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The effect of salinity on the iodine metabolism and standard metabolic rates of coastal and inland prickly… Bohn, Arne 1964

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THE EFFECT OF SALINITY ON THE IODINE METABOLISM AND STANDARD METABOLIC RATES OF COASTAL AND INLAND PRICKLY SCULPINS COTTUS ASPER RICHARDSON by ARNE BOHN B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1961  A THESIS SUBMITTED IN PARTIAL FULFILMENT.OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the DEPARTMENT OF ZOOLOGY  We a c c e p t t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1964  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 a v a i l a b l e f o r reference  and  the L i b r a r y s h a l l make i t f r e e l y  study.  I f u r t h e r agree that .per- ••  m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the Head of my Department or  his representatives.  I t i s understood that,'copying or p u b l i - -  c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not without my  written  Department  of  permission*  The U n i v e r s i t y .of B r i t i s h . Columbia, Vancouver 8, Canada s Date  •  0aV7. 7 /C ^  ~~  by  T"—•  "  be  allowed  ABSTRACT The  lower B r i t i s h  Columbia mainland waterways  are  i n h a b i t e d by  two  r a c e s o f the p r i c k l y s c u l p i n Cottus asper  Richardson.  One  r a c e l i v e s i n the  estuaries during  and  the  spends at l e a s t p a r t of i t s l i f e , p a r t i c u l a r l y  spawning season, under t i d a l i n f l u e n c e .  r a c e i s the confined  "coastal".  The  o t h e r race,  This  c a l l e d "inland", i s  e n t i r e l y t o f r e s h water where i t i s found i n s c a t -  t e r e d , more or l e s s i s o l a t e d By  radioiodine,  i t was  fish.  on the u t i l i z a t i o n  found t h a t s a l i n i t y  i o d i n e metabolism and, r a t e s o f the  populations.  i n v e s t i g a t i n g the e f f e c t s o f ambient s a l i n i t y  the oxygen consumption and  of  on  injected  profoundly e f f e c t s  the  t o a l e s s e r degree, standard m e t a b o l i c  In f r e s h water the t h y r o i d s  appear r e l a t i v e l y q u i e s c e n t but ticularly  lower p a r t of r i v e r s or i n  with i n c r e a s i n g  o f both r a c e s salinity,  par-  i n hyperosmotic environment, t h e r e i s a marked i n -  c r e a s e i n t h y r o i d uptake o f t r a c e r i o d i n e and l e v e l s of c i r c u l a t i n g t h y r o i d hormone.  The  a l s o i n the  i n l a n d f i s h demon-  s t r a t e a greater  a b i l i t y than c o a s t a l f i s h t o r e t a i n i o d i d e i n  f r e s h water, and  i n sea water t h e i r t h y r o i d s  The  appear more a c t i v e .  i n l a n d f i s h a l s o have a lower oxygen consumption i n f r e s h  water than do c o a s t a l  fish.  Viewed t o g e t h e r , the r e s u l t s l e a d t o the t h a t the  inland f i s h , with respect  conclusion  t o t h e i r osmoregulating  ability,  show g e n e t i c d i v e r g e n c e from!the c o a s t a l forms.  l a t t e r p r o b a b l y more c l o s e l y resemble the a n c e s t r a l  form.  The  ACKNOWLEDGMENTS  My s i n c e r e g r a t i t u d e i s due t o Dr. W. S. Hoar who suggested t h e problem and o f f e r e d i n v a l u a b l e encouragement and numerous h e l p f u l suggestions  d u r i n g t h e development o f  the i n v e s t i g a t i o n . I am a l s o indebted  t o Dr. C. P. Hickman J r . , t o  Dr. P. A. Dehnel and t o Dr. C.C. L i n d s e y and c r i t i c i s m .  for their interest  Dr. S. W. Nash o f f e r e d a d v i c e on t h e  s t a t i s t i c a l problems and Mr. L. Heger conducted t h e r e g r e s s i o n analyses  a t t h e U n i v e r s i t y Computing Centre;  their  assistance  i s g r a t e f u l l y acknowledged. Thanks a r e due t o D r . J . G. E a l e s f o r i n s t r u c t i n g me i n t h e use o f r a d i o l o g i c a l methods and i t s a p p l i c a t i o n t o thyroid physiology.  A l s o , I am indebted  t o Mr. W. L. Woodall  f o r h i s keen a s s i s t a n c e on many f i e l d t r i p s c o l l e c t i n g t h e fish,  and t o my f e l l o w graduate students  f o r many s t i m u l a t i n g  discussions. F i n a n c i a l support  was r e c e i v e d from t h e N a t i o n a l  Research C o u n c i l through g r a n t s - i n - a i d o f r e s e a r c h t o W. S. Hoar.  TABLE OF CONTENTS  I. II.  INTRODUCTION MATERIALS AND METHODS A.  III.  1  C o l l e c t i o n and c a r e o f f i s h .  6  B. I Blood t o n i c i t y d e t e r m i n a t i o n s .  7  C.  Techniques f o r e v a l u a t i n g  8  D.  Metabolic rates.  13  A.  Blood t o n i c i t i e s .  16  B.  Iodine metabolism.  thyroid a c t i v i t y .  RESULTS  1. E x c r e t i o n o f r a d i o i o d i n e . a. E f f e c t o f s i z e on e x c r e t i o n o f radioiodine.  19  b. E f f e c t o f environment on e x c r e t i o n of r a d i o i o d i n e .  28  c. Comparisons between e x c r e t i o n  rates  o f c o a s t a l and i n l a n d f i s h . 2. T h y r o i d  32  uptake o f r a d i o i o d i n e .  a. E f f e c t o f s i z e on t h y r o i d a c t i v i t y .  40  b. E f f e c t o f environment on t h y r o i d activity.  40  c. Comparisons o f t h y r o i d  activity  i n c o a s t a l and i n l a n d f i s h .  47  3. C i r c u l a t i n g radiohormone. a. D e t e r m i n a t i o n o f c o n v e r s i o n r a t i o s as a measure o f t h y r o i d a c t i v i t y .  50  ii  b.  E f f e c t s of s a l i n i t y ^ a n d - h a b i t a t ! on c o n v e r s i o n r a t i o s .  4. C.  E f f e c t s o f sex on i o d i n e metabolism  1.  General c o n s i d e r a t i o n s .  58  2.  E f f e c t of s a l i n i t y  62  3.  E f f e c t o f abrupt changes i n s a l i n i t y  4.  on oxygen consumption.  VI. VII.  on 66  E f f e c t o f h a b i t a t o f f i s h on oxygen consumption.  V.'  56  Standard m e t a b o l i c r a t e s .  oxygen consumption.  IV.  55  68  DISCUSSION  70 •  SUMMARY  76  LITERATURE CITED APPENDIX  .  79 82  LIST OF  TABLES  Table I. II.  Page F r e e z i n g p o i n t depressions  C o r r e l a t i o n s between percent r a d i o i o d i n e and  III. IV.  o f blood  serum.  excretion  19  of  l o g weighty  28  E f f e c t of s i z e on e x c r e t i o n of r a d i o i o d i n e .  27  Values of Student's t t e s t s and  conclusions  from comparisons between e x c r e t i o n r a t e s i n d i f f e r e n t environmental s a l i n i t i e s . V„  38  Comparisons by Student's t t e s t s between e x c r e t i o n r a t e s of c o a s t a l v e r s u s i n l a n d fish.  VI.  38  C o r r e l a t i o n s between l o g s i z e and  thyroid  r e t e n t i o n of I ^ \ VII. VIII.  C o r r e l a t i o n s between l o g s i z e and  Contributions TUF  X.  TUF  values.  S t a t i s t i c s from r e g r e s s i o n l i n e s of TUF time i n f i s h a c c l i m a t e d  IX.  41  values  versus  to d i f f e r e n t s a l i n i t i e s .  42  of t h y r o i d uptake t o c a l c u l a t e d  24 hours a f t e r i n j e c t i o n .  C o n t r i b u t i o n of lower jaw t i s s u e s t o  46 total  body weight. XI.  41  S t a t i s t i c s from r e g r e s s i o n l i n e s of TUF  47 versus  time i n c o a s t a l f i s h compared t o c o r r e s p o n d i n g s t a t i s t i c s from i n l a n d f i s h . .  48  iv  XII. XIII.  Variability  i n TUF values„  Weights o f f i s h used f o r  50 interracial  comparison of metabolic r a t e s i n d i f f e r e n t salinities. XIV.  69  Data from c o a s t a l f i s h used f o r i n t e r s e x comparisons.  XV.  85  Data from i n l a n d f i s h used f o r i n t e r s e x comparisons.  XVI.  Statistics  and c o n c l u s i o n s from  86 intersex  comparisons o f data from c o a s t a l f i s h . XVII.  Statistics  87  and c o n c l u s i o n s from i n t e r s e x  comparisons o f data from i n l a n d f i s h .  88  L I S T OF  Respirometer Freezing  FIGURES  (diagram).  point  depression  curves. 131 .  The e f f e c t o f s i z e on e x c r e t i o n sea  water a c c l i m a t e d acclimated  m  of I  131 . in  fish.  . The e t f e c t o t s i z e on e x c r e t i o n fish  of I  t o f r e s h water, t o i s o t o n i c ,  and t o h y p e r t o n i c  sea water. 131  T o t a l body  retention  acclimated  to three  of I  i n coastal  fish  salinities. 131  T o t a l body r e t e n t i o n acclimated  to three  of I  i n inland  fish  salinities.  131 Retention of I as  a function  200 h o u r s a f t e r i n j e c t i o n of  salinity, 131 .  T o t a l body  retention  acclimated  coastal  T o t a l body  retention  inland  fish  of I  i n fresh  and i n l a n d of I  acclimated  water  fish. i n coastal  t o l l ° / o o and  and  26°/oo  salinities. 131 Extrathyroidal acclimated  retention  to fresh  of I  in fish  water.  131 Extrathyroidal retention of I in fish o t ° / • a c c l i m a t e d t o 11 /oo and 26 /oo s a l i n i t i e s .  VI.  12.  E f f e c t of sxze on t h y r o i d r e t e n t i o n o f I  13„  Rate of i n c r e a s e  1 31  .  i n TUF a f t e r i n j e c t i o n of  coastal fish. 14o  Rate o f i n c r e a s e  44 i n TUF a f t e r i n j e c t i o n o f  inland f i s h . 15.  Rate o f i n c r e a s e  45 i n TUF a f t e r i n j e c t i o n of  c o a s t a l and i n l a n d f i s h a c c l i m a t e d t o A. watery B, ll°/oo s a l i n i t y 16.  fresh  and C. 26°/oo s a l i n i t y .  Conversion r a t i o s from c o a s t a l and i n l a n d  Conversion r a t i o s from c o a s t a l and i n l a n d  54  Oxygen consumption of c o a s t a l and i n l a n d  fish  exposed t o i n c r e a s i n g and d e c r e a s i n g s a l i n i t y . 19.  53  fish  a c c l i m a t e d t o ll°/oo s a l i n i t y . 18.  49  fish  a c c l i m a t e d t o f r e s h water and 26°/oo s a l i n i t y . 17.  39  Oxygen consumption i n c o a s t a l and i n l a n d  64  fish  o when the s a l i n i t y  i s changed  from 10 /oo t o  2b /oo.  20.  21„  fob  The e f f e c t o f i n c r e a s i n g  salinity  on oxygen  consumption i n s t a r v e d  coastal f i s h .  The e f f e c t o f s a l i n i t y  on r a t e of l 31  i n TUF a f t e r i n j e c t i o n with I"*"  .  67  increase .  74  I.  INTRODUCTION  I n f r e s h water, f i s h tend t o l o s e s a l t s i n t h e copious f l o w o f d i l u t e u r i n e e x c r e t e d by t h e k i d n e y s .  This i s  compensated f o r by a c t i v e uptake o f i o n s a g a i n s t an osmotic g r a d i e n t i n the g i l l s the f o o d .  (Krogh,  1937), and by i n g e s t i o n o f i o n s i n  With i n c r e a s i n g ambient s a l i n i t y ,  d i f f u s e i n t o the body by osmosis, u n t i l , would assume no osmotic environmental  l e s s water w i l l  a t i s o t o n i c i t y , one  a c t i o n t o take p l a c e as a r e s u l t o f  differences i n tonicity.  I f t h e ambient medium  becomes h y p e r t o n i c w i t h r e s p e c t t o t h e f i s h , t h e osmotic  flow o f  water w i l l take t h e o p p o s i t e d i r e c t i o n , t h a t i s , outwards from the f i s h i n t o t h e s u r r o u n d i n g s . must change a c c o r d i n g l y .  The osmoregulatory  mechanisms  I n sea water, s a l t e x c r e t i o n by t h e  g i l l s has been demonstrated,  (Keys, 1931, 1932; Smith, 1930),  t h i s i s a r e s u l t o f the f i s h d r i n k i n g t h e h y p e r t o n i c medium. C o n s i d e r a b l e a t t e n t i o n has been focussed i n r e c e n t years on the endocrine f a c t o r s i n v o l v e d i n t h e osmoregulation of  teleost fishes.  P i c k f o r d and A t z (1957) d i s c u s s e d t h e r o l e  of  the p i t u i t a r y g l a n d i n osmoregulation  of  t h e knowledge i n t h e f i e l d .  Holmes,  and summarized much (1963) d i s c u s s e d t h e  r o l e s o f s t e r o i d s from the a d r e n a l c o r t e x d u r i n g  osmoregulation  i n the European e e l ( A n g u i l l a a n g u i l l a ) and i n t h e rainbow trout  (Salmo g a i r d n e r i ) .  The a c t i o n o f a d r e n o c p r t i c o s t e r o i d s  2  is  b e l i e v e d t o be a s s o c i a t e d w i t h r e n a l and e x t r a r e n a l c e l l s  c o n t r o l l i n g the e l e c t r o l y t e balance of the body through r e g u l a t i n g the e x c r e t i o n and a b s o r p t i o n o f sodium. Koch and Heuts  (1942),  and Olive.re.au (1948,  demonstrated the importance o f t h y r o i d hormone i n in fish.  1950)  osmoregulation  The v a r i e d a c t i v i t i e s o f t h i s hormone have been  s u b j e c t t o i n t e n s i v e study and a number o f p o s s i b l e b i o c h e m i c a l r o l e s i n c e l l metabolism f o r t h i s hormone have been c o n s i d e r e d . In f i s h , the t h y r o i d g l a n d seems t o p l a y a r o l e d u r i n g changes i n a v a r i e t y o f i n t e r n a l and environmental these are s e x u a l m a t u r i t y , temperature I960? Fortune,  1955  factors.  (Leloup and  and 1956), p h o t o p e r i o d  Some o f Fontaine,  (Hoar, 1959?  S w i f t , I960? E a l e s , 1963), m i g r a t o r y behaviour  (Hoar and  Bell,  1950? Baggerraan, 1963), season,  and ambient i o d i n e c o n c e n t r a t i o n  (McNabb, 1963? Hickman, 1959).  The r o l e o f the t h y r o i d i n  osmoregulation  has been the b a s i s of s e v e r a l s t u d i e s .  Koch and  Heuts (1942) showed t h a t when the t h r e e s p i n e d s t i c k l e b a c k (Gasterosteus aculeatus) was osmoregulatory  a b i l i t y was  t h a t t h y r o x i n e treatment f o r f r e s h water.  f e d d e s i c c a t e d t h y r o i d gland, i t s  impaired.  (1959) found  o f t h i s s p e c i e s produced p r e f e r e n c e  In 1959,  Hickman, u s i n g r a d i o l o g i c a l  methods, s t u d i e d osmoregulation (Platiehtys s t e l l a t u s ) .  Baggerman  i n the s t a r r y f l o u n d e r  H i s r e s u l t s showed t h a t sea water  3  a c c l i m a t i o n was  associated with greater t h y r o i d a c t i v i t y  than was  f r e s h water a c c l i m a t i o n .  increase  i n metabolic rates with i n c r e a s i n g s a l i n i t y  s u g g e s t e d t h a t t h i s may  a l s o found an and  be a demonstration of c a l o r i g e n i c  a c t i o n not p r e v i o u s l y obtained (1962)  He  i n work w i t h t e l e o s t s .  showed t h a t t h y r o i d a c t i v i t y i n c r e a s e d  Wiggs  in sticklebacks  t r a n s f e r r e d i n the l a b o r a t o r y from f r e s h t o sea water; he a l s o was  u s i n g r a d i o l o g i c a l methods. The  p a t t e r n emerging from these s t u d i e s has-been::  d i s c u s s e d by Hoar (1959, 1963), Leloup and F o n t a i n e others.  (1960) and  Most authors seem t o f e e l t h a t the t h y r o i d hormone  i n i t s a c t i v e form must a f f e c t c e l l u l a r metabolism at some r a t h e r fundamental p o i n t ; i t has may  have more than one On  even been suggested t h a t i t  such fundamental f u n c t i o n .  the b a s i s o f these c o n s i d e r a t i o n s ,  we  felt i t  w o u l d be o f i n t e r e s t t o i n v e s t i g a t e t h y r o i d a c t i v i t y  and  m e t a b o l i c r a t e s i n another s p e c i e s o f e u r y h a l i n e t e l e o s t . The  primary aim o f t h i s i n v e s t i g a t i o n was  to  s t u d y the e f f e c t o f ambient s a l i n i t y on i o d i n e metabolism and  on standard  metabolic r a t e s .  The  environmental  salinity  i n xvhich f i s h are i n osmotic e q u i l i b r i u m w i t h the medium  was  f i r s t e s t a b l i s h e d ; comparisons were then made between the e f f e c t s o f f r e s h water and metabolism and  o f i s o t o n i c and h y p e r t o n i c  on standard  metabolic r a t e s .  sea water on  iodine  4  The  animal chosen f o r t h e work was t h e p r i c k l y  s e u l p i n , Cottus asper Richardson. obtained  T h i s f i s h may be  d u r i n g most months o f t h e year, i t i s t o l e r a n t t o  elevated salinities, t h e laboratory.  and i t i s r e l a t i v e l y easy t o keep i n  The l i f e c y c l e o f t h e p r i c k l y s e u l p i n i n  B r i t i s h Columbia has been d e s c r i b e d by K r e j s a  (1964.)  It  occurs v e r y commonly and i n l a r g e numbers i n most o f t h e lower F r a s e r R i v e r waterways, p a r t i c u l a r l y t h e l a k e s and i n slow f l o w i n g streams as f a r down as t h e e s t u a r i e s where t h e comp o s i t i o n o f t h e water i s under t i d a l i n f l u e n c e . o c c u r s d u r i n g A p r i l and May.  Spawning  F i s h l i v i n g close t o the  r i v e r o u t l e t s tend t o have c o n s i d e r a b l y  less prickly  texture  on t h e i r d o r s a l and l a t e r a l s u r f a c e s than have f i s h found f u r t h e r i n l a n d , as f o r i n s t a n c e  i n Cultus  and H a t z i c Lakes,  British  Columbia. In t h e t r i b u t a r i e s t o H a t z i c Lake, t h e source o f a l l t h e f i s h used f o r our t h y r o i d and m e t a b o l i c  rates  experiments, s c u l p i n s spawn upstream from t h e l a k e .  It is  d i f f i c u l t t o say how e f f e c t i v e t h i s i s o l a t i o n from t h e c o a s t a l form i s , b u t t h e f a c t t h a t t h e r e appears t o be a demonstrable g e n e t i c d i f f e r e n c e i n t h e t e x t u r e o f t h e s k i n between t h e two forms  (Krejsa, 1964), i n d i c a t e s t h a t t h e i s o l a t i o n has been  s u f f i c i e n t t o cause g e n e t i c d i v e r g e n c e .  The e v o l u t i o n o f  t h e i n l a n d type i s t h e r e s u l t o f upstream m i g r a t i o n adaption  and t h e  t o new c o n d i t i o n s o f e a r l y s c u l p i n s d i v e r g i n g from an  5  a n c e s t r a l marine stock? i t has p r o b a b l y been e v o l v i n g  since  these e a r l y forms s t a r t e d t o e x p l o i t f r e s h water h a b i t a t s . secondary aim o f our study was t o look i n t o the o f a demonstrable g e n e t i c the c o a s t a l and i n l a n d  possibility  d i f f e r e n c e i n osmoregulation  fish.  A  between  II.  A.  MATERIALS AND METHODS  C o l l e c t i o n and c a r e o f f i s h .  Experiments were c a r r i e d out d u r i n g t h e autumn o f 1963 and w i n t e r and s p r i n g o f 1964.  The c o a s t a l f i s h were  c o l l e c t e d from Campbell R i v e r i n White Rock, B r i t i s h approximately 500 f t above the r a i l w a y t r e s t l e . forms came from t h r e e d i f f e r e n t l o c a t i o n s .  Columbia,  The i n l a n d  A l l the inland  f i s h used f o r the t h y r o i d work were c o l l e c t e d from Lagace Creek which runs i n t o H a t z i c Lake.  For determinations o f  standard m e t a b o l i c r a t e s , t h e f i s h were c o l l e c t e d  from  C h i l q o a Creek which a l s o runs i n t o H a t z i c Lake? f o r t h e b l o o d t o n i c i t y experiments f i s h were o b t a i n e d from C u l t u s Lake. C o l l e c t i n g from r i v e r s was done by two men u s i n g a p o l e s e i n e o f 5 mm mesh h e l d down by a l e a d l i n e . and g r a v e l bottoms t h i s proved q u i t e e f f i c i e n t ,  At sandy  particularly  where t h e s e i n e c o u l d be pushed up a g a i n s t a moderately swift current.  Where the f i s h were h i d i n g under an o v e r -  hanging bank, the s e i n e c o u l d be p l a c e d downstream and h e l d i n p o s i t i o n by one man w h i l e the o t h e r s c a r e d t h e f i s h t h e i r h i d i n g places i n t o the seine.  from  I n C u l t u s Lake t h e f i s h  were caught i n minnow t r a p s which were lowered t o the bottom from permanent f l o a t s near t h e entrance t o Sweltzer Creek. The t r a p s were checked each week f o r f i s h .  7  F o r the t y r o i d work t h e f i s h were a c c l i m a t e d a t 11.5 ± 0.3°C and p l a c e d i n 15 l i t e r g l a s s j a r s , t h r e e q u a r t e r s f i l l e d w i t h water; these were f l o a t i n g i n a c o n s t a n t bath.  No more than t w e n t y - f i v e f i s h  temperature  (average weight about 7 gra)  were p l a c e d i n each j a r ; the water was changed every 48 hours. The f i s h were allowed t o a c c l i m a t e t o t h e i r r e s p e c t i v e e n v i r o n ments f o r one week b e f o r e i n j e c t i o n w i t h r a d i o i o d i n e . These o d i f f e r e n t environments were useds f r e s h water, l l o / o o  salinity  sea water and 26o/oo s a l i n i t y sea water. For t h e experiments  on standard m e t a b o l i c r a t e s and  on b l o o d t o n i c i t i e s the f i s h were a c c l i m a t e d i n a c o n s t a n t environment room a t 9.6 ± 0.4°C i n p l a s t i c boxes c o n t a i n i n g f i v e l i t e r s o f water.  No more than seven f i s h were k e p t i n each box  and the water changed every 48 hours. one week f o r a c c l i m a t i o n .  The f i s h were allowed  No f i s h were f e d d u r i n g the  experiments. B.  Blood t o n i c i t y d e t e r m i n a t i o n s .  F i v e t o s i x f i s h were used f o r each sample a f t e r a c c l i m a t i o n t o the d e s i r e d s a l i n i t y f o r one week.  The b l o o d  was o b t a i n e d from t h e d o r s a l a r t e r y by c u t t i n g the body j u s t a n t e r i o r t o the c a u d a l peduncle.  i  C a p i l l a r y tubes were used f o r  c o l l e c t i n g t h e b l o o d which was subsequently pooled and c e n t r i -  8  fugedo  The  osmolarity  o f the b l o o d  serum was  then measured by  f r e e z i n g p o i n t d e t e r m i n a t i o n s i n a F i s k e Osmometer, c a l i b r a t e d f o r measurements i n the 0 t o 1000 freezing point depression average of t h r e e separate  C.  m i l l i o s m o l s range.  o f each serum sample was  The  taken as  the  determinations.  Techniques f o r e v a l u a t i n g t h y r o i d a c t i v i t y .  On  the f i r s t day  o f an experiment, a l l the f i s h  of  t h a t experiment were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h a s o l u t i o n 131  of c a r r i e r f r e e x  i n d i s t i l l e d water.  e x a c t l y 20 u l s o l u t i o n c o n t a i n i n g maximum o f 0.4 0.04  Each f i s h  1 uc o f t r a c e r .  uc/gm f o r the s m a l l e s t and  received T h i s gave a  a minimum of about  uc/gm f o r the l a r g e s t f i s h y both v a l u e s  were w e l l  within  the l i m i t s o f s a f e t y from r a d i a t i o n damage t o the t h y r o i d on the one hand, and Eales  f o r d e t e c t a b i l i t y on the o t h e r .  (1963) measured the dependence o f c o n v e r s i o n  Hoar  r a t i o on  dose of i n j e c t e d r a d i o i o d i n e i n g o l d f i s h , l a k e chub and s p e c i e s o f salmonid f i s h . l e s s than 0.5  uc/gm per  and the  three  T h e i r r e s u l t s i n d i c a t e d t h a t a dose of  f i s h would have no d e t r i m e n t a l  t h y r o i d t i s s u e s i n the s p e c i e s  a d j u s t a b l e t o d e l i v e r a constant  e f f e c t on  studied.  I n j e c t i o n s were d e l i v e r e d by a 0.1  f i s h were r e p l a c e d  gland  volume.  ml  microsyringe,  A f t e r i n j e c t i o n s the  i n the 15 l i t e r g l a s s j a r s and k e p t f o r the  9  r e s t o f the experiments under c o n d i t i o n s acclimation period. s e l e c t e d from the the n e a r e s t  0.1  b l o o d sample was  subjected  Blood was  weighed t o  t o the f o l l o w i n g treatment.  taken up i n a 75 mm  c a p i l l a r y tube and  plasticene. containing  and  Each f i s h i n the sample was  c o l l e c t e d from the d o r s a l a o r t a by c u t t i n g  c a u d a l peduncle. heparinized  one  end  the red b l o o d c e l l s was  tube w i t h the serum was  c e n t r i f u g e tube.  The  the c e n t r i f u g e tube. weight o f the serum by  radioiodine  (TCA)  gently  i n a 10  ml  serum  without s p l a s h i n g on the w a l l s  of  Weighing the empty c a p i l l a r y tube gayexithe difference.  )'., were c e n t r i f u g e d  and  once w i t h 1.5  and  the washings were combined w i t h 0.5  prevent a b s o r p t i o n  the  s m a l l mouthpiece which would f i t  precipitated proteins, containing (PBI  with  r e s t of  over the c a p i l l a r y tube and by blowing c a r e f u l l y , the c o u l d be pushed i n t o the TCA  the  mm  then the serum was  trichloracetic acid  By u s i n g  A  o f each c a p i l l a r y tube  broken o f f .  weighed, and  f o r c e d i n t o 4 ml o f 12.5%  by 1.0  o f the tube s e a l e d  A f t e r c e n t r i f u g i n g , the end  The  the  At i n t e r v a l s , samples o f e i g h t f i s h were  jars.  gm  i d e n t i c a l with  ml o f a 2.5%  and washed once w i t h 2 ml  s o l u t i o n o f TCA.  o f the r a d i o i o d i n e  the p r o t e i n bound  The  supernatant  ml o f c a r r i e r s o l u t i o n t o (1^1)  o n  the g l a s s .  Each  131 t o t a l o f 8-ml  s o l u t i o n would c o n t a i n  a l l the morganxc I  each o r g i n a l b l o o d sample, except what may or adhering t o the red b l o o d c e l l s .  The  of  have been p r e s e n t i n  carrier solution  was  10  prepared a c c o r d i n g t o Chase I  (1960) t o g i v e a l a r g e s u r p l u s o f  p r e s e n t i n each sample.  were l e f t f o r some days.  A t t h i s p o i n t , t h e blood  fractions  U s u a l l y , the r a d i o a c t i v i t y measure-  ments o f t h e b l o o d was done a t t h e end o f each experimental r u n , i . e . from one week t o t e n days a f t e r i n j e c t i o n s .  Thentfche  p r o t e i n a c e o u s p r e c i p i t a t e s were d i s s o l v e d i n 2 ml o f NaOH and t r a n s f e r r e d t o s p e c i a l l y designed c o u n t i n g tubes, made t o f i t the w e l l s c i n t i l l a t i o n counter used  i n most o f t h i s study.  The  c e n t r i f u g e tubes were r i n s e d twice with 1 ml p o r t i o n s o f 1 N NaOH each and t h e p o r t i o n s t r a n s f e r r e d t o t h e i r r e s p e c t i v e c o u n t i n g tubes f o r a t o t a l o f 4 ml i n each tube. From t h e 8-ml p o r t i o n s o f supernatant c o n t a i n i n g t h e 131 inorganic I  , 4 ml was t r a n s f e r r e d t o another s e t o f c o u n t i n g  tubes a f t e r thorough  stirring.  The r a d i o a c t i v i t y o f each  sample f r a c t i o n was measured i n a Nuclear Chicago Model DS5 W e l l S c i n t i l l a t i o n D e t e c t o r i n c o n n e c t i o n With a P h i l l i p s PW 4022 h i g h v o l t a g e supply a m p l i f i e r u n i t and a PW 4035 E l e c t r o n i c counter.  Counting o f t h e r a d i o a c t i v i t y was always  done by measuring t h e time taken t o reach a p r e s e t t o t a l number Of counts.  When t h e p r e s e t number o f counts had been reached,  the E l e c t r o n i c Counter would a u t o m a t i c a l l y switch i t s e l f o f f and t h e time c o u l d be read t o the n e a r e s t t h r e e hundredth o f a second on a c l o c k Which would a u t o m a t i c a l l y s t a r t ! whenever t h e c o u n t i n g mechanism was a c t i v a t e d .  The f i g u r e s were converted  11 t o counts per minutes samples.  t o form the b a s i s o f comparisons  between  F o r the b l o o d f r a c t i o n s t h e counter was p r e s e t t o  3  3x10  counts, making t h e c o u n t i n g time o f each sample u s u a l l y  somewhere from 5 t o 15 minutes. A f t e r a b l o o d sample was taken, the f i s h were k i l l e d and the t h y r o i d t i s s u e d i s s e c t e d out.  In Cottus asper, the  t h y r o i d f o l l i c l e s were found t o be l o c a t e d i n the f l o o r o f the mouth between the f i r s t and t h e t h i r d g i l l  arch.  t h y r o i d t i s s u e o f each f i s h was d e p o s i t e d d i r e c t l y  The  into  c a l i b r a t e d c o u n t i n g tubes c o n t a i n i n g 4 ml 10% f o r m a l i n . was found e a r l i e r  (Wiggs,  1962) t h a t the observed  It  radiation  from a p o i n t source on the bottom o f a c o u n t i n g tube i s n o t s i g n i f i c a n t l y d i f f e r e n t from t h a t o f a source of  solvent.  dispersedi.dn4,4mml  The t i s s u e lumps were measured f o r r a d i a t i o n a t  the e a r l i e s t convenience, t o g e t h e r with t h e PBI^ - - and i n o r g a n i c 3  jl31  f  r a c  tions.  A standard s o l u t i o n o f I  1  3  1  1  was prepared by  i n j e c t i n g a mixture o f 90.0 ml d i s t i l l e d water and 10.0 ml c a r r i e r twice w i t h 1 y.c? u s u a l l y t h i s was done a t the time when the f i s h were i n j e c t e d . to  Four ml o f t h i s were t r a n s f e r r e d  a c o u n t i n g tube which c o n s e q u e n t l y would, a t any time,  emit  a r a d i a t i o n equal t o 8.0% o f t h e i n j e c t e d dose a t t h e same time. of  A f t e r d e d u c t i n g t h e background  r a d i a t i o n , the a c t i v i t y  each sample c o u l d be c o n v e r t e d t o per c e n t o f t h e i n j e c t e d  dose by comparison  with t h i s standard.  12  The  r a d i o a c t i v i t y c o n t a i n e d i n t o t a l body mass  measured d i r e c t l y , u s i n g  a Harshaw Nal  was  s c i n t i l l a t i o n c r y s t a l and  a m p l i f i e r probe which c o u l d be plugged i n t o the same a m p l i f i e r s c a l e r u n i t as the w e l l c o u n t e r . l y and  The  probe was  p r o t r u d e d i n t o a 20 x 20 x 14.5  castle.  The  l e a d , the  r o o f and w a l l s  cm  mounted v e r t i c a l -  chamber i n a l e a d  of the c a s t l e were o f 51 mm  f l o o r , a l s o l e a d , was  thick  27 ram t h i c k .  A f t e r removal o f the t h y r o i d t i s s u e , each f i s h s l i c e d and  the s l i c e s p l a c e d  from 5 t o 10 mm  i n thickness,  depending on the s i z e o f the f i s h , the diameter o f  d i s c would v a r y from 30 t o 60 mm. were p l a c e d ,  one  d i r e c t l y under the nation  o f the  e x a c t l y 140  mm.  The  largest fish  f l o o r , the f i s h were p l a c e d  from the g l a s s p l a t e t o the probe  At t h i s d i s t a n c e ,  15.0  d i s c s was  neglegible.  was  (30 mm)  and  A standard which was  pared at the time of i n j e c t i o n would c o n t a i n I t was  plate,  any measurable v a r i a t i o n i n  large  10 ml  (over  on a low g l a s s  t o s i z e d i f f e r e n c e between s m a l l  i n j e c t e d dose.  the  To prevent contami-  r a d i a t i o n due (60 mm)  fish.  a t a time, i n the c e n t r e o f the chamber  scintillation crystal.  so t h a t the d i s t a n c e  was  i n a c i r c u l a r d i s c which measured  A l s o depending on the s i z e o f the  gm)  -  made by adding two  200%  of  pre-  the  1 uc i n j e c t i o n s t o  10% c a r r i e r s o l u t i o n i n a f l a t g l a s s d i s h p r o v i d e d w i t h a  t i g h t f i t t i n g removable g l a s s l i d . The d i s h measured 48 mm  and  the t h i c k n e s s  diameter of the  glass  o f the l i q u i d l a y e r 5.5  mm.  13  R a d i a t i o n from each f i s h , and from the standard, was e i t h e r t o 3x10  4  counts or t o 1x10  4  measured  counts depending on the  activity.  Do  I t was  Metabolic rates.  necessary t o c o n s t r u c t an apparatus  i n which  the f i s h would be exposed t o a change of the ambient  salinity  without undue d i s t u r b a n c e .  Winkler t i t r a t i o n s  were used f o r  oxygen d e t e r m i n a t i o n s and the oxygen consumption o f the c a l c u l a t e d as the average  o f s e v e r a l measurements.  fish  It  was  d e s i r a b l e t o keep the f i s h a t a c o n s t a n t oxygen t e n s i o n t o a v o i d the e f f e c t s of depressed oxygen t e n s i o n on metabolism. The  f i s h were kept i n the l a b o r a t o r y i n f r e s h water  f o r one week without f e e d i n g . f u r t h e r f e c a l m a t e r i a l was  At the end o f t h i s p e r i o d no  d i s c h a r g e d i n the water and  the  f i s h were presumed t o be i n the p o s t a b s o r p t i v e s t a t e . were then p l a c e d i n e i t h e r one o f two (2.5 l i t e r c a p a c i t y ) each 15 cm c o a s t a l and one were connected i n F i g . 1.  respirometer b o t t l e s  i n diameter?  for inland material.  one was  used f o r  The r e s p i r o m e t e r b o t t l e s  t o the same c o n s t a n t f l o w water supply as shown  Both groups of f i s h would then be s u b j e c t t o  i d e n t i c a l c o n d i t i o n s w i t h r e s p e c t t o environmental salinity,  They  and oxygen content of the incoming water.  temperature, One  bottle  14  F i g u r e 1.  Respirometer. current„  Arrows i n d i c a t e d i r e c t i o n o f  CONSTANT HEAD (b) OVERFLOW Water aerated flowinq back to reservoir  rjt  I  (  0 \ A  2 5L -15 CM —  2 5L k l 5 CM-*1  RESPIFIOMETER CHAMBERS SAMPLES TAKEN AT • a *  (a)*«  RESERVOIR  \  15  c o u l d be used s e p a r a t e l y i f o n l y one group o f f i s h was studied.  t o be  The f l o w of water through each r e s p i r o m e t e r c o u l d be  r e g u l a t e d by r a i s i n g or l o w e r i n g the o u t l e t s ( a ) . Samples were c o l l e c t e d i n 30-ml Winkler b o t t l e s with t i g h t f i t t i n g ground g l a s s stoppers and t i t r a t e d  immediately  with 0.025 N sodium t h i o s u l p h a t e a c c o r d i n g t o the standard Winkler oxygen t i t r a t i o n t e c h n i q u e .  C o n t r o l samples g i v i n g  oxygen c o n t e n t of the i n f l o w i n g water were o b t a i n e d d i r e c t l y from the r e s e r v o i r .  The range of v a r i a t i o n i n the c o n t r o l  samples o f the same s a l i n i t y o c c a s i o n a l l y reached a maximum of about two  and a h a l f p e r c e n t , but was  and two p e r c e n t .  u s u a l l y between  The oxygen t e n s i o n o f the water i n the  r e s e r v o i r d i d not f a l l below 97 p e r c e n t o f s a t u r a t i o n .  one  the  16 III. A. One  RESULTS  Blood  tonicities.  o f the aims o f t h i s study was  t o compare the  v a r i o u s aspects o f t h y r o i d metabolism of f i s h a c c l i m a t e d t o and living to  i n an i s o t o n i c environment with those o f f i s h a c c l i m a t e d  and l i v i n g i n f r e s h water and h y p e r t o n i c sea water.  achieve t h i s , of  i t was  necessary t o determine  f i s h from d i f f e r e n t s a l i n i t i e s .  the b l o o d  More than one  To tonicity  salinity  was  d e s i r a b l e , s i n c e the osmotic p r e s s u r e of the b l o o d i n many t e l e o s t s has been shown t o depend on the ambient osmotic  pressure  (Black, 1957). F r e e z i n g p o i n t d e p r e s s i o n s and m i l l i o s m o l v a l u e s f o r t h r e e groups o f f i s h are compiled  i n Table I .  F i g . 2 compares  the t h e o r e t i c a l f r e e z i n g p o i n t s of water o f d i f f e r e n t  salinities  w i t h the f r e e z i n g p o i n t s of t h r e e groups o f s e u l p i n b l o o d . v a l u e s f o r water were c a l c u l a t e d from the two  and  A  =  0.0966 C l +  Salinity  =  0.03 +  0.0000052 C l  3  o  and  d e p r e s s i o n of the sea water s o l u t i o n  In  equations;  l 805 X Cl  where C l i s the c h l o r i n i t y i n °/oo  and Fleming,  The  A.  i s the f r e e z i n g p o i n t  (from Sverdrup,  Johnson,  1942). J u l y 1963,  isotonic salinity  b e f o r e s t a r t i n g the thyroid... experiments,  of a group of c o a s t a l f i s h was  determined  as  17  Figure  2.  Freezing  p o i n t d e p r e s s i o n curves f o r  —$  #—  s sea water  —  X—  s c o a s t a l s c u l p i n s caught i n A p r i l  — Q- A -  -Q-A ••  1964  % i n l a n d f i s h caught i n February and March s c o a s t a l f i s h caught i n J u l y  1963  % freezing point depression i n centigrade degrees.  1964  IO SALINITY  2 0 C%o)  18  11.0i9/oo by e x t r a p o l a t i o n from f r e e z i n g p o i n t s of b l o o d 139/oo and  268/oo  s a l i n i t y acclimated f i s h  (Fig. 2 ) . . This  used as the v a l u e f o r i s o t o n i c i t y throughout experiments.  l i m i t e d and  needed, no b l o o d t o n i c i t y  t i o n s were made o f the i n l a n d f i s h a t t h i s t i m e . and March 1964,  In  I t was  determinaFebruary  determinations  d i s c o v e r e d t h a t t h e i r b l o o d was  more d i l u t e than t h a t o f the c o a s t a l f i s h .  slightly  A repeat experiment  on c o a s t a l f i s h caught i n Campbell R i v e r lat® i n May, approximately h a l f a m i l e upstream, gave v a l u e s almost c a l t o those o b t a i n e d f o r the i n l a n d f i s h . two  a  a s u f f i c i e n t number o f i n l a n d f i s h were caught  i n minnow t r a p s i n C u l t u s Lake f o r b l o o d t o n i c i t y t o be made.  was  f o r the t h y r o i d  S i n c e the supply of i n l a n d f i s h was  r e l a t i v e l y l a r g e number was  from  experiments were i d e n t i c a l t o the f i r s t ,  1964, identi-  Both of these  last  except t h a t the  i n t e r m e d i a t e v a l u e s the f i s h were k e p t i n l l 8 / o o i n s t e a d o f 138/oo  salinity. The  reason  f o r the d i s c r e p a n c y i n the d a t a from Camp-  b e l l River f i s h i s d i f f i c u l t  t o p i n p o i n t s i n c e t h e r e are a t  l e a s t two  unknown v a r i a b l e s p r e s e n t , one  graphic.  The  f i s h i n the 1963  s e a s o n a l and one  geo-  group were caught l a t e r i n the  year a f t e r the spawning season was  over, they were a l s o caught  c l o s e r t o the e s t u a r y than were the f i s h f o r the 1964  experiment.  Both l o c a t i o n s are under t i d a l i n f l u e n c e , however. A l l the c o a s t a l f i s h used f o r the t h y r o i d and meta-  19  b o l i c r a t e experiments came from t h e downstream l o c a t i o n . F o r the t h y r o i d experiments, t h e f i s h were captured and  i n t h e l a t e summer  f a l l o f 1963, f o r t h e m e t a b o l i c r a t e d e t e r m i n a t i o n s they were  captured  d u r i n g t h e s p r i n g o f 1964.  Table I . F r e e z i n g p o i n t d e p r e s s i o n s  o f blood  serum from  p r i c k l y sculpins a f t e r acclimation t o d i f f e r e n t salinities. A  =. F r e e z i n g p o i n t  F.W. Coastal,  ll9/oo  raosm  J u l y , 1963.  Coastal,  A  0  mosm  May, 1964.  Inland  A  mosm A  c  139/oo  265>/oo  316  319  587  .593  290  298  304  .539  .555  .565  286  302  304  531  B.  1„  depression.  .561  Iodine  .565  metabolism.  Excretion of radioiodine. a.  E f f e c t s o f s i z e on e x c r e t i o n o f r a d i o i o d i n e .  Radioiodine continuously  excreted  i n j e c t e d i n t o t h e coelom o f s c u l p i n s was f o r t h e e n t i r e d u r a t i o n o f t h e experiments.  20  Hickman (1959) d e s c r i b e d  the movement of r a d i o i o d i n e from the  coelom of the s t a r r y f l o u n d e r  ( P l a t i c h t y s s t e l l a t u s ) i n t o the  v a r i o u s body compartments o f these f i s h .  There i s l i t t l e  t o b e l i e v e t h a t the path o f i n j e c t e d i o d i n e i n the seulpin i s e s s e n t i a l l y d i f f e r e n t . I ^ l  ^  s  a t  fi  r  s  t  E x c r e t i o n o f the i n j e c t e d  When t h e body i s permeated and  i s a t t a i n e d , e x c r e t i o n continues  instantaneous c o n c e n t r a t i o n  f a c t o r s s the a b s o l u t e  equilibrium  at a r a t e which i s p r o p o r t i o n a l  t o the i n s t a n t a n e o u s c o n c e n t r a t i o n  fish.  prickly  v e r y r a p i d as the r a d i o i o d i n e d i f f u s e s i n t o the  t i s s u e s o f the f i s h .  The  reason  of r a d i o i o d i n e  of I 131  131  amount o f I  ( F i g . 5 and  i n a f i s h depends on  present  and  In our experiments the i n j e c t e d dose was,  two  the s i z e of  the  i n each case,  i d e n t i c a l , w h i l e the s i z e o f the f i s h v a r i e d from about 2 gm 27 gm  at the extremes, the modal s i z e b e i n g  sequently,  the c o n c e n t r a t i o n  6).  about 7 gm.  to  Con-  at the time o f e q u i l i b r i u m would  vary. Comparison between s c u l p i n s of d i f f e r e n t s i z e s show t h a t e x c r e t i o n depends on the weight of the f i s h acclimated  t o 26o/oo s a l i n i t y  c o r r e l a t i o n between percent  (Fig. 3).  would show a s i g n i f i c a n t  e x c r e t i o n and  l o g weight  That t h e r e i s a l i n e a r r e l a t i o n s h i p between e x c r e t i o n and  the l o g a r i t h m  the untransformed v a l u e s range use.  Fish  negative  (Table I I ) . percent  o f body s i z e r a t h e r than between  o f these two  i s not obvious from the s i z e  However, t h e o r e t i c a l c o n s i d e r a t i o n s do not r e a d i l y  21  permit a l i n e a r r e l a t i o n s h i p between the untransformed v a l u e s  even  i n t h i s s m a l l range s i n c e t h i s would imply zero e x c r e t i o n a f t e r 200 hours i n animals l a r g e r than f o r t y - f i v e grams  0  Comparable  experiments done on animals where a l a r g e r s i z e range available  (Hickman, 1959)  c o n f i r m t h a t the b e s t s t r a i g h t l i n e  r e l a t i o n s h i p w i t h i n the range was logarithm  of body s i z e along the Fig.  salinities  (Qu/oo, l l o / o o and  from one  according  obtained  by p l o t t i n g the  abscissa.  4 shows the e x c r e t i o n of r a d i o i o d i n e i n t h r e e  groupsg s m a l l , medium and obtained  was  26o/oo) f o r f i s h o f t h r e e s i z e  large.  Each p o i n t r e p r e s e n t s  i n d i v i d u a l f i s h or from two  data  where p o s s i b l e  t o Table I I I . When e v a l u a t i n g the body s i z e - e x c r e t i o n of i - ^ l  relationship,  i t must be r e c o g n i z e d  t h a t the i n i t i a l  of r a d i o i o d i n e i n the a v a i l a b l e i o d i n e space would be i n the s m a l l f i s h . doeses may  I n i t i a l l y a l a r g e r percent  concentration higher  o f the i n j e c t e d  accumulate i n the t h y r o i d s of s m a l l f i s h than i n the  t h y r o i d s o f l a r g e r f i s h , t h i s may e x c r e t i o n r a t e s i n the s m a l l e r  initially  tend t o b i a s  animals towards lower  the  values.  131 However, the a b s o l u t e  amounts o f I  increases with time.  Since,  accumulated i n any  thyroid  presumably, the l a r g e r a t h y r o i d  is,  the more r a d i o i o d i n e i t can b i n d , one  may  c o n t r i b u t e t o a decreased r a t e o f t o t a l e x c r e t i o n o f i - ^ l £  the l a r g e r f i s h r e l a t i v e t o the s m a l l e r  c o u l d argue t h a t  this n  f i s h with i n c r e a s i n g time  22  after  injection. To t e s t the h y p o t h e s i s t h a t t h y r o i d s i z e per  se  131 a f f e c t s e x c r e t i o n r a t e s , the t h y r o i d r e t e n t i o n s of I 200  hours were p l o t t e d a g a i n s t the l o g a r i t h m  of f i s h from each s a l i n i t y group tions calculated  (Table V I ) .  ( F i g . 12),  In a l l cases,  of the body s i z e and  the c o r r e l a -  In f r e s h water the s l o p e of  r e g r e s s i o n l i n e i s p o s i t i v e , i n i s o t o n i c and negative.  Q  however, i t i s c l o s e t o zero and  cannot be r e j e c t e d i n any The  f a c t t h a t the s m a l l e r  the  sea water i t i s  the c o r r e l a t i o n s are too s m a l l t o be s i g n i f i c a n t , the i s that H  after  since  conclusion  case. f i s h excrete  radioiodine  f a s t e r than l a r g e r f i s h i s i n t e r p r e t e d as i n d i c a t i v e of a more r a p i d e l e c t r o l y t e metabolism i n the s m a l l e r  animals.  This i s i n  agreement with the g e n e r a l l y v a l i d statement t h a t s m a l l e r mals have h i g h e r Care was  ani-  m e t a b o l i c r a t e s than comparable l a r g e r animals.  taken, consequently, t o make comparisons between  ex-  c r e t i o n r a t e s i n d i f f e r e n t s a l i n i t i e s and between e x c r e t i o n r a t e s of c o a s t a l and  i n l a n d f i s h s t r i c t l y on the b a s i s of a n i -  mals o f comparable s i z e s .  23  F i g u r e 3.  The e f f e c t o f s i z e on the e x c r e t i o n of I 26< °/oo s a l i n i t y l  acclimated f i s h .  f i t t e d by method o f l e a s t  in  Regression  lines  squares.  Upper graph  s 24 hours a f t e r  i n j e c t i o n with  1^1.  Lower graph  s 200 hours a f t e r i n j e c t i o n with I  131  G RAM S  24  F i g u r e 4.  The e f f e c t o f s i z e on e x c r e t i o n of I  in fish  a c c l i m a t e d t o f r e s h water, t o i s o t o n i c and t o hypertonic  sea water.  average of two  Left figure  Each p o i n t r e p r e s e n t s the  fish.  s f r e s h water a c c l i m a t e d  fish.  Middle f i g u r e s l l 9 / o o s a l i n i t y  acclimated  fish.  Right f i g u r e  acclimated  fish.  A —  s 26?/oo s a l i n i t y  10 - 15 gm  fish  -®  6.0  - 8.0 gm  fish  - 0 * - 0 - -  2.5 - 4.5 gm  fish  HOURS  25  F i g u r e 5.  T o t a l body r e t e n t i o n of  i n coastal fish  acclimated to three s a l i n i t i e s < >  Regression  l i n e s were f i t t e d by t h e method o f l e a s t  —@ —Q— —  @- O— )(—  s f r e s h water a c c l i m a t e d  squares.  fish.  s ll#/oo s a l i n i t y  acclimated  fish.  s 26^/oo s a l i n i t y  acclimated  fish.  HOURS  26  F i g u r e 6.  T o t a l body r e t e n t i o n o f I  in.inland  acclimated t o three s a l i n i t i e s .  fish  Regression  were f i t t e d by the method o f l e a s t  lines  squares.  —©  @—  s f r e s h water a c c l i m a t e d  - 0  0 -  s 110/oo s a l i n i t y  acclimated  fish.  X  % 26B/oo s a l i n i t y  acclimated  fish.  X  fish.  HOURS  Table I I I .  E f f e c t of body s i z e on e x c r e t i o n of r a d i o i o d i n e i n t h r e e s a l i n i t i e s . value i s the average  of two  Each  f i s h except those marked w i t h * which are  from one f i s h o n l y . Hours  Av. wto  Small (gm) Av. E x c r . {%]  Av. wt.  Medium (gm) Av. E x c r .  24  13.1  .9  6.8  9.6  48  13.7  9.5  7.5  9.0  120  11.0  12.1  196  10.5  13.9  7.1  24  13.2  1.2  0 48 o 0_, 72  11.6  (%)  Large Av.wt. (gm)  Av.Excr.(%)  2.8  15.3  2.4  17.1  21.1  2.8  18.7  7.0  17.0  4.5*  18.2  12.7  6.8  22.8  3.3*  26.1  11.8  18.7  7.1  26,8  4.5*  38.2  150  12.4  28.0  4.3  47.0  200  11.7  29.7  7.1  43.3  2.9*  58.5  24  11.5*  27.3  6.5  29.6  3.7  33.5  48  12.0  39.6  7.1  43.8  13.5*  43.4  6.9  47.3  3.1  51.9  6.9  61.3  fa  8 %  7 2  96  CM  150  12.4  59.5  7.0  71.7  4.7  76.8  200  11.5*  61.5  7.2*  74.6  4.5  79.0  28 Table I I .  C o r r e l a t i o n s between percent e x c r e t i o n o f r a d i o i o d i n e and l o g weight o f f i s h a f t e r a c c l i m a t i o n t o 26S/oo s a l i n i t y .  H = Q  no s i g n i f i c a n t c o r r e l a t i o n ,  d f = degrees o f freedom. Time a f t e r injection  df  24 hours  =-.624  14  200 hours  -.866  14  Conclusion Highly s i g n i f i c a n t . Each H rejected. Q  b. E f f e c t o f environment on e x c r e t i o n o f r a d i o i o d i n e . As e a r l y as i n 1932, Keys had e s t a b l i s h e d t h a t t e l e o s t f i s h e s would l o s e sodium by way o f the g i l l s hypertonic  media.  ocean, h y p e r t o n i c  To compensate  when exposed t o  f o r osmotic water l o s s i n the  sea water i s swallowed.  Water and sodium  c h l o r i d e , and presumably other u n i v a l e n t c a t i o n s and h a l i d e s can move across  the w a l l o f the i n t e s t i n e i n t o t h e blood  while  most o f t h e d i v a l e n t ions are e x c r e t e d . In f r e s h water, however, the t u r n o v e r o f ions i s considerably  slower.  membranous s u r f a c e s an osmotic g r a d i e n t , as d i l u t e u r i n e .  In t h i s medium, water flows across the o f t h e f i s h , p a r t i c u l a r l y the g i l l s , and i s subsequently e x c r e t e d  along  by the k i d n e y s  When r a d i o i o d i n e i s i n j e c t e d i n t o s c u l p i n s  acclimated  t o media r e p r e s e n t i n g  containing  the same amount o f i n o r g a n i c i o d i n e , then the ex-  c r e t i o n rates of i ^ l f i e c t r e  a range o f s a l i n i t i e s but  the i n c r e a s e d  t u r n o v e r o f serum  29  e l e c t r o l y t e s at h i g h e r there and  salinities  ( F i g s . 5 and  6).  Initially  i s a very r a p i d l o s s of r a d i o i o d i n e which i s soon reduced  t h e r e a f t e r f o l l o w s a l o g a r i t h m i c p a t t e r n a f t e r the content of  r a d i o i o d i n e i n the v a r i o u s body compartments has brium.  reached  T h i s appears t o have been e s t a b l i s h e d w i t h i n the  24 hours i n a l l s a l i n i t i e s .  first  At approximately i s o t o n i c concen-  t r a t i o n s the e x c r e t i o n r a t e s are i n t e r m e d i a t e f r e s h water and  equili-  26o/oo s a l i n i t y  sea water.  assumed t h a t r a d i o i o d i n e i s excreted of other h a l i d e s , the i n t e r m e d i a t e  between those of  Since  i t can  be  along the common pathways  excretion of r a d i o i o d i n e i n  i s o t o n i c medium i n d i c a t e s t h a t t h e r e i s a c o n s i d e r a b l e  excre-  t i o n o f c h l o r i d e from the t i s s u e s of the f i s h i n the i s o t o n i c environment.  The major pathway o f c h l o r i d e e x c r e t i o n i n sea  water i s v i a the g i l l s ,  a f t e r d r i n k i n g o f the h y p e r t o n i c  During d i s s e c t i o n o f s c u i p i n s i t was the stomachs o f a l l f r e s h water a c c l i m a t e d  medium.  n o t i c e d that while  f i s h appeared com-  p l e t e l y c o l l a p s e d , as might have been expected f o r f i s h i n the post  absorptive  state, a considerable  f i s h from both the other l e s s distended t h i s may t o n i c but  and  two  number (but not a l l ) o f  groups had  the  t h e i r stomachs more or  f i l l e d w i t h a watery f l u i d .  The  cause o f  be t h a t the s c u i p i n s swallow water not o n l y i n hypera l s o i n i s o t o n i c media.  assume a g r a d u a l  Furthermore, s i n c e we  must  change i n osmoregulatory behaviour i n going  from f r e s h water t o i s o t o n i c i t y t o sea water, i t appears t h a t  30  swallowing o f t h e medium i n s m a l l q u a n t i t i e s takes p l a c e even i n hypotonic  medium.  Simple i n t e r p o l a t i o n between the body r e t e n -  t i o n o f r a d i o i o d i n e i n f r e s h , i s o t o n i c and sea water  ( F i g . 7)  i n d i c a t e s a r a p i d i n c r e a s e i n e x c r e t i o n r a t e o f r a d i o i o d i n e and a l s o , presumably, o f c h l o r i d e , both commencing immediately on the presence o f e l e c t r o l y t e s i n the water. One c o u l d i n t e r p r e t t h i s as decreased a b s o r p t i o n o f i o n s by t h e k i d n e y t u b u l e s with d e c r e a s i n g  hypotonicity.  How-  ever, Holmes e t a l . (1963) p o i n t out t h a t rainbow t r o u t i n sea water have c o n s i d e r a b l y reduced u r i n e output b u t s t i l l t u b u l a r a b s o r p t i o n o f about 90 percent by glomerular  filtration.  show a  of the c h l o r i d e excreted  A more l i k e l y i n t e r p r e t a t i o n o f t h e  e x c r e t i o n r a t e s o f s c u i p i n s appears t o be t h a t w i t h the appearance o f e l e c t r o l y t e s i n t h e ambient medium, t h e b a r r i e r a g a i n s t outward flow o f ions through-'c.the • giil^lsiubecome^ l«ssiie£^icien± and ions are permitted  t o leak o u t . T h i s i s then compensated f o r by  d r i n k i n g and by e x c r e t i o n through t h e k i d n e y s o f excess water; the k i d n e y s a r e now r e l i e v e d o f some o f t h e waterload osmotic d i f f u s i o n The  caused by  alone.  r e g r e s s i o n s i n F i g . 5 and 6 were t e s t e d f o r s i g -  n i f i c a n t d i f f e r e n c e s by means o f Student's t t e s t s .  The s l o p e  of each l i n e was compared with the s l o p e o f each o f the other l i n e s r e p r e s e n t i n g d i f f e r e n t environmental s a l i n i t i e s o f f i s h from the same g e o g r a p h i c a l  region.  In each i n s t a n c e the  31  F i g u r e 7«,  The  e f f e c t of s a l i n i t y  hours a f t e r i n j e c t i o n . and  r e p r e s e n t s the  inland  data.  on  e x c r e t i o n of I  The  curve was  average of  (*)  200  f i t t e d by  coastal  and  eye (•)  CM  C°/<0  ^  <C  CD  NOI13«DX3  32  s t a t i s t i c a l v a l u e i s s i g n i f i c a n t on the 1% l e v e l and  H  (no  Q  s i g n i f i c a n t d i f f e r e n c e between the e x c r e t i o n r a t e s i n the s a l i n i t i e s compared) was  rejected  (Table  two  IV)»  In other words, the i n f l u e n c e of the environment on jl31  e x c r e  r a t e of  tion  i s such t h a t a h i g h e r s a l i n i t y causes a h i g h e r  excretion.  c.  Comparisons between e x c r e t i o n r a t e s c o a s t a l and  inland  of  fish.  Coastal p r i c k l y sculpins excrete radioiodine but  slightly  s i g n i f i c a n t l y f a s t e r than the i n l a n d ones i n f r e s h water  (Fig.  8 and  9).  In sea water o f ll°</oo and  26$/oo s a l i n i t y  d i f f e r e n c e s i n e x c r e t i o n r a t e s a f t e r the f i r s t significant groups do initial  (Table v ) .  24 hours are  However, i n these s a l i n i t i e s ,  the  the not  two  show a d i f f e r e n c e , namely t h a t t h e r e i s a more r a p i d  e x c r e t i o n i n c o a s t a l than i n i n l a n d f i s h .  w i t h i n 24 hours, and  This l e v e l s o f f  a f t e r t h a t time the e x c r e t i o n r a t e s  are  approximately the same. To t e s t the assumption t h a t the slower i n i t i a l in  excretion  i n l a n d f i s h adapted t o a s a l i n e environment i s at l e a s t i n 131  p a r t a r e f l e c t i o n of a more r a p i d uptake of I  by the  thyroids  o f these f i s h , the e x t r a t h y r o i d a l e x c r e t i o n r a t e s were compared (Fig.  10 and  11)„  In a l l environments the r e l a t i v e p o s i t i o n s of  the e x t r a t h y r o i d a l r e g r e s s i o n l i n e s are approximately the same as  33  Figure  8.  T o t a l body r e t e n t i o n o f I f r e s h water.  i n f i s h acclimated to  Regression l i n e s f i t t e d by the method  of l e a s t squares.  —® — 0—  @— -0 —  s coastal  ° inland  fish. fish.  60h  o r  AO -  UJ uJ  o:  20 I SO  I IOO  _l ISO  HOURS  L_  2 0 0  34  131 F i g u r e 9.  T o t a l retention of I water  0  i n f i s h a c c l i m a t e d t o sea  Regression l i n e s f i t t e d by t h e method  o f l e a s t squares. —A  &—  s coastal fish,  ll8/oo  —A  A-  s inland fish,  —H§  JH—  s coastal fish,  —0—  ~D—  g  inland f i s h ,  salinity  ll°/oo s a l i n i t y 269/oo s a l i n i t y  26#/oo  salinity  HOURS  35  Figure  10.  E x t r a t h y r o i d a l r e t e n t i o n of I t o f r e s h water.  in fish  acclimated  Regression l i n e s f i t t e d by  method o f l e a s t squares.  —®  @—  s coastal  -O  O—  t inland  fish. fish.  the  IOO  HOURS  ISO  2 0 0  36  131 Figure  11.  Extrathyroidal retention of I to l l 8 / o o s a l i n i t y (lower c u r v e s ) .  (upper) and 26oVoo  acclimated  salinity  Regression l i n e s f i t t e d by t h e  method of l e a s t squares. —#-—fc-  % coastal  —0  s inland  0-  in fish  fish. fish.  HOURS  37 those r e p r e s e n t i n g  t o t a l excretions.  The  t r e n d t o slower  initial  e x c r e t i o n i n the c o a s t a l f i s h i n s a l i n e environment i s s t i l l  evi-  dent . Wiggs (1962) d i s c u s s e d d i u r e s i s i n the t h r e e spined aculeatus).  the p o s s i b i l i t y o f  stickleback  (Gasterosteus  He proposed t h a t the s t r e s s induced by  and d r a s t i c changes i n environment may  back, although h i s experiments d i d not o f f e r any  ferences being  due  I t may  treatment  be the reason f o r h i g h  i n i t i a l e x c r e t i o n r a t e s observed e x p e r i m e n t a l l y  evidence f o r t h i s .  laboratory  i n the  stickle-  exclusive  be p o s s i b l e t o i n t e r p r e t our  dif-  i n excretion rates i n inland versus c o a s t a l f i s h  as  t o d i f f e r i n g s e n s i t i v i t y t o l a b o r a t o r y s t r e s s , i f we  assume t h a t l a b o r a t o r y d i u r e s i s i s the cause o f the h i g h excretion rates.  initial  I t i s a l s o p o s s i b l e t h a t i n the i n l a n d f i s h  the  i n j e c t e d i o d i n e w i l l d i f f u s e i n t o the t o t a l a v a i l a b l e i o d i n e space f a s t e r than i n the c o a s t a l f i s h , be so, i s hard t o  say.  although why  this  should  38 Table IV.  Values o f Student's t t e s t s clusion  about H  and a p p r o p r i a t e con-  (no s i g n i f i c a n t d i f f e r e n c e ) from  0  comparisons between e x c r e t i o n r a t e s i n d i f f e r e n t environmental s a l i n i t i e s .  Fish  Compared  Coastal  FW  -  P <^ .01  t  II8/00  6.041  ; highly  significant.  df  Conclusion  17  Highly s i g n i f i c a n t . H rejected. Q  ll-8/oo  -  26 /oo Q  7.769  54  Highly s i g n i f i c a n t . H rejected. Q  Inland  FW  -  ll8/oo  26  8.585  Highly s i g n i f i c a n t . H rejected. Q  ll8/oo  -  268/00  20  9.151  Highly s i g n i f i c a n t . H rejected. Q  Table V.  Comparisons by Student's t t e s t s between e x c r e t i o n rates o f c o a s t a l versus c o n c l u s i o n s about H  Salinity FW  .01 s h i g h l y  q  inland f i s h with appropriate  (no s i g n i f i c a n t  P  (  P  <^ .05 s not s i g n i f i c a n t .  t 2.779  difference).  significant.  Conclusion  df 47  Highly s i g n i f i c a n t . H rejected. Q  llo/oo  1.010  52  Not not  significant. rejected.  H  Q  268/00  .826  20^df^48  Not not  significant. rejected.  H  Q  39  F i g u r e 12.  E f f e c t o f s i z e on t h y r o i d hours a f t e r  retention  injection. A.  26#/oo,  B.  ll°/oo, and  C.  0°/oo s a l i n i t y .  of I  200  GRAMS  40 2.  Thyroid  uptake o f r a d i o i o d i n e ,  a»  E f f e c t o f s i z e on t h y r o i d  The  s i z e e f f e c t on t h y r o i d r e t e n t i o n was e v a l u a t e d by  by c a l c u l a t i n g r e g r e s s i o n c.  activity.,  l i n e s as presented i n F i g . 12a, b and  C o r r e l a t i o n c o e f f i c i e n t s , p r e s e n t e d i n Table VI, are not  significant.  In small  f i s h , presumably r a p i d e x c r e t i o n i s  compensated f o r by a c o r r e s p o n d i n g l y r a p i d t h y r o i d uptake w h i l e i n l a r g e r f i s h t h e slower e x c r e t i o n  l e a v e s more  radioiodine  a v a i l a b l e b u t t h e t h y r o i d metabolism i s a l s o slower.  Since  comparisons between d i f f e r e n t s a l i n i t y groups and between c o a s t a l and i n l a n d f i s h a r e based on TUF v a l u e s  (see page 41)  r a t h e r than on t h y r o i d uptake per se, a d e t e r m i n a t i o n was made o f t h e i n f l u e n c e o f s i z e on c o r r e s p o n d i n g TUF v a l u e s .  In fresh  131 water low,  adapted f i s h , where t h e I  t h e r e was no evident  uptake by t h e t h y r o i d s i s  c o r r e l a t i o n between TUF and s i z e .  In  s a l i n e environments, however, t h e t h y r o i d uptake f a c t o r s were found t o be s i z e dependent  (Table V I I ) .  Comparisons between  t h y r o i d uptake f a c t o r s i n d i f f e r e n t groups o f f i s h  therefore,  were made s t r i c t l y on t h e b a s i s o f f i s h o f comparable s i z e . b.  E f f e c t o f environment on t h y r o i d  Radiological estimation bases on two f a c t o r s ? uptake o f I and  activity.  o f t h y r o i d a c t i v i t y may be by t h e t h y r o i d  follicles  s e c r e t i o n o f radio-hormone from t h e f o l l i c l e s i n t o t h e  b l o o d stream.  Hoar and E a l e s  (1963) compared t h e r a t i o s between  41 Table VT.  C o r r e l a t i o n s between l o g s i z e and  thyroid  reten-  131 tions of I  200  hours a f t e r i n j e c t i o n i n pooled  d a t a from c o a s t a l and f r e s h , i s o t o n i c and H  0  Medium  no  inland f i s h acclimated  26°/oo s a l i n i t y sea  to  water.  significant correlation. r  df  Freshwater  -.200  20  ll°/oo s a l i n i t y  -.334  20  Conclusion  Not s i g n i f i c a n t . H 26°/oo s a l i n i t y  Table VII.  -.365  not  rejected,  14  C o r r e l a t i o n s between l o g s i z e and pooled d a t a from c o a s t a l and  TUF  values i n  i n l a n d f i s h 200  hours  131 a f t e r i n j e c t i o n with I df  Conclusion  F r e s h water  .287  20  Not s i g n i f i c a n t . H accepted.  llR/oo s a l i n i t y  -.514  20  Highly s i g n i f i c a n t . rejected.  H  Q  26°/oo s a l i n i t y  -.724  14  Highly s i g n i f i c a n t . rejected.  H  Q  t h y r o i d c o n t e n t and  t o t a l body content of r a d i o i o d i n e  f i s h exposed t o v a r i a b l e s o t h e r than s a l i n i t y changes. r a t i o they c a l l e d t h y r o i d uptake f a c t o r  (TUF)s  Q  i n goldThis  42  per cent of injected do>s© found i n thyroid  T U F  per cent of injected dose found i n body including thyroid Regressions of thyroid uptake factors on time i n coastal f i s h acclimated to three s a l i n i t i e s are compared i n F i g . 13 and the regression c o e f f i c i e n t s compared i n Table VIII.  The  rate of thyroid uptake of radioiodine i n fresh water and i s o tonic sea water i n these f i s h i s not s i g n i f i c a n t l y d i f f e r e n t . Table VIII.  S t a t i s t i c s from regression  l i n e s of TUF versus time  i n f i s h acclimated to d i f f e r e n t s a l i n i t i e s compared by Student's t t e s t s . ence.  Q  s no s i g n i f i c a n t d i f f e r -  P <^ ,o/ % highly s i g n i f i c a n t , b i s regres-  sion c o e f f i c i e n t ,  Environment  H  Parameter Compared  a i s intercept of regression  t  line,  Conclusion  FW - I I 8 / 0 0 1  b  +JFW - l l o / o o  a  2.347  0 i r 9 / o o - 26c9/oo  Highly s i g n i f i c a n t . H "o rejected.  b  7.56  Highly s i g n i f i c a n t . H rejected.  1.779  No s i g n i f i c a n t difference. H accepted. Q  Q  "OFW - llf?/oo ft 5ll'9/oo - 26<9/oo  b  4.89  b  7.90  Highly s i g n i f i c a n t . Ho rejected. Highly s i g n i f i c a n t , H rejected. Q  However, i n the group acclimated to i s o t o n i c medium, the TUF values increased  considerably  than i n the fresh water group.  faster during the f i r s t 24 hours This i s i n part a r e f l e c t i o n of  43 the f a c t t h a t TUF  a f t e r 24 hours of e x c r e t i o n i s c a l c u l a t e d  on  the b a s i s o f twelve per cent more r a d i o i o d i n e remaining i n the f r e s h water group than i n the i s o t o n i c group. t r i b u t i o n t o the h i g h e r TUF  But the main con-  values i n i s o t o n i c acclimated  fish  comes from a more r a p i d accumulation of r a d i o i o d i n e i n the r o i d f o l l i c l e s during  the f i r s t  24 hours a f t e r i n j e c t i o n (Table  S i n c e the t h y r o i d f o l l i c l e s are d i s p e r s e d jaw,  and must be d i s s e c t e d out  thy-  i n the  i n a mass w i t h t h i s q u i t e  IX).  lower  vascular  t i s s u e , some o f the r a d i a t i o n observed i n t h y r o i d measurements w i l l have o r i g i n a t e d i n e x t r a t h y r o i d a l i n o r g a n i c Consequently, the observed r a p i d i n c r e a s e the f i r s t  radioiodide.  i n t h y r o i d uptake w i t h i n  24 hours i s i n p a r t o n l y apparent? some o f t h i s r a d i a -  t i o n i s background r a d i a t i o n from e x t r a t h y r o i d a l t i s s u e . timate o f the background r a d i a t i o n was  made, on the  An  es-  assumption  t h a t the e x t r a t h y r o i d a l t i s s u e o f the lower jaw w i l l c o n t a i n same amount o f r a d i o i o d i n e per mg does on the average.  the  weight as the r e s t of the body  Compiled i n T a b l e X are o b s e r v a t i o n s  of  the  p e r c e n t c o n t r i b u t i o n o f the lower jaw t o the t o t a l body weight. I t w i l l be  seen t h a t the background r a d i a t i o n which may  t e d from e x t r a t h y r o i d a l t i s s u e of the lower jaw  be  expec-  i s a t the most a  l i t t l e over one h a l f p e r c e n t . Presumably a l l t h r e e  s a l i n i t y groups o f c o a s t a l  accumulate r a d i o i o d i d e i n t h e i r t h y r o i d s a t approximately same r a t e f o r the f i r s t  f i v e t o ten hours a f t e r i n j e c t i o n .  fish the The  44  F i g u r e 13.  Rate o f i n c r e a s e i n TUF a f t e r i n j e c t i o n o f radioiodine into coastal three s a l i n i t i e s . method o f l e a s t  f i s h acclimated i n  Regression l i n e s f i t t e d by  squares.  —®—•—©—  % fresh  —O —  s HP/oo  salinity  s 26°/oo  salinity  — ^  -0-  water  45  F i g u r e 14  0  Rate o f i n c r e a s e o f TUF a f t e r iodine into salinitieso of l e a s t  — ©  inland  %  f i s h acclimated i n three  R e g r e s s i o n l i n e s f i t t e d by method  squares.  ®—  — 0—  i n j e c t i o n of radio-  s fresh  — -O — X—  §  water  ' ll°'/oo  salinity  26°/oo  salinity  46 26°/oo s a l i n i t y  ;  group o f f i s h c o n t i n u e a t approximately t h i s same  r a t e throughout the experiment, w h i l e t h e f r e s h water f i s h and the  i s o t o n i c group b e g i n t o slow down a f t e r about 10 and 20 hours  respectively.  The time o f supernormal t h y r o i d uptake  with the period  of very high concentration  blood, before establishment of r a d i o i o d i n e the v a r i o u s  coincides  of radioiodine  i n the  e q u i l i b r i u m between  body compartments.  Fig  0  14 shows the t h y r o i d uptake p a t t e r n  of inland  fish.  There i s one important d i f f e r e n c e from the t r e n d s e x h i b i t e d by coastal f i s h .  The d i f f e r e n c e i n slopes  l i n e s representing  between the r e g r e s s i o n  f r e s h water and the i s o t o n i c a c c l i m a t e d f i s h i s  somewhat l a r g e r i n t h e i n l a n d f i s h .  Student's t t e s t s show t h a t  the d i f f e r e n c e i s l a r g e enough t o be accepted as r e a l , w h i l e i n the case o f the c o a s t a l f i s h the c o r r e s p o n d i n g d i f f e r e n c e i s not significant Table I X o  (Table  VIII).  Contributions  o f t h y r o i d uptake t o c a l c u l a t e d TUF  v a l u e s a t 24 hours a f t e r i n j e c t i o n . sents t h e group average.  Group " ' F r e s h water  TU  Isotonic  3.5  2.7  repre-  TU s t h y r o i d uptake i n p e r -  c e n t o f t o t a l i n j e c t e d dose. factor  Each v a l u e  TUF § t h y r o i d uptake  (see t e x t ) . % retention ' ' ~~ ' 2.7 91 — — - — — . 9 JL ""(' !2 © 7 3.5 80 80 + 3.5  TUF " " " ' x 100 = 2.9%  X  IQQ ^ 4 2 % D  ~~~  47 T a b l e X,  Contributions  o f lower jaw t i s s u e s t o t o t a l body-  weight „ Weight o f t h y r o i d  Weight o f thyroid tissue  Weight o f fish  .098 gm  17.1 gm  .063 gm  9. 3 gm  . 68%  .060 gra  8.9 gm  .68%  .039  gm  6.9 gm  .57%  .037 gra  6.5 gm  .57%  .029  gm  5.4 gm  .54%  .015 gm  2.1 gm  .72%  x  c.  4.34%  tissue  x 100  Weight o f f i s h .58%  x  0.62%  Comparisons o f t h y r o i d a c t i v i t y i n c o a s t a l versus inland  Although the t r e n d  fish.  t o f a s t e r t h y r o i d uptake i n h i g h e r  s a l i n i t y i s demonstrated by both c o a s t a l and i n l a n d f i s h , the i n l a n d f i s h do, on the average, tend t o have more a c t i v e thyroids  i n s a l i n e water than do the c o a s t a l ones  (Fig,, 15) .  Most o f t h i s h i g h e r a c t i v i t y seems t o m a n i f e s t i t s e l f the  first  sions  24 hours a f t e r i n j e c t i o n .  from c o a s t a l  f i s h with regressions  comparative s a l i n i t y . rate of increase  Table XI compares  within regres-  from i n l a n d f i s h o f  The o n l y s i g n i f i c a n t d i f f e r e n c e  i n the  o f TUF i s between the two f r e s h water groups.  48 However, d u r i n g  the f i r s t  24 hours the i n l a n d f i s h from  environment appear t o accumulate r a d i o i o d i n e faster  in their  saline  thyroids  than the c o a s t a l f i s h . The d i f f e r e n c e s are not s u f f i c i e n t l y l a r g e t o g i v e  s i g n i f i c a n t d i f f e r e n c e s i n the i n t e r c e p t s o f the r e g r e s s i o n lines.  There i s , however, both i n i n l a n d as w e l l as i n c o a s t a l  f i s h an i n c r e a s i n g v a r i a b i l i t y i n the r e g r e s s i o n s  Table X I .  S t a t i s t i c s from r e g r e s s i o n  of thyroid  l i n e s o f TUF versus time  i n c o a s t a l f i s h compared by Student's t t e s t s t o corresponding s t a t i s t i c s from i n l a n d forms, b = regression c o e f f i c i e n t , . 05^> P)> .01 % s i g n i f i c a n t , H  c  = no s i g n i f i c a n t  a - intercept. P^>. .05 s not s i g n i f i c a n t .  difference.  Environment  Parameter  t  Conclusion  F r e s h water  b  2.398  F r e s h water  a  0.666 Not s i g n i f i c a n t . H  Q  not r e j e c t e d  lltf/oo s a l i n i t y  b  1.130 Not s i g n i f i c a n t . H  Q  not r e j e c t e d  ll°/oo s a l i n i t y  a  0.715 Not s i g n i f i c a n t . H  Q  not r e j e c t e d  26°/oo s a l i n i t y  b  0.562 Not s i g n i f i c a n t . H  Q  not r e j e c t e d  26°/oo s a l i n i t y  a  0.437 Not s i g n i f i c a n t . H  Q  not r e j e c t e d  Significant.  H  rejected.  Q  49  F i g u r e 15.  Rate o f i n c r e a s e i n TUF a f t e r a c c l i m a t e d tos A. f r e s h and C. 268/oq  Solid  injection  water; B. ll°/oo  salinity.  lines  § coastal  Broken l i n e s , s i n l a n d  fish. fish.  of f i s h salinity  50 uptake f a c t o r s on time with i n c r e a s i n g estimate o f the t r u e p o p u l a t i o n  parameters and t h e v a l u e s o f  Student's t i n c r e a s i n g l y u n c e r t a i n . mates f o r each r e g r e s s i o n  Table X I I .  Variability fish,  s a l i n i t y which makes the  Standard e r r o r s o f the e s t i -  l i n e are compared  i n Table X I I .  i n TUF v a l u e s i n c o a s t a l and i n l a n d  s s standard e r r o r o f the e s t i m a t e .  Group  Environment  s o f TUF  Sample  Coastal  F r e s h water  1.1219  17  Coastal  llB/oo s a l i n i t y  2.3115  26  Coastal  26°>/oo s a l i n i t y  3.9380  29  1.2073  33  Inland  F r e s h water  Inland  ll°/oo s a l i n i t y  4.2685  27  Inland  26°/oo s a l i n i t y  9.0502  23  size  3. C i r c u l a t i n g radiohormone. a. Determination o f c o n v e r s i o n r a t i o s as a measure of t h y r o i d  activity.  The use o f t h e c o n v e r s i o n r a t i o  (CR) o f r a d i o i o d i n e as  an index o f t h y r o i d metabolism was d i s c u s s e d 1961) and by Hoar and E a l e s  (1963).  by Hickman  (1959,  I t i s c a l c u l a t e d as the 131  ratio  o f p r o t e i n bound r a d i o i o d i n e ,  iodine %  (PBI  ), t o t o t a l plasma  51 PQJ-13X Q p m p  B I  ux  c.p.m. -f Serum l  i  J  A  Cop.ra.  where c.p.m. i s counts p e r minute. I d e a l l y , the change i n c o n v e r s i o n i n j e c t i o n of radioiodine gives t i n g t h y r o i d hormone.  r a t i o with time a f t e r  an index o f t h e l e v e l o f c i r c u l a -  I t i s not e s t a b l i s h e d which i s t h e a c t i v e  form o r forms o f t h y r o i d hormone.  Both t e t r a - and t r i i o d o t h y -  r o s i n e as w e l l as d i - and monoiodothyrosine have been demonstrated t o possess t h y r o i d hormone l i k e p r o p e r t i e s t o some degree i n various  species.  attached  A l l these f o u r compounds have been  reported  t o p r o t e i n as t h y r o g l o b u l i n from i n v e s t i g a t i o n s o f t e l e -  ost f i s h e s .  P r e c i p i t a t i o n o f the plasma p r o t e i n s with  tri-  c h l o r a c e t i c a c i d thus removes t h e compounds w i t h t h y r o i d hormone l i k e p r o p e r t i e s The  from t h e supernatant.  separation  o f PBI  by Wiggs (1963), t o be s u b j e c t  from serum I  t o error unless  was shown  care  i s taken  immediately t o remove the f r e s h l y p r e c i p i t a t e d p r o t e i n from the supernatant.  He found t h a t , i n t h e t h r e e s p i n e d  (Gasterosteus a c u l e a t u s ) , auratus),  as w e l l as i n g o l d f i s h  the v a l u e o f c o n v e r s i o n  with time a f t e r a d d i t i o n o f TCA. the v a l u e o f the c o n v e r s i o n hours.  stickleback  r a t i o s increase  (Carassius exponentially  In one s e r i e s o f s t i c k l e b a c k s  r a t i o doubled i n approximately t e n  I n view o f t h i s , c a r e was taken t o e f f e c t t h e s e p a r a t i o n  of p r e c i p i t a t e d p r o t e i n and PBI as f a s t as p o s s i b l e from t h e  52  supernatant c o n t a i n i n g r e l a t i v e l y large amounts o f inorganic  According  t o Leloup and F o n t a i n e  (1960),  i o d i d e i s l o o s e l y bound t o p r o t e i n i n the b l o o d  inorganic  o f the l i v i n g  t e l e o s t , and t h i s bond i s broken when the p r o t e i n s are dena-  tured w i t h TCA.  However, i t i s not i m p o s s i b l e  removal o f i n o r g a n i c  that the  i o d i d e from serum p r o t e i n d u r i n g t r e a t -  ment w i t h TCA i s l e s s complete i n some s p e c i e s than i n other specieso I t w i l l be noted from the i l l u s t r a t i o n s and  ( F i g s . 16  17), t h a t i n no case does e x t r a p o l a t i o n o f t h e r e g r e s s i o n  l i n e s of conversion at zero.  r a t i o s on time, i n t e r c e p t the a b s c i s s a  T h i s may p o s s i b l y be due t o serum i o d i d e  p r e c i p i t a t e d together  being  with FBI so t h a t some o f t h e i n o r g a n i c  r a d i o i o d i n e contributes t o the r e g i s t e r e d r a d i o a c t i v i t y of the PBi^31  s i n c e the serum l e v e l o f i n o r g a n i c I""" ^ decreases 3  o  with time, the e f f e c t would be l a r g e r i n samples taken e a r l i e r than i n samples taken l a t e r and hence would c o n t r i b u t e t o negative  regression c o e f f i c i e n t s of the per.cent  conversion  r a t i o versus time p l o t . Another e x p l a n a t i o n  i s possible.  The r e l a t i v e l y  r a p i d accumulation o f r a d i o i o d i n e by the t h y r o i d s o f f i s h from most o f the experimental groups, n o t i c e d e a r l i e r , may have been the cause o f a r a p i d flow o f r a d i o a c t i v e hormone i n t o the b l o o d  53  F i g u r e 16.. C o n v e r s i o n r a t i o s f o r c o a s t a l and i n l a n d '  " a c c l i m a t e d t o f r e s h water and 26°/oo sea water.  fish  salinity  R e g r e s s i o n l i n e s c a l c u l a t e d by the  method of l e a s t squares * — --()•-  —HJ" - • -  s c o a s t a l f i s h , f r e s h water. —o  § inland,  fissb, f r e s h water.  S — 8 c o a s t a l f i s h , 26°/oo s a l i n i t y . £ i n l a n d f i s h , .26°/oo  salinity.  SO  IOO  ISO  HOURS  2 0 0  54  F i g u r e 17.  Conversion r a t i o s f o r c o a s t a l and i n l a n d a c c l i m a t e d t o 11-/oo s a l i n i t y .  Regression l i n e s  f i t t e d by the method o f l e a s t squares.  S  A— s c o a s t a l  -A — — —  s  inland  fish.  fish.  fish  55  stream o f these f i s h , the f i r s t  24 hours.  a flow which had reached  i t s peak w i t h i n  I f the t h y r o i d goes i n t o abnormally  p r o d u c t i o n of hormone i n response  high  t o the s t r e s s caused by  injec-  t i o n , t h i s might cause the c o n v e r s i o n r a t i o s t o decrease  with  time i f the hormone p r o d u c t i o n f a l l s t o a near zero l e v e l  after  the s t r e s s c o n d i t i o n has been overcome.  b.  E f f e c t s o f s a l i n i t y and h a b i t a t on c o n v e r s i o n ratios.  The per cent c o n v e r s i o n r a t i o i n c r e a s e s w i t h time i n sea water a c c l i m a t e d f i s h , while i n f r e s h water a c c l i m a t e d t h e r e i s , on the average,  a slow d e c l i n e .  fish  In f r e s h water, as  w e l l as i n sea water, the P B I ^ l l e v e l i s somewhat h i g h e r i n i n l a n d s c u l p i n s than i n the c o a s t a l groups.  At 72  hours,  r a t h e r h i g h v a l u e s were o b t a i n e d f o r the c o a s t a l sea water group. line,  These were not i n c l u d e d i n c a l c u l a t i o n of the r e g r e s s i o n s i n c e they p r e s e n t a r a t h e r l a r g e d e v i a t i o n from  straight line  any  relationship.  F i g . 17 r e p r e s e n t s the v a l u e s o b t a i n e d from the i n t e r m e d i a t e group.  In an experiment done on c o a s t a l f i s h  caught i n the middle  of J u l y , extremely  high conversion r a t i o s  o f about 15 t o 20% were o b t a i n e d a t 24 and 48 hours f o r the f r e s h water and i n t e r m e d i a t e groups, but not f o r the sea water group.  Repeat experiments on f i s h caught t h r e e months l a t e r  56 f a i l e d completely t o c i n f i r m these r e s u l t s . experiments gave low v a l u e s throughout  Instead,  these  f o r t h e f r e s h water f i s h ,  but f o r the i n t e r m e d i a t e group v a l u e s which i n c r e a s e with  time  a t a r a t e which i s s u r p r i s i n g l y h i g h when compared t o the r a t e s o b t a i n e d from t h e o t h e r groups. In summary, t h e c o n v e r s i o n r a t i o s show t h a t t h e r e i s a markedly h i g h e r l e v e l o f c i r c u l a t i n g t h y r o i d hormone i n sea water a c c l i m a t e d f i s h than i n f r e s h water a c c l i m a t e d f i s h .  There  i s some i n t e r r a c i a l d i f f e r e n c e i n t h a t the i n l a n d group shows h i g h e r P B I ^ ^ l e v e l s , p a r t i c u l a r l y i n 26°/oo s a l i n i t y . 3  In i n t e r m e d i a t e s a l i n i t y t h e r e i s an i n t e r m e d i a t e r a t e o f i n c r e a s e i n p e r cent c o n v e r s i o n r a t i o i n t h e i n l a n d  fish,  while the c o a s t a l group shows a s u r p r i s i n g l y h i g h r a t e o f increase.  4.  The reason  f o r t h e l a t t e r i s not understood.  E f f e c t s o f sex on i o d i n e metabolism. F i s h o f o p p o s i t e sex and o f no more than 0.5 gm  d i f f e r e n c e i n weight were matched i n p a i r s .  Each p a i r was  s e l e c t e d from the same group w i t h r e s p e c t t o treatment  (accli-  mation s a l i n i t y and time o f s a c r i f i c e ) so t h a t the o n l y w i t h i n p a i r d i f f e r e n c e was sex and  0.5 gm weight.  o f males e q u a l l e d t h a t o f females  The average weight  (Tables XIV and XV,  Appendix).  Seventeen p a i r s o f c o a s t a l f i s h and seventeen i n l a n d f i s h were used.  S i x n u l l hypotheses were formeds  pairs of  57  no 1)  excretion  female f i s h  b.  the  the c o a s t a l  race  the  c o n v e r s i o n r a t i o s from males and the c o a s t a l  a.  The c a l c u l a t e d and  i n l a n d race  females of the  i n l a n d race  i n t e r s e x comparisons performed by (Table XVI  H l b and Q  i n the  b.  females of  d i f f e r e n c e s w i t h i n each matched p a i r o f f i s h were  signed rank t e s t  implies  race  of  i n l a n d race  t h y r o i d uptake f a c t o r s from males and a.  3)  r a t e s from male and  the c o a s t a l r a c e  a. 2)  s i g n i f i c a n t d i f f e r e n c e between  Wilcoxon's  and XVII, Appendix).  H 2b are r e j e c t e d , Q  the othersrnot. < ThisT' L  a d i f f e r e n c e between i n l a n d and  c o a s t a l f i s h i n that,  i n l a n d f i s h , t h e r e i s a sex d i f f e r e n c e i n i o d i n e meta-  b o l i s m , w h i l e i n the c o a s t a l f i s h no  such d i f f e r e n c e c o u l d  be  demonstrated. In our experimental samples the f i s h was was  1.25.  c l o s e t o 0.5 In the  c  ^  sex r a t i o o f  , w h i l e i n the c o a s t a l f i s h the  i n l a n d f i s h the males have h i g h e r  r a t e s and h i g h e r t h y r o i d uptakes than the females. d i f f e r e n c e s between the c o a s t a l and i n l a n d group has uptakes. may  lower e x c r e t i o n  With r e s p e c t  inland ratio  excretion The  main  i n l a n d groups are t h a t  the  r a t e s and h i g h e r t h y r o i d  to excretion  r a t e s , the  i n l a n d males alone  not be d i f f e r e n t from the c o a s t a l group, but  their  thyroid  58  uptakes must be even more d i f f e r e n t from the c o a s t a l f i s h than are the pooled v a l u e s of both males and i n the s t a t i s t i c a l e v a l u a t i o n  o f our  females which were used  i o d i n e metabolism r e s u l t s .  Consequently, both male and  female i n l a n d p r i c k l y  s c u i p i n s are s i g n i f i c a n t l y d i f f e r e n t from c o a s t a l p r i c k l y s c u i p i n s i n at l e a s t some aspects of t h e i r i o d i n e metabolism. In a d d i t i o n , t h e r e  i s a demonstrable sex d i f f e r e n c e i n the  i o d i n e metabolism of i n l a n d f i s h but not  C.  1. General  fish.  Standard M e t a b o l i c Rates  considerations. The  r e s p i r a t i o n i n aquatic  depend on s e v e r a l f a c t o r s . feeding,  i n coastal  The  animals has  a c t i v i t y o f the  ambient temperature, s a l i n i t y and  a c c l i m a t i o n temperature and  salinity,  d e t e r m i n i n g the m e t a b o l i c r a t e  been found to animal,  oxygen t e n s i o n ,  a l l may  play a role in  (Fry, 1957).  T h i s p l a c e s c e r t a i n r e s t r i c t i o n s on the way m e t a b o l i c r a t e s may  and  be determined.  Fry  that, an animal's s t a t e of a c t i v i t y may extremes of m e t a b o l i c r a t e , one  i n which  (1957) suggested be used t o d e f i n e  two  "standard" m e t a b o l i c r a t e which  i s the minimum r a t e at which the animal can  sustain i t s l i f e  p r o c e s s e s i n the p o s t a b s o r p t i v e  one  s t a t e , and  metabolic r a t e which i s the maximum r a t e t o be  "active" achieved at peak  59  activity. For p r a c t i c a l purposes, an a c t i v e m e t a b o l i c r a t e i s the more d i f f i c u l t one t o swim a g a i n s t exhausted, and mate standard at  to a s c e r t a i n .  A f i s h that i s required  a c u r r e n t at maximum speed w i l l q u i c k l y become the r e s p i r a t o r y r a t e w i l l f l u c t u a t e .  metabolism, on the c o n t r a r y ,  An  approxi-  can be maintained  an i n d e f i n i t e p e r i o d of time, the main problem being  many organisms r e f u s e t o remain m o t i o n l e s s f o r extended  that periods  of time. Some workers  (Ege  and Krogh, 1914), t r i e d t o overcome  t h i s d i f f i c u l t y by a n e s t h e t i z i n g the animal.  T h i s , however,  h a r d l y foe s a t i s f a c t o r y s i n c e other v a r i a b l e s are introduced  w i t h the degree o f a n e s t h e s i a  biochemical  automatically  t o be used and  s i d e r e a c t i o n s o f the a n e s t h e t i z i n g  can  possible  drug.  With the p r i c k l y s c u l p i n t h i s problem i s l e s s severe than with other, a bottom d w e l l i n g time and  pelagic, species.  The  p r i c k l y s c u l p i n , being  f i s h , w i l l remain m o t i o n l e s s f o r hours at a  o n l y d a r t away at the appearance o f food or i f  frightened. To i n v e s t i g a t e standard apparatus shown i n F i g . 1.  The  metabolic r a t e s , we used  m e t a b o l i c r a t e of f i s h  the  was  determined from the decrease i n oxygen t e n s i o n between i n f l o w i n g and  outflowing  water.  The  oxygen consumption o f an animal i s the  most widely used index of m e t a b o l i c r a t e , p a r t i c u l a r l y i n work  60  with aquatic  animals.  I t i s considerably  t h a n the more f u n d a m e n t a l c a l o r i c the  construction  Oscygea  exchange,  o f a complete c a l o r i m e t e r  consumption c a n be measured q u i t e  standard  easier  Winkler t i t r a t i o n  to investigate  which.would  require  for i t s investigation.  s i m p l y b y means o f t h e  technique.  S i n c e t h e o x y g e n c o n s u m p t i o n o f t e l e o s t s t o some  extent  d e p e n d s on t h e a m b i e n t o x y g e n t e n s i o n ,  t o make i n v e s t i g a t i o n s  constant for  of metabolic  ambient oxygen t e n s i o n .  the p r i c k l y seulpin,  approximately  occur  saturation. burette  a l t h o u g h some v a r i a t i o n d i d d r o p a s l o w a s 85% o f  e r r o r of 20  of five  On d i f f e r e n c e s  o f ten per cent  (sample and c o n t r o l ) , t h i s g i v e s  p e r c e n t on e a c h  Each p o i n t  a micro-  o f 10 ml e a c h , t h e a c c u r a c y o f e a c h 1%.  b e t w e e n two t i t r a t i o n s ,  an  difference.  on t h e f i g u r e s  titrations  r e p r e s e n t s t h e mean  o f sample and f i v e  an a c c u r a c y on e a c h mean d i f f e r e n c e  of control,  o f 20/5 — 4 p e r c e n t .  The s i z e d e p e n d e n c e o f o x y g e n c o n s u m p t i o n c a n b e expressed  symbolically Q  0  u  2  bys =  used  was k e p t a t  W i t h t h e u s e o f 30 ml W i n k l e r b o t t l e s ,  d e t e r m i n a t i o n was a b o u t  giving  I n t h e r e s p i r a t i o n chamber  would o c c a s i o n a l l y  and two t i t r a t i o n s  difference  a t a c o m p a r a b l e and  t h e oxygen t e n s i o n  90% o f s a t u r a t i o n ,  and t h e t e n s i o n  rate  i t i s preferable  b-1  aW  61  where Qg^ i s the oxygen consumption per u n i t time, a i s a constant W i s the weight o f t h e animal, and b i s an exponent which e m p i r i c a l l y has been found u s u a l l y t o be approximately 0.7. T h i s simple formula has been found t o be remarkably v a l i d throughout t h e animal kingdom, w i t h o n l y the c o n s t a n t "a" changing from group t o group b u t h a v i n g t h e same n u m e r i c a l v a l u e w i t h i n each group o f comparable a n i m a l s .  In each o f our  experiments, t h e same animals were used throughout a s e r i e s o f different salinities.  Consequently, the p o s s i b i l i t y o f s i z e  e r r o r was a u t o m a t i c a l l y e l i m i n a t e d .  When i n t e r r a c i a l com-  p a r i s o n s were made, c a r e was taken t o s e l e c t f i s h o f matching s i z e s from each group, so t h a t not o n l y the average s i z e , b u t a l s o the sample v a r i a n c e f o r t h e two samples was v e r y s i m i l a r (Table X I I I ) . A l l experiments were performed a t an i d e n t i c a l env i r o n m e n t a l temperature o f 10±1.0°C.  The experiments were p e r -  formed i n t h e same c o n t r o l l e d environment room where a c c l i m a t i o n was c a r r i e d out a t 10°C. I n t h e i r n a t u r a l environments, the c o a s t a l f i s h would a t t h e time o f t h e y e a r when these experiments were performed, l i v e i n water which was f i v e t o t e n degrees c e n t i g r a d e warmer than the water where the i n l a n d were c o l l e c t e d .  Because o f t h i s ,  and t o a l l o w the f i s h t o  fish  62 reach t h e p o s t a b s o r p t i v e s t a t e , they were a c c l i m a t e d f o r one week p r i o r t o b e i n g used.  Any l o n g e r a c c l i m a t i o n p e r i o d was  avoided s i n c e s t a r v a t i o n might c o n t r i b u t e t o d i s t u r b a n c e o f the metabolic r a t e .  Feeding w i t h chopped beef h e a r t , b r i n e shrimp  or d r y f i s h food was attempted, b u t without much s u c c e s s .  The  o n l y food which seemed p a l a t a b l e t o the c a p t i v e s c u l p i n s was other s m a l l e r members o f t h e same s p e c i e s which they would eat o c c a s i o n a l l y . Only f i s h o f s u f f i c i e n t l y make mutual devouring periments  similar sizes to  i m p o s s i b l e were used, s i n c e i n these ex-  a l l f i s h o f each group were t o g e t h e r i n a r e s p i r o -  meter. A f t e r a group o f f i s h were p l a c e d i n s i d e a r e s p i r o meter, t h e water c i r c u l a t i n g system was s t a r t e d and allowed t o run f o r 24 hours b e f o r e the f i r s t sample was taken. each change t o a h i g h e r o r lower to  salinity,  After  t h e f i s h were allowed  a c c l i m a t e f o r 65 hours b e f o r e a new s e r i e s o f samples was  taken. Care was taken t o draw samples a t t h e same time every day t o a v o i d b i a s due t o d i u r n a l a c t i v i t y samples were taken i n t h e evenings  pattern.  The  from 7 p.m. t o midnight  s i n c e t h i s was t h e time o f day w i t h t h e l e a s t d i s t u r b a n c e i n the laboratory. 2. E f f e c t o f s a l i n i t y  on oxygen consumption.  I n 1958, Houston demonstrated t h a t t h e locomotor  63  activity  o f j u v e n i l e salmon decreases w i t h i n c r e a s i n g ambient  salinity.  T h i s was  e x p l a i n e d as due t o h i g h e r demands on the  metabolism o f these animals i n h i g h e r s a l i n i t i e s , energy was  a v a i l a b l e f o r locomotion.  with l e s s  Evidence t o the same e f f e c t  presented by Hickman (1959), who  found t h a t the  respira-  t i o n of the s t a r r y f l o u n d e r ( P l a t i c h t y s s t e l l a t u s ) i n c r e a s e d w i t h i n c r e a s i n g environmental In  salinity.  the p r i c k l y s c u l p i n , the v a r i a t i o n i n oxygen  consumption with s a l i n i t y up t o 20°/oo was i n F i g . 18.  found as shown  I n the c o a s t a l f i s h t h e r e i s i n i t i a l l y a con-  s i d e r a b l y h i g h e r oxygen consumption i n f r e s h and d i l u t e water than i n h y p e r t o n i c medium. ment when the s a l i n i t y did  At the end o f the e x p e r i -  a g a i n lowered,  the m e t a b o l i c r a t e  not go back up t o i t s p r e v i o u s h i g h l e v e l s .  coastal fish for  was  sea  When s t a r v e d  ( F i g . 19) which had been kept i n the l a b o r a t o r y  f o u r weeks without f e e d i n g were p l a c e d i n the r e s p i r o -  meter and exposed t o i n c r e a s i n g s a l i n i t i e s ,  t h e r e was  also a  drop i n oxygen consumption going from f r e s h water t o i s o t o n i c although t h i s drop was u n s t a r v e d animals.  v e r y s m a l l compared t o t h a t shown by  From 10°/oo t o 15°/oo t h e r e was  no change,  and from 15°/oo t o 35°/oo the r a t e showed a l i n e a r i n c r e a s e . In  i n l a n d f i s h , the t r e n d i s t o a s l i g h t l y  h i g h e r oxygen o/  consumption i n the s a l i n e medium.  But a t 20 /oo  i t had dropped  from i t s p r e v i o u s v a l u e a t 15°/oo so t h a t f o r these f i s h ,  oxygen  64  F i g u r e 18.  Oxygen consumption o f c o a s t a l and i n l a n d exposed t o i n c r e a s i n g  and d e c r e a s i n g  —•  ©— : coastal  —0  0~ : i n l a n d  fish. fish.  fish  salinities.  QC I  2 IOO  O 80 2  ^  60h  IO  20  10  SALINITY C%<0  65  F i g u r e 19.  The e f f e c t o f i n c r e a s i n g s a l i n i t i e s  on oxygen  consumption  f o r one  i n c o a s t a l f i s h starved  month p r i o r t o the experiment.  66  consumption does not seem t o be r e l a t e d t o s a l i n i t y w i t h i n the range used. In  summary: the oxygen consumption o f i n l a n d  appears t o be o n l y v e r y s l i g h t l y ,  fish  i t a t a l l , a f f e c t e d by  sali-  o n i t y change below 20 /oo.  The c o a s t a l f i s h , on the other hand,  were s e n s i t i v e t o low s a l i n i t i e s e a r l y i n t h e experiments and had maximum oxygen consumption at t h i s s t a g e . salinity to  Later,  and f r e s h water d i d not cause t h e oxygen  increase to i t s previous l e v e l .  low  consumption  At t h i s time, however,, the  f i s h had been s t a r v e d f o r seventeen days i n the r e s p i r o m e t e r i n a d d i t i o n t o the one week a c c l i m a t i o n p e r i o d .  When another  group o f s t a r v e d c o a s t a l f i s h were exposed t o h i g h e r  salini-  t i e s , the oxygen consumption i n c r e a s e d w i t h i n c r e a s i n g  salinity  between 15°/oo and 35°/oo. 3. E f f e c t o f abrupt changes i n s a l i n i t y on oxygen Fig.  20 i l l u s t r a t e s  consumption.  the r e s u l t s o f i n c r e a s i n g the  o ,  o ,  s a l i n i t y d i r e c t l y from 10 /oo t o 20 /oo.  A f t e r a sudden  i n c r e a s e i n oxygen consumption, a p e r i o d o f heavy m o r t a l i t y would f o l l o w .  S u r v i v o r s from the c o a s t a l group, a f t e r h a v i n g  been r e p l a c e d i n 10°/oo s a l i n i t y  and l e f t f o r 65 hours, showed  25% h i g h e r oxygen consumption than d u r i n g the f i r s t exposure t o o, 10 /oo  salinity. The reason f o r t h i s m o r t a l i t y i n 25°/oo s a l i n i t y can  67  F i g u r e 20.  Oxygen consumption i n c o a s t a l and i n l a n d f i s h when o ° s a l i n i t y i s changed from 10 /oo t o 25 /oo.  —•  — Q.  •— g_  : coastal . inland  fish. fish.  68 not  a t p r e s e n t be s a t i s f a c t o r i l y  explained.  Under normal l a b -  o r a t o r y c o n d i t i o n s , p r i c k l y s c u i p i n s c o u l d be t r a n s f e r r e d d i r e c t l y from i s o t o n i c sea water i n t o 25°/oo s a l i n i t y without i l l e f f e c t s .  Presumably the combination o f s e v e r a l  f a c t o r s may have c o n t r i b u t e d slightly  t o the m o r t a l i t y : osmotic s t r e s s ,  lowered oxygen t e n s i o n and i n c r e a s e d  s e a s o n a l f a c t o r may were c a p t u r e d d u r i n g  sea water  CO2 t e n s i o n .  a l s o have been o f importance.  These  A  fish  spawning season and although the use o f  f i s h which appeared r i p e or r e c e n t l y spent was avoided, the s c u i p i n s may  at t h i s time o f the year be i n a c o n d i t i o n o f  decreased r e s i s t a n c e t o sudden osmotic s t r e s s .  4. E f f e c t o f h a b i t a t o f f i s h on oxygen consumption. The oxygen consumption i n u n s t a r v e d c o a s t a l f i s h i s h i g h e r i n f r e s h water than t h a t o f i n l a n d f i s h i n f r e s h water (Fig.  17).  T h i s d i f f e r e n c e i s as l a r g e as 52%? but a t the end  of the run a f t e r seventeen days o f s t a r v a t i o n i n the r e s p i r o meter the d i f f e r e n c e had dropped t o o n l y 14%.  In i s o t o n i c media  the d i f f e r e n c e vanishedY but a t 20°/oo the c o a s t a l f i s h  again  had a 16% h i g h e r oxygen consumption than the i n l a n d group. The e x i s t e n c e  of this i n i t i a l difference  was  confirmed by the two p r e v i o u s experiments ( F i g . 20), where a group o f c o a s t a l f i s h o f the average weight 5.71 gm,  had  40% h i g h e r oxygen consumption than a group o f i n l a n d f i s h with an average weight o f 6.63  gm.  69  The c o a s t a l f i s h seem more s u b j e c t s t a r v a t i o n than t h e i n l a n d f i s h .  During t h e time o f e x p e r i -  ment t h e f r e s h water oxygen consumption fell  t o the e f f e c t s o f  of the coastal  fish  from 100 ± 5 ug 0 /gm f i s h / h r t o 64 r 3 jxg 0 /gm f i s h / h r 2  2  and t h a t o f t h e i n l a n d f i s h from 66 ± 3 ug 02/gm f i s h / h r t o 54 t 2 ug 0 /gm f i s h / h r . 2  Table X I I I .  Weights o f f i s h used f o r i n t e r r a c i a l of metabolic rates  Coastal  fish  i n different  Inland  salinities.  fish  #1  7.5 gm  7.5 gm  #2  6.8 gm  7.0 gm  #3  4.8 gm  5.1 gm  #4  4.8 gm  4.9 gm  #5  4.8 gm  4.4 gra  Totals  28.7 gra  Average  5.74 gm  28.9  comparison  gm  5.78 gm  IV.  The  DISCUSSION  importance o f i o d i n e balance i n r a d i o l o g i c a l  work of the k i n d undertaken i n t h i s study has been s t r e s s e d by Hickman  (1961) and McNabb (1963).  When f i s h  brought i n t o the l a b o r a t o r y from a low the t h y r o i d may  be  i n a goiterous  a v a i l a b l e environmental i o d i d e . and  are  i o d i d e environment  s t a t e due  t o l a c k of  I n j e c t i o n with r a d i o i o d i d e  subsequent r a d i o l o g i c a l measurements may  i n t h i s case  l e a d t o erroneous i n t e r p r e t a t i o n o f t h y r o i d a c t i v i t y  due  t o abnormal accumulation of r a d i o i o d i d e i n the t h y r o i d . counteract  this,  Hickman (1959) suggested t h a t  brought i n from the f i e l d be p l a c e d with i o d i n e so t h a t i t c o n t a i n s  i n water  To  fish  enriched  the same amount o f i n o r -  127 ganic  I  as sea water.  T h i s procedure was  followed  i n our  experiments. A goiterous  c o n d i t i o n i n any  be r e f l e c t e d i n a c o r r e s p o n d i n g l y  of the f i s h would  increased  accumulation of  r a d i o i o d i n e at some p o i n t or p o i n t s i n the m e t a b o l i c pathway. On  the other hand, any p h y s i o l o g i c a l d i f f e r e n c e i n osrao-  r e g u l a t i n g c a p a c i t y between i n l a n d and a l s o r e v e a l i t s e l f as i n c r e a s e d i n the t h y r o i d s of one  coastal fish  accumulation o f r a d i o i o d i e  race of f i s h r e l a t i v e t o the  race, and/or as i n c r e a s e d p e r i p h e r a l hormone I t i s not known how  could  other  utilization.  r a p i d i s the accumulation of  71  environmental i o d i d e i n the p r i c k l y s e u l p i n , nor known how  is i t  f a s t accumulated i o d i n e can be u t i l i z e d  t h y r o i d hormone.  But  evidence obtained  in  i n t h i s study shows 131  t h a t the t h y r o i d w x l l begin t o absorb i n j e c t e d I  within  24 hours, and p r o b a b l y even i n c o n s i d e r a b l y  l e s s time.  Radio-  a c t i v e p r o t e i n bound i o d i d e i s a l s o present  in this seulpin  24 hours a f t e r i n j e c t i o n , at l e a s t i n some c a s e s . Presumably, then, a one i o d i d e r i c h medium should  week a c c l i m a t i o n p e r i o d i n  go f a r towards e s t a b l i s h i n g i o d i d e  balance, both i n f r e s h water where the demand on i s low,  and  i s higher.  thyroxine  i n s a l i n e water where the demand on the hormone In s a l i n e water the e l e c t r o l y t e turnover  f i s h i s a l s o higher  and  i n the  i n o r g a n i c i o d i d e removed from the  blood by the t h y r o i d can be r e p l e n i s h e d more r a p i d l y by i o d i d e taken up from the surrounding medium together  with  other e l e c t r o l y t e s . A simple e x p l a n a t i o n  of the observed d i f f e r e n c e i n  f r e s h water e x c r e t i o n r a t e s i s t h a t the  i n l a n d f i s h have  developed a h i g h e r degree o f e f f i c i e n c y i n t h e i r a b i l i t y r e t a i n i o d i d e i n h a l i d e poor f r e s h water. t h y r o i d uptake f a c t o r s , c o n v e r s i o n of sea water a c c l i m a t e d between c o a s t a l and  fish,  any  to  When examining  r a t e s and  excretion  rates  fundamental d i f f e r e n c e  i n l a n d f i s h seems t o m a n i f e s t i t s e l f i n  r e a c t i o n s t a k i n g p l a c e w i t h i n the f i r s t  24 hours.  There i s ,  i n general, r e l a t i v e l y higher excretion rates, faster  thyroid  131 uptake and h i g h e r l e v e l s o f PBI than l a t e r .  produced  i n this period  The magnitude of these i n i t i a l r a t e s , however,  depends on the o r i g i n o f the  fish.  In c o n t r a s t t o the i o d i n e metabolism, the standard m e t a b o l i c r a t e s as measured i n our experiments  only  t o be s l i g h t l y a f f e c t e d by s a l i n i t y up t o 20°/oo. (1959) argued  appeared Hickman  t h a t the t h y r o i d hormone p l a y s a c a l o r i g e n i c  r o l e i n m e t a b o l i c demands a s s o c i a t e d w i t h e l e c t r o l y t e i n the s t a r r y f l o u n d e r ( P l a t i c h t y s s t e l l a t u s ) .  balance  In the  p r i c k l y s c u l p i n , however, t h e r e seems t o be l i t t l e  correla-  t i o n between oxygen consumption and s a l i n i t y below 25°/oo salinity. In f r e s h water, c o a s t a l f i s h have 40 t o 50% h i g h e r r e s p i r a t o r y r a t e s than comparable i n l a n d f i s h a t the b e g i n n i n g o f an experiment.  Two  had decreased but was C„  and a h a l f weeks l a t e r the d i f f e r e n c e still  apparent  ( F i g . 18).  asper p r o b a b l y has evolved from marine a n c e s t o r s .  From t h i s e v o l u t i o n a r y viewpoint, the i n l a n d forms may developed  the a b i l i t y t o osmoregulate  i n f r e s h water w i t h  l e s s expense o f m e t a b o l i c energy than the c o a s t a l f i s h . experiments  support t h i s  have  Our  theory.  Comparison of i o d i n e metabolism i n the two  races lends  more support t o a c o n c l u s i o n c o n c e r n i n g g e n e t i c d i f f e r e n c e .  The  i n i t i a l l y lower e x c r e t i o n  r a t e s and h i g h e r t h y r o i d  uptakes i n s a l i n e waters observed i n the i n l a n d f i s h a r e an i n d i c a t i o n of genetic conclusive  d i f f e r e n c e b u t can not be accepted as  evidence s i n c e the reason f o r these  i n i t i a l r a t e s can not be s a i t s f a c t o r i l y  exceptional  explained.  However,  the i n l a n d f i s h a l s o show a s i g n i f i c a n t d i f f e r e n c e i n the rate of increase water.  i n TUF i n f r e s h as compared t o i s o t o n i c  The c o a s t a l f i s h do n o t .  F i g . 21 r e p r e s e n t s an a t -  tempt t o i n t e r p o l a t e t h y r o i d uptakes w i t h changing  salinity  from t h e v a l u e s o b t a i n e d i n the p r e s e n t experiments 200 hours after injection.  The c o a s t a l f i s h appear c o n s i d e r a b l y  sensitive to salinity inland f i s h . excretion  changes a t low s a l i n i t i e s  A l s o t h e i n l a n d f i s h have slower  less  than do t h e iodide  i n f r e s h water than have the c o a s t a l  fish.  On the b a s i s o f these c o n s i d e r a t i o n s ,  the conclusion  t h a t the i n l a n d f i s h used i n our experiments have d i v e r g e d g e n e t i c a l l y from t h e main, o r c o a s t a l , p o p u l a t i o n aspects o f t h e i r metabolism, seems  i n some  logical.  That the t h y r o i d hormone i s i n v o l v e d  i n osmoregula-  t i o n i n the p r i c k l y s e u l p i n seems beyond doubt.  F i g . 21,  however, i n d i c a t e s t h a t the r a t e o f t h y r o i d uptake i s not proportional  t o the s a l i n i t y ,  l a g s behind as t h e s a l i n i t y  rather  increases  the t h y r o i d metabolism up t o 25°/oo.  c o n v e r s i o n r a t i o s shows t h a t although the t h y r o i d s  Study o f o f both  74  F i g u r e 21.  The  e f f e c t of s a l i n i t y  i n TUF  on r a t e of  a f t e r i n j e c t i o n with I t coastal s inland  fish, fish.  131  .  increase  SALINITY C%<0  i n l a n d and c o a s t a l  f i s h accumulate i  i J J  -  i n f r e s h water, the  r e l e a s e o f radiohormone a t l e a s t a f t e r the i n i t i a l p e r i o d , i s slow o r n o n - e x i s t e n t .  24 hour  I n sea water, however, both  c o n v e r s i o n r a t i o s and TUF v a l u e s are h i g h . How t h i s can be i n t e r p r e t e d utilization  and m e t a b o l i c demand f o r t h y r o i d hormone by  the f i s h i s s u b j e c t t o doubt. are found high.  i n terms o f p e r i p h e r a l  Presumably, h i g h PBI l e v e l s  i n f i s h where t h e demand on t h y r o i d hormone i s  But s i n c e n o t h i n g a t p r e s e n t i s known o f the h a l f  l i f e o f t h y r o i d hormone i n t h e p r i c k l y s c u l p i n , a c l o s e r a n a l y s i s o f t h e r e l a t i o n s h i p between c o n v e r s i o n r a t i o s and p e r i p h e r a l  u t i l i z a t i o n may not be c a r r i e d o u t .  V.  SUMMARY  Some aspects o f osmoregulation i n two l o c a l Columbia r a c e s o f the p r i c k l y s e u l p i n were i n v e s t i g a t e d .  (Cottus  asper Richardson)  Groups Of f i s h from each race were a c c l i -  mated t o f r e s h water, i s o t o n i c and h y p e r t o n i c i n j e c t e d with the r a d i o a c t i v e iodine isotope intervals,  British  sea water and 1^1,  &t  samples o f f i s h were s e l e c t e d a t random and t e s t e d  f o r e x c r e t i o n o f t h e r a d i o i o d i n e , f o r t h y r o i d uptake and f o r conversion  t o t h y r o i d hormone.  A n a l y s i s o f the data showed  the e x c r e t i o n r a t e s t o be e x p o n e n t i a l l y the t h y r o i d uptake f a c t o r s  r e l a t e d t o time, and  (TUF) and c o n v e r s i o n  r a t i o s t o be  approximately l i n e a r l y r e l a t e d t o time. Standard metabolic r a t e s i n d i f f e r e n t s a l i n i t i e s were determined a f t e r a c c l i m a t i o n o f the f i s h t o a c o n s t a n t temperature.  A standard Winkler technique was used t o measure  oxygen consumption a f t e r t h r e e days o f a d d i t i o n a l t o each new s a l i n i t y . 1.  Increasing  The f o l l o w i n g c o n c l u s i o n s  acclimation were reached?  s a l i n i t y results i n increasing  e l e c t r o l y t e t u r n o v e r , measurable by t h e i n c r e a s i n g  excretion  131  of I  i n both r a c e s o f f i s h . 2.  Increasing  s a l i n i t y results i n increasing thyroid  uptake of i o d i n e . 3.  The h i g h e r t h y r o i d uptake o f i o d i n e i n h y p e r t o n i c  sea water r e f l e c t s h i g h e r l e v e l s o f c i r c u l a t i n g t h y r o i d  77 hormone and  t h i s i s confirmed by the c o n v e r s i o n  Presumably t h i s i s d i c t a t e d by  ratios.  i n c r e a s i n g needs o f the  fish  f o r t h y r o i d hormone when i t i s exposed t o i n c r e a s i n g l y s a l i n e environments, and  i t i s presumably a l s o connected with i n -  c r e a s e d p e r i p h e r a l u t i l i z a t i o n o f t h y r o i d hormone. 4. during  The  the f i r s t  s c u i p i n s have more r a p i d i o d i n e metabolism 24 hours a f t e r i n j e c t i o n than l a t e r .  In  t h i s e a r l y p e r i o d a f t e r i n j e c t i o n , the b l o o d c o n c e n t r a t i o n  of  radioiodine i s r e l a t i v e l y high u n t i l iodine equilibrium i s e s t a b l i s h e d between the d i f f e r e n t body compartments. 5.  The  i n l a n d f i s h i n c r e a s e t h e i r t h y r o i d uptake  o f r a d i o i o d i n e s i g n i f i c a n t l y f a s t e r with i n c r e a s i n g i n hypertonic the TUF  media than do c o a s t a l f i s h .  fish.  In f r e s h water  v a l u e s f o r i n l a n d f i s h are lower, and  medium h i g h e r than f o r c o r r e s p o n d i n g l y  salinity  in isotonic  acclimated  coastal  T h i s i s i n t e r p r e t e d as a d a p t i o n t o fresh-water  ment on the p a r t o f the i n l a n d 6. excretion  The  environ-  race.  i n l a n d f i s h have s i g n i f i c a n t l y slower  i n f r e s h water than do the c o a s t a l f i s h .  iodine  This  pre-  sumably r e f l e c t s a h i g h e r c a p a c i t y f o r r e t a i n i n g i o d i n e i n iodine-poor 7.  environments. In the c o a s t a l f i s h there  i s no sex  difference  with r e s p e c t t o i o d i n e metabolism as measured i n t h i s study. In i n l a n d f i s h , however, the male f i s h have h i g h e r  excretion  78 r a t e s and h i g h e r t h y r o i d uptakes than do the females. 8. gen  The standard m e t a b o l i c r a t e as measured by oxy-  consumption i s not c o n s i s t e n t l y a f f e c t e d by an i n c r e a s e  i n ambient s a l i n i t y fish.  Starvation  from zero t o 20°/oo i n e i t h e r group o f  appears t o o v e r r u l e  changing s a l i n i t i e s  during  the i n f l u e n c e o f  the f i r s t two or t h r e e weeks  a f t e r t h e f i s h have been brought i n t o the l a b o r a t o r y .  Evidence  from a group o f c o a s t a l f i s h which had undergone prolonged s t a r v a t i o n i n d i c a t e p o s i t i v e c o r r e l a t i o n between oxygen c o n o . sumption and s a l i n i t y  when t h e l a t t e r  increases  from 15 /oo t o  35 /oo. 9.  Coastal  f i s h have a h i g h e r m e t a b o l i c r a t e i n  f r e s h water than do i n l a n d f i s h .  This d i f f e r e n c e vanishes  in d i l u t e sea water o f 10°/oo t o 15°/oo 10.  Consideration  the c o n c l u s i o n  of points  salinity.  5, 6, 7 and 9 leads t o  t h a t our samples o f i n l a n d f i s h show g e n e t i c  d i v e r g e n c e as compared w i t h the c o a s t a l form.  In t h e i n l a n d  f i s h the i o d i n e r e t e n t i o n and a b i l i t y t o osmoregulate i n f r e s h water appears t o be more e f f i c i e n t  than i n the c o a s t a l  fish.  LITERATURE CITED  1. BAGGERMAN, B. The r o l e of e x t e r n a l f a c t o r s and hormones i n m i g r a t i o n of s t i c k l e b a c k s and j u v e n i l e salmon. Symp. Comp. End. e d i t e d by A. Gorbman, Wiley and Sons Inc. 24-37 (1959). 2. BAGGERMAN» B „ The e f f e c t of TSH and a n t i t h y r o i d substances on s a l i n i t y p r e f e r e n c e and t h y r o i d a c t i v i t y i n j u v e n i l e p a c i f i c salmon. Can. J o u r . Z o o l . 41, 307-319 (1963). 3. BLACK, V.S. E x c r e t i o n and osmoregulation, i n The phys i o l o g y of f i s h e s . E d i t e d by M.E. Brown, Academic Press Inc., New York. 1 (1957). 4. CHASE, G.D. P r i n c i p l e s of r a d i o i s o t o p e gess P u b l i s h i n g Comp., Minnesota.  methodology. (1963).  Bur-  5. EALES, J.G. A complete study of t h y r o i d f u n c t i o n i n mig r a n t j u v e n i l e salmon. Can. J o u r . Z o o l . 41, 811824 (1963). 6. EGE,  R., and KROGH, A. On the r e l a t i o n s h i p between the temperature and r e s p i r a t o r y exchange i n f i s h e s . I n t e r n . Rev. ges. H y d r o b i o l . Hydrog. 7, 48-55 (1914). C i t e d by F r y (1957).  7. FORTUNE, P.Y. Comparative s t u d i e s of the t h y r o i d f u n c t i o n i n t e l e o s t s of t r o p i c a l and temperate h a b i t a t s . Jour. Exp. B i o l . 32, 504 (1955). 8. FORTUNE, P.Y. auratus. 9. FRY,  An i n a c t i v e t h y r o i d g l a n d i n C a r a s s i u s Nature 178, 98 (1956).  F.E.J. A q u a t i c r e s p i r a t i o n of f i s h , i n The p h y s i o l o g y of f i s h e s . E d i t e d by M.E„ Brown, Academic Press Inc., New York. 1 (1957).  10.  HICKMAN, C.P. J r . The osmoregulatory r o l e of the t h y r o i d gland i n the s t a r r y f l o u n d e r , P l a t i c h t y s s t e l l a t u s . Can. J o u r . Z o o l . 37, 997-1960 (1959).  11.  HICKMAN, C.P. J r . The c o n v e r s i o n r a t i o as a d i s c r i m i n a t o r y test for thyroid activity in f i s h . Nature 189, 10121013 (1961).  80 12. HOAR, W.S. and BELL, G.M. The t h y r o i d g l a n d i n r e l a t i o n t o t h e seaward m i g r a t i o n o f p a c i f i c salmon. Can. J o u r . Res. D, 28, 126-136 (1950). 13. HOAR, W.S. Endocrine f a c t o r s i n t h e e c o l o g i c a l adaption of f i s h e s . In Comparative e n d o c r i n o l o g y . E d i t e d by A. Gorbman, John Wiley & Sons, New York. (1959). 14. HOAR, W.S. The endocrine r e g u l a t i o n o f m i g r a t o r y behav i o u r i n anadroumous t e l e o s t s . P r o c . XVI I n t . Cong. Z o o l . 3, 14-20 (1963). 15. HOAR, W.S. and EALES, J.G. The t h y r o i d g l a n d and low temperature r e s i s t a n c e i n g o l d f i s h . Can. J o u r . Z o o l . 41, 653-669 (1963). 16. HOLMES, W.N., PHILLIPS, J.G., and CHESTER JONES, I . Adrenoc o r t i c a l f a c t o r s a s s o c i a t e d with a d o p t i o n o f v e r t e b r a t e s t o marine environments. Recent Progress i n Hormone Research, 19, 619-672 (1963). 17. HOUSTON, A.H. Locomotor performance and osmoregulation i n j u v e n i l e anadromous salraonids f o l l o w i n g abrupt e n v i r o n mental s a l i n i t y change. Ph.D. T h e s i s , Univ. B r i t i s h Columbia, (1958). 18. KEYS, A.B. C h l o r i d e and water s e c r e t i o n and a b s o r p t i o n by the g i l l s o f t h e e e l . Z. V e r g l e i c h . P h y s i o l . 15, 364-388 (1931). 19. KEYS, A.B. The mechanism o f a d a p t i o n t o v a r y i n g s a l i n i t y i n t h e common e e l and t h e g e n e r a l problems o f osmotic regulation i n fishes. P r o c . Royal Soc. B112, 184-199 (1933). 20. KOCH, H.J. and HEUTS, M.J. I n f l u e n c e de l'hormone t h y r o i dienne s u r l a r e g u l a t i o n osmotique chez G a s t e f o s t e u s a c u l e a t u s L., forme gymnurus. Cuv. Ann. Soc. Royal Z o o l . B e l g . 73, 165-172 (1942). 21. KREJSA, R.J. The r e p r o d u c t i v e behaviour o f t h e p r i c k l y s c u l p i n Cottus asper Richardson. In manuscript. (1964) 22. KROGH, A. Osmotic r e g u l a t i o n i n f r e s h water f i s h e s by active absorption of c h l o r i d e ions. Z. V e r g l e i c h . Physiol. 24, 656-666 (1937).  81 23. LELOUP, J . and FONTAINE, M„ I o d i n e metabolism i n lower v e r t e b r a t e s . Ann. N.Y. Acad. S c i . 86, A r t . 2, 316353 (1960) . 24. McNABB, R.A. C o n t r i b u t i o n o f d i e t a r y and environmental i o d i n e t o i o d i n e balance o f rainbow t r o u t (Salmo g a i r d n e r i ) . M . S c , Univ. o f A l b e r t a . (1963). 25. OLIVEREAU, M. I n f l u e n c e d'une d i m i n u t i o n de s a l i n i t e s u r 1 ' a c t i v i t y de l a glande t h y r o i d e de deux t e l e o s t e e n s marinss Muraena h e l e n a L. e t Labrus b e r g y l t a . A s c . Compt. Rend. Soc. B i o l . 142, 176-177 (1948). 26. OLIVEREAU, M„ I n f l u e n c e d'une augmentation de s a l i n i t y sur 1 ' a c t i v i t e t h y r o i d i e n n e des d i v e r s e s teleosteens d'eau douce. Compt. Rend. Soc. B i o l . 144, 775-776 (1950). 27. PICKFORD, G.E. and ATZ, J.W. The p h y s i o l o g y o f the p i t u i t a r y gland of f i s h e s . N.Y. Z o o l . S o c , New York, N.Y. (1957). 28. SMITH, H W. The a b s o r p t i o n and e x c r e t i o n o f water and s a l t s by marine t e l e o s t s . Am. J o u r . P h y s i o l . 93, 480-505 (1930). 0  29. SVERDRUP, H.U., JOHNSON, M W „ , and FLEMING, R.H. P r e n t i c e H a l l I n c . (1963). 0  The oceans.  30. SWIFT, D.-R. C y c l i c a l a c t i v i t y of the t h y r o i d gland of f i s h i n r e l a t i o n t o environmental changes. Symp. Z o o l . Soc. London 2, 17-27, (1960). 31. WIGGS, A J Some f a c t o r s a f f e c t i n g r a d i o i o d i d e metabolism i n the t h r e e s p i n e s t i c k l e b a c k . M . S c , Univ. o f B r i t i s h Columbia. (1962). 0  0  32. WIGGS, A . J . Notes on t h e use of t h e c o n v e r s i o n r a t i o as an index o f t h y r o i d a c t i v i t y . Can. J o u r . Z o o l . 41, 1176-1177 (1963).  VII.  APPENDIX  To make a s t a t i s t i c a l approach t o e v a l u a t i o n  of  the r e s u l t s p o s s i b l e , the assumptions were made t h a t both values  and  conversion  exponentially,  r a t i o s are l i n e a r l y ,  r e l a t e d t o time.  and  excretion  TUF rates  These assumptions enabled  us  t o perform s t a t i s t i c a l comparisons between b o d i e s o f data with unequal means and  variances.  Whether or not the use o f l i n e a r r e g r e s s i o n s s t r i c t l y v a l i d as a matter f o r d i s c u s s i o n . r a t e o f e x c r e t i o n o f r a d i o i o d i d e a t any momentary  is  T h e o r e t i c a l l y , the  time depends on  the  concentrations KV  where V i s the amount of I By  present  i n the f i s h at any  integrations InV  hence l o g t r a n s f o r m a t i o n  —  kt  + c,  o f V y i e l d s the l i n e a r r e l a t i o n s h i p .  B i o l o g i c a l l y speaking, the s i m p l i c i t y of t h i s i s dubious s i n c e  (a) t h e r e i s an uptake o f i o d i d e by  t h y r o i d and perhaps by the o v a r i e s i n female f i s h , may  time t .  be more than one  d i l u t e sea water, and  theory  the  (b)  there  pathway o f e x c r e t i o n , p a r t i c u l a r l y i n (c) t h e r e i s an inknown amount o f  b i n d i n g of i n o r g a n i c i o d i d e t o b l o o d p r o t e i n s . pragmatic approach works i n t h a t s e m i l o g a r i t h m i c  However, the transforma-  83 t i o n s o f the e x c r e t i o n data y i e l d p l o t s which c o n f i r m r a t h e r c l o s e l y t o s t r a i g h t l i n e s and a r e s i g n i f i c a n t l y with  correlated  time. For TUF v a l u e s , and even more f o r c o n v e r s i o n r a t i o s ,  our mathematical treatment be expected iodide  1J  i s entirely pragmatic  I t i s to  t h a t as long as t h e r a t e o f t h y r o i d uptake o f  97  (I - ") i s constant, t h e r a t e o f t h y r o i d uptake o f I i  131  should be l o g a r i t h m i c f o r t h e same reason as e x c r e t i o n r a t e s are l o g a r i t h m i c a l l y r e l a t e d t o time.  The v a r i a t i o n o f TUF  v a l u e s w i t h time, b e i n g based on the d i v i s i o n o f two l o g a r i t h mic  f u n c t i o n s w i t h another,  would be expected  t o be l i n e a r ,  131 However, both t h y r o i d uptake r a t e s o f I  , due t o p r o d u c t i o n  and r e l e a s e o f radiohormone, and e x c r e t i o n r a t e s , are s u b j e c t t o d e v i a t i o n from t h e simple t h e o r y .  T h e r e f o r e , t h e data were  f i r s t p l o t t e d and examined f o r resemblance t o mathematically definable curves. For c o n v e r s i o n r a t i o s , a simple mathematical r e l a t i o n s h i p may o r may not be expected.  I t i s d i f f i c u l t to  a n t i c i p a t e the p a t t e r n o f hormone p r o d u c t i o n i n a r e l a t i v e l y u n i n v e s t i g a t e d animal.  What can be s a i d i s t h a t a constant  f l o w o f hormone from t h e t h y r o i d should be r e f l e c t e d i n conv e r s i o n r a t i o s which i n c r e a s e l i n e a r l y with time as long as the TUF v a l u e s i n c r e a s e l i n e a r l y . S i n c e i t seemed j u s t i f i a b l e t o use s t r a i g h t  line  84 r e l a t i o n s , r e g r e s s i o n l i n e s were c a l c u l a t e d by the method o f l e a s t squares, and used as the b a s i s f o r f u r t h e r examination. I f the use o f r e g r e s s i o n l i n e s i s v a l i d , t h e c o r r e l a t i o n between the two v a r i a b l e s must be s i g n i f i c a n t , consequently the c o r r e l a t i o n c o e f f i c i e n t s were examined.  For regression  lines  with r e g r e s s i o n c o e f f i c i e n t s approximately equal t o zero, the c o r r e l a t i o n c o e f f i c i e n t : between x and y w i l l a l s o mate z e r o .  F o r t h i s reason, whenever t h e r e g r e s s i o n  approxicoef-  f i c i e n t o f a l i n e was c l o s e t o zero, t h e c o r r e l a t i o n c o e f f i c i e n t was o v e r r u l e d and t h e p o s s i b i l i t y o f u s i n g a r e g r e s s i o n l i n e was determined by v i s u a l i n s p e c t i o n o f t h e graph. A l l r e g r e s s i o n l i n e s were c a l c u l a t e d a t the U n i v e r s i t y o f B r i t i s h Columbia Computing Centre, standard  program.  y, standard coefficient,  following a  T h i s program g i v e s the mean v a l u e s o f x and  d e v i a t i o n s o f x and y, c o r r e l a t i o n , r e g r e s s i o n i n t e r c e p t , and standard  e r r o r o f estimate.  T a b l e XIV.  Weight i n 0* 7.9 5.4 6.2 8.8 8.8 6.8 12.4 7.0 5.4 2.8 2.3 12.1 4.5 10.8 10.8 3.1 10.8  Data from c o a s t a l f i s h used f o r i n t e r s e x comparisons of i o d i n e  gm  ? 7.5 5.6 5.7 9.3 9.3 6.9 12.4 7.0 5.3 2.8 2.5 11.5 4.6 10.4 11.0 3.3 10.6  Total difference  Excretion Diff. -.4 4.2 -.5 + .5 4.5 4.1 0 0 -.1 0 4.2 -.6 4.1 -.2 4.2 4.2 -.2  0.0  81.3 84.4 45.8 43.1 43.1 43.5 32.5 24.1 35.8 94.0 81.5 93.2 76.9 86.5 86.5 73.2 60.1  TUF  metabolism.  CR  ?  Diff.  d*  o  Diff.  82.7 74.0 42.3 47.7 36.6 28.0 37.1 18.7 43.4 86.5 78.2 91.5 91.5 83.4 86.1 68.9 71.9  41.4 -10.4 -3.5 +4.6 46.5 -15.5 44.6 -5.4 +7.6 -7.5 -3.3 -1.7 +14.6 -3.1 -.4 -4.3 +11.8  3.76 3.92 6.54 2.32 2.32 3.80 9.16 7.22 5.06 3.20 3.71 2.25 4.93 3.66 3.66 3.27 2.95  4.18 2.46 5.47 6.37 4.03 2.06 3.96 6.64 8.88 2.56 2.37 2.81 2.89 3.47 3.36 3.64 5.07  + .42 -1.46 -1.07 +4.05 +1.71 -1.74 -5.20 -.58 + 3.82 -.64 -1.34 4.56 -2.04 -.19 -.30 + .37 + 2.12  c?  8.12 9.52 9.52 10.4 4.94 4.03 3.03 3.20 1.98 2.03 1.47 1.47 1.47 1.10 9.41  Diff.  4.97 4.98 6.05 2.18 3.19 4.40 3.73 4.78 1.94 .96 2.25 1.62 3.12 1.84 3.24  -3.15 -3.54 -3.47 -8.2 -1.75 + .37 + .70 +1.58 -.04 -1.07 + .78 + .15 + 1.65 + .74 -6.17  Table XV.  Data from inland f i s h used for intersex comparisons of iodine metabolism.  Weights i n gm  % 5.8 5.0 4.2 4.6 5.8 5.0 6.0 3.3 3.3 3.1 3.8 3.8 2.3 3.8 3.1 3.8 3.7  5.9 4.8 3.7 4.6 6.1 4.8 6.5 3.6 3.0 3.2 4.1 3.6 2.6 3.7 3.3 3.3 3.7  Diff. 4.1 -.2 -.5 0 4.3 -.2 4.5 4.3 -.3 4.1 4.3 -.2 4.3 -.1 4.2 -.5 0  Total difference+0.1  Excretion  TUF  o*  ?  Diff.  81.7 38.0 67.1 79.1 65.3 32.6 25.3 45.6 45.6 17.2 78.1 78.1 73.9 35.4 8.5 10.2 8.99  93.0 53.7 62.5 85.0 82.4 18.9 31.6 42.7 51.9 14.6 83.5 76.1 78.7 40.8 14.3 14.3 16.5  411.3 415.7 -4.6 45.9 417.1 -14.7 46.3 -2.9 +6.3 -2.6 45.4 -2.0 44.8 45.4 45.8 44.1 47.5  <T  1.65 5.27 6.06 5.25 6.72 8.08 14.3 3.14 3.14 8.54 3.08 3.08 1.95 9.69 11.8 13.0 6.87  CR  ? 1.66 4.70 6.64 2.52 3.29 4.26 4.89 5.05 3.01 8.16 3.30 2.78 2.70 9.48 10.3 10.3 4.67  Diff. 4.01 -.57 4.58 -2.73 -3.43 -3.82 -9.40 41.91 ^-.13 -.38 4.22 -.30 4.75 -.22 -1.50 -2.70 -2.20  4.96 2.36 2.12 1.09 1.87 2.93 6.63 2.22 2.22 6.23 1.69 1.69 2.11 3.33 9.40 9.55 7.36  ?  Diff.  1.27 3.99 2.08 1.07 1.75 9.93 8.36 2.10 2.34 4.40 1.72 1.45 1.53 2.05 9.78 9.78 8.66  -3.69 +1.63 -.04 -.02 -.12 47.00 41.73 -.12 4.12 -1.83 4.13 -.24 -.58 -1.28 4.38 4.23 41.30  87  T a b l e XVI o  S t a t i s t i c s and c o n c l u s i o n s parisons  from i n t e r s e x  o f d a t a from c o a s t a l f i s h .  are based on Wilcoxon's signed no  significant difference.  Q  uptake  ft  i s true.  Conversion  ratio*  Diff.  Rank  Diff.  Rank  -.4 •f-1.4 -1.7 -3.1 -3.3 -3.5 -4„3 +4.6 +4.6 -5.4 +6.5 -7.5 +7.6 -10.4 +11.8 +14,6 -15.5  -1 +2 -3 -4 -5 -6 -7 +8 +9 -10 +11 -12 + 13 -14 +15 +16 -17  -.19 -.30 + .37 + .42 + .56 -.58 -.64 -1.07 -1.34 -1.46 + 1.71 -1.74 -2.08 + 2.12 +3.82 +4.05 -5.20  -1 -2 +3 +4 +5 -6 -7 -8 -9 -10 +11 -12 -13 +14 +15 +16 -17  -.04 + .15 + .37 + .70 + .74 + .78 -1.07 +1.58 +1.65 -1.75 -3.15 -3.47 -3.54 -8.2  -1 +2 +3 +4 +5 +6 -7 +8 +9 -10 -11 -12 -13 -14  +72  +68  +37  72  68  37  P > . 05 not r e j e c t e d  P> • 05 H not r e j e c t e d Q  * only  s  P % p r o b a b i l i t y of  Rank  C r i t i c a l numbers  Q  H  Diff.  S m a l l e s t sums  Conclusions H  Thyroid  Conclusions  rank t e s t .  the c a l c u l a t e d c r i t i c a l number i f H  Excretion  com-  H  0  .05 not r e j e c t e d  14 p a i r s a v a i l a b l e .  Table XVII.  S t a t i s t i c s and c o n c l u s i o n s from i n t e r s e x comp a r i s o n s o f data from i n l a n d f i s h .  Conclusions  are based on Wilcoxon's signed rank t e s t . no s i g n i f i c a n t d i f f e r e n c e .  Q  i s true.  Conversion r a t i o  Diff.  rank  Diff.  rank  Diff.  rank  -2.0 -2.6 -2.9 + 4.1 -4.6 + 4.8 + 5.4 + 5.4 + 5.8 + 5.9 + 6.3 + 6.3 + 7.5 +11.3 -14.7 + 15.7 + 17.1  -1 -2 -3 +4 -5 +6 +7 +8 +•9 + 10 + 11 +-12 + 13 + 14 -15 + 16 + 17  + .01 -.13 + .22 -.22 -.30 -.38 -.57 + .58 + .75 -1.50 + 1.91 -2.20 -2.70 -2.73 -3.43 -3.82 -9.40  +1 -2 + 3.5 -3.5 -5 -6 -7 +8 +9 -10 + 11 -12 -13 -14 -15 -16 -17  -.02 -.04 -.12 -.12 +-. 12 +-.13 + .23 -.24 + .38 -.58 -1.28 1.30 1.63 1.73 -1.83 -3.69 + 7.00  -1 -2 -4 -4 +•4 +6 +7 -8 +9 -10 -11 12 13 14 -15 -16 + 17  S m a l l e s t sums C r i t i c a l numbers  Conclusions  T h y r o i d uptake  -26  +•32.5  -71  26  32.5  71  .02> P ) .01 .05) P > .02 H rejected H rejected Q  D  Q  P s p r o b a b i l i t y of  the c a l c u l a t e d c r i t i c a l number i f H  Excretion  H  H  Q  P> .05 not r e j e c t e d  :  

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