In addition, important quantitative differences i n the effect of s a l i n i t y between small and large f i s h may come to l i g h t . In 1his discussion, the terms metabolism, tota l metabolism, oxygen con-sumption, oxygen uptake and respiration w i l l refer to "oxygen consumption per f i s h per hour". The terms metabolic rate, respiratory rate, respiratory intensity, weight-specific oxygen consumption and rate of oxygen consumption w i l l mean "oxygen consumption per gram body weight per hour." 32 A., DETERMINATION OF STANDARD METABOLIC RATE 1. Apparatus. Standard metabolism was measured i n an apparatus u t i l i z i n g the constant flow principle described by Keys (1930). This method i s presently gaining wide favor for oxygen consumption determinations i n f i s h (Job. 1955} Shepard, 1955; Fry. 1957). The f i s h i s placed i n a chamber through which water continuously flows and the oxygen consumption determined by analysing the oxygen content of the inflowing and outflowing water and the rate of water flow through the chamber. Because a variety of experimental s a l i n i t i e s was needed, the apparatus used here was modified so that the water of the desired s a l i n i t y could be continuously recirculated. The respiratory chambers, containing individual f i s h , were sub-merged side by side i n an insulated trough, 264 cm. long by 25 cm. wide. Water from an elevated 120 l i t e r barrel entered the trough at one end through a constant-level bottle and l e f t by an overflow at the other. Overflow water and outflowing water from the respirometers was collected i n a 100 l i t e r tank below and pumped to the reservoir above. The water level i n the reservoir was maintained by a fl o a t and mercury switch which controlled the operation of the pump below. Water was cooled i n the collecting tank with a series of glass cooling tubes through which cold fresh water flowed, and then held thermostatically at the desired temperature. During an entire experimental run (about 24 hours) the temperature varied less than 0.4°C. and less than 0.1°C. from one end of the respiration trough to the other at any one time. The circulating water was vigorously aerated i n the collecting tank and repeated analyses showed that the oxygen tension i n the trough was always at a i r saturation. Because f l a t f i s h are laterally-compressed they present a special problem i n the selection of a proper respiration chamber. The clear Lucite refrigerator boxes manufactured by Tri-State P l a s t i c s , L o u i s v i l l e , Kentucky, proved to be admirably suited for t h i s purpose. From a variety of obtainable sizes, four were 33 selected which would accomodate a size range of flounder up to 300 grams and lemon sole to 200 grams. The dimensions and capacities of the chambers were: 26.3 x 19 x 10.2 mm. (5.1 l i t e r s ) , 18.6 x 13 x 10.2 mm. (2.47 l i t e r s ) , 16.7 x 11.8 x 6.7 mm. (1.32 l i t e r s ) , 11.8 x 8.2 x 6.6 mm. (0.64 l i t e r s ) . The boxes were d r i l l e d on one end and a two—hole rubber stopper was f i t t e d with inflow and outflow tubes. Water entering the respirometer through the longer inflow tube passed the length of -the box to the opposite end. The outflow tube was short and was connected to a length of tygon tubing which syphoned the effluent over the edge of the trough and into a sample bottle (30 ml. glass-stoppered Erlenmeyer f l a s k s ) . Rate of water flow through each respirometer was con-t r o l l e d by raising or lowering the sample bottle with a number of thi n plywood shims. This method was found superior to regulating the flow with a clamp, which tended to trap air bubbles or excreta, resulting i n tube blockage. Just prior to collecting water samples for oxygen analysis, flow rates were measured by collecting the overflows from the sample bottles i n graduate cylinders. The error between duplicate flow measurements was usually less than one percent for the large respirometers with a fast flow and up to 3 percent for the small respirometers with a r e l a t i v e l y slow flow. A view of part of the apparatus i s shown i n Figure 5. The respirometer used corresponded roughly to the size of f i s h , with a volume of water i n the chamber 15 to 100 times the volume of f i s h . In accord with Geyer and Mann (1939), who found that a respiration chamber with a volume smaller than 10 times that of the f i s h caused over-excitement of Perca  f l u v i a t i l i s . i t was noted that a chamber at least 10 times the volume of flounder was necessary to avoid heightened respiration. A layer of washed, screened sand was placed i n the bottom of each respirometer. The f l a t f i s h usually buried themselves i n the sand, leaving only the upper surface of the head and operculum exposed. 34 Figure 5. A view of part of the apparatus for standard metabolism measurements. Shown from above i s the insulated trough containing individual respirometers. Tygon tubing leads from each respirometer to a sampling flask set i n a small plastic dish and arranged such that the overflow can be collected i n graduate cylinders below for flow measurements. 35 2. Chemical Analysis Dissolved oxygen was determined by a semi-micro modification of Winkler's iodometric technique, observing the precautions discussed by Ohle (1953). A 20 ml. aliquot part of the sample was t i t r a t e d , using a microburette and a dilute (.01N) thiosulphate solution. The error i n repeated t i t r a t i o n s of one fixed sample was less than one percent. 3. Experimental Procedure A number of workers (e.g. Keys, 1930; Wells, 1932; and Black, Pry, and Scott, 1939) have demonstrated that the handling of f i s h necessary to place them i n the experimental chamber affects a greatly heightened oxygen consumption which subsides only gradually to a standard rate several hours later. For this reason i t i s necessary to maintain the f i s h several hours under controlled con-ditions before the f i r s t samples are drawn. Fish were introduced into the respirometers the evening before the day that measurements of metabolic rate were made. They thus had a period of 18 to 22 hours i n the chambers under constant experimental conditions (darkness, quiet, and constant temperature, s a l i n i t y and water flow). Another influence which tends to supplement the physiological variation i n metabolic rate already present i s that of endogenous 24 hour cycles i n res-piratory intensity. Endogenous cycles have been found i n f i s h respiration by a number of workers (e.g. Clausen, 1936; Spoor, 1946 and Higgenbothan, 1947). P i l o t experiments showed the presence of such a diurnal rhythm i n starry flounder, with consumption high i n the morning, decreasing measurably u n t i l about 1:00 p.m., and leveling off during the remainder of the afternoon u n t i l about 6 p.m., when i t increased sharply. To minimize this influence and ensure basal conditions a l l samples were collected during the afternoon. The oxygen consumption of 12 to 18 f i s h was determined during each experimental run. After adjusting the water i n the apparatus to the desired ex-36 perimental s a l i n i t y and temperature, the f i s h were transferred with a minimum of handling from the holding tank to the respirometers. The respirometer tops were then sealed with a heavy, non-toxic grease (Nevastane, Heavy X, Keystone Co.). When a l l the f i s h were i n place, the trough was covered and flow rates adjusted so that under conditions of standard metabolism the concentration of dissolved oxygen i n the water leaving the respirometers would be between 70 and 85 percent a i r saturation. At 1 and 3 p.m. of the following afternoon, flow rates were measured and samples of outflowing water from each respirometer were immediately collected, fixed and analyzed for dissolved oxygen. A thi r d sample was analyzed i f the f i r s t two differed i n excess of about 5 percent. At these same times three samples were drawn for oxygen content analyses of the water at both ends and i n the center of the trough (inflowing water). After completion of the experiment, the f i s h were removed from respirometers, weighed (wet weight with bodies blotted) and returned to the holding tanks. 4. S t a t i s t i c a l Procedures Rate of oxygen consumption was plotted as a function of body weight (weight-specific) i n a double logarithmic coordinate system. This method was selected over the other common mode of presentation, i . e . , logarithm of t o t a l oxygen consumption against logarithm of body weight, because i t graphically emphasizes the weight dependence of respiratory intensity. When plotted on a double logarithm gr i d , weight-specific oxygen consumption of animals over an adequate size range depicts a straight or nearly straight line with a negative slope. Regression of total oxygen consumption against body weight i s of the form: or l o g 0 2 s l o g a + b l o g W where 0 2 i s t o t a l oxygen consumpt ion o f t h e f i s h i n mg O V g m . / h r . , W i s body 37 weight i n grams, a the intercept, and b the slope of the l i n e . Since rate of oxygen consumption was preferred for presentation here, the equation was trans-formed to weight-specific by dividing by W. Thus: ¥ or: log 0 2 - log W = log a + b log W - log W The problem here was to test whether s t a t i s t i c a l l y significant differences existed between two regression l i n e s , representing the metabolic rates of f l a t -f i s h measured i n two different s a l i n i t i e s . Usually oxygen consumptions of 12 to 24 f i s h were determined for each experimented s a l i n i t y during a test series and often considerable v a r i a b i l i t y existed around each calculated regression l i n e . Data of th i s nature are readily adapted to s t a t i s t i c a l treatment by analysis of covariance. The procedure was to test by analysis of covariance the n u l l hypothesis that no true differences existed i n the effect of two s a l i n i t i e s on oxygen consumption, i . e . , that the two regression lines could be represented just as well by a single regression l i n e . To eliminate negative log X values i n the s t a t i s t i c a l analysis, oxygen consumption rates were multiplied by 100 before con-verting to common 5-place logarithms. A standard method of covariance analysis outlined by Ostle (1954) for a randomized design was followed. B. RESULTS 1. Standard Metabolic Rates of Starry Flounder, Lemon Sole  and Speckled Sand Dab a. Interspecific comparison In Figure 6a a graphic comparison i s made of standard metabolic rates of flounder, sole and sand dab i n sea water of 25°/oo. The data are given i n Table I I I . Measurements were made at a temperature of 15°C. and at a time when 38 Figure 6a. Standard metabolic rate of Platichthys stellatus (open c i r c l e s ) . Parophrys vetulus (closed circles) and Citharichthys stigmaeus (triangles) i n sea water of 20-25 °/oo. Lines are f i t t e d by the method of least squares. Estimated body weight at the time of f i r s t maturity i s indicated as approximately 20 grams for the sand dab and 200 grams for the flounder and sole. Experimental temperature 14-15°C. Data of Table I I I . Figure 6b. Standard metabolic rate of winter Platichthys stellatus showing the departure from the linear log weight-log rate relationship seen i n summer flounder. Line f i t t e d by eye. Experimental temperature 10°c ,' s a l i n i t y 24.7-26.0 °/oo» Table I I I . Standard metabolic rates of Platichthys stellatus. Parophrys vetulus and Citharichthys stigmeaus i n sea water. X = body weight i n grams, Y = rate of oxygen consumption i n mgm. 0?/gm./hr. (average of 2-3 determinations). Parophrys Platichthys Citharichthys vetulus stellatus stigmaeus X Y X Y X Y 3.5 .123 9.0 .099 3.6 .092 3.5 .165 9.9 .114 3.8 .082 4.3 .145 10.1 .109 4.9 .102 5.3 .121 11.3 .092 5.2 .075 5.7 .155 16.0 .100 5.8 .103 8.0 .117 45.6 .084 7.0 .095 10.8 .094 45.6 .080 7.1 .074 24.1 .099 62.4 .081 10.2 .088 24.7 .101 65.6 .089 11.6 .075 24.7 .089 78.0 .084 12.3 .071 29.8 .086 79.0 .088 13.1 .070 39.1 .092 132.3 .077 19.1 .071 53.8 .095 139.1 .064 22.9 .086 77.7 .085 212.2 .062 28.1 .080 92.1 .084 223.6 .071 28.3 .074 93.4 .077 245.6 .065 38.0 .076 97.5 .075 110. .082 113.0 .075 117.8 .080 124.3 .077 143.8 .083 191.6 .071 217.0 .082 Exp. temp. 15.3i.2°C. Exp. temp. 15.0-.1°C. Exp. temp. 14.0-.2°C. Exp. s a l . 20.0%o Exp. s a l . 22.8 /bo Exp. s a l . 24.4 /oo June 30, 1956 May 13, 1957 July 5, 1957 Slope = -.158 Slope = -.141 Slope = -.095 40 a l l species were i n a similar nutritional state (3-4 days of fasting). Weight-specific oxygen consumption of flounder and sole are essentially the same throughout the size range examined but greater than the metabolic rate of sand dab at an equivalent body size. In addition, there exists a more rapid depression of metabolic rate with increasing size of flounder and sole (b = -.141 and -.158 respectively) than of sand dab (b = -.095). However, i t i s important to recognize that one i s dealing with an inter-specific comparison of fish that d i f f e r markedly with respect to ultimate size attained and size at sexual maturation. In-cluded i n Figure 6a i s an indication of the size of each species at f i r s t maturity. In both starry flounder and lemon sole, males usually reach maturity a year ahead of females (a general phenomena among fishes) — roughly at 200—250 grams body weight (Orcutt, 1950j Ketchum, 1947, 1956). Starry flounder grow very large, i n excess of 9,000 grams and lean sole to perhaps 3,000 grams. The sand dab, on the other hand, is a small species. Sexually mature females of 20 grams were occasionally found, and ihe largest individuals of the species rarely exceed 50 grams. Hence, with respect to the ultimate sizes of three species, i t i s clear that altogether different periods of ontogeny are represented i n the metabolism curves. . Figure 6a shows that the respiratory intensity of sand dab at matura-tion (about .075 mgm. 0Vgm./hr.) i s essentially the same as that of sole and starry flounder at maturation (about .070 mgm. O^gm./hr.). Whether or not the metabolic rate of these species i s actually more dependent on the rate of growth and physiological age than upon body size, per se, w i l l have to await further experimentation. b. Significance of the slope of regression of metabolic rate The relationship of metabolism to body size as expressed by the formulae 41 implicates a weight or allometric dependence of oxygen consumption of animals* According to the surface concept, established by Rubner (1883). metabolism i s a 2/3 power of body weight. Plotting oxygen consumption as a function of body weight on a double logarithmic grid, the slope of a surface proportional curve i s 0.67 as opposed to a slope of 1.0 for weight proportionality. Plotted as weight-specific metabolism as expressed by: T = aW the slopes of regression for weight and surface proportionalities are 0 and -0.33 respectively. Slopes of weight-specific metabolism of f l a t f i s h studied here i n -variably gave values f a l l i n g between weight and surface proportionality. Con-siderable intraspecific variation i n (b-l) was witnessed which could not always be attributed to any specific environmental influence. However, as w i l l be shown, important shifts i n slope occurred concomitant with transfers of flounder to a changed osmotic gradient. Considerable significance has been attached to the exponent b for a species i n spite of recent reviews (Zeuthen, 1947, 1953, 1955) pointing out the wide range of values that the slope, b, can take. Bertelanffy (1951, 1957) has categorized a large number of animals, including f i s h , into "metabolic types" depending on the proportionality (surface, weight or intermediate) of metabolism. Notwithstanding numerous examples to the contrary (Fry, 1957) Bertalanffy has lumped f i s h as a group into one "metabolic type" — surface proportionality. The assumption made but not stated i s that the slope neither changes during the ontogeny of the species nor i s influenced by physiological alterations affected by environmental stress. Neither of these assumptions i s tenable on the basis of experimental evidence. Zeuthen (1953) has presented several examples of animals which show significant changes i n metabolic rate during the i r ontogeny. Fishes form no 42 exception. Shamardina (1954) found that metabolic rate of pike increased with growth of larvae, then inflected sharply and f e l l during post-larval, juvenile and adult growth. The same author sites similar findings of three other Russian workers: Bezler woking with carp and bream, Korzhuiev with sturgeon and Privolnev with salmon. Lindroth (1942) and Zeuthen (1947) also report significant changes i n slope during growth of f i s h . Therefore, i t i s impossible to neglect onto-genetic changes i n the percentage decrease i n metabolic rate with increasing body size of f i s h . Usually no ontogenetic change i n slope was apparent i n the present studies on flounder and sole. A l l of the spring and summer determinations yielded data that conformed to a straight line on logarithmic paper. However, data collected on winter flounder measured at 10°C. strongly suggest a percentage increase i n slope towards weight proportionality with increasing body size. These data are shown i n Figure 6b. Why such an effect should appear i n the winter (10°C.) but not i n the summer (15°C.) i s d i f f i c u l t to understand, but i t i s not inconceivable that thepercentage increase i n slope reflects heightened metabolic demands with the approach of sexual maturity and the spawning season (December through March). For purposes of s t a t i s t i c a l comparison of data collected with different s a l i n i t y treatments, the points were assumed to conform to a linear log weight-log rate relationship with no change i n slope. The data i n Figure 6b, for instance, i s f i t t e d with a straight l i n e i n Figure 12b for convenience of treatment comparisons. Sometimes changes i n slope were noted i n comparing weight-rate curves obtained from flounder measured at the same s a l i n i t y and temperature but at different times of the year. For example, the slopes of the metabolic rate of flounder i n sea water measured on May 13 and June 29 at the same experimental temperature (15°C.) were -0.141 (Fig. 13a) and -0.311 (Fig. 11a) respectively. The results imply seasonal effects. Though seasonal influences or cycles were 43 not a part of this work and were not studied, the few observations made show the need to account for seasonal effect before placing any significance i n the slope of regression of metabolic rate. 2. Diurnal Rhythm i n the Metabolic Rate of Starry Flounder In the p i l o t experiments an endogenous cycle was indicated i n the metabolic rate of starry flounder. Successive samples drawn for dissolved oxygen analysis during the morning hours showed a gradual decrease i n oxygen consumption u n t i l early afternoon, when oxygen consumption appeared to level off. Accordingly, for a l l subsequent experiments, samples for snalysis were collected i n the afternoon when fluctuations i n metabolic rate were minimal. I t i s emphasized that the f i s h were under controlled experimental conditions i n excess of 12 hours, even for the morning measurements, certainly an adequate delay to ensure that the high morning values were not the result of the stimulation of handling i n placing the flounder i n the respirometers. To assess ihe complete diurnal a c t i v i t y cycle, 14 flounder of a l l sizes, from 6.7 to 225 grams, were simultaneously carried through one 24 hour period. After ihe i n i t i a l 18 hour delay, oxygen consumption measurements were begun and continued for 24 hours, with measurements every 3 hours. During this period the f i s h were completely protected from external stimuli such as l i g h t , noise and vibration. The temperature varied 0.1°C. The f i s h had been acclimated to temperature (15°C.) and s a l i n i t y (26.5°/°°)» conditions closely approximating those i n the environment from which the f i s h were captured at that time of year (late September). They were starved four days prior to the experiment. The results of the experiment are summarized i n Table IV. In Figure 7 the weight-specific oxygen consumptions of the 14 flounder are plotted for each of the 8 sampling periods during the 24 hours and lines of best f i t calculated by the method of least squares. The data were tested by analysis of covariance 44 Figure 7. Metabolic rates of 14 Platichthys stellatus measured at 3-hour intervals over one 24 hour period. Each point represents one determination for a f i s h at the indicated time of day. Lines are f i t t e d by the method of least squares. Experimental temperature 15.0°C, s a l i n i t y 26.6 °/oo, September 26-28, 1957. Data of Table IV*. Table IV. Diurnal variation of metabolic rate i n Platichthys stellatus. Body weights and rates of oxygen consumption i n mgm. O^/gm./hr. of 14 flounder followed through one 24 hour period (September 25-26, 1957). Determinations made at 3 hour intervals. Time of Day 0200 0500 0820 1100 1400 1710 2000 2300 , Rate of Oxygen Consumption Weight 6.7 .152 .0735 .101 .127 .113 .0995 .125 .117 7.1 .104 .085 .082 .100 .105 .0995 .109 .102 8.6 .093 .102 .0995 .091 .100 .081 .0995 .0995 12.1 .088 .093 .0975 .093 .105 .167 .129 .177 12.3 .131 .152 .149 .139 .148 .115 .147 .153 27.7 .0775 .115 .080 .066 .069 .098 .0995 .104 28.1 .091 .101 .082 .083 .084 .076 .0905 .104 34.0 .09 .0925 .11 .075 .075 .0645 .083 .081 48.0 .0745 .065 .0565 .0565 .064 . .069 .0515 .053 65.0 .081 .065 .054 .065 .065 .061 ,088 ,069 69.0 .071 .068 .072 .057 .064 .051 .070 .069 80.0 .054 .058 .118 .070 .057 .0645 .067 .058 138.5 .0895 .0625 .0555 .0495 .0517 .039 .0572 .063 225.0 .0485 .051 .046 .045 .051 .0425 .0457 .0465 Exp. temp. 15.0-15.1°C Exp. s a l . 26.6°/oo Days unfed = 4 VJ1 46 to determine whether true differences existed between the 8 regression l i n e s . The hypothesis that there are no differences between effect of the time periods i s rejected at the lfo probability level (Appendix - table I ) , indicating that a true diurnal rhythm i s present. Considerable v a r i a b i l i t y i s present around the regression l i n e , the small flounder i n particular showing rather wide departures from the mean. Part of this v a r i a b i l i t y i s due to the error i n the method because i t was possible to analyze only one oxygen sample for each f i s h at each time period, whereas at least two and often three samples were collected for each f i s h i n the s a l i n i t y effect experiments and the results averaged. But most of the variation i s due to true physiological differences i n the metabolic rates of the flounder them-selves. A close examination of Figure 7 shows that certain of the flounder i n particular hold definite positions above or below the regression l i n e . The 12.3 gram flounder, for instance, would appear to have an inherently high total metabolism, the 7.1 and 81.6 gram animals, inherently low metabolic rates. This sort of i n t r i n s i c variation i s well known i n lower vertebrates, i s normal but troublesome, and necessitates using a rather large sample size for proper s t a t i s t i c a l treatment. Figure 8 represents a summary of the experiment. From each regression line two points were taken, one representing a 10 gram, the other a 100 gram flounder, and plotted on a semi-logarithmic grid. The cycle i s i l l u s t r a t e d best by the lower plot (for 100 gram flounder) since less v a r i a b i l i t y existed i n the large than i n the small f i s h . Oxygen consumption i s highest at night. With the approach of daylight hours (though these experimental animals were maintained i n darkness throughout) the rate of oxygen consumption declines and, at least for the large f i s h , reaches a low point i n late afternoon before increasing sharply i n the evening. The small flounder did not show a similar decline during the daylight hours but i t i s possible that any true changes were masked by the large v a r i a b i l i t y of the small f i s h . 477 Figure 8. Diurnal variations i n metabolic rate of Platichthys stellatus. The "10 gram f i s h " and "100 gram f i s h " rates are derived from the points i n Figure 7 where the lines of best f i t cross the 10 gram and 100 gram lines of each of the 8ltime periods• i—i—i—III—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r r r 10 Gram fish 01 CD ^ 0.81 O <2 0.6 100 Gram fish 0.4 J—I—I—I—I—I—I I I I I I I I • • * • * ! • • » • • 0 2 4 6 8 10 12 14 16 18 20 22 24 HOURS, PACIFIC DAYLIGHT TIME 48 3. Effect of Starvation on the Standard Metabolic  Rate of Starry Flounder Two experiments were performed to assess the effect of starvation on the rate of oxygen consumption. As with the previous experiment on diurnal rhythm, this experiment was not designed i n any way as a definitive study of caloric starvation effects on metabolic rate but rather to evaluate the influence of th i s variable on the s a l i n i t y effect experiments to be presented i n the next section. Both spring (May) and winter (December) f i s h were studied. Spring experiment The oxygen consumption of a selected size range of flounder was determined on the fourth, seventh and twentieth days after collection. During t h i s period, the flounder were held unfed under controlled conditions of temperature (15°C.) and sa l i n i t y (25±2°/°0)* As with a l l the metabolism experiments, the animals were introduced into the respirometers 20 hours before water analyses were begun. The results tabulated i n Table V are plotted individually i n Figure 9. The three starvation periods were s t a t i s t i c a l l y treated by analysis of covariance two at a time to determine whether true decreases i n oxygen consumption occurred. In both cases, the tests showed significant drops i n standard metabolism (Appendix-table I I ) . Winter experiment The temperature of the environmental locale of the winter f i s h collected i n mid-December was 10.5°, thus the experiments were performed at 10°C. In other respects, experimental conditions were the same i n both the winter and spring studies. Standard metabolic rate of a selected range of small to medium sized flounder was determined on thesecond and eleventh days of starvation and are given i n Table VI and Figure 10a. The adjusted means of regression of respiratory rate on body weight were compared by co variance analysis (Appendix-table III) and the difference i n the means found highly significant. 49 Figure 9. Standard metabolic rate of spring Platichthys stellatus starved 4, 7 and 20 days. Respiratory rates axe f i t t e d by the method of least squares. Experimental temperature 14.9 *.2°C, s a l i n i t y 22.8-26.4 °/oo. May, 1957. Data of Table V. STARVED 7 DAYS • J I 1 ' I ' ' ' I I I I I I I I I I —I 1— 4 10 4 0 100 4 0 0 B O D Y W E I G H T IN G R A M S 50 Figure 10a« Standard metabolic rate of winter Platichthys stellatus starved 2 and 11 days* Respiratory rates are f i t t e d by the method of least squares* Experimental temperature 10 * 1°C*, s a l i n i t y 24,7 - 26.0 °/°°* December, 1956. Data of Table VI. Figure 10b. Decrease i n standard metabolic rate of Platichthys stellatus due to starvation. Points represent adjusted means of regression lines (adj. Tj[ - b( X i - X ) ) . shown i n Figures 9 and 10a. I I I I — I I I I I I I 2 -X Q 3 h J 1 I I • • • • • l_ 2 4 7 II 20 DAYS FASTED Table V. Effect of starvation on the standard metabolic rate of summer Platichthys stellatus. X = body weight i n grams, Y = rate of oxygen consumption i n mgm. O^/gm./hr. (average of 2-3 determinations). Days Fasted 4 X Y 9.0 .099 9.9 .114 10'il .109 11.3 .092 16.0 .10 45.6 .0895 45.6 .084 62.4 .081 65.6 .0893 78.0 .0845 79.0 .088 132.3 .0765 139.2 .0645 212.1 .0618 223.6 .071 245.6 .065 7 X Y 8.8 .093 9.8 .111 10.0 .097 10.6 .146 15.3 .089 21.3 .0825 26.6 .085 44.8 .071 45.7 .077 61.1 .079 64.5 .079 75.0 .065 76.6 .0775 131.7 .072 131.8 .0575 205.5 .049 215.7 .06 236.9 .0595 20 X Y 8.2 .0844 9.0 .098 9.5 .075 10.3 .0762 14.0 .085 20.1 .080 25.6 .0825 44,8 .0575 59.9 .0655 62.4 ,0577 71.6 .0615 75.0 .0566 90.5 .062 130.7 .0515 137.3 .0595 98.8 .056 213.9 .048 236.9 .052 Exp. temp. 15.oi.l°C. Exp. temp. 14.8i,l°C. Exp. temp. 14.9-.2°C. Exp. s a l . 22.8 /oo Exp. s a l . 26.4o/oo Exp. s a l . 25*1 /oo May 13, 1957 May 16, 1957 May 29, 1957 Slope = -.141 Slope = -.203 Slope = -.161 Table VI. Effect of starvation on the standard metabolic rate of winter Platichthys stellatus. X = body weight i n grams, I = rate of oxygen consumption i n mgm. 02/gm./hr. Days Fasted 2 11 X I X I X I 2.5 .103 49.7 .0355 4.0 .0455 3.9 .085 54.0 .0435 5.6 .0415 4.8 .0655 57.0 .0465 5.7 .0435 5.2 .058 70,1 .035 8.5 .042 5.5 .076 80.7 .0435 9.7 ,041 6.5 .055 81.0 .047 12.0 .0375 7.8 .051 91.0 .046 13.0 .0375 8.1 .063 100 .0345 48 .0315 9.2 .0525 134 .0345 51 .0365 9.8 .079 138 .0435 71 .0308 10.3 .047 157 .044 79 .034 12.1 .0505 240 .0435 80 .0435 13.4 .051 264 .0355 130 .0355 21.8 .063 284 .0385 Exp. temp. 10-,1°C. Exp. s a l . 24.7-26.0°/oo December 15, 1956 and December 20, 1956 (2 groups) Exp. temp. 10-.1°C. Exp. s a l . 25.7 /oo December 24, 1956 53 The adjusted mean rates of oxygen consumption for each regression of summer and winter f i s h were calculated from the covariance analyses by the formula (adj Y i = Y i - b (Xi - X)). The drop i n standard metabolic rate of the spring f i s h i s essentially logarithmic and the adjusted means of regression may be f i t t e d with a straight line when plotted on a logarithmic grid (Fig. 10b)« The means of the winter rates are included for comparison, though i t can only be assumed that the drop i s logarithmic because of the absence of a t h i r d time period i n this group. Since no fed controls were followed along with the fasted flounder, i t i s uncertain whether the systematic f a l l i n respiratory rate i s due entirely to starvation. A small part of the chase might be caused by thermal acclimation during this period i f discrepancies existed between the true environmental temperature and the laboratory temperature. As pointed out before, i t was never possible, because of unknown ve r t i c a l movements of the f i s h population sampled, and t i d a l changes with corresponding temperature variation at the surface, to measure either the true mean environmental temperature or the range of temperatures i n the environment. However, discrepancies between true acclimation temperature and laboratory temperature must be small at most (less than 1.5°C.). I t seems l i k e l y that essentially a l l of the progressive f a l l i n metabolic rate can be attributed to caloric starvation. Regardless of whether the drop i n metabolic rate i s due entirely to starvation or p a r t i a l l y to starvation and p a r t i a l l y to some other influence such as thermal acclimation, the drop w i l l henceforth be referred to as a starvation effect. Data collected for the effect of s a l i n i t y on metabolic rate was corrected for starvation by referring any two group comparisons to the same day of fasting. For instance, i f the metabolic rates of fresh water adapted flounder fasted four days are being compared to a control group of sea water flounder fasted three days, the individual metabolic rates of the four-day fasted group are divided by a fraction graphically derived from an enlarged graph of Figure 10b to bring the metabolic rates to a three-day fasted l e v e l . 5 4 The question arises as to whether or not the depletion of stored energy reserves during fasting with the systematic f a l l i n metabolic rate i n any way interferes with normal osmotic exchange i n the flounder. In total starvation (abrupt and complete food deprivation) the body must f a l l back on stored food reserves to provide energy for metabolic a c t i v i t y . At the outset, carbohydrates (mostly l i v e r glycogen) are consumed to provide calories, but the quantity i s small; within a short time the body must rely entirely on f a t and tissue proteins for energy. The result i s a progressive drop i n weight and, i n poikilotherms, a f a l l also i n respiratory intensity. However, the diminution of metabolic rate and weight do not f a l l i n the same manner. Smith (1935 a,b) has followed the respiration of fasted African lungfish, Frotopterus arthiopicus for periods i n excess of 600 days. Whereas weight f e l l slowly at f i r s t and i n a nearly linear fashion, oxygen consumption f e l l exponentially to 50$ of the i n i t i a l value within the f i r s t 7 days of the fast. His data depict an approximately straight l i n e when plotted on double logarithmic paper. By following mixed and non-protein B.Q. throughout, Smith found that the amount of carbohydrate burned was small and largely consumed within a few days. For the remainder of the fast u n t i l near death over a year and a half l a t e r , the lungfish subsisted on stored fat reserves. The amount of tissue protein consumed was very small u n t i l late i n the fast when a gross combustion of t h i s component occurred concomitant with a sharp terminal increase i n oxygen consumption just before death. Starvation appears to have l i t t l e effect on the work capacity of f i s h , at least u n t i l the terminal stages of inanition. Barrett (unpublished, cited by Fry, 1957) demonstrated that fasted Salmo gairdnerii can perform muscular work as well as fed controls and consume essentially as much oxygen i n active metabolism experiments as did the controls. Many f i s h cease feeding entirely during spawning migration with no apparent decrease i n swimming a b i l i t y . However, careful measure-ments have shown a progressive though small decrement i n the swimming a b i l i t y of 55 fasting Pacific salmon as they migrate up the Columbia River (Paulik and DeLacy, 1958). Thus, there seems l i t t l e doubt that sufficient energy exists for osmo-regulation, for even the capacity to perform muscular work suffers l i t t l e or no impairment. In this investigation the s a l i n i t y effect experiments were carried out during the f i r s t few days of the fast. During this time, carbohydrates and fats supply nearly a l l of the energy needed. I t i s not u n t i l late i n starvation, whem combustion of tissue proteins commences, that i t i s possible to tax severely homeostatic mechanisms. For this reason, i t seems a reasonably safe assumption that the few days of starvation imposed on the experimental f l a t f i s h had a com-pletely insignificant effect on normal osmotic exchange. 4. Effect of S a l i n i t y on the Standard Metabolic Rate of Starry Flounder These experiments were carried out during the summer and winter of 1956 and spring of 1957. Three experimental s a l i n i t i e s were chosen: the "normal" environmental s a l i n i t y of about 25°/oo» concentrated sea water of 45°/°°. and fresh water. P i l o t experiments revealed that moderate changes i n the osmotic gradient were inadequate to demonstrate differences i n standard metabolism. An example i s shown i n Figure 11a and the data are summarized i n Table VII. In this experiment the standard metabolism of 18 flounder was measured i n an experimental s a l i n i t y of 20°/oo ( A 1.076°C), the prevailing environmental s a l i n i t y on that date. Two days la t e r , oxygen consumption measurements were repeated on 12 f i s h of the same group i n a s a l i n i t y of 8.0°/oo ( A 0.43°C.) after the standard 20 hour adaptation period i n the respirometers. The results, corrected for the systematic f a l l i n metabolic rate due to starvation, were compared by covariance analysis and found to be non-significant, (Appendix-table IV). Essentially no change occurred either i n the adjusted treatment means or i n the slopes of the f i t t e d l i n e s . Flounder introduced into fresh water show a significant drop i n meta-56 Figure 11a. Comparisons of standard metabolic rate of Platichthys stellatus i n 20 °/oo (closed circles) and 8 °/oo (open circles) sea water. Respiratory rates f i t t e d by the method of least squares. Experimental temperature 15.2 ±.2°C. The regression lines are not sig n i f i c a n t l y different. Data of Table VII. Figure l i b . Comparison of standard metabolic rate of Platichthys stellatus i n 20 /OO sea water (closed circles) and after 20 hours i n fresh water (open c i r c l e s ) . Respiratory rates f i t t e d by the method of least squares. The regression lines are significantly different at the 5fo level of proba-b i l i t y . Experimental temperature 15.2 **.2°C. Data of Table VII. "I 1—I— I l i l t "I 1 I—I—I—I I I I -I 1 I X o v. CM o H 0.6| 0.3 h J J I I I I I I J 1—I i i i i i • ' i i i 10 40 100 BODY WEIGHT IN GRAMS 400 3h " i 1 — i — i i i i i i i 2 0 % IT X 2 CVJ O 3 o.e| Fresh 0.3 h J 1 I I I I I 1 I | I I I I I I i l l 4 10 40 100 400 BODY WEIGHT IN GRAMS Table VII. The standard metabolic rate of Platichthys stellatus i n 20°/oo sea water and fresh water. 1956 summer studies. X = body weight i n grams, Y = rate of oxygen consumption i n mgm. O^/gm./hr, (average of 2-3 determinations). 20/oo sea water 8 /00 sea water Corrected X Y X Y Y 23.4 .117 13.9 .18 .194 38.4 .13 28.9 .079 .0854 40.3 .0845 41,0 .094 .1017 41.1 .091 41.4 .0765 .0827 48.2 .103 43.3 .1005 .1087 51.6 .078 63.3 ,079 .0854 62.9 .0765 68.0 .075 .0811 67.2 .074 86.6 .087 .0941 68.4 ,097 87.7 .084 .0909 84.1 .097 143.8 ,057 .0616 84.4 .0885 146.1 .053 .0573 101.9 ,066 282.5 .059 .0638 120.1 ,079 142.2 ,07 148.2 .056 187.6 .055 188.6 .053 283.7 .065 Exp. temp. 15.3-.2°C. Exp. s a l . 8.0°/oo Days unfed, 5 July 1, 1956 Slope - .331 Starvation correction to day = Y/.924 Exp. temp. 15.0-,2°C. Exp. s a l . 20,0°/oo Days unfed, 3 June 29, 1956 Slope - .311 Table VII (Continued) Fresh water, 20 hr. adaptation Corrected X T T 13.9 .075 .0834 28.9 .071 .0789 41.0 .08 .0889 41.4 .066 .0734 43.3 .063 .0700 63.3 .065 .0723 68.0 .072 .0800 86.6 .068 .0756 87.7 .061 .0678 143.8 .064 .0711 146.1 .049 .0545 282.5 .055 .0611 Exp. temp. 15.1*.1°C. Exp. s a l . 0 Days unfed, 6 July 2, 1956 Slope = -.126 Starvation correction to day 3 = T/.899 Fresh water, 4 day adaptation Corrected X I T 11.7 .074 .0774 19.0 .074 .0774 19.5 .068 .0712 24.1 .069 .0722 47.8 .055 .057 59.7 .0615 .0643 80.4 ,059 .0617 81.7 .0495 .0518 84.5 .055 .0575 90.0 .049 .0513 94.7 .058 .0607 145.5 .057 .0596 171.2 .0525 .0549 196.2 .0525 .0549 Exp. temp. 15.0±.2°C. Exp. s a l . 0 Days unfed, 6 July 9, 1956 Slope = -.135 Starvation correction to day 3 = Y/.955 59 bolic rate* The decrease i n oxygen consumption appears to be gradual and becomes increasingly marked after the f i s h have adapted to fresh water. In Figures l i b and 12a the oxygen consumption of two groups of flounder i n fresh water are compared with a control group i n 20°/oo sea water and with each other. In the f i r s t group (Fig. l i b ) , measurements were made 20 hours after abrupt transfer of the flounder to fresh water. Although the data are sign i f i c a n t l y different at the 5$ probability level (F Q 1 - 7.68 > F = 7.5 > F 0 j = 4.21), the difference i s primarily a prominent change i n slope due to a drop i n t o t a l metabolism of the small f i s h only. In Figure 12a, these fresh water flounder are again compared with another group previously adapted to fresh water for 4 days before metabolic rates were determined. In this interval a highly significant drop i n metabolic rate has occurred (F = 29.1>F ^ = 7.88). The drop (16$ difference between the treatment means) i s represented i n both small and large flounder, for there was essentially no change i n slope. These experiments were carried out i n June and July, 1956. The data are summarized i n Table VII and the s t a t i s t i c a l tests are given i n Appendix-table IV. A l l data were corrected for the effect of starvation so that treatment comparisons are of adjusted data representing flounder of the same nutritional state. In December, 1956, an experiment f i r s t was conducted to measure the effect of high s a l i n i t y on oxygen consumption of flounder. In Figure 12b, Table VIII, metabolic rates of 13 flounder determined after 20 hours i n 49°/oo sea water show a marked increase above the control group of 28 flounder i n 25°/°° s e a water. The differences are highly significant at the 1$ probability level (F = 55.1>F 0 Q 5 = 8.83; Appendix-table V). Thus, the metabolic demands for osmoregulation of flounder i n sea water increases with increasing s a l t content of the water. Another series of experiments was performed i n May, 1957. which i n efftot serve to replicate the studies already described. These data are given 60 Figure 12a« Effect of adaptation time i n fresh water on the standard metabolic rate of Platichthys stellatus* 20 hour adaptation rates are represented as closed c i r c l e s and 4 day adaptation as open c i r c l e s . Respiratory rates are f i t t e d by the method of least squares. The regression lines are signifi c a n t l y different at the ifo level of probability. Data of Table VII. Figure 12B. Comparison of standard metabolic rate of Platichthys stellatus i n 25 °/oo sea water (closed circles) and i n 49 °/oo sea water (open c i r c l e s ) . Respiratory rates are f i t t e d by the method of least squares. The regression lines are significant at the 1% level of probability. Experimental temperature 9.9 ±.2°C. Data of Table VIII. Table VIII. Standard metabolic rate of Platichthys stellatus i n 25°/oo and 49°/oo sea water. 1956 winter studies. X = body weight i n grams, T = rate of oxygen consumption i n mgm. 0 /gm./hr. (average of 2-3 determinations). 25°/oo sea water 49°/oo sea water Corrected X I X T Y Y 2.5 .103 49.7 .0356 2.5 .142 .1525 3.9 .085 54 .0435 4.8 .073 .0784 4.8 .0655 57 .0465 6.5 .125 .1342 5.2 .058 70.1 .035 7.8 .064 .0687 5.5 .076 80.7 .0435 9.0 .0659 .0707 6.5 .055 81 .047 9.2 .0645 .0692 7.8 .051 91 .046 9.8 .0795 .0853 8.1 .063 100 .0347 21.8 .0741 .0795 9.2 .0527 134 .0346 57.0 .0505 .0542 9.8 .079 138 .0433 100 .057 .061 10.3 .047 157 .044 138 .0566 .0607 12.1 .0505 240 .0435 157 .0455 ,0488 13.4 .051 264 .0354 264 .0346 .0371 21.8 .0628 284 .0384 h-1 Exp. temp. = 10.oi.l°C. Exp. temp. = 9.8i.l°C. Exp. s a l . 24.7 - 26.0%o Exp. s a l . = 49.2°/oo December 15, 1956 and Days unfed = 3 December 20, 1956 (two groups) December 21, 1956 Days unfed = 2 Slope = -0.203 Slope = -0.16 Starvation correction to day 2 = Y/.931 62 i n Table DC. Standard metabolism studies were conducted on flounder at two fresh water adaption periods, f i r s t at 20 hours (Fig* 13a) and again at 5 days (Fig. 13b) after introduction into fresh water from sea water. Similar measurements were carried out on flounder 20 hours after introduction into a s a l i n i t y of 43°/oo from 25°/°° (Fig. 14). Since the observations were not carried out simultaneously, but over a period of several days, each experiment represents groups of f i s h each i n d i s t i n c t nutritional states. As before, the data were adjusted by reference to the exponential slope representing the mean rate of f a l l of metabolic rate due to starvation (see Figure 10b). The starvation effect thus removed, the data can be compared s t a t i s t i c a l l y and graphically for changes due to s a l i n i t y . Figure 13a shows that a small decrease i n standard metabolism has occurred i n flounder 20 hours i n fresh water as compared with sea water controls of the same nutritional state (8$ decrease i n the mean rate of oxygen consumption). The decrease i s significant at the 10$ but not at the 5$ probability level (F = 4.17>F = 3.04>F = 2.88). After 5 days i n fresh water, (Fig. 13b), the standard metabolism of fresh and salt water flounder i n the same nutritional state have diverged to a 10.5$ difference i n mean rates. This drop i n metabolic rate (difference i n the regression means) i s highly significant (Appendix-table VI). These results are i n accordance with those of the summer, 1956, series which showed that flounder adapted 4 days to fresh water had r e l a t i v e l y lower mean metabolic rates than those i n fresh water for only 20 hours. The percentage decrease i n metabolic rate of flounder transferred to fresh water i s dependent on body size and i s greater i n small than i n large animals. This observation i s based on the decrease i n slope of the regression l i n e through the oxygen consumptions of fre3h water flounder as compared with their salt water controls. The slope of the 20 hour adapted fresh water 63 Figure 13a. Comparison of standard metabolic rate of Platichthys stellatus i n 22.8 /oo sea water (closed circ l e s ) and i n fresh water, 20 hour adapta-tion (open c i r c l e s ) . Respiratory rates f i t t e d bjr the method of least squares. The regression lines are signifi c a n t l y different at the 10$ but not at the 5$ probability l e v e l . Experimental temperature 15.0 ±.1°C. Data of Table EC. Figure 13b. Comparison of standard metabolic rate of Platichthys stellatus i n 25 °/oo sea water (closed circles) and i n fresh water, 5 day adaptation (open c i r c l e s ) . Respiratory rates are f i t t e d by the method of least squares. The regression lines are significantly different at the 1$ probability l e v e l . Experimental temperature 14.8 ±.1°C. Data of Table IX. I I I I I I T 1 1 1 1—I - P T r 2 o CM O 0.6\ 2 2.8 %, Fresh water, 20 hour adaptation 0.3r J I I ' I I l I l J I L 10 40 100 BODY WEIGHT IN GRAMS 400 -i 1 1 — i — i — i i i i i 1 i i i i i 1 1 I 3h or x o v. CM O e> 2 0 ^ 26.4 %, n iii n II Fresh water, 5 day adaptation 0.ZY J I ' l I I I I J L J L 10 40 100 BODY WEIGHT IN GRAMS 400 22.8°/°o sea water X Y 9.0 .099 9.9 .114 10.1 .109 11.3 .092 16.0 .10 45.6 .084 45.6 .0894 62.4 .081 65.6 .0893 78.0 .0845 79.0 .088 132.3 .0766 139.2 .0645 212.1 .0618 223.6 .071 245.6 .065 Exp. temp. = 15.0°C. Exp. s a l . = 22.8%>o Days unfed = 4 May 13, 1957 Slope = -0.141 TX. Standard metabolic rate of Platichthys stellatus i n fresh water, normal sea water and concentrated sea water. 1957 spring studies. X = body weight i n grams, Y = rate of oxygen consumption i n mgm. 02/gm./hr. (average of 2-3 determinations). Fresh water, 20 hour adaptation 43.2°/oo sea water, 22 hour adaptation X Corrected Y Corrected Y 7.6 .0975 .1008 9.2 .0855 .0884 12.6 .0775 .0801 16.6 .097 .1003 16.8 .082 .0848 17.1 .102 .1054 24.6 .0925 .0956 36.9 .090 .093 48.0 .0835 .0863 59.5 .0736 .0761 67.7 ,0565 .0584 83.5 .0785 .0811 97.4 .0544 .0562 116.0 .071 .0734 128.0 .0795 .0822 175.8 .073 .0754 197.7 .0603 .0623 306.5 .059 .061 Exp. temp. = 15.Q±.1°C* Exp. s a l . = 0 Days unfed = 5 May 14, 1957 Slope = -0.124 Starvation correction to day 4 = Y/.967 5.3 .134 .1308 7.1 .123 .1201 8.4 .143 .1396 11.0 .118 ,1152 18; i .105 .1025 18.4 .098 .0957 23.0 .122 .1191 31.0 .100 .0976 48.4 .077 ,0751 63.2 .096 .0937 68.0 .090 .0878 94.3 .070 .0683 148.1 .105 .1025 204.4 .067 .0654 229.5 .0755 .0736 286,4 .0775 .0756 Exp, temp. = 14.0±.1°C. Exp. s a l . = 43.2°/oo Days unfed = 6 May 22, 1957 Slope = -0.151 Starvation correction to day 7 = Y/l.024 Table IX. (Continued). 25-26.4%o sea water X Y X Y 4.4 .14 64.5 .079 8.8 .093 75.0 .065 9.2 .091 76.6 .0775 9.8 .111 81.5 .071 10.0 .097 85.9 .076 10.6 .156 131.7 .058 11.7 .116 131.7 .072 13.6 ,088 131.8 .075 15.3 .089 136.7 .0596 16.5 .081 145.7 • .073 21.3 ,0825 146.1 .068 26.6 .085 205.5 .049 44.8 .071 215.7 .06 45.7 .077 236.9 .0595 61.1 .079 Exp. temp. = 14.8±.1°C. Exp. s a l . = 25.0 and 26.4°/oo Days unfed = 7 May 16, 1957 and May 23, 1957 (two groups) Slope = -0.192 Fresh water, 5 day adaptation Corrected X Y Y 7.1 .086 .0898 9.1 .0734 .0766 11.9 .069 .072 16.1 *075 .0783 16.2 .096 > .1002 16.5 .079 .0825 47.0 .068 .071 57.9 ,066 .0689 66.1 .0645 .0673 79.4 .069 .072 92.5 .0604 .063 114.9 .055 .0574 127.5 .054 .0564 166.4 .0543 .0567 194.3 .045 .047 299.5 .0542 .0566 Exp. temp. = 14.7±.1°C Exp. s a l . = 0 Days unfed = 9 May 18, 1957 Slope = -0.142 Starvation correction to day 7 = Y/,958 665 flounder i s -.124 and -.140 for the control group. For the 5 day fresh water adapted flounder the slope i s -.142 as compared to -.192 of the sa l t water controls. The decrease i s small but consistent and i s i n agreement with the 1956 series which showed even greater decreases i n slope. The mean standard metabolic rate of flounder i n 43°/oo (Fig. 14, Table IX) i s 15.1$ above the mean of the control group. The difference i s highly significant, with a variance ratio of 17.3 (Appendix-table VI). These results agree with the winter, 1956, experiment (Fig. 12b) though the mean increase i n metabolic rate i s not as great (15.1$ i n the summer experiment as opposed to a 25$ increase i n the winter). The slopes of regression, though different from the controls i n both cases, are inconsistent i n the direction of change and preclude any generalization as to a possible interaction between body size and s a l i n i t y effect so far as the high s a l i n i t y i s concerned. In the spring 1957 experiment, the slope i s less than that of the control groups (-.151 and -.192 respectively) and may indicate a r e l a t i v e l y greater energy expenditure for osmotic regulation i n the large than i n the small animals. In the winter experiment, however, the slope became steeper i n the high s a l t concentration (-.203 at 49%>o and -.16 at 25°/oo) but the results represent only 13 f i s h with considerable v a r i a b i l i t y i n the oxygen consumption rates. Thus, these results, while unquestionably demonstrating a higher expenditure of energy i n the high s a l t concentration for a l l f i s h , are inadequate with respect to showing whether the energy demands i n the high s a l i n i t y are rela t i v e l y greater for small than large flounder, or vice versa. In fresh water, on the other hand, energy demands for osmoregulation are signifi c a n t l y less. The results as well show a consistent decrease i n slope i n the weight-specific oxygen consumption of flounder i n fresh water, implying that osmo-regulatory demands for energy are r e l a t i v e l y less for small than large flounder i n fresh water as compared with the energy expenditure of small and large salt water flounder. 67T Figure. 14. Comparison of standard metabolic rate of Platichthys stellatus i n 25 °/oo sea water (closed circ l e s ) and i n 43.2 °/oo sea water (open c i r c l e s ) . Respiratory rates f i t t e d by the method of least squares. The regression lines are significantly different at the 1% probability l e v e l . Experimental temperature 14.0 - 14.8°C. Data of Table IX. J 1 I I I I 11 I I I I I I I 11 I I I 4 10 4 0 100 4 0 0 B O D Y W E I G H T IN G R A M S 68 5. The Effect of S a l i n i t y on the Standard Metabolic Rate  of Lemon Sole and Speckled Sand Dab Because an adequate supply of lemon sole (Parophrys vetulus)and speckled sand dab (Citharichthys stigmaeus) was unpredictable at best and actually unavailable most of the time, i t was not possible to replicate complete-l y with these species the s a l i n i t y effect experiments carried out with flounder. Only scattered experiments were conducted when an adequate size range of either species was collected. The two experiments here presented, one on the sole, the other, the sand dab, are of some value as a comparison of the influence of s a l i n i t y on the respiration of these stenohaline forms with the euryhaline starry flounder. Figure 15a, Table X shows that an increase i n the ambient salt con-centration i s accompanied by a marked increase i n tot a l metabolic rate of the speckled sand dab. The experiment was carried out after a 3 day adaptation period i n concentrated sea water of 43 p.p.t. ( A 2.275°C.) and i s compared with a control group of the same nutritional state (fasted 3-4 days) i n 23°/oo sea water ( A 1.24°C.). The difference i n the means of regression are highly significant with a variance ratio of 44.6 (F . = 7.56, Appendix-table VII). There i s also an increase i n slope i n the high s a l i n i t y (b = -.203 as com-pared with -.095 for the controls). This suggests that the actual amount of energy expended for osmoregulation as a percentage of the to t a l metabolic rate i s influenced by body size, with a greater increase i n energy expenditure for osmotic work i n small than i n large sand dab. This interpretation must be accepted with caution because i t was not possible to replicate the experiment. I t should be noted -that the respiratory response of sand dab exposed to increased s a l i n i t y i s the same as that of starry flounder i n increased s a l i n i t y -an overall increase i n total metabolic rate. As pointed out i n the introduction, the measurement of metabolic changes attending the transfer of a marine f i s h into a sea water d i l u t i o n below 69 figure 15a. Comparison of standard metabolic rate of Citharichthys stigmaeus i n 23 "/oo sea water (closed circles) and i n 43.4 /oo sea water (open c i r c l e s ) . Respiratory rates are f i t t e d by the method of least squares. The regression lines are significantly different at the 1$ level of probability. Experimental temperature 14.9 £.2°C. Data of Table X. Figure 15b. Comparison of standard metabolic rate of Parophrys vetulus i n 24.3 Voo sea water (closed circles) and i n 5.8 /oo (open c i r c l e s ) . No significant change i n metabolic rate occurred although individual v a r i a b i l i t y was greatly increased i n the group i n the low s a l i n i t y . Experimental temp-; erature 15.0 ±.2°C. Data of Table XI. I i 1 1 1—i—i—r—i 1 1 1 1 i i i r 4 10 40 100 BODY WEIGHT IN GRAMS Table X, Standard metabolic rate of Citharichthys stigmaeus i n 24.4°/oo and 43.4°/oo sea water. X = body weight i n grams, T = rate of oxygen consumption i n mgm. C^/gm./hr. (average of 2-3 determinations). 24.4°/oo X I 3.6 .092 3.8 .082 4.9 .102 5,2 .0748 5.8 .103 7.0 .0955 7.1 .074 10.3 ,082 11.6 .0746 12.3 .071 13,1 .070 19.1 .071 22.9 .086 28.1 ,080 28.3 .074 38.0 .0706 43.4°/oo X Y 4.55 .176 5.05 .118 6.0 .154 7.25 .137 8.1 .101 8.3 .134 9.3 .113 9.8 .094 13.5 .093 13.9 .116 20.3 .108 24.4 .0905 24.5 *088 31.4 .076 35.2 .115 47.9 .101 Exp. temp. = 14.8±.1°C. Exp. s a l . = 24*4°/oo July 5 and 6, 1957 Days unfed = 3 and 4 Slope = -0.095 Exp. temp. = 15.0±.1°C. Exp. s a l . = 43.4°/oo July 15 and 16, 1957 Days unfed = 4 and 5 Slope = -0.203 Table XI. Standard metabolic rate of Parophrys vetulus i n 24.35 0/ 0 0 ^ d 5.8°/oo sea water. X = body weight i n grams, Y = rate of oxygen consumption i n mgm. O^/gm./hr. (average of 2-3 determinations). 24.35%o 5.8%o X Y X Y 3.5 .165 2.1 .145 3.5 .123 2.5 .101 4.3 .145 4.1 .23 5.3 .123 4.2 ,152 5.7 .155 4.2 .149 8.0 .117 5.2 .169 10.8 .094 6.5 .101 26.9 .101 6.9 ,088 8.4 .121 12.3 .157 17.8 .095 24.1 *094 25.3 .126 33.4 .095 -4 Exp, temp. = 15.1±,1°C. Exp. s a l . = 5.8°/oo, adapted 3 days Days unfed = 4 and 5 July 22 and 23, 1957 Slope = -0,15 Exp* temp. = 14,9^.1 C. Exp. s a l . = 24.35°/oo Days unfed = 3 July 21, 1957 Slope = -0.153 72 the incipient lethal s a l i n i t y level has questionable physiological significance. Nevertheless, the procedure offers an interesting comparison between the metabolic responses of euryhaline and comparatively stenohaline marine fishes introduced into low s a l i n i t i e s . Such an experiment was per-formed and i s i l l u s t r a t e d i n Figure 15b (Table XI). The standard metabolic rates of a group of lemon sole were determined three days after transfer from normal sea water to a s a l i n i t y of 5.8°/oo ( 0.31). The results show that essentially no change has occurred i n the mean metabolic rate of these sole as compared with the controls, but that there has been a large increase i n individual v a r i a b i l i t y . Lemon sole generally survive less than a week i n 6°/oo sea water. In this experiment some mortality occurred at the time of the oxygen consumption determinations; the attendant v a r i a b i l i t y i n metabolic rate reflects the moribund condition of the sole. C. COMMENT It has been shown that the starry flounder i s both a hypotonic and hypertonic regulator, possessing compensating mechanisms for internal osmotic regulation i n the face of abrupt and drastic alteration or reversal of osmo— l a r i t y of the external environment. These alterations were characterized by significant changes i n metabolic a c t i v i t y of the flounder which are assumed equivalent to changed energy needs for osmotic work. An understanding of osmotic energetics necessitates consideration of a l l processes u t i l i z e d to maintain internal equilibrium regardless of external osmotic vicissitudes. Starry flounder consumed less oxygen i n fresh water than i n normal sea water and s t i l l greater energy demands were made i n supernormal s a l i n i t i e s . These relationships are shown diagrammatically i n Figure 16. Any proposed ex-planation for these events must of necessity explain the apparent increasing 73 Figure 16. Diagrammatic representation of relative energy demands of starry flounder for osmotic regulation i n hypotonic and hypertonic media. Oxygen consumption of "basal*1 c e l l u l a r metabolism other than processes connected with osmoregulation are represented by the lower cross-hatched portion of the bars. This portion i s assumed to be unaffected by changes i n s l i n i t y . The upper clear portion of the bars represents variable oxygen demands for osmoregulation de-pending on the environmental s a l i n i t y . hyperregulation fresh water hyporegulation >-74 osmotic work with increasing salt content of the environment. The true explanation l i e s i n relative metabolic demands for the transportation of electrolytes and water i n the principal organs of exchange, the g i l l s , the kidney and the gut. The importance of the integument i n li m i t i n g permeability to salts and water cannot of course be overemphasized, but permeability of f i s h skin i s a passive attribute of scales and mucus, rather than an active energy consuming process. A " d i f f e r e n t i a l " permeability may or may not exist but u n t i l such a phenomena i s demonstrated i t must be assumed that the diffusion of water through the body surface i s dependent only on the concentration and direction of the gradient on either side. Therefore, the relative osmotic work loads of the individual organs of exchange must be considered, but here one i s hampered by our meager know-ledge of the interrelationships of these organs, the relative osmotic demands made on them and their efficiency i n doing the job. While no direct measure-ments have been made of the oxygen consumption of the perfused teleost kidney or g i l l doing osmotic work, the recent disclosures of Ussing (1958) are of great value i n predicting energy demands of these organs for ion transport. In his study of active ion transport by the frog skin, Ussing found that i t costs as much oxygen to transport equivalent amounts of sodium across a low concentration gradient as across a high concentration gradient. Oxygen consumption i s dependent on the amount of sodium transported regardless of whether the movement i s u p h i l l or not. Ussing demonstrated further that 16 to 20 sodium ions were transported for each molecule of oxygen consumed. Thus, with the frog skin at least, and very probably i n a l l biological membranes, an inseparable quantitative relationship exists for energy demands of the ion transport mechanism. This new quantitative thermodynamic concept of active transport pro-vides a convenient basis to a possible explanation for the observed lower 75 oxygen consumption of flounder i n fresh water than i n sea water. Since the s a l i n i t i e s used i n these experiments represent t o n i c i t i e s of essentially equal though opposite osmotic gradient (fresh water of A 0 < body f l u i d of A 0.65 < sea water of A 1.35) i t can be assumed that the hydrating and dehydrating effects of fresh and salt water respectively are nearly equal i n their d i s -turbing influence on water balance. The difference i s found i n salt metabolism. In order to maintain osmotic equilibrium the fresh water f i s h excretes osmotic water i n a copious urine of low specific gravity. Small amounts of salt are continually lost i n the urine and are regained from ingested food and by ion absorbing c e l l s i n the g i l l s and mucous membranes (Krogh, 1937; Copeland, 1948, 1950; Wikgren, 1953). The marine f i s h , however, has adopted a much more circuitous method for regaining lost osmotic water, that of drinking sea water and excreting the salts v i a the g i l l s . This method entails more "handling'' of salts by the organs of exchange. Salt must be transported across the gas-trointestinal mucosa to the blood, carried to the g i l l s and again secreted across membranes to the external milieu. The process i s wasteful of precious body water since the ingested hypertonic sea water must be diluted to isotonicity i n the gut (Smith, 1930, and unreported observations i n this laboratory on the starry flounder). While univalent ions (sodium, chloride and small quantities of potassium) are absorbed from the gut and excreted by the g i l l s , most divalent ions are retained and concentrated i n the intestinal residue (Smith, 1953). Some divalents do enter the circulation and are excreted by the kidney, accompanied by an obligatory water loss since the flounder i s unable to concentrate urine even to isotonicity (Figs, l a and l b ) . Again active transport i s necessary, either to resorb univalent ions from the glomerular f i l t r a t e or, what i s probably more important i n view of the relative unimportance of the la t t e r (Forster, 1953), to secrete selectively divalents into the urine by the tubules from the renal portal circulation. A l l of this 76 complex ion movement i s necessitated by the i n a b i l i t y of marine f i s h to separate sp e c i f i c a l l y water molecules from sea water for direct absorption. I t appears, i n fact, that no animal c e l l can pump water into the c e l l against an osmotic gradient (though water can be pumped out of a c e l l against an osmotic gradient after i t has passively diffused i n ) . Therefore, the d i s t i l l a t i o n of sea water by marine teleosts obligates much electrolyte transfer with a concomitant expenditure of energy. In contrast to t h i s , fresh water fishes have only the r e l a t i v e l y simple task of replacing the small amount of salt l o s t i n the urine. Water i s free for the fresh water teleost and t h i s , of course, causes i t s major osmotic problem - disposing of water without losing s a l t . I t i s generally assumed that since fresh water f i s h with but one known exception possess glo-merular kidneys, the dilute and copious urine i s formed from the glomerular f i l t r a t e , an u l t r a f i l t r a t e of the a r t e r i a l blood supply to the kidney. However, no measurements of either renal blood flows or glomerular f i l t r a t i o n rates have been carried out on fresh water f i s h . In view of the importance of the renal portal supply to the teleost kidney, i t i s altogether possible that a greater or lesser portion of the urine represents tubular secretion of water or solutes from this venous supply. The one exception to the apparent universality of glomerular kidneys of fresh water teleosts, the aglomerular pipefish, Microphis  boaja, i s significant, for i t means that tubular secretion alone i s adequate for l i f e i n fresh water. Numerous measurements of oxygen consumption by the mammalian kidney have been reported and are summarized by Smith (1951). In general, oxygen consumption correlates d i r e c t l y with renal blood flow so that as the l a t t e r i s decreased so follows the oxygen consumption. Water diuresis has no influence on renal oxygen consumption. These findings would appear to have new meaning i n the l i g h t of Ussing's disclosure of the precise relationship between the amount of sodium transported and the energy ( i n terms of oxygen consumption) 7.77 needed for the process. Increased renal blood flow means an increased f i l t r a t i o n rate and greater ion reabsorptive a c t i v i t y by the tubule, hence the greater oxygen consumption. Water diuresis would have no effect since the work of f i l t r a t i o n i s supplied by the heart through the a r t e r i a l pressure. As long as the f i l t r a t i o n rate remains the same, no quantitative alteration i n ion reabsorption would occur i n spite of the greater urine flow. Since the rate of glomerular f i l t r a t i o n almost certainly increases when the euryhaline flounder moves from salt to fresh water (though studies of this interesting change i n renal function are wholly lacking), a con-comitant increase i n tubular reabsorptive a c t i v i t y for the formation of hypo- . tonic urine would be expected. Whether this process of the fresh water flounder kidney consumes more energy than the secretion of divalent ions by the kidney of the marine flounder i s an open question. Our present lack of knowledge on kidney function i n fishes i s a prominent obstacle i n the way of a clear understanding of teleost osmoregulation. The work of Forster and Berglund (Forster, 1953; Forster and Berglund, 1956; Berglund and Forster, 1958) has contributed much to an understanding of renal function i n marine fishes, but i n fresh water fishes the picture remains obscure. The results of the standard metabolism experiments show an. apparent contradiction, v i z : the oxygen consumption of flounder i s less i n fresh water than i t i s i n 8°/oo, a s a l i n i t y approaching isotonicity (Fig. 11a). Pre-sumably energy demands should be minimal i n the absence of an osmotic gradient to tax energy consuming homeostatic mechanisms. However, there i s no a p r i o r i reason to believe that a l l osmotic mechanisms cease when a euryhaline f i s h enters isotonic brackish water. I t i s conceivable that marine starry flounder continue to drink water after entering low s a l i n i t i e s , although the process would no longer seem to be efficacious. As long as salt i s transported through membranes regardless of the osmotic gradient, energy demands for osmoregulation 78 continue* Again, the lack of experimental evidence precludes further specula-tion as to the reason for the observed re l a t i v e l y high oxygen consumption i n brackish water. In supernormal s a l i n i t i e s (45—50°/oo) the metabolic rate of starry flounder and speckled sand dab was found to increase considerably above the rate i n normal sea water (25°/°°)» As the external s a l t concentration increases, the osmotic loss of body water through mucous membranes also increases. To restore f l u i d balance, more sea water must be ingested, the salt separated and excreted. Since the ingested water i s more saline, a much greater quantity of salt must be transported for each gram of water absorbed by the gut. Not only are extrarenal water losses augmented, but i t becomes increasingly d i f f i c u l t to replace these losses. In addition, the flounder must excrete more divalent ions v i a the kidneys and because the urine remains hypotonic even i n concentrated sea water (Fig. l b ) , the obligatory renal water losses are greater. Though no measurements have been made, we would theoretically expect an increase i n urine flow with a rise i n the external osmotic gradient. A certain r e l i e f from t h i s mushrooming problem i n water con-servation i s obtained by allowing the concentration of the body f l u i d s to r i s e somewhat (Fig. l b ) . I f careful metabolism studies were made over a range of s a l i n i t i e s , i t would probably be found that the oxygen consumption increases exponentially rather than l i n e a r l y with an increasing osmotic gradient. The net result of high s a l i n i t i e s i s that a greater portion of the animal's basal energy requirements must be devoted to maintaining homoiosmoticity. Since fishes are s t r i c t l y limited i n the amount of oxygen available for c e l l u l a r respiration both by g i l l limitations and because of the low oxygen tensions i n the aquatic environment, any increase i n basal metabolic demand i s d i s t i n c t l y detrimental to a species, since i t decreases the respiratory reserve for a c t i v i t y * Maximal oxygen consumption (active metabolism) i s never far above 79 standard metabolic rates, even i n active species such as trout or salmon (Job, 1955; Fry, 1957). In a high s a l i n i t y , the "scope for a c t i v i t y " (Fry, 1947) or the difference between active and standard metabolic rates, i s considerably decreased as a result of augmented energy demands for osmotic work and also because oxygen s o l u b i l i t y i n water decreases with increasing salt content. Decreases i n a c t i v i t y of salmon smolt (Salmo salar) moving into salt water from fresh water have been observed (Huntsman and Hoar, 1939). In this laboratory, Houston (1958) has measured significant decreases i n locomotor a c t i v i t y of chum salmon (Oncorhynchus keta) moved from fresh to sea water. Although i t i s uncertain whether decreased a c t i v i t y i n sea water i s the result of lowered "scope for a c t i v i t y " or i s due to a direct inhibitory action on muscle fibers of changes i n electrolyte composition of the blood, i t i s evident that high s a l i n i t i e s reduce the physiological reserve of f i s h for other environmental demands. 8 0 V THE EFFECT OF ENVIRONMENTAL SALINITY ON THYROID ACTIVITY AND RADIOIODIDE METABOLISM OF THE STARRY FLOUNDER The frequently implicated role of the teleost thyroid i n water and electrolyte metabolism has remained obscure. The problem has been approached both with respect to measured alterations i n thyroid a c t i v i t y effected by s a l i n i t y changes and to induced changes i n s a l i n i t y tolerance by administering thyroid preparations. The literature on th i s subject has been reviewed by Fontaine (1953, 1956), Hoar (1951, 1957), Smith (1956) and Pickford and Atz (1957). I f a generalization can be ventured from the existing evidence, some of which i s contradictory, i t appears that thyroid a c t i v i t y decreases with increasing s a l i n i t y of the external environment. Freshwater species trans-ferred to dilute sea water frequently show a transitory thyroid inactivation more noticeable i n those species with particularly active glands i n fresh water (Olivereau, 1950, 1954). Conversely, marine species maintained i n hypotonic s a l i n i t i e s usually develop hyperactive thyroids. Hoar (1952) found that landlocked smelt (Osmerus mordax) and alewives (Pomolobus pseudoharengus) always had more active thyroids than individuals collected from coastal estuaries. The landlocked alewives had extremely hyperplastic glands and experienced heavy mortality during the reproductive season when demands on thyroid hormone are evidently increased. Leloup (1948) and Olivereau (1948, 1954) noted a transitory decrease i n thyroid a c t i v i t y of two marine species, Muraena helena and Labrus bergylta. subjected to dilute sea water followed by a gradual return to normal a c t i v i t y i n the lowered s a l i n i t y . Transfer of the marine k i l l i f i s h , Fundulus majalis, from sea water of 25.5%>o to dilute sea water of 5.1°/<x> 131 caused a small (25$) increase i n thyroid uptake of 1 (G-orbman and Berg, 1955), while the same treatment with the brackish water Fundulus heteroclitus 81 131 resulted i n a very much greater (150$) increase i n the peak 1 uptake of f i s h i n the lower s a l i n i t y . The most convenient explanation for these findings i s that fresh water f i s h have a greater physiological demand for thyroid hormone i n a direct or adjunctive osmoregulatory role. A more l i k e l y explanation, however, l i e s i n the low iodine levels of fresh water as compared with sea water. Since Marine and Lenhart i n 1910 demonstrated that thyroid hyperplasia i n brook trout, Salvelinus fontinalis could be completely abolished by adding small amounts of iodine to the water, several authors have shown that thyroid hyperactivity i n fresh water species i s frequently a goitrogenic reaction to the low iodine content of fresh water. Both the uptake of radioiodine (Berg and Gorbman, 1953, 1954; La Roche, 1953) and histological c r i t e r i a (Hamre and Nichols, 1926; La Roche, 1950; Hoar, 1952; Robertson and Chaney, 1953; Schlumberger, 1955) have been used to demonstrate hyperplasia and to show that the condition was immediately alleviated by adding even minute amounts of iodine to the water. Lack of iodine prevents normal production of thyroid hormone, resulting i n a lowered t i t e r of hormone i n the blood. With the normal inhibiting action of thyroid hormone removed, more thyroid-stimulating hormone from the anterior pituitary i s produced with the result that the thyroid f o l l i c l e s become distended with an incomplete form of thyroglobulin or " c o l l o i d " . Not a l l experimental findings can be explained on the basis of the iodine content of the water. Among the most interesting experiments are those of Koch and Heuts (1943) and Heuts (1943) who found that feeding thyroid to the euryhaline stickleback, Pygosteus markedly decreased the resistance of this species to sea water. The treatment appeared to have a direct effect on mineral metabolism; chlorides accumulated i n the blood and the animals eventually died. Fresh water sticklebacks were unaffected by thyroid feeding. Similarly, Baggerman (1957) found that thyroxine administration induced a 82 fresh water preference and thiourea a salt water preference i n the three-spined stickleback. Gasterosteus. Fontaine and Baraduc (1934) found that while a single feeding of iodinated casein decreased the s a l i n i t y resistance of Salmo salar, prolonged treatment with thyroxine or iodinated casein induced certain morphological changes (pseudo-smoltification) and a concomitant increase i n s a l i n i t y resistance. Thus the length of treatment appears to be an important factor i n inducing changes i n s a l i n i t y tolerance. Baggerman (1957) also demonstrated a reversal i n the induced s a l i n i t y preference of Gasterosteus. While thyroxine induced fresh water preference during the f i r s t few days of treatment, a return to s a l t water preference occurred on the seventh day. Similarly, thiourea treatment caused sticklebacks to pass transitionally through a period of fresh water preference, then return to s a l t water preference (Exps. 38A and 38B, Fig. 20, of Baggerman's paper). However, i n other experiments, the reversal i n induced s a l i n i t y preference did not occur during the period of treatment. These results and those of Fontaine and Baraduc (1954) may possibly explain the results of Koch and Heuts (1942) who reported decreased s a l i n i t y tolerance of sticklebacks after a single feeding of thyroxine. The recent work of Burden (1956) and Smith (1956) strongly suggests that at least f o r the species studied, k i l l i f i s h and trout, an osmoregulatory role of the thyroid, i f present, i s co l l a t e r a l or subsidiary to some other endocrine influence. Smith's work with Salmo trutta showed that while thyroxine i n high dosages promoted s a l i n i t y resistance, growth hormone was far more effective i n this respect. Burden's work indicates the involvement of an unknown pituitary factor i n osmoregulation of Fundulus. Thyroxine therapy of hypophysectomized f i s h had no effect on the i n a b i l i t y of these animals to survive i n fresh water. Gorbman ( i n discussion following paper by Smith,, 1956) found that thiourea treatment had no effect on the normal 83 euryhalinity of Fundulus heteroclitus. Treated f i s h with inhibited thyroid glands resisted transfer to either fresh or salt water as well as controls. I t i s doubtful that either the administration of thyroid material or the i n h i b i t i o n of thyroid function with chemical inhibitors are effective or j u s t i f i a b l e ways to demonstrate an osmoregulator role of the thyroid gland. One cr i t i c i s m i s that any therapy may have unknown side-effects that could a l t e r the normal physiology of the species. U n t i l fundamental research, presently lacking, reveals the effects of thyroid inhibitors, such as thiourea or thio u r a c i l , on a l l aspects of the fishes' metabolism, they should be used with caution. Similarly the feeding or injection of thyroid substance may have f a r -reaching effects not immediately apparent. For one thing, thyroid adminis-tration inhibits secretion of TSH resulting i n a marked hypoactivity of the animal's own thyroid gland. Another argument against such treatment i s that i t i s d i f f i c u l t to know when or whether the treatment has been effective. As already discussed, the apparent effect induced by treatment may actually reverse i f the treatment i s continued long enough. The teleost thyroid i s perhaps the most l a b i l e of the endocrine glands. Just about a l l of the environmental factors' known to influence the total metabolism of the animal, such as season, body size, sex, sexual maturity, reproductive cycles and temperature have been shown i n f l u e n t i a l on thyroid a c t i v i t y . These factors have been discussed i n several reviews such as those by Hoar (1951, 1957), Lynn and Wachowski (1951) and Pickford and Atz, (1957) and w i l l not be reiterated here. To evaluate thyroid function with respect to one specific effect, such as the role of the thyroid i n osmotic regulation, demands a careful appraisal of many other environmental influences. Factors such as temperature, body size and iodine content of the water can be experi-mentally controlled. Seasonal and reproductive cycles cannot be controlled but can be accounted for i n the experimental design. Therefore i t i s im-possible to divorce the study of an osmoregulatory role of the thyroid gland 84 from other direct or collateral effects. Several c r i t e r i a for evaluating thyroid a c t i v i t y are available, but only two, histological and radiological, have been used extensively by f i s h physiologists. Of the radiological methods, the simple uptake of radio-iodine by the thyroid gland has become a research tool of considerable im-portance. Tests usually attempt to quantitate the amount of radioiodine trapped by the thyroid either by measuring with counting instruments the pro-portion of the dose accumulated by the excised gland or by some quantitative interpretation of autoradiographs prepared from the gland. At least two dist i n c t mechanisms are responsible for the uptake of iodine by the thyroid gland. The f i r s t i s the "iodide trap" and the second i s the u t i l i z a t i o n of the accumulated inorganic iodide to synthesize organic thyroid hormone. Radio-iodine tests of thyroid a c t i v i t y of fishes most commonly have been of the form 131 of uptake curves, where the percentage fraction of a single tracer dose of 1 accumulated by the thyroid i s plotted against the time after administration of 131 the dose. "In vivo" measurements of 1 accumulation using external counting arrangements have not yet been developed for use with fishes, so that i n prac-t i c e , a large number of f i s h must be given a standard dose simultaneously, sacrificed at intervals of time thereafter, the thyroids removed and their radioactivity counted. The percentage thyroid uptakes of individual f i s h are plotted against time of sacrifice to form a composite uptake curve. Injections are usually made intraperitoneally. These curves appear i n most cases to be a reliable estimate of the true thyroid a c t i v i t y (the rate of secretion of thyroid hormone) under many experimental conditions. Two very important factors, how-ever, l i m i t the diagnostic accuracy of radioiodine tracer studies i n fishes. One i s the amount of elemental iodide i n the ambient water already mentioned. The other equally important factor i s the disappearance rate of the tracer dose from the blood. I t i s clear that collection of radioiodine by the thyroid gland i s 85 t o t a l l y dependent on the amount of isotope delivered to the gland by the blood 131 stream. Other things equal, less 1 w i l l accumulate i n the thyroid when the injected dose i s rapidly removed from the body than when removal i s slower. I f the excretion rates of radioiodine d i f f e r under different experimental con-131 ditions, i t may not be j u s t i f i a b l e to use thyroid 1 uptake curves as a com-parison of thyroid a c t i v i t y between experimental treatments. S a l i n i t y varia-tions particularly would be expected to produce changes i n iodide behavior i n the body f l u i d s because of the marked quantitative and directional alterations i n electrolyte and water movements across the organs of exchange - the kidney, g i l l s , oral membranes and alimentary canal. Temperature variations should also influence iodide excretion rates, since metabolic rate and concomitantly the rate of electrolyte exchange of poikilothermic animals such as f i s h are d i r e c t l y dependent on the environmental temperature. Por these reasons i t must be con-131 eluded that thyroid 1 uptake i n i t s e l f i s an unreliable parameter for eva-luating thyroid a c t i v i t y i f either s a l i n i t y or temperature are altered as experimental treatments. To evaluate thyroid a c t i v i t y of flounder i n fresh and s a l t water, another parameter, that of thyroid clearance has been chosen. This appears to satisfy the requirements for a test that i s essentially uninfluenced by variations i n the disappearance rate of radioactive iodide from the blood. Thyroidal radioiodide clearance, expressed as the volume of blood cleared of i t s radioiodide per minute, i s actually a modification of the standard renal clearance formulae: 131 l 1 3 1 Clearance, thyroid = ^ o i d 1 uptake during t minutes mean blood cone, of ,131 during t minutes. Since the dose of radioiodide i s disappearing exponentially from the blood and 131 because following the early phase of iodine uptake 1 -labeled hormonal iodine begins to appear i n the blood, only the f i r s t few hours after injection are suitable for clearance calculations. 86 In the present study, thyroid a c t i v i t y of the starry flounder as influenced by s a l i n i t y has been studied with radioiodine, paying particular attention to the behavior of the tracer dose i n the body f l u i d s . With the exception of some preliminary work by Chavin (1956b) essentially nothing i s known of iodine metabolism i n fishes. A large portion of the research was devoted, therefore, to radioiodine movements - i t s excretion, distribution i n the body and behavior i n the blood — i n both fresh and salt water flounder. In comparing thyroid a c t i v i t y i n fresh and salt water, flounder were pre-adapted to fresh water containing an added amount of iodide equivalent to the iodine content of sea water at the experimental s a l i n i t y used. As with the foregoing sections of this thesis on body f l u i d concentration and metabolic rate, attention has been given to the effect of size on thyroid a c t i v i t y . A. METHODS: DETERMINATION OF IODIDE MOVE- MENT AND THYROID ACTIVITY WITH RADIOIODINE 1. Injections f i Radioiodine i n the form of carrier-free sodium iodide was diluted with saline or d i s t i l l e d water for injection. For any one experimental series, 131 the dosage of 1 injected into each f i s h was the same, usually 5 microcuries per f i s h . Itfhen s e r i a l sampling of blood was carried out, larger doses (20-30 microcuries per fish) were administered to ensure adequate radioactivity of the 131 small samples for counting. The volume of injected f l u i d containing the 1 was always 0.05 ml. per f i s h . Injections were made intraperitoneally with a 0.25 ml. tuberculin syringe and 27 gauge needle. To prevent leakage of the injected from the blind side by passing the needle at an acute angle through the ventral musculature and into the posterior portion of the coelom. The muscle thus acted as a seal against f l u i d leakage after withdrawal of the needle. 87 Five aliquots of each dose were reserved as standards and given appro-priate dilutions with s l i g h t l y alkaline water for counting with the samples. 2. Collection, Treatment and Counting of Samples. a. Thyroid As with most teleosts, the thyroid of the flounder i s a diffuse gland with thyroid f o l l i c l e s scattered widely about the ventral aortae. By section-131 ing this area of the lower jaw of flounder previously injected with 1 and counting the radioactivity of individual small portions, i t was determined that the thyroid always lay anterior to the t h i r d branchial arch with some extension of f o l l i c l e s a short distance l a t e r a l l y along the g i l l bars. Hence, i n c o l l e c t -i n t thyroid samples as much non-thyroidal tissue as possible was trimmed from the lower jaw area without infringing on the region where thyroidal tissue was known to l i e . During the course of the study three separate counting instruments were used. I n i t i a l l y a Geiger-Mueller counter was employed. Later, a s c i n t i l l a -t i o n well counter and f i n a l l y an end-probe s c i n t i l l a t i o n counter became available. Consequently, treatment of the thyroid sample i n preparation for counting depended on the counting instrument used. For beta-ray counting with the end-window Geiger-Mueller counter, the thyroid samples were placed i n 15 ml. test tubes, calibrated at 10 ml. and wet-ashed with 5 ml. of 2N NaOH. Following ashing and cooling, the solution was diluted with water to the 10 ml. mark, stir r e d and allowed to sett l e . A one ml. aliquot of the ash solution was transferred to a stainless-steel planchet 25 mm. i n diameter and with a 7 mm, edge. The samples were allowed to evaporate to near-dryness at room temperature, then dried thorough-l y under an infrared lamp. Standard samples were prepared by pipetting into planchets 1 ml. aliquots representing 1/100 of the dose. Using a predetermined count scaling unit with the thin mica-end-window Geiger counter, two measure-ments were made of each sample and standard to 3000 counts. Glassware was 88 cleaned thoroughly with detergent and potassium iodide carrier between uses and checks showed that there was no transfer of radioactivity between sample solutions* Since the Geiger-rMueller counter detects mostly beta radiation, " s e l f -absorption" of these particles by the residue i n the ashed sample i s an important factor which must be accounted for* The effect i s particularly important when sample mass varies as i s the case when evaluating thyroid a c t i v i t y of f i s h of different sizes. To quantitate the self-absorption effect a set of standards was prepared using different masses of thyroid samples but with constant amount of radioactivity i n each sample. These were ashed i n sodium hydroxide, aliquots pipetted to steel planchets, dried and counted with the Geiger-Mueller counter and referred to a standard with no tissue or a l k a l i , representing zero s e l f -absorption. The results (Fig. 17) show that the observed counting rate decreases with increasing thyroid mass i n a non-linear (but not exponential) manner. Forty to 50 percent of the radiation may be absorbed by the tissue mass of thyroids of large flounder (100-200 grams). Thyroids of small f i s h show much less self-absorption, but the effect never approaches zero because of the deposit of sodium hydroxide i n the planchet which i t s e l f absorbs about 10$ of the emitted radiation. 131 In comparing thyroid 1 uptake of f i s h of the same size, the s e l f -absorption effect can for most purposes be ignored. Serious errors w i l l , how-ever, be introduced i f the effect i s ignored when studying thyroid a c t i v i t y of 131 f i s h of different sizes. Since the counting rate of 1 i s essentially i n -dependent of sample mass when counting gamma rays with a s c i n t i l l a t i o n counter, this instrument i s much to be preferred over the Geiger counter when working with variable sample masses. No correction for self-absorption i s necessary with gamma ray counting. Most of the studies associated with body size effect on thyroid a c t i v i t y were carried out using the well-crystal s c i n t i l l a t i o n counter as the 89 Figure 17. The effect of self-absorption on the observed counting rate of 131 I i n thyroid samples as measured with an end-window Geiger-Mueller counter. 5000 zero self-absorption 4000 30001-2000 © 0 0 .6 .8 1.0 12 1.4 1.6 IB ZO 2.2 2.4 2J6 2.8 3.0 THYROID WEIGHT 100 £ H90 > H80 < -J70 g" 60 H 50 z U J 40 g U J a. 90 counting instrument.''' Thyroid samples were collected, trimmed and ashed i n sodium hydroxide as before. One ml. aliquots of the samples were pipetted into 5 ml. plastic v i a l s ("Clearsite," 1? dram size) and diluted to 4 ml. with water. Counting was then accomplished with the s c i n t i l l a t i o n well counter (l£M x 2" Nal (Tl) crystal) and a predetermined count scaler. During the studies on s a l i n i t y effect on thyroid a c t i v i t y , counts were made with an end-probe s c i n t i l l a t i o n counter, using a I f " diameter x I 2 " thick Nal (Tl) crystal. Total sample digestion was unnecessary with this counter, provided the samples and standards were similar i n mass and geometry. T^byroid samples were placed directly into steel plane he ts (25 mm. x 7 mm.) with about 1 ml. of hot 2N NaOH and allowed to set overnight i n a warm (70°C.) oven. This simple treatment effectively digested the tissue to spread the radio-a c t i v i t y evenly over the planchet. Standards representing l/lOO dose were prepared and allowed to spread evenly over the planchet bottom. Counting was carried out with the crystal 6 cm. distant from the samples. b. Blood Blood was collected at the time each flounder was k i l l e d by puncturing the dorsal aortae above the coelom and withdrawing the blood with a clean pipette as i t welled up from the wound. The blood sample, amounting to about 1$ of the body weight, was placed i n a steel planchet with Parafilm cover, tared to 4 decimal places, and the cover firmly folded over the planchet top to pre-vent evaporation from the blood. The sample was then weighed, the Parafilm cover discarded and 1 ml. 2N NaOH added to the blood i n the planchet to dissolve 1, The s c i n t i l l a t i o n well counter was made available by the B r i t i s h Columbia Medical Research Institute through the kindness of Dr. Peter Solvonuk. 2. The end-probe s c i n t i l l a t i o n counter was made available through the generosity of Dr. Harold Copp and Dr. Carl P. Crammer of the University of B r i t i s h Columbia Department of Physiology. 91 the clot and spread, the radioactivity evenly over the planehet bottom. After drying, samples and standards representing l/lOO dose were counted with the end-probe s c i n t i l l a t i o n counter with the crystal 2 to 6 cm. distant depending on the a c t i v i t y of the sample. Serial sampling of blood was carried out on three flounder (103, 154 and 213 grams) by direct needle puncture of the caudal artery with a ice. syringe and 27 gauge needle. With some practice, the artery could be readily located by introducing the needle into the caudal hypaxial musculature just ventral to the l a t e r a l line and passing the needle inward and forward u n t i l the artery was entered. A very small sample of blood (0.03 to 0.1 ml.) was withdrawn, trans-ferred to a tared Parafilm-covered planchet, weighed and digested with NaOH as before. Dryed samples and standard were counted with the end-probe s c i n t i l l a -t i o n counter. c. Urine Urine samples were collected at irregular intervals. After blotting the body of the flounder dry, urine was expressed into a long, thin pipette placed against the urinary papillae by gently pressing the body wall over the urinary bladder. The amount collected varied considerably - between 0.1 and ifo of the body weight. The sample was placed into a tared, Parafilm-covered planchet, sealed, weighed and counted as described for blood samples. d. Body To measure the rate of disappearance of radioactivity from the bodies of l i v i n g flounder, f i s h were counted individually with the end-probe s c i n t i l l a -t i o n counter. At intervals after injection, flounder were removed from the experimental aquarium, placed on a Lucite tray and the a c t i v i t y counted with the s c i n t i l l a t i o n crystal 18 cm. from the f i s h . The flounder never struggled during this period i f covered with a wet paper towel. After counting, the 92 flounder was immediately returned to the aquarium. 131 Disappearance rates of 1 from the bodies of flounder were usually compiled from single body counts of individual flounder k i l l e d at intervals after injection. After collecting blood and urine samples and removing the thyroid gland, the body of each flounder was placed i n a plastic p e t r i dish (90 mm. i n diameter with 13 mm. sides) and covered. A flounder too large to f i t d i r e c t l y was cut into pieces and f i t t e d into the dish,' or, i n the case of a very large flounder, divided among 2 or 3 dishes. Standards containing the entire dose were prepared either by injecting f i s h of representative sizes with the standard dose and immediately k i l l i n g and placing them i n p e t r i dishes or by cutting layers of f i l t e r paper to represent roughly the shape of the f i s h , 131 and adding the standard 1 dose to the paper i n the p e t r i dish. Bodies and standards of similar mass and geometry were then counted i n the end-probe s c i n t i l l a t i o n counter with the crystal 18 cm. from the samples. 3. Expression of Results The simplest expression of the amount of radioactivity i n the thyroid gland i s counts per minute, corrected for background count. However, this expression of concentration i s adequate as a comparative measure only during one specific dosage and counting arrangement. The usual method for expressing 131 the uptake of 1 by the thyroid gland, and the method here adopted, i s 131 percentage of dose of 1 accumulated by the gland per unit of time after injection. This i s the most convenient and certainly the most easily inter-preted way to present the results. In these experiments, a standard dose was administered to a l l flounder regardless of body weight. Hence, the mean a c t i v i t y of the body f l u i d s of a 10 gram f i s h i s 10 times that of a 100 gram f i s h . How-ever, i f the true a c t i v i t y of the thyroids of the 10 and the 100 gram f i s h are the same they w i l l both take up the same percentage of the dose, since the measurement i s of the entire gland rather than a similar unit of mass from both 93 animals. The actual weight of the trimmed thyroid tissue as a percentage of the body weight varied considerably, but there appeared to be no unconscious tendency toward.more thorough trimming i n small or i n large f i s h . The average weight of the trimmed tissue was about 1.3$ of the tot a l body weight and varied between 0.935$ and 1.535$ of the body weight. This variation i n the amount of non-thyroided tissue could contribute a significant error to the results, 131 because the thyroid tissue i s heavily vascularized and 1 quickly diffuses throughout the extracellular space. However, the error i s probably significant only during the f i r s t one or two hours following injection when the a c t i v i t y of 131 blood and i n t e r s t i t i a l tissue i s high and the actual uptake of 1 by -(he 131 f o l l i c l e s i s s t i l l low. Later the effect becomes insignificant as 1 i s rapidly removed from the extracellular space (renal and extrarenal excretion and thyroidal uptake) while the level trapped by the f o l l i c l e s steadily increases. The amount of radioactivity i n the body i s also conveniently expressed 131 as percentage of dose. The total excretion of 1 between the times of injection and sacrifice of the flounder i s then the difference between 100$ and the percentage of dose remaining i n the body. The expression of blood and urine a c t i v i t y presents a different sort of problem since we are no longer dealing with entire organs or glands. A ml. blood sample from a 10 gr. flounder would contain 10 times the percentage of dose as a blood sample from a 100 gr. flounder when both f i s h are given the same dose. Thus the body weight must be taken into account. This i s done by mul-tip l y i n g the percentage of dose per gram of blood or urine by the body weight i n grams. The expression has been called the "biological concentration coefficient" (Comar, 1955). In t h i s study, the concentration coefficient i s divided by 100 to give a value more easily compared to thyroid uptake values. The calculation of "excretory" clearance (representing a l l routes of 94 131 1 excretion) and thyroidal clearance are simple modifications of the standard renal clearance formula, usually expressed as: concentration of A i n the urine x volume of urine concentration of A i n the blood or: rate of excretion of A concentration of A i n the blood 131 Substance A i n this case i s 1 and the formula becomes: 131 ,131 . rate of 1 excretion during t minutes L Clearance, excretory = r-;—, • .—r-. zraTTT~rr~-— * ' * mean blood concentration of ]_131 during t minutes and 131 ^131 Q i e a r a n c e -a _ rate of thyroid 1 uptake during t minutes , ^nyr - m e a n D i 0 0 ^ concentration of ^L31 during t minutes 131 1 i s disappearing exponentially from the blood and i t s mean con-131 centration may be calculated graphically by plotting 1 concentration on a log scale against time on a linear scale, or by the formula: B 1 " B 2 B = In B 1 - In B 2 131 where B i s the concentration of 1 i n percentage of dose (concentration 131 coefficient) i n the blood. Thyroidal and renal clearance of 1 i s discussed by Myant and Pochin (1949); Myant, Pochin and Goldie (1949); Berkson, e t . a l . (1950); Keating, e t . a l . (1950), and Berson e t . a l . (1952). 95 B. RESULTS 1. Factors Influencing the Excretion of Radioiodide a. Effect of radioisotope reentry Fish injected with a labeled substance excrete the isotope d i r e c t l y into the environment i n which they l i v e . Unless the water i s continually renewed, the concentration of the isotope w i l l increase i n the ambient media and reenter the body of the f i s h . I t was not possible to keep flounder i n constantly renewed water, since experimental treatment necessitated con-t r o l l i n g the s a l i n i t y , temperature and elemental iodine content of the water. ,131 I t was necessary therefore to assess the effect of i reentry on the rate of excretion of the isotope to determine whether normal behavior of the isotope i n the body was upset by the effect. Two groups of 3 flounder each were maintained i n separate aquaria, each containing 7.5 l i t e r s of water, and under the same conditions of s a l i n i t y (19°/°°) a"0** temperature (18.0 * .3°C). In the f i r s t group, the water was changed at very frequent intervals after injection of the f i s h to prevent any 131 accumulation of excreted I i n the water. Water was never changed i n the 131 second group of flounder to allow accumulation and reentry of the I . After injection, the disappearance of radioiodine from the bodies was followed by measuring the radioactivity of the entire l i v i n g animal at intervals with an end-probe s c i n t i l l a t i o n counter. The results are shown i n Figure 18 with composite curves of the two groups shown i n the inset. In this experiment, 131 reentry of I did not become apparent i n the excretion curves u n t i l 20 hours after injection. 131 131 The rapidity of which I reentry becomes apparent i n I behavior 131 i n the body i s a direct function of the rapidity of which excreted I accumulates i n the surrounding water. This i n turn i s dependent on the volume of water i n the experimental tank. In this experiment the three flounder i n 96 Figure 18. Effect of reentry into the body of excreted isotope on the I excretion curves. Individual curves show the tota l excretion of a single dose 131 of I from the bodies of 6 flounder (22.9 to 48.6 grams body weight). The three flounder shown by the upper curves were maintained i n continually renewed sea water. The other three flounder shown by the lower curves were kept i n un-renewed water. The effect of reentry f i r s t becomes apparent at about 20 hours after injection, as shown by inset composite curves. Experimental temperature 18.0 ± .3°C, s a l i n i t y 19.0%o. June 7, 1958. 97 the reentry group were contained i n 7.5 l i t e r s of sea water, or 2.5 l i t e r s per f i s h . In none of the experiments to be described were less than 2 l i t e r s of water per f i s h provided; usually the experimental tank contained 3 to 5 131 l i t e r s or more per f i s h . Reentry of excreted I was therefore without effect 131 131 on the i n i t i a l portions of the thyroid I uptake and blood I disappearance curves although measurements made after 36 to 48 hours probably contain some 131 error i n the form of heightened a c t i v i t y due to I reentry. Since only the events of the f i r s t few hours following injection are important for thyroid 131 clearance calculations, return of excreted I into the body cannot be con-sidered an important source of error. b. Effect of s a l i n i t y For these experiments, carried out during February, 1958, small flounder weighing 2.5 to 20 grams were selected. They were maintained within 1°C. of the environmental temperature at the time of their capture i n February (en-vironmental temperature 6.8°C, experimental temperature 7.1 — 7«5°C.). The fresh water group were given a 5 day preadaptation to fresh water with added elemental iodine equivalent to the amount present i n 30°/°° s e a water - 43 ug. iodine per l i t e r . 131 A comparison of the cumulative t o t a l excretion of I of fresh and sa l t water flounder i s shown i n Figure 19. I n i t i a l l y , fresh water flounder 131 lose I at a much faster rate, but after the f i r s t few hours t o t a l excretion i s more rapid i n the salt water group. Since radioiodine i s being trapped and held by the thyroid gland for long periods, this portion of the dose i s essentially removed from the free inorganic iodide phase available for renal and extra-renal excretion. Thus the excretion curve i s expected to reach an asymptote sometime before the whole dose i s excreted. The difference between 131 the asymptote and 100$ excretion represents I trapped by the thyroid gland. 131 Work on man (Keating, Power, Berkson and Haines, 1947) shows that I excretion 98 131 Figure 19. Effect of s a l i n i t y on the cumulative tota l excretion of I by Platichthys stellatus. Each point represents the proportion of a standard dose of 1^31 excreted by an individual f i s h . Experimental temperature 7.3 i .2°C. February, 1958. HOURS AFTER INJECTION 99 rates give reliable estimates of thyroid a c t i v i t y . The present experiment on f i s h was not extended long enough to determine where the curves plateau, though a real plateau may never be attained because of the slow turnover from the thy-131 roid of organically bound I which w i l l slowly be broken down. The inorganic 131 I i s returned to the iodide pool and f i n a l l y excreted. A comparison of the change i n rate of radioiodide removal i n fresh and salt water i s shown i n Figure 20. Rates were derived graphically from the eye-fitted cumulative excretion curves i n Figure 19 and plotted with excretion rates as percentage of dose per hour on a log scale against time on a linear scale. During the f i r s t 2 hours after injection of salt water flounder, there i s an i n i t i a l very rapid f a l l i n excretion rate. The change i n rate then assumes a linear f a l l for several hours, which strongly suggests a simple exponential 131 removal of I from a stable iodide compartment. Twenty hours after injection the rate of change (10.49$ per hour)* decreases rather rapidly to another rate of 1.73$ per hour, then decreases even further to an almost steady excretory rate at about 0.27$ of dose per hour. 131 The excretion of I from fresh water flounder i s d i s t i n c t l y different from that of salt water flounder (Fig. 19). The primary difference i s that the fractional rate of change of excretory rate (Fig. 20) never assumes an exponen-t i a l f a l l , but rather i s continuously changing i n a fashion that cannot be treated by the usual mathematical formulae for first-order reactions. The ex-cretory rate at f i r s t i s considerably above that of salt water flounder, but 131 1. The constant k i s the f ctional ra e of change of I with time and i s calculated by the equation 1^ = I* e~k or ^ = l/t(ln(l£ with Ig)) where I j amount of I present at the time t and IQ amount of present at zero time. The half-value time, t representing the time for removal of half the I*-'* present is calculated by: + i 2.3 log i 0.693 T>2 —' _______ = _____ k k 100 Figure 20. Rate of removal of I from the bodies of Platichthys stellatus. Rates were derived graphically from the fresh water and sa l t water excretion curves shown i n Figure 27 by the relation: i f — I*e"^ where I* = amount of X3X 13X I present at time t and I 0 = amount of I present at zero time. 101 f a l l s very rapidly, so that between the f i r s t and twenty-fifth hour, the excre— 131 tory rate of I i s less i n fresh than i n salt water. At 35 hours after i n -jection, however, the rate appears to level at 0.5$ of dose per hour, at which point the experiment was terminated. 131 Since the fractional rate of change of I excretion rate with time does not proceed as a logarithmic function, i t can only be assumed that the volume of distribution of the tracer dose (radioiodide space) never stabilized during the experiment. The excretory organs, primarily the kidney and g i l l s , 131 127 presumably do not distinguish between I and I • Assuming equilibrium of the tracer dose i n the body f l u i d s , the proportion of the dose excreted i s also the proportion of a l l the iodide i n the body which i s excreted. However, i f 131 the I dose does not rapidly come into equilibrium with the body f l u i d s , but continues to penetrate slowly, there w i l l result a continual enlargement of the 131 volume of distribution of the I . This appears to be the case i n the fresh water flounder and to some extent i n salt water flounder. While the true reason for these observations must await further research, a probable explanation i s that the adaptation time of the flounder to fresh water was inadequate to ensure a r r i v a l at a stable level of osmotic regulation. I t was shown i n a previous section (Fig. la) that the osmotic concentration of the blood of starry flounder transferred abruptly to fresh water dropped appreciably but reached an essentially stable level within 3 days following transfer. These experiments were carried out at 15°C., whereas the present studies on thyroid a c t i v i t y were carried out i n February at a much lower temperature (7.1 - 7.5°F.), which very probably slowed the a r r i v a l of the body fl u i d s at osmotic equilibrium i n the new environ-ment. This hypothesis i s given considerable support by an examination of the disappearance rate of radioiodine from the blood i n the same group of fresh water 131 flounder. As shown i n Figure 23 the concentration of I i n the blood decreases hardly at a l l between the third and ninth hour following injection, even though 102 10.5$ of the injected dose was excreted during this period (Pig. 19). The most 131 plausible explanation i s that the volume of distribution of the injected I has shrunk! This i s exactly what would be expected i f the measurements were made during the period that the body f l u i d s were shifting toward a new level of stable osmoregulation. These events were shown i n Figure 4. I t was pointed out that a decrease i n the extracellular space (essentially the same as the iodide space) was frequently observed i n flounder after transfer to fresh water from sea water, an effect apparently caused by a s h i f t of water from without to within the c e l l s . 131 Whatever the exact cause may be, a decrease i n the volume of I distribution seems a probable explanation for both the very slow disappearance rate of radio-iodide from the blood and the gradual decrease i n the fractional rate of change 131 of I excretion rates. 131 c. Relative importance of renal and extrarenal excretion of I 131 Excretory clearances of I calculated from the same data for the f i r s t 5 hours after injection are given i n Table XII. These clearance estimates represent the proportion of the radioiodide space which i s cleared per hour by the organs of excretion. Although usually presented as the volume of blood or plasma cleared, i t i s understood that the total volume of inorganic iodide i s being cleared by the kidney or g i l l s or other pathway of removal. As expected from the slower excretory rates (Fig. 20) i n the fresh water flounder, excretory clearances also are less i n the l a t t e r than i n salt water flounder. Averages of the clearance estimates, omitting the f i r s t hour 131 when the concentration of I i n the blood i s changing rapidly during absorption and distribution of the dose, give 0.242 grams of blood i n the salt water flounder and 0.141 grams of blood i n the fresh water flounder. Thus, i n spite of the greater urine flow of the fresh water f i s h , t o t a l removal i s less rapid 1 0 3 Table XII Excretory clearance of radioiodine from the blood of flounder i n salt and fresh water. Clearances adjusted for body weight and expressed as grams of blood cleared of radioiodine per hour i n a 10 gram flounder. Salt Water Starry Flounder Hour io dose excreted Blood concentration Clearance per hour fo dose per gram x Body weight/100 1 5.4 2.25 0.24 2 4.3 1.8 0.238 3 3.7 1.43 0.258 4 3.3 1.36 0.242 5 3.0 1.29 0.232 Fresh Water Starry Flounder Hour fo dose excreted „, , .. er hour Blood concentration Clearance • p e r ° % dose per gram x Body weight/100 1 4.8 2.45 0.196 2 3.3 2.45 0.134 3 2.55 1.51 0.169 4 2.12 1.51 0.141 5 1.8 1.48 0.121 Direct data are not available to indicate the relative importance of the several possible pathways of exchange of iodide with the external environ-ment i n either fresh or salt water. However, i t i s possible to make a rough 131 estimate of the volume of urine needed to remove a l l of the I being excreted assuming that there i s no extrarenal excretion of any kind. By comparing these hypothetical urine flows with average urine flow values for fresh and salt water 104 species reported i n the literature, one arrives at a rough indication of the 131 proportion of I removed by renal and extrarenal pathways. Urine flows were calculated by dividing the proportion of the dose excreted per hour by the 131 urinary concentration of I expressed as the biological concentration coefficient (fo dose per gram of urine x body weight / 100). The calculated values are summarized i n Table XIII. These values are far above actual urine flow measure-ments reported i n the literature (summarized i n Table I I I of Black's review paper, 1957). Values reported for salt water species range from about 3 to 30 ml; of urine per kg. body weight per day as compared to the average of 118.7 ml./kg./day calculated for salt water flounder assuming a l l the radioiodide was excreted renally. Similarly, reported urine flows for freshwater species range between 7 and 106 ml./kg./day as compared to the 623 ml./kg./day average urine flow calculated for the fresh water flounder. I t i s evident that a very large pro-131 portion of the excreted I i s being removed extrarenally i n both salt and fresh water flounder. The possible pathways of extrarenal, iodide removal are the g i l l s , the mucous membranes of the mouth and with the fecal wastes. In man only minute quantities of iodide are. lost extrarenally, that i s , i n the feces (Nelson, e t . a l . , 1947? Keating and Albert, 1948) the expired a i r and the perspiration (Riggs, 1952). In fishes i t i s also doubtful that appreciable amounts of iodide leave i n the feces, particularly i n fasted animals such as were used i n these experi-ments. Hence, the g i l l s and mucous membranes must constitute the major organs of extrarenal iodide exchange. 131 Measurements of the concentration of I i n the g i l l lamellae of flounder i n both fresh and salt water were undertaken i n the hope of providing an indication of the relative importance of the g i l l s i n iodide exchange. The results are given i n Table XIV. With the exception of the f i r s t 6 hours i n the case of fresh water flounder, the radioiodide concentration i s appreciably higher i n the g i l l lamellae than i n the blood, indicating a c e l l u l a r concentra-105 Table XIII Calculated urine flows of individual fresh and salt water flounder, 131 assuming no extrarenal excretion of I . Urinary concentration expressed as fo of dose per gram of urine x body weight/100. Salt Water Flounder Time Urinary % dose excreted Calculated urine flow Concentration per hour ml./lOO gm./hr. ml./kg./day 3.52 3.73 3.2 0.9005 225 4:55 6.05 3.25 0.496 119 6:03 4.99 2.43 0.537 129 7:00 3.42 2.1 0.701 168 7:05 4.91 2.15 0.489 117 7:20 5.92 2.0 0.392 94 10:00 2.8 0.8 0.282 67.7 24:25 4.42 0.35 0.124 29.7 Average 118.7 Fresh Water Flounder Time Urinary Concentrated % dose excreted per hour Calculated urine flow ml./lOO gm./hr. ml./kg./dt 12:10 0.504 1.35 .90 427 13:00 0.388 1.88 .85 525 18:11 0.232 • 0.95 .66 681 23:00 0.35 0.73 .57 391 35:35 0.11 0.50 .50 1090 Average 623 106 t i o n of the isotope. The interpretation, however, i s d i f f i c u l t , for i t i s impossible to say whether the iodide i s being concentrated i n the g i l l s prior to secretion to the exterior media or whether i t represents simply a c e l l u l a r accumulation with no net movement i n either direction. Marine flounder may secrete the univalent iodide ions together with univalent sodium and chloride ions v i a the "chloride secreting c e l l s " to the exterior. If so, a concentration of iodide i n the g i l l s i s not surprising. Fresh water flounder are not expected normally to secrete iodide, since i t s concentration i s far lower i n fresh water than i n the blood. There may possibly exist an active iodide absorption mechanism i n the g i l l s of fresh water flounder (hence the high concentrations of radioiodide i n the g i l l lamellae), but Krogh (1938) found that no such mechanism existed i n goldfish and that iodide was lost slowly from the body by diffusion. I t should be possible to quantitate experimentally the relative proportions of iodide transferred by each of the pathways of exchange with external environment. Table XIV 131 The concentration of I i n the g i l l lamellae of starry flounder ex-131 pressed as percentage of the concentration of I i n the blood. Body weight 24 to 105 grams. Hours after injection 1 3 6 12 24 48 100 Fresh water flounder 83.6 40.4, 88.3 61.7, 69.2 147 210, 309 264, 474 208, 212 Average 83.6 64.3 65.4 147 259.5 369 210 Salt water flounder (25°/oo sea water) Average 109, 267 188 83.4, 108 95.7 123, 214 168.5 79, 157 118 133, 175 154 128, 175.5, 202 168.5 1 0 7 d. Effect of size When a number of starry flounder of a l l sizes are simultaneously injected with tracer iodide, k i l l e d together several hours later and measure-ments made of the proportion of the dose excreted, i t i s seen that small flounder lose the isotope more rapidly than large flounder. An example i s shown i n Figure 21a, i n which the percentage of dose excreted i s plotted against body weight for three sampling periods after injection. The points best conformed to a linear relationship with body weight placed on a log scale. The results of each of three s a l i n i t y treatments -46°/oo, 25°/oo and fresh water — were plotted on semilogarithmic paper and f i t t e d with a straight line by the method of least squares. From each of these lines of best f i t , three points were graphically derived representing the t o t a l excretion of flounder weighing 5, 15 and 50 grams and plotted against time after 131 injection (Fig. 21b). In a l l three s a l i n i t i e s the tracer dose of I i s more rapidly excreted by small f i s h . Since the body does not distinguish between stable and radioactive iodide and since the intake, of iodide i s assumed to balance i t s loss from the body, i t i s evident that the net turnover of iodide i s greater i n small than i n large flounder. The relationship between body size and rapidity of iodide exchange i s yet another indication of the more intensive metabolic rate of small flounder. 2. Factors Influencing Uptake of Radioiodide By the Throid Gland a. Effect of iodide content of the water The effect of the quantity of elemental iodide i n the ambient water on the uptake of radioiodine by the thyroid i s shown i n Figure 22. I t i s apparent 131 from Figure 22a. that the rate of accumulation of I by the thyroid of flounder adapted to fresh water i s much greater i n natural fresh water than i n fresh water reinforced with 50 ug iodine per l i t e r (40 ug iodine as KIO3 and 10 ug iodine as KI), 108 Figure 21a # Effect of body size on the excretion of I from Platichthys stellatus i n sea water of 25 /oo* Each point represents the proportion of 131 a standard dose of I excreted by an individual f i s h at the indicated time after injection* Points f i t t e d by the method of least squares* Experimental temperature 15*0 i*2°G. Figure 21b. Effect of body size on the excretion of I from Platichthys stellatus i n fresh water, normal sea water and concentrated sea water* The three points for each curve representing 5, 15 and 50 gram flounder were graphically derived from lines of best f i t through individual t o t a l excretion values as shown i n Figure 21a. Experimental temperature 15*0 £*2°C, 100 75 h 50 25 0 75 50 25 ——I 1 1—i i i I I I 1 1 1—l I I I l I 12 Hours Q UJ t-UJ ce o x UJ ' I I I i i i i i j i • i I 1 1 1 1 26 Hours u w O o O 0 ' i i i i i i i i j i I I I I 1 I I 51 Hours 75 50 25 J I I i I I I I I i • i i i i I 4 10 40 BODY WEIGHT IN GRAMS 100 Fresh water Sea water, 25 °/oo Sea water, 46 9. eo[ 0 20 40 0 20 HOURS AFTER INJECTION 60 109 Figure 22. Effect of elemental iodine content of the water on thyroid 131 I uptake of Platichthys stella t u s . 22a. Individual thyroid uptakes of flounder i n fresh water are shown by s o l i d c i r c l e s and the average by the solid l i n e * Uptakes of flounder i n iodine reinforced water are. shown by open c i r c l e s with a broken l i n e average. Uptake i s stimulated i n iodine deficient fresh water* 22b. Flounder i n iodine deficient sea water have greater uptakes (solid c i r c l e s , s o l i d l i n e average) than do flounder i n natural sea water which contains about 50 ugm. io d i n e / l i t e r (open c i r c l e s , broken l i n e average). 110 the normal iodine content of undiluted sea water (Strickland, J.D.H., 1956, pers. comm.). To assess the effect of low iodine i n salt water, a r t i f i c i a l sea water was prepared following the formula of Brujewicz (cited i n Table 37, Sverdrup, Johnson and Fleming) using analytical grade reagents. The solution contains the major elements found i n sea water (chloride, sodium, magnesium, calcium, potassium, bromide, sulphate and bicarbonate) without iodine. However, iodine undoubtedly i s present i n small amounts as a contaminant i n the reagents used, so that the prepared solution cannot be considered iodine free. As shown i n Figure 22b, thyroidal uptake of radioiodine by starry flounder i s greater i n iodine deficient a r t i f i c i a l sea water than i n natural sea water of the same s a l i n i t y (30°/oo). The increased rate of thyroidal accumulation of radioiodine by flounder i n fresh water and a r t i f i c i a l sea water as compared to flounder i n iodine r e i n -forced fresh water and natural sea water are both interpreted as the familiar compensatory response of the iodide trapping mechanism to iodine deficiency. As already discussed i n the introductory remarks to this section, thyroid hyper-plasia as a result of iodine deficiency i n the water has been demonstrated i n f i s h by several authors. I t i s evident that the quantity of iodine available to a f i s h i s an extremely important factor influencing the v a l i d i t y of the radio-iodine uptake method as a measure of thyroid a c t i v i t y . Low levels of iodine i n the environment produce a hyperactivity of the "iodide trap" resulting i n a high rate of radioiodide accumulation that does not represent an overall increase i n release of hormone into the blood stream. This effect i s of course particularly important i n comparing thyroid a c t i v i t y of flounder i n fresh water containing less than one microgram per l i t e r and i n sea water containing about 50 micrograms per l i t e r (at s a l i n i t y 35°/oo)» ^or t h i s reason, fresh water was always r e i n -forced with iodide to bring i t s iodide concentration equivalent to sea water. I l l b. Effect of s a l i n i t y 131 In the foregoing section i t was shown that the thyroid uptake of I by flounder adapted to fresh water i s greater than that of marine flounder, but that essentially a l l of "the difference was due to iodine deficiency i n fresh water. With elemental iodide added to the fresh water, both fresh water and sea water flounder were found to have about the same thyroidal uptakes. Prom the i n i t i a l efforts to separate differences i n thyroid a c t i v i t y of starry flounder i n different s a l i n i t i e s , i t became apparent that thyroidal 131 uptake of I was inadequate as a c r i t e r i o n for a c t i v i t y evaluation for two reasons; a) the method i s insensitive and cumbersome because of the rather large 131 v a r i a b i l i t y between individual f i s h i n the percentage uptake of I by the thyroid, necessitating large sample sizes for each test, and b) the v a l i d i t y of the method rests on the assumption that the disappearance rate from the blood 131 of a single injected dose of I i s uninfluenced by s a l i n i t y . I t has already been pointed out that there was good reason to doubt this l a s t assumption because of marked differences i n electrolyte behavior and the different excretion rates of radioiodide between fresh water and salt water f i s h . For these reasons, thyroidal clearance of radioiodide from the blood has been used instead of thy-ro i d uptakes to evaluate thyroid a c t i v i t y . 131 For the determination of thyroidal clearance of I from the blood, 131 simultaneous measurements of the rate of increase of concentration of I i n 131 the thyroid and the I concentration i n the blood are taken. This was done 131 by injecting standard tracer doses of I into the body cavities of two groups of flounder, one group maintained i n sea water and the other previously adapted to iodine-reinforced fresh water. Individual f i s h were sacrificed at intervals of time after injection and the thyroid and a blood sample removed from each for counting. The results are given i n Figure 23, where the s o l i d c i r c l e s represent the thyroidal I 1 " 3 1 uptakes and the open ci r c l e s the blood I 1 ^ * con-112 centration as the biological concentration coefficient. i . The behavior of radioiodide i n the blood Figures 23 and 24 show that several components are present i n the blood 131 I curves. In both fresh and s a l t water flounder, the concentration i n the blood increases rapidly after the intraperitoneal injection as the dose i s absorbed into the blood stream. The rate of absorption di f f e r s considerably i n the two groups and w i l l be discussed separately. Marine Flounder In small marine f i s h (2.5 - 20 grams), absorption from the body cavity i s rapid (Fig. 23). The blood appears to reach a peak concentration of nearly 5.5$ of dose''' at one-half hour after injection, followed by a rapid f a l l to about 2.2$ within a few minutes. This rapid r i s e and f a l l i s interpreted as a rapid 131 absorption of I into the blood stream v i a the vascular peritoneum followed by a less rapid diffusion into the extracellular space or, more accurately, the iodide.space. At one hour after injection, the disappearance rate becomes ex-ponential. Between the f i r s t and t h i r d hours the disappearance rate has a half-value time ( t j ) of 3.45 hours (D = 20$/hour) but decreases after 3 hours to a slower rate of removal ( t i =12.8 hours, K = 5.4$/hour). The reason for the change i n disappearance rate at 3 hours i s not known. In mammals, the removal of radioiodine from the iodide space remains exponential u n t i l the appearance of plasma—bound radioiodine i n the blood causes a gradual decrease i n the apparent removal rate (McConahey, Keating and Power, 1949; Riggs, 1952). Because of the r e l a t i v e l y low a c t i v i t y and iodine turnover rate of the teleost thyroid i t i s very unlikely that plasma-bound radioiodine appears i n appreciable amounts u n t i l 1* Blood I concentration i s calculated as the percentage of administered dose per gram of blood x body weight/100 (biological concentration coefficient), but for brevity i n this discussion blood Jr^ concentration w i l l be expressed simply as percentage of dose. 113 Figure 23. Effect of s a l i n i t y on thyroid I uptake and blood I disap-pearance rate of Platichthys stella t u s . Thyroid uptake ( ) i s expressed as percentage of dose ( 5 microcuries carrier-free Nal per f i s h , i n t r a -peritoneal injection) accumulated by the whole gland; blood I i s ex-pressed as percentage of dose per gram ofblood x body weight/lOO. I i s concentrated more rapidly i n the thyroids of sea water flounder i n 131 spite of the more rapid disappearance of I from the blood. S a l i n i t y of sea water 29 °/oo; fresh water reinforced with 40 micrograms/ l i t e r o of iodine. Experimental temperature 7.5 *.3 C. 10 8 6 1 1 1 I r -SEA WATER -i 1 1 r i r 1 1 1 1 1 1 r-I .8 .6 CO O | 0 H 8 z HI 6 o cc FRESH WATER r Thyroid A. J L ' Blood J I I L 6 J I I I I L. 8 9 / / 10 20 30 40 50 TIME AFTER INJECTION, HOURS 114 Figure 24. Behavior of radioiodine i n the blood of 3 large (102 to 213 gm Platichthys stellatus i n sea water. Blood s e r i a l l y sampled at intervals from the caudal artery. A composite curve of the three individual curves i s shown. Sal i n i t y 29 °/oo, experimental temperature 7.5 -•3°C« HOURS AFTER INJECTION 115 at least 24 hours after injection of a tracer dose. I t i s also very doubtful that 131 reentry of excreted I into the body from the ambient water can be cause for the 131 change i n rate of I removal (Fig. 18). I t seems probable that some change has 131 occurred i n the volume of distribution of the I i n the body f l u i d , the explanation of which must await further experimentation. Serial sampling of blood was carried out on three large marine flounder over a period of nearly 50 hours (Fig. 24). Absorption of the dose from the 131 coelom i s considerably less rapid than i n the small flounder and I appears not to enter the blood stream more rapidly than i t diffuses into the iodide space since i t s concentration does not reach the high levels seen i n small flounder. 131 The slower absorption of I into the blood stream appears to be another example of the lower metabolic rate of large flounder. I t w i l l be recalled that d i s -appearance rate of iodine from the body of flounder was more rapid i n small than i n large flounder, indicating a more rapid body turnover of elemental iodine i n smaller individuals. Disappearance of radioiodine from the blood i s slower i n large than 131 i n small flounder. After equal diffusion of the I with the body fl u i d s at about 6 hours after injection, i t assumes an exponential removal curve with a half-time of 11.95 hours (K = 5.8$/hour) as calculated from the composite curve i n Figure 24. About 13 hours after injection, the rate of removal slows to a half-time of 52.8 hours (K = 1.3$/hour). As expected, because of the slower 131 131 I tot a l excretion from the bodies of large flounder, the removal of I from blood of large flounder i s less rapid than from the blood of small flounder. 131 The upswing of the blood I concentration curve of small flounder at 40 to 50 hours after injection may indicate the appearance of plasma-bound 131 radioiodine i n the blood at this time. By this time I which has been trapped by the thyroid w i l l have been synthesized into hormonal iodine, released into the blood stream and bound with plasma proteins. In man, protein-bound 116 iodine appeaxs i n the blood within a few hours after injection (McConahey, Keating and Power, 1949), particularly i n hyperthyroidism. No measurements appear to have been made of plasma-bound iodine among the fishes, but i t s appearance i n the blood i s probably very slow because of the re l a t i v e l y slow uptake and release of radioiodine by the thyroid gland. Fresh Water Flounder The absorption of radioiodine from the body cavity of small fresh water flounder i s much slower than i n small marine flounder (Figure 23). 131 After diffusion throughout the iodide space the I disappears from the blood at an extremely slow rate (t£ = 63.6 hours, K = 1.09$/hour)., a slower rate than 131 would be expected from the t o t a l excretion of I (about 10.5$ of the dose between the th i r d and ninth hours after injection, (Fig. 19). The excretion 131 of the I dose i n fresh water flounder has already been discussed i n a pre— 131 vious section, where i t was postulated that the volume of I distribution i n these flounder decreased somewhat after injection indicating that the body fl u i d s were s t i l l shifting toward a new stable level of osmoregulation after transfer to fresh water. I t i s emphasized that such a change i n the volume of 131 the iodide space i s without effect on the thyroid clearances of I from the blood since this function depends on the a c t i v i t y of the "iodide trap" of the 131 thyroid f o l l i c l e s and the level of I i n the blood passing through the gland at any one time; i i . The thyroidal clearance of radioiodide from the blood 131 In Table XV, thyroidal clearance of I from the blood of fresh water and salt water flounder are calculated from the data shown i n Figure 23 131 131 for the period when the uptake of I by the thyroid and removal of I from the blood were both nearly exponential. I t i s apparent that the average thyroid 131 clearance of I i n fresh water flounder (about .00135) i s several times less 117 than the average clearance calculated for salt water flounder (about .0197). Since the level of elemental iodine i n the environment was the same for both groups, these results appear to be conclusive evidence that thyroid a c t i v i t y i s greater i n s a l t water flounder than i n fresh water flounder. The experiment from which these values were derived (Fig. 23) i s a replicate of an earlier experiment which also showed much greater clearance rates for s a l t water flounder. Table XV Thyroid clearance of radioiodide from the blood of Platichthys stellatus. Clearance rates expressed for a 10 gram flounder as grams of blood 131 cleared of I per hour. Clearance rates were calculated from the data shown i n r l 3 l Figure 23 during the period of exponential disappearance of I by the formulae: rate of uptake by the thyroid mean blood concentration of I J " > i Fresh Water Flounder from the blood Hours after i n j ection 3 - 4 4 - 5 5 - 6 Average blood j l - * l concentration 1.500 1.485 1.470 Thyroid uptake .02 .02 .02 Clearance .00133 .00135 .00136 Salt Water Flounder Hours after injection 2 - 3 3 - 4 4 - 5 rl31 Average blood concentration 1.6 1.38 1.32 Thyroid uptake .27 .28 .29 Clearance .0169 .0202 .022 118 c. Effect of body size on thyroid a c t i v i t y of  starry flounder and speckled sand dab Starry Flounder The effect of body size on thyroid a c t i v i t y was assessed by injecting 131 a large number of flounder of variable body size with a standard dose of I and sampling groups of f i s h at predetermined intervals of time after injection. Thyroid samples were removed, prepared for counting, and their a c t i v i t y measured with the well-crystal s c i n t i l l a t i o n counter, thereby avoiding self—absorption problems due to variable sample mass. In Figure 25 an example i s shown of the effect of body size on thyroid 131 I uptake of flounder i n sea water of 25°/oo. The diphasic eye—fitted l i n e placed through the individual thyroidal uptakes i s included only to indicate a trend between uptake and body size and i s not to be interpreted as having high significance i n i t s e l f . Uptakes are r e l a t i v e l y high i n small flounder of 2 to 6 grams, are much less i n 20-30 gram flounder and increase again with increasing body size above 30 grams. The same interaction between body size apH thyroidal uptakes was equally prominent i n fresh water adapted flounder and i n flounder adapted to concentrated sea water (45°/°°)» 131 Since I i s excreted more rapidly from the body i n small than i n large flounder (Figs. 21a and 21b), the isotope i s also disappearing more rapidly 131 from the blood stream. Hence, thyroidal trapping of I by small flounder i s 131 proceeding against a lower concentration of I i n the blood than i n large flounder. This means that the actual thyroid a c t i v i t y of a 2 gram flounder i s even greater than indicated by the measured uptakes as compared to the a c t i v i t y of a 20 gram flounder. For the same reason, some small part of the measured 131 increase i n thyroid I uptake of larger flounder (100-200 grams) may be due to the slower excretion of I 1 3 1 i n -these individuals. Speckled Sand Dab 131 The effect of body size on thyroid I uptake of sand dab i s shown i n 119 Figure 25. Effect of body size on thyroid a c t i v i t y of Platichthys stella t u s . 131 Indicated times represent hours after injection of a tracer dose of I . Lines f i t t e d by eye. Experimental temperature 15°C., s a l i n i t y 25 °/oo» 120 Figure 26 and 27. Measurements were carried out with sand dab under experimental conditions identical to those used with the flounder ( s a l i n i t y 25°/°°. temperature 15°Ce), Thyroid uptake increases proportional to body size, apparently over the entire size range studied (4 to 28 grams). I t was not possible to obtain sand dab smaller than about 3 grams and i t appears probably that the true picture of the relationship between size and thyroid a c t i v i t y i s incomplete i n this species for this reason. I t w i l l be noted from Figure 26 that there i s a suggestion of a diphasic curve at the 24 hour sampling period, indicating that thyroid a c t i v i t y may be greater i n individuals smaller than about 4 grams. As already mentioned, the speckled sand dab i s a small species rarely exceeding 50 grams i n weight and probably reaching maturity at 20 grams. I t i s therefore not j u s t i f i a b l e to compare di r e c t l y thyroid uptakes of a flounder and a sand dab of the same body weight because the physiological age of the two individuals i s entirely different. A 30 gram sand dab i s a mature adult, whereas a 30 gram flounder i s an immature juvenile. The increase i n thyroid a c t i v i t y with body size of flounder above 25 grams implicates the beginning of some reproductive function of the thyroid hormone associated with gonad maturation. Because of the early maturity and small body size of sand dab, gonad maturation probably begins i n very small individuals, possibly i n 3 or 4 gram f i s h , as suggested by ihe 24 hour sampling period (Fig. 26). If so, a 4 gram sand dab may be the physiological age of a 25 gram flounder. Although t h i s suggestion i s more i n the nature of speculation than deduction, i t does offer a possible explanation to what would otherwise be a puzzling contradiction between the effect of body size on thyroid a c t i v i t y of flounder and of sand.dab. 121 Figure 26. Effect of body size on ifcroid a c t i v i t y of Citharichthys s t i g -131 maeus. Times indicate hours after injection of a tracer dose of I . Lines f i t t e d by eye. Experimental temperature 15°C., s a l i n i t y 25 °/oo# WEIGHT IN GRAMS 122 Figure 27. Effect of body size on thyroid a c t i v i t y of Citharichthys s t i g - maeus and Platichthys stellatus. Curves representing f i s h of the indicated body weight were graphically derived from Figures 25 and 26. HOURS A F T E R INJECTION 123 VI DISCUSSION This investigation of the osmoregulatory metabolism of the starry flounder demonstrates two significant facts and suggests an interdependence between them. The f i r s t point i s that energy demands are greater i n more saline aquatic environments. The second i s that the thyroid gland i s more active i n flounder l i v i n g i n marine habitats. I f these factors are interrelated i t argues strongly for a calorigenic action of thyroid hormone i n at least one species of f i s h - a fact not yet generally recognized. These points w i l l be discussed i n turn. Some ecological implications w i l l f i n a l l y be considered. The theoretical energy requirements for osmoregulation i n fresh and salt water have already been discussed (Section TV C). I t was pointed out that i n fresh and salt water of equal though opposite osmotic gradient with respect to the flounders body f l u i d s , the hydrating and dehydrating effects are essen-t i a l l y equal. Fresh water f i s h excrete excess osmotic water i n a dilute and copious urine and regain lost salts i n food and by means of ion-absorbing c e l l s i n the g i l l s * Marine f i s h , however, employ a r e l a t i v e l y complex osmoregulatory mechanism. To replace water lost osmotically they must drink sea water. Water and univalent ions are absorbed, the salts transported to the g i l l s and actively secreted to the external environment. Divalent ions are removed with the intestinal residue and i n the urine with further loss of body water. This method of regaining water lost osmotically to the environment by drinking hypertonic sea water entails considerable "handling" of salts by the organs of exchange and, hence, much active ion transport. Reference was made to the recent research of Ussing (1958) who has been able to quantitate oxygen demands for active cation transport i n frog skin. Ussing showed that these energy demands were determined wholly by the amount of ions transported rather than the osmotic gradient present. 124 If this quantitative relationship exists for a l l biological membranes, i t follows that oxygen demands are greater i n sea water because of the r e l a t i v e l y circuitous osmoregulatory mechanisms employed by marine f i s h . Precise measurements of stand-ard metabolic rate i n different s a l i n i t i e s have demonstrated that flounder consume more oxygen i n sea water and thus strongly support the theory of greater energy demands i n this medium. Quantitative differences i n active ion transport would appear to be the origin of greater demands for thyroid hormone by marine flounder. A possible point of action of thyroid hormone would be a direct effect on the c e l l s doing osmotic work. However, the question of a calorigenic action of thyroid hormone on the cellu l a r metabolism of fishes i s by no means clear. The many attempts made to stimulate oxygen consumption of f i s h with thyroxine or thyroid treatment and the conflicting results obtained are discussed i n several reviews (e.g. Hoar, 1951, 1957j Pickford and Atz, 1957). Using both fresh water and marine fishes, most authors have reported negative results (Drexler and Issekutz, 1935; Root and Etkin, 1937; Etkin, Root and Kofshin, 1940; Hasler and Meyer, 1942; Smith and Everett, 1943; Punt and Jongbloed, 1945; Baraduc, 1954; Hoar, 1958). Chavin and Rossmoore (1956) also found thyroxine to have no effect on goldfish respiration, but obtained significant increases i n oxygen consumption with thyrotropin treatment. However, Pickford (Pickford and Atz, 1957) points out that the increase may have been due to contaminating gonadotropin rather than to a direct thyroid stimulation. In spite of these negative reports, a well controlled experiment carried out on goldfish by Muller (1953) showed highly significant increases i n oxygen consumption after single injections of thyroxine. Haarmann (1936) found that an optimum dose of thyroxine stimulated the res-piration of isolated muscle of carp. Studies of the effects of thyroidectomy and thyroid inhibition also have given conflicting results. Neither surgical removal nor radiological 125 destruction of the thyroids of parrot f i s h (Matty, 1957) and rainbow trout (Fromm and Reineke, 1956) affected their respiration. The use of thyroid chemical inhibitors has frequently yielded negative results (Matthews and Smith, 1947;• Chavin and Rossmoore, 1956) although some authors have reported a depression i n respiratory rate with the use of these drugs (Osborn, 1951; Zaks and Zamkova, 1952; Muller, 1953). Hoar (1957) has urged that these results be interpreted with caution because of the strong col l a t e r a l anti-oxidant effect of some antithyroid materials. I t i s questionable whether feeding thyroid or immersion i n thyroxine i s the most effective way to study the calorigenic action of thyroid hormone. These cla s s i c a l procedures are certainly effective i n producing a l l manner of other morphogenetic and metabolic effects i n fishes (Hoar, 1957). However i t i s possible that thyroxine i s not the active derivative participating i n oxidative metabolism. While the iodinated precursors to thyroxine appear to be the same i n a l l vertebrates (Gorbman, Lissitsky, Micheal and Roche, 1952; Berg and Gorbman, 1953; Gorbman, and Berg, 1955) there i s evidence that i n mammals thyroxine must be converted to triiodothyronine before i t acquires hormonal a c t i v i t y (Barker, 1955; Gross, 1955). Another c r i t i c i s m of the use of administered thyroid compounds or antithyroid drugs i s that selection of optimum dosage borders on pure guesswork. In the present study, a correlative decrease i n thyroid a c t i v i t y and i n metabolic rate has been demonstrated i n flounder exposed to a normal physiological situation - a decrease i n environmental s a l i n i t y . This appears to be the f i r s t positive demonstration of a calorigenic action of the thyroid hormone i n f i s h not using administered materials such as thyroid hormone or antithyroid compounds. At this point, reference could be made to the work of Olivereau and Francotte-Heriry (1956) who have suggested that low metabolic rate and slow growth of African blind cavefish (Caecobarbus geertsi) may be correlated with the inactive thyroid of this animal. 126 There i s yet another deficiency i n the experimental procedure of many reported experiments. Frequently inadequate attention has been paid to the im-portance of the method used i n determining oxygen consumption of f i s h . Fish respiration as measured by the usual method of placing the animal i n a closed container and recording the amount of dissolved oxygen consumed per unit of time i s notoriously variable. However, i t has not been always recognized that much of this v a r i a b i l i t y can be eliminated by measuring standard metabolism following the procedures developed by Fry and his students (Fry, 1957). Changes i n respiratory intensity effected by treatment such as feeding thyroxine or dessi-cated thyroid may be very small and could easily be overshadowed by individual v a r i a b i l i t y unless the precautions outlined by Fry are followed. In this investigation the changes i n standard metabolic rate resulting from s a l i n i t y alterations were not of a large magnitude. The differences could easily be lo s t i n individual v a r i a b i l i t y with a less sensitive method. A case i n point i s the recent work of Matty (1957) who was unable to demonstrate any change i n oxygen consumption of parrot f i s h (Pseudoscarus guacamaia) after surgical removal of the encapsulated thyroid gland. Matty used the constant-flow principle for his oxygen consumption measurements and was careful to delay sampling for 18 hours to remove the effect of heightened respiration due to handling. However, four deficiencies i n procedure may be recognized. F i r s t the sample size of 3 f i s h was far too small. Second, only large f i s h of 3.0 to 3.2 kg. were used. Third, no effort was made to measure a possible diurnal, respiration rhythm, hence, there i s no assurance that the measurements represent standard metabolic rate. F i n a l l y , the respirometers may have been too small (8.5 times the volume of f i s h instead of the minimal 1:10 ratio suggested by Geyer and Mann, 1939). The importance of body size i n metabolism experiments i s frequently overlooked. Reference to Figure l i b shows that the s a l i n i t y treatments were inadequate to effect an obvious difference i n metabolic rate among large flounder. 127 Only by inclusion of a large size range of flounder did differences effected by s a l i n i t y become s t a t i s t i c a l l y apparent. Thus, attention to body size i s important not only because metabolic rate i s weight dependent but also because changes produced by experimental treatment may show up only among small animals. For the reasons discussed above i t would appear that the many f r u i t -less attempts to show a calorigenic action of the f i s h thyroid hormone should by no means be regarded as conclusive evidence that such an action i s not present. For one 'tiling i t i s d i f f i c u l t to see how thyroid hormone can have such apparent effects on protein and carbohydrate metabolism of fishes without influencing i n any way oxidative metabolism. Moreover, the well controlled experiment of Muller,j (1953) has demonstrated that thyroid hormone can stimulate oxygen consumption of f i s h . In view of these facts, the writer would agree with Pickford (Pickford and Atz, 1957) that " i t becomes increasingly d i f f i c u l t to deny that thyroid hormone plays some role i n the respiration of fishes". I t i s usually stated that the primary action of thyroid hormone i s i t s effect on energy metabolism and that the many other effects are secondary results of this primary function. Comparative physiologists, however, tend to dissociate metabolic and morphogenetic effects of thyroid hormone and often argue that the developmental function i s quite independent of the calorigenic function (Fleischmann, 1947; Hoar, 1957; and earlier references cited therein). This question cannot be resolved at the present time but several observations made during the course of this study on metabolic rate and thyroid a c t i v i t y of flounder are apropos. As with essentially a l l animals, metabolic rate of small flounder was greater than large flounder. As a result of their more intense metabolism, smaller flounder excrete a tracer dose of radioiodine considerably faster than do larger individuals (Fig. 21a and 21b). Also disturbances i n the osmoconcentra-tion of the body fl u i d s resulting from abrupt s a l i n i t y alterations were more rapid i n smaller flounder (Fig. 3). This i s p a r t i a l l y due to a more rapid turn-128 over of electrolytes and water by the organs of exchange with the environment and p a r t i a l l y due to the proportionately greater surface area of small flounder exposed for osmotic movement of water and loss or gain of salts. Thyroid a c t i v i t y , on the other hand, forms a striking exception to the proportional decrease i n metabolic a c t i v i t y of physiological processes with increasing body size, for i t was shown (Fig. 25) that thyroidal radioiodine uptakes increased i n flounder above 30 grams i n weight. These relations are summarized diagrammatically i n Figure 28. The decrease i n thyroid a c t i v i t y with increasing body size of small flounder correlates with a concomitant decrease i n metabolic rate. The relation may be a morphogenetic one with thyroid a c t i v i t y decreasing with the decreasing rate of growth with increasing body size. This would be i n agreement with the theory that the growth regulating function of the thyroid i s more or less i n -dependent of the calorigenic function. The effect of thyroid hormone i n growth and development i s well documented (see reviews by Lynn and Wachowski, 1951; Hoar, 1957 and Pickford and Atz, 1957) and there i s general agreement that thyroid a c t i v i t y i s high during periods of metamorphosis. Hoar (1951) examined histologically the thyroids of starry flounder i n various stages of development. Thyroids appeared active i n metamorphosing flounder, while i n f u l l y metamorphosed individuals the thyroids had undergone involution. Hoar's findings corresponded with those of Sklower (1939) on the European flounder Pleuronectes platessa. These histological observations strongly indicate that the decrease i n thyroid a c t i v i t y of starry flounder with increasing body size i s associated with declining rate of growth. I t i s not clear to what extent thyroid hormone i s involved i n growth changes, although i t i s generally f e l t that i t plays a second-ary role to the growth hormone but i s necessary for the normal expression of the l a t t e r . I t was suggested earlier that the systematic increase i n thyroid a c t i v i t y i n flounder above 30 grams was associated with gonad maturation. Several 129 Figure 28. Diagrammatic representation of the weight dependency and inter— relationships of thyroid a c t i v i t y ($ uptake of I ), excretion ($ dose of I excreted 25 hours after injection) and metabolic rate of starry flounder. 10 (A O Q 5* 100 E X C R E T I O N o o 50 M E T A B O L I C R A T E • 2 I 2 (9 CM O (9 2 .08 .06 .04 1 • • • . _J_ • • • 1 10 100 B O D Y W E I G H T IN G R A M S 1000 130 investigators have reported that thyroid hormone i s necessary for gonad maturation. Thyroid inhibition retards gonad development (Goldsmith e t . a l . , 1943; Barrington and Matty, 1952; Hopper, 1952; Smith, Sladek and Kellner, 1953) while thyroid or thyroxine treatment stimulates the development of second-ary sexual characters (Gaiser, 1952; Hopper, 1952). Increased thyroid a c t i v i t y prior or during spawning i s well known (Olivereau, 1948, 1949, 1954; Buchmann, 1940; Barrington and Matty, 1954). Hoar, (1951) reported that thyroids of adult starry flounder appeared histologically as active as those metamorphosing flounder. These findings and others are suggestive of some correlate between thyroid a c t i v i t y and the sexual cycle although i t s significance i s presently obscure. The point of particular interest to be noted from Figure 28 i s that the increase i n thyroid a c t i v i t y i n flounder above 30 grams i s without effect on t o t a l metabolic rate. I f the increase i n thyroid radioiodine uptake i s t r u l y indicative of greater hormone secretion into the blood stream, i t i s significant that oxygen consumption i s not stimulated. This suggests that the thyroid hormone may have rather specific effects on c e l l s of the body. The increased thyroid a c t i v i t y i n sea water i s also suggestive of hormone s p e c i f i c i t y , i n this case a direct effect on oxidative metabolism of c e l l s doing osmotic work. The work of Barker (Barker, 1951; Barker and Schwartz, 1953) indicates that mammalian tissues respond very selectively to thyroid hormone with respect to metabolic rate: l i v e r , kidney, gastric mucosa, salivary glands, pancreas and various muscle tissues responded to thyroid hyper- or hypo-activity, whereas brain, spleen, thymus and various reproductive organs did not respond. Unfortunately, few studies have been carried out on the effect of thyroid hormone on the meta-bolism of tissues of cold-blooded vertebrates. In the present studies the i n -f l e c t i o n of the thyroid a c t i v i t y curve i n 20-30 gram flounder suggests some direct or indirect involvement between thyroid hormone and gonadal development with no effect on the oxidative metabolism of these tissues. I f subsequent research on 131 the thyroid-reproductive relationship i n fishes supports t h i s explanation, the results, together with the suggestion of a specific effect on energy meta-bolism of osmoregulatory tissues, appear to support the thesis that i n lower vertebrates, the developmental and calorigenic actions of thyroid hormone are two independent effects. The tendency for starry flounder to move into fresh water has certain ecological implications that w i l l now be considered. Because standard metabolic rate of starry flounder i s less i n fresh water than i n sea water, i t i s easier, at least i n this respect, for this species to l i v e i n the fresh water environ-ment. The movement of flounder several miles up the Eraser River into entirely fresh water may represent adaptive radiation of this species into a r e l a t i v e l y unexploited environment where energy demands are less. I t i s generally con-ceded that food i s more abundant i n the sea than i n inland waters. However, the demand for available food i s much greater i n the sea. Particularly i n the coastal area of B r i t i s h Columbia adjacent to r i v e r s , the starry flounder i s undoubtedly the most abundant resident species. Intraspecific competition and interspecific competition with the other species such as the common euryhaline armoured sculpin, Leptocottus armatus must be very keen. Movement into fresh water would offer a certain r e l i e f from food competition not only with respect to food a v a i l a b i l i t y but also because less food must be consumed to meet the lower energy demands for osmotic regulation i n fresh water. However, whereas movement into fresh water i s simple and perhaps advantageous, the permanent establishment of a fresh water race of starry flounder i s a much greater problem. Starry flounder are marine pelagic spawners as are a l l the Family Pleuronectidae. Any eggs shed i n fresh water would be swept downstream by the current or, i f i n a more placid situation, sink to the bottom. Even i f some larvae succeeded i n hatching, i t i s unlikely that any would survive i n an environment to which, as larvae, they are morphologically and physiolo-132 g i c a l l y unsuited. I t i s evident that there must be drastic changes i n mode of reproduction before there can occur the establishment of a residing race of fresh water flounder. Another point for consideration i s the variable a b i l i t y of the thyroid gland of f i s h to trap iodine i n fresh water. There i s evidence that considerable variation exists among fishes i n the relative efficiency of the "iodide trap". The alewife (Pomolobus pseudoharengus) and the smalt (Osmerus mordax) are both capable of osmotically regulating i n fresh water as well as i n their normal marine habitat (Hoar, 1952). However, the thyroids of landlocked alewives were found to be extremely hyper-plastic and showed signs of tot a l exhaustion. At • the time of spawning when demands for thyroid hormone are increased, there occurs a spectacular annual mortality of this species. Smelt, on the other hand, have active (although not extremely hyper-plastic) glands i n fresh water but experience no mortality at spawning. Because of the very low iodine levels i n the Great Lakes region where these f i s h were taken, the extreme hyper-plasia of the alewife thyroid i s probably a goitrogenic reaction to iodine deficiency. Thus, there appears to be a difference i n the a b i l i t y of these two species to trap iodine i n fresh water. I t i s indicated that the development of an e f f i c i e n t iodide trap i s an important prerequisite to invasion of fresh water. The development of euryhalinity and penetration of brackish water by marine fishes i s not uncommon. Gunter (1942), after a thorough study of the matter, reports that there are nine times as many marine fishes invading low s a l i n i t i e s as fresh water fishes penetrating brackish or marine waters i n Middle and North America. He found proportionately more euryhalinity among the phylogenetically primitive orders of fishes. U n t i l recently the theory that the vertebrates originated i n fresh water as developed by Romer and Grove (1935) has been generally accepted. Denison (1956) and Robertson (1957), however, present evidence to show that many of the arguments used to support the theory of fresh 133 water origin are either erroneous or improbable and that a marine origin theory-i s more s o l i d l y supported by existing evidence. I f the views of Denison and Robertson are accepted, the development of euryhalinity among the early marine fishes must have preceded freshwater invasion. Perhaps the movement of starry flounder into fresh water exemplifies some of the events which must have occurred eons ago when primitive fishes f i r s t developed fresh water tolerance and began invasion of this new habitat. *34 VII SUMMARY AND CONCLUSIONS 1. Energy demands for osmotic regulation and the possible osmoregulatory role of the thyroid gland were investigated i n the euryhaline starry flounder, Platichthys stellatus. 2. Using a melting-point technique to measure changes i n serum osmolarity, i t was established that starry flounder possessed e f f i c i e n t osmoregulatory mechanisms i n s a l i n i t i e s between 0°/oo (fresh water) and 45°/oo (concentrated sea water). 3. After abrupt s a l i n i t y changes, small flounder experienced more rapid d i s -turbances of serum osmolarity than large flounder. This i s related at least i n part to the rel a t i v e l y greater surface area of small flounder exposed to osmotic exchange. 4. Standard metabolic rate of starry flounder decreased after transfer to fresh water from sea water. The decrease became more evident with increased time of adaptation i n fresh water. Transfer from normal sea water to concen-trated sea water resulted i n increased metabolic rate of starry flounder and speckled sand dab (Citharichthys stigmaeus). These findings support the theory that energy demands are greater i n more saline waters because of the comparative complexity of regulatory mechanisms employed by marine f i s h . 5. Using the thyroidal uptake of single intraperitoneal doses of radioiodine 131 as a measure of thyroid a c t i v i t y , I uptake was found to be greater i n fresh water adapted flounder. However, reinforcement of iodine deficient fresh water with elemental iodine i n amounts equivalent to that present i n sea water reduced 131 thyroid uptake of I to approximately the same as the uptake of sea water flounder. 131 6. Absorption of I from the body cavity and blood disappearance rate were more rapid i n marine than i n fresh water flounder. 135 7. The decrease i n thyroid a c t i v i t y of small flounder with increasing body size appears to be associated with decreasing rate of growth and metabolic rate. 8. Flounder larger than about 30 grams show thyroid a c t i v i t y increasing with body size. This increase i n a c t i v i t y has no effect on metabolic rate and may possibly be correlative with gonad maturation i n larger flounder. 9. Percentage uptake of radioiodine by the thyroid was shown to be an insen-s i t i v e and inaccurate c r i t e r i o n for evaluating thyroid a c t i v i t y i n different s a l i n i t i e s because removal rates of radioiodine from the body and blood differed between fresh water and marine flounder. Using thyroid clearance of radioiodine form the blood as a measure of a c t i v i t y salt water flounder were shown to have much greater thyroid clearance rates and, hence, more active thyroid glands than flounder adapted to fresh water. 10. The greater a c t i v i t y of the thyroid of marine flounder correlates with greater oxygen demands i n sea water and indicates a direct or adjunctive osmo-regulatory role of the"thyroid gland of f i s h . 136 APPENDIX Table I Analysis of covariance of differences between treatments (times) i n the metabolic rate of Platichthys stellatus measured over one 24 hour period. Data of Table IV. Source of Variation Sum of Squares Degrees of Freedom Mean Square Variance Ratio Differences between times Total 0.72001 0.87612 103 110 .00699 3.18 *** Significant at the 1$ l e v e l . Table I I Analysis of covariance for the effect of starvation on the standard metabolic rate of spring Platichthys stellatus. Data of Table V. Source of Variation Sum of Squares Degrees of Freedom Mean Square Variance Ratio Differences between 4 days fasted and 7 days fasted 0.08210 31 .00264 ** 5.113 Total 0.9564 32 Differences between 7 days fasted and 20 days fasted 0.08557 35 .00245 *** 20.6 Total 0.13592 36 ** Significant at the 5$ l e v e l . *** Significant at the 1$ l e v e l . 137 Table I I I Analysis of covariance for the effect of starvation on the standard metabolic rate of winter Platichthys stellatus. Data of Table VI. Source of Variation Sum of Squares Degrees of Freedom Mean Square Variance Ratio Difference between 2 and 11 days of fasting 0.1757 40 .00439 *** 37.2 Total 0.3392 41 *** Significant at the 1% l e v e l . Table IV Analysis of covariance of the effect of various s a l i n i t y treatments on the standard metabolic rate of Platichthys stellatus. Data of Table VII. Source of Variation Sum of Squares Degrees of Freedom Mean Square Variance Ratio Differences between 20°/oo and 8°/oo s a l i n i t y treatments 0.13135 27 .00486 N.S. Total 0.13218 28 Differences between 20°/oo and 0°/oo (20 hour adaptation) s a l i n i t y treatments 0.10826 27 .00401 ** 7.45 Total 0.13832 28 Adaptation time i n fresh water (20 hour vs. 4 day adapta-tion) 0.02945 23 .00128 *** 29.15 Total 0.06675 24 ** Significant at the 5$ l e v e l . *** Significant at the lfo l e v e l . 138 Table V Analysis of covariance of effect of increased s a l i n i t y on the standard metabolic rate of winter Platichthys stellatus. Data of Table VIII. Source of Variation Sum of Squares Degrees of Freedom Mean Square Variance Ratio Differences between 25°'/oo and 49°/oo s a l i n i t y treatments Total 0.16592 0.40647 38 39 .00437 *** 55.1 *** Significant at the 1$ l e v e l . Table VI Analysis of covariance of the effect of various s a l i n i t y treatments on the standard metabolic rate of Platichthys stellatus. Data of Table IX. Source of Variation Sum of Squares Degrees of Freedom Mean Square Variance Ratio Differences between 22.8°/oo and 0°/oo (20 hour adaptation) s a l i n i t y treatments Total 0.07219 0.07925 31 32 .00232 * 3.04 Differences between 26°/oo and 0°/oo (5 day adaptation) s a l i n i t y treatments Total 0.12139 0.15335 42 43 .00289 11.1 Differences between 25°/oo and 43.2°/oo s a l i n i t y treatments Total 0.13670 0.19301 42 43 .00325 17.3 * Significant at the 10$ l e v e l . *** Significant at the 1$ l e v e l . 139 Table VII Analysis of covariance of the effect of increased s a l i n i t y on the standard metabolic rate of Citharichthys stigmaeus* Data of Table X. 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