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The effects of photoperiod and temperature on the daily pattern of locomotor activity in juvenile sockeye… Byrne, John Edward 1968

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THE EFFECTS OF PHOTOPERIOD AND TEMPERATURE ON THE DAILY PATTERN OF LOCOMOTOR A C T I V I T Y I N J U V E N I L E SOCKEYE SALMON, ONCORHYNCHUS NERKA (WALBAUM).  by JOHN EDWARD BYRNE B.A., U n i v e r s i t y M.Sc,  o f H a w a i i , 1962  U n i v e r s i t y o f H a w a i i , 1964  A THESIS SUBMITTED I N PARTIAL FULFILMENT  OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of ZOOLOGY  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g required  tothe  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA December, 1968  In p r e s e n t i n g an  this  thesis  in partial  advanced degree a t the U n i v e r s i t y  the  Library  I further for  shall  make i t f r e e l y  agree that  permission  s c h o l a r l y p u r p o s e s may  by  his representatives.  of  this  thesis  f u l f i l m e n t of the requirements f o r of B r i t i s h  Columbia,  a v a i l a b l e f o r r e f e r e n c e and f o r extensive  It i s understood gain  shall  that  copying or p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t  John E. Byrne  of  Zoology  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Date  16 December,  1968  Columbia  thesis  Department o r  wr i t t e n . p e r m i s s i o n .  Department  that  Study.  copying of this  be g r a n t e d b y t h e Head o f my  for financial  I agree  my  i Supervisor:  N.  R.  Liley  ABSTRACT The to  e n d o g e n o u s and e x o g e n o u s f a c t o r s  a d i e l rhythm of locomotor  sockeye  activity  salmon Oncorhynchus nerka  i n the l a b o r a t o r y . i n v e s t i g a t i o n was frequency  sound  The  contributing  i n juvenile  (Walbaum) w e r e e x a m i n e d  b a s i c measure employed i n t h e  spontaneous locomotor (800 kHz)  was  used  activity.  High  as a m o n i t o r i n g  technique to c o l l e c t continuous a c t i v i t y  records.  Three major areas r e c e i v e d a t t e n t i o n .  First,  a  d e s c r i p t i o n and a n a l y s i s w e r e p r o v i d e d f o r t h e e n t r a i n e d diel activity  p a t t e r n under t h r e e d i f f e r e n t  (5°,  15°C)  10°,  (8L 16D,  and 12L  temperature  12D,  and and  three d i f f e r e n t  16L  8D).  The  temperatures  photoperiods  combined e f f e c t s  and p h o t o p e r i o d u p o n t h e b a s i c 24 h o u r  w e r e r e c o r d e d and  salmon were n o c t u r n a l l y  i m m e d i a t e l y a f t e r emergence from t h e g r a v e l . p a t t e r n was  14 d a y s and was  active A  diurnal  g r a d u a l l y acquired d u r i n g the  m a i n t a i n e d f o r 12 m o n t h s .  following  Photoperiod  the primary environmental synchronizer f o r e i t h e r or  response  analyzed.  J u v e n i l e sockeye  activity  of  was  diurnal  nocturnal activity. The  e n d o g e n o u s component o f t h e a c t i v i t y  rhythm  examined i n c o n s t a n t e n v i r o n m e n t a l c o n d i t i o n s . light  (34.4  response  l u x a t 10°C)  was  Constant  f a c i l i t a t e d the free-running  w h i l e constant dark i n h i b i t e d  f r e q u e n c y i n c o n s t a n t l i g h t was  i t .  The  spontaneous  23.30 h o u r s b u t t h i s c o u l d  be  altered The  between  by  light  final  the  response.  The  experiments  environmental  physiological  sensory  The  mediating  intensity  eyes  not  When  the  sockeye  injections serotonin  were  pineal  of  amplitude  with  the  was  period  between  in  dark  photoperiodic transmission (melatonin  might thus  the  photoreceptors  light-dark  environment.  d i s a p p e a r e d when  removed  by  the  salmon  light  possess  which  The  the  fish  are  daylength  resulting  juvenile  Intraperitoneal  the  and  dark  the  endogenous the  light  active,  emergence. can  Mediation  v i a the  by  or  respectively.  temperature  pathways.  activity  i n the  an  altered  generally  responses.  nerve  or  after  serotonin) produced  control  shaded,  i s s y n c h r o n i z e d by  immediately  active  optic  or  selectively  information occurs  and  and  (N-acetyl-5-hydroxy-tryptamine)  rhythm  cycle.  f o r the  temporary  relationship  the e n t r a i n e d  increased activity.  i n either  interactions  dark,  body  sockeye  activity  photoperiodic  the  (photoperiod)  primary  (5-hydroxy-tryptamine)  circadian  pineal  the  response  melatonin  Juvenile  except  stimulus  upon  feeding.  illuminated.  responded  activity  focused  i n f o r m a t i o n about  was  periodic  mechanisms m e d i a t i n g  entrained activity  retina  or  retina, Chemical  the  retina  amplitude  characteristic  However, result  of  the  but  without  agents and/or  in light entrained  and pattern.  ERRATA Bunning should read  Blinning  D a n i e l e v s k i i should read p.  36,  line  15,  p.  87,  line  23,  p.  105,  line  22,  p. 106,  line  24,  p.  10 7,  line  1,  p.  112,  line  30,  Danilevskii  significant locations Baggerman, B. principles rhythms division  19 57  iii  TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  i  i  L I S T OF TABLES  i v i  L I S T OF FIGURES  v i i  ACKNOWLEDGEMENTS.  x  1.  INTRODUCTION  1  2.  MATERIALS AND METHODS  6  2.1  WATER SUPPLY  6  2.2  A C T I V I T Y CHAMBERS  7  2.3  TEMPERATURE CONTROLS  9  2.4  PHOTOPERIOD CONTROLS  10  2.5  A C T I V I T Y MONITORING SYSTEM  11  2.6  L I V E MATERIAL  18  2.7  EXPERIMENTAL DESIGN  20  2.8  ANALYTICAL PROCEDURE  22  3.  RESULTS  24  3.1  24  DETERMINATION OF A B A S I C ENTRAINED RESPONSE. 3.11 3.12  The R e l a t i o n s h i p o f A g e t o t h e B a s i c E n t r a i n e d Response The S y n c h r o n i z i n g E f f e c t o f t h e Environmental L i g h t Cycle a t a Constant Temperature  3.2  28  29  THE EFFECTS OF TEMPERATURE AND PHOTOPERIOD ON THE DAILY A C T I V I T Y PATTERN OF J U V E N I L E SOCKEYE SALMON  33  iv  Page 3.21  The E n t r a i n e d R e s p o n s e a t 5°C t o Three D i f f e r e n t Photoperiods (8L 16D, 12L 12D, 16L 8D)  33  The E n t r a i n e d R e s p o n s e a t 10°C t o Three D i f f e r e n t Photoperiods (8L 16D, 12L 12D, 16L 8D)  36  The E n t r a i n e d R e s p o n s e a t 15°C t o Three D i f f e r e n t Photoperiods (8L 16D, 12L 12D, 16L 8D)  38  3.24  The E f f e c t o f T e m p e r a t u r e o n t h e E n t r a i n e d Response  40  3.25  The E f f e c t o f P h o t o p e r i o d on t h e E n t r a i n e d Response  40  3.26  The I n t e r a c t i o n o f T e m p e r a t u r e a n d Photoperiod  44  3.22  3.23  3.3  ENDOGENOUS A C T I V I T Y EXPRESSED I N A CONSTANT ENVIRONMENT 3.31  3.32  3.33  3.34  The E n d o g e n o u s R e s p o n s e i n C o n s t a n t D a r k n e s s (DD) a t a C o n s t a n t T e m p e r a t u r e o f 10°C  50  The E n d o g e n o u s R e s p o n s e i n C o n s t a n t L i g h t (LL) a t a C o n s t a n t T e m p e r a t u r e o f 10°C  54  The R e s p o n s e o f t h e E n d o g e n o u s Component t o I n c r e a s i n g L i g h t Intensities ;  58  The E n t r a i n i n g E f f e c t s o f P e r i o d i c Feeding i n Constant Light  3.4  49  a t 10°C  58  THE P R I N C I P A L SENSORY RECEPTORS INVOLVED I N THE MEDIATION OF THE ENTRAINED RESPONSE. 3.41 The E n t r a i n e d R e s p o n s e i n Pinealectomized Fish  60 62  V  Page 3.42  The E n d o g e n o u s R e s p o n s e i n Pinealectomized Fish  66  3.43  The E n t r a i n e d R e s p o n s e w i t h Opaque o r T r a n s p a r e n t P l a s t i c Shields over t h e s i t e of the Pineal.  66  3.44  The E n t r a i n e d R e s p o n s e i n Blinded Fish  3.5  THE EFFECTS OF MELATONIN, SEROTONIN AND TELEOST SALINE ON THE ENTRAINED RESPONSE  4.  DISCUSSION 4.1  69  ;  THE B A S I C A C T I V I T Y PATTERN 4.11  79 83 84  The I n f l u e n c e o f T e m p e r a t u r e a n d Photoperiod  85  4.2  THE ENDOGENOUS A C T I V I T Y RESPONSE  88  4.3  MECHANISMS OF ENTRAINMENT  89  4.4  THE ADAPTIVE SIGNIFICANCE OF A DAILY  4.5  A C T I V I T Y CYCLE  93  4.41  Emergence and M i g r a t i o n  94  4.42  P e l a g i c Stage  96  4.43  Feeding  97  4.44  Diel Vertical Migrations  98  SEASONAL REGULATION OF THE ENTRAINED RESPONSE  '99  5.  SUMMARY AND CONCLUSIONS  102  6.  LITERATURE CITED.  105  APPENDIX - C i r c u i t D i a g r a m s a n d S u r g i c a l T e c h n i q u e s . . . 114  vi  L I S T OF TABLES  Table I  Page A comparison  of activity  p e r 10  minute i n t e r v a l b e f o r e , d u r i n g and after II  instrument operation  A comparison  17  o f v i s u a l and i n s t r u m e n t a l  recording techniques III  17  Summary o f t e m p e r a t u r e a n d photoperiod conditions  IV  Summary o f c o n s t a n t  21  environmental  conditions V  Periodicity darkness  VI  21 expressed  i n constant  a t 10°C  Periodicity  53  expressed i n constant  l i g h t a t 10°C VII  Periodicity light  expressed w i t h  i n t e n s i t i e s a t 10°C  57 increasing 59  vii  LIST OF FIGURES  Figure  Page  1  Top and s i d e views o f a c t i v i t y chambers  2  S o n i c t r a n s d u c e r and components  13  3  B l o c k diagram o f m o n i t o r i n g and c i r c u i t r y  15  4  5  6  7  8  9  10  11  8  instruments  The e n t r a i n e d response o f one i n d i v i d u a l a t 8L 16D, 5°C  26  The mean d a i l y a c t i v i t y p a t t e r n s o f j u v e n i l e L a k e l s e and Kamchatka sockeye  26  The mean d a i l y a c t i v i t y p a t t e r n s a t d i f f e r e n t s t a g e s o f development  30  The mean d a i l y a c t i v i t y p a t t e r n and periodogram a n a l y s i s o f f i v e i n d i v i d u a l s recorded simultaneously  32  The mean d a i l y a c t i v i t y p a t t e r n s f o r t h r e e p h o t o p e r i o d s a t 5°C  34  The mean d a i l y a c t i v i t y p a t t e r n s f o r t h r e e p h o t o p e r i o d s a t 10°C  37  The mean d a i l y a c t i v i t y p a t t e r n s f o r t h r e e p h o t o p e r i o d s a t 15°C  39  The mean d a i l y a c t i v i t y p a t t e r n s a t 12L 12D f o r 5°C, 10°C, and 15°C  41  viii  Figure 12  13  14  15  16  17  18  19  20  21  22  Page P l o t s o f a c t i v i t y and temperature at three d i f f e r e n t photoperiods  42  P l o t s o f a c t i v i t y and p h o t o p e r i o d a t t h r e e d i f f e r e n t temperatures  43  Three d i m e n s i o n a l i l l u s t r a t i o n o f t h e mean t o t a l a c t i v i t y a t each temperature and p h o t o p e r i o d  45  Mean d a i l y a c t i v i t y p a t t e r n s f o r two i n d i v i d u a l s a t 8L 16D, 15°C  46  Percent occurrence of n o c t u r n a l a c t i v i t y a t 5°C, 10°C, and 1 5 ° C  ..  48  P e r c e n t a c t i v i t y d u r i n g l i g h t and dark a t t h r e e p h o t o p e r i o d s  48  The endogenous response i n c o n s t a n t dark a t 10°C  51  Periodogram a n a l y s i s o f one i n d i v i d u a l i n c o n s t a n t dark  ;.  52  A c o n t i n u o u s 12 day r e c o r d o f one i n d i v i d u a l i n c o n s t a n t l i g h t a t 10°C  55  Periodogram a n a l y s i s o f t h e d a t a p r e s e n t e d i n F i g u r e 20  56  The mean a c t i v i t y p a t t e r n i n c o n s t a n t l i g h t to periodic feeding  61  ix  Figure 23  24  25  26  27  28  29  30  31  Page The a c t i v i t y r e s p o n s e o f one i n d i v i d u a l i m m e d i a t e l y a f t e r p i n e a l e c t o m y and a g a i n two weeks l a t e r  63  The mean d a i l y a c t i v i t y p a t t e r n s f o r p i n e a l e c t o m i z e d a n d sham pinealectomized f i s h  65  Periodogram analysis of a s i n g l e pinealectomized f i s h i n constant light..  67  Mean d a i l y a c t i v i t y p a t t e r n s o f b l a c k s h i e l d and c l e a r s h i e l d t e s t s  70  Mean d a i l y a c t i v i t y p a t t e r n s f o r b l i n d e d and p i n e a l e c t o m i z e d , b l i n d e d , a n d b i l a t e r a l l y enucleated groups  73  Mean d a i l y a c t i v i t y p a t t e r n s f o r b l a c k and c l e a r eye cap groups  76  12L 12D, 5°C r e s p o n s e i n n o r m a l f i s h and f i s h t e s t e d a f t e r removal o f b l a c k eye caps.  78  Mean t o t a l a c t i v i t y f o r s e r o t o n i n , m e l a t o n i n and t e l e o s t s a l i n e groups  81  Mean d a i l y a c t i v i t y p a t t e r n s f o r t h r e e c o n s e c u t i v e days a f t e r i n j e c t i o n  82  X  ACKNOWLEDGEMENTS T h i s s t u d y was f i n a n c e d b y t h e F i s h e r i e s  Research  B o a r d o f C a n a d a t h r o u g h g r a n t s made t o D r . N.R. L i l e y a n d t h e Department  of Zoology, U n i v e r s i t y of B r i t i s h  Columbia.  I o f f e r my s i n c e r e t h a n k s t o D r . C. G r o o t f o r t h e initial  encouragement,  s u g g e s t i o n s and l a b o r a t o r y space a t  t h e Nanaimo B i o l o g i c a l S t a t i o n .  D r . J.R. B r e t t p r o v i d e d  many h e l p f u l comments o n t h e w o r k a n d o b t a i n e d s t o c k s o f j u v e n i l e sockeye salmon whenever needed.  I t was.a p l e a s u r e  t o w o r k u n d e r t h e g u i d a n c e o f D r . N.R. L i l e y , my m a j o r s u p e r v i s o r , t o whom I owe a g r e a t d e a l f o r t h e c o m p l e t i o n of t h i s  study.  D r s . W.S. H o a r , N . J . W i l i m o v s k y , N.R. L i l e y > N o r t h c o t e a n d J.R. B r e t t c r i t i c a l l y o f f e r e d many u s e f u l s u g g e s t i o n s .  T.G.  r e a d t h e t h e s i s and D r . H.C. N o r d a n p r o v i d e d  l a b o r a t o r y s p a c e o n t h e U.B.C. campus.  H i s i n t e r e s t and  s u g g e s t i o n s were h e l p f u l i n e s t a b l i s h i n g t h e c o n t r o l l e d laboratory  environment.  P e r s o n a l f u n d s w e r e made a v a i l a b l e t h r o u g h a t h r e e y e a r s c h o l a r s h i p f r o m t h e F i s h e r i e s R e s e a r c h B o a r d o f Canada and a U.B.C. T e a c h i n g A s s i s t a n t s h i p . I would a l s o l i k e t o acknowledge  my w i f e ' s  assistance  i n counting t h e "endless m i l e s " of event r e c o r d e r charts o b t a i n e d d u r i n g t h e i n v e s t i g a t i o n , and t h e v a r i o u s p e o p l e i n the Department  o f Z o o l o g y who h e l p e d make my s t a y a t t h e  U n i v e r s i t y o f B r i t i s h Columbia a p l e a s a n t one.  1  1. The an  INTRODUCTION  primary objective  of t h i s thesis i s to provide  a c c u r a t e d e s c r i p t i o n and a n a l y s i s o f t h o s e endogenous  and  exogenous f a c t o r s c o n t r i b u t i n g  to the expression of  a d a i l y rhythm o f l o c o m o t o r a c t i v i t y by j u v e n i l e salmon, Oncorhynctius n e r k a The life  c y c l e which these f i s h before migrating  duration  two,  of t h i s period  and o c c a s i o n a l l y  lakes  (Walbaum).  term " j u v e n i l e " r e f e r s t o the i n t e r v a l i n the  water lakes The  sockeye  spend as r e s i d e n t s  i n fresh-  t o a marine environment. i s usually  one,  frequently  t h r e e y e a r s , b u t i n t h e more n o r t h e r n  of t h e i r geographical d i s t r i b u t i o n the longer  o f r e s i d e n c e a r e more common ( F o e r s t e r ,  terms  1968).  I t appears t h a t b i o l o g i c a l r h y t h m i c i t i e s  are  ubiquitous  i n t h e a n i m a l kingdom, and c a n have a p r o f o u n d e f f e c t upon a d i v e r s i t y o f p h y s i o l o g i c a l and b e h a v i o u r a l  processes.  d i s p l a y o f p e r i o d i c phenomena a t b o t h t h e c e l l u l a r  The  and i n t a c t  o r g a n i s m l e v e l s has been w e l l documented, and endogenous rhythms a r e a p p a r e n t l y a t t h e base of.numerous e x p r e s s i o n s of b e h a v i o u r and development r e c e i v i n g a t t e n t i o n years  (reviews:  1965;  H a r k e r , 1964; F a r n e r , 1965; B u n n i n g , 1 9 6 7 ) .  and  lunar  i n recent  Cloudsley-Thompson, 1960, 1961; D a n i e l e v s k i i , Circadian  p e r i o d i c i t i e s have been i d e n t i f i e d i n s i n g l e  neurons  (Strumwasser, 1 9 6 5 ) , and a t t h e s u b - c e l l u l a r l e v e l t h e rhythmic production of nucleic acids attention  ( E l i n g , 1967; J e r u s a l e m ,  has r e c e i v e d  1967).  limited  D a i l y p e r i o d i c i t i e s have been i n v e s t i g a t e d a t t h e i n t a c t organism l e v e l the protozoa  i n a number o f s p e c i e s r a n g i n g  to the higher vertebrates.  d i n o f l a g e l l a t e Gonyaulax polyedra i n bioluminescence  The  from  unicellular  e x h i b i t s a d i e l rhythm  and c e l l d i v i s i o n  (Sweeney a n d  Hastings,  1957, 1 9 5 8 ) , whereas t h e h i g h e r v e r t e b r a t e s i n c l u d i n g b i r d s , rodents  a n d man  have been d e m o n s t r a t e d t o p o s s e s s  c h a r a c t e r i s t i c d i e l c y c l e s which are synchronized s p e c i f i c environmental and  stimuli  (DeCoursey, 1960; Hauty  Adams, 1 9 6 5 ; M e n a k e r , 1 9 6 5 ; M e n a k e r and E s k i n , P h o t o p e r i o d i c mechanisms r e c e i v e f r e q u e n t  a t i o n i n those  with a characteristic an i m p o r t a n t  consider-  H e r e , an endogenous  rhythmici  24 h o u r p e r i o d i s s u g g e s t e d t o p l a y  r o l e i n the d i r e c t i o n f i n d i n g of a v a r i e t y of  organisms, i n c l u d i n g f i s h  (Schwassmann, 196 0; Schwassmann  B r a e m a r , 1 9 6 1 ; G r o o t , 1 9 6 5 ; H a s l e r , 1967) . A survey  of the l i t e r a t u r e reveals that only  a t t e n t i o n has been f o c u s e d in teleosts.  upon p h o t o p e r i o d i c  Most e f f o r t s have c o n s i d e r e d  s i g n i f i c a n c e of the response t o daylength and  1967).  i n v e s t i g a t i o n s p e r t a i n i n g t o the f i e l d s of  o r i e n t a t i o n and n a v i g a t i o n .  and  by  mechanisms  the  Baggerman, Harrington,  1 9 5 7 , 1959, 1 9 6 0 a , b; F a r n e r ,  adaptive  and t e m p e r a t u r e ,  i t s r e l a t i o n s h i p t o t h e t i m i n g and o n s e t o f  or the c o n t r o l of reproductive behaviour  limited  migrations  (reviews: 1961a, 1965;  1959; H o a r , 1951, 1953, 1965).  C y c l i c p a t t e r n s o f l o c o m o t o r a c t i v i t y have been  3  r e p o r t e d i n a.few t e l e o s t s , i n c l u d i n g s a l m o n i d s  (Northcote,  1962;  B l a h m , u n p u b l i s h e d ; D a v i s and B a r d a c h ,  1965;  1965;  T h i n e s , e t a l . , 1965;  1966;  L i c h t e n h e l d , 1967; 1968).  The  Bohun and W i n n ,  V e r h e i j e n and D e G r o o t , 1967;  p a t t e r n i s e x p r e s s e d by  salmon d u r i n g a l l s t a g e s o f t h e i r l i f e  In  Chaston,  evidence a v a i l a b l e to date suggests that a  rhythmic d i e l a c t i v i t y  Johnson,  Groot,  1961;  Groot,  cycle  sockeye  (Hoar,  1958;  1965).  t h i s i n v e s t i g a t i o n the locomotor a c t i v i t i e s  j u v e n i l e sockeye  of  salmon were m o n i t o r e d w i t h o u t t h e u s u a l  v i s u a l and t a c t i l e  s t i m u l i t h a t have.been a s s o c i a t e d w i t h  f o r m e r t e c h n i q u e s f o r r e c o r d i n g t h e movements o f s m a l l f i s h or  other a q u a t i c organisms.  S o n i c . t r a n s d u c e r s were  i n t r o d u c e d as s e n s i n g d e v i c e s t o d e t e c t f i s h movement. Supporting e l e c t r o n i c instruments transformed the f r o m t h e s e u n i t s t o an e l e c t r i c a l  output  impulse a c t u a t i n g  pens  on an e v e n t r e c o r d e r . The  first  experiments provide a d e s c r i p t i o n  and  a n a l y s i s of e n t r a i n e d l o c o m o t o r - a c t i v i t y under t h r e e d i f f e r e n t p h o t o p e r i o d s and t h r e e d i f f e r e n t The  design of the experiments  temperatures.  enabled the i n v e s t i g a t o r  r e c o r d and a n a l y z e t h e c o m b i n e d e f f e c t s o f t e m p e r a t u r e p h o t o p e r i o d upon t h e . b a s i c t w e n t y - f o u r hour The  to and  cycle.  s e c o n d p h a s e f o c u s e s u p o n an a n a l y s i s o f t h e  e n d o g e n o u s c o m p o n e n t , as e x p r e s s e d by t h e f r e e - r u n n i n g rhythm  of f i s h h e l d under c o n s t a n t e n v i r o n m e n t a l  conditions.  4 This i s considered.necessary since i t i s t h i s which  component  i s e n t r a i n e d by t h e e n v i r o n m e n t a l s t i m u l i .  ment r e s u l t s i n t h e c h a r a c t e r i s t i c d i e l apparent The  under normal t h i r d and  Entrain-  periodicity  o s c i l l a t i n g environmental  conditions.  f i n a l p h a s e i n v e s t i g a t e s some r e l a t i o n -  s h i p s b e t w e e n t h e e n v i r o n m e n t a l s t i m u l i , and t h e  physiological  s e n s o r y mechanisms m e d i a t i n g t h e e n t r a i n e d r e s p o n s e .  The  m o s t s t r i k i n g c h a n g e s i n m o t o r a c t i v i t y d u r i n g any  twenty-  f o u r hour p e r i o d o c c u r r e d i n r e l a t i o n t o the onset  and  t e r m i n a t i o n of l i g h t .  Emphasis i s f o c u s e d upon t h e  role  o f t h e e y e s a n d / o r p i n e a l b o d y as t h e p r i n c i p a l r e c e p t o r organs  f o r d e t e c t i n g changes i n l i g h t i n t e n s i t y .  Both  s u r g i c a l and p h a r m a c o l o g i c a l t e c h n i q u e s w e r e u t i l i z e d t h i s phase of the The  in  investigation.  f o l l o w i n g q u o t a t i o n i s a p p l i c a b l e to the problems  a t hand. " I f any a t t e m p t i s t o be made t o c o n t r o l a c t i v i t i e s of f i s h through a manipulation of e n v i r o n m e n t a l c o n d i t i o n s i t w i l l be n e c e s s a r y t o u n d e r s t a n d t h e p h y s i o l o g i c a l b a s i s o f some of the behaviour. This i s p a r t i c u l a r l y true w i t h r e s p e c t t o m i g r a t o r y phenomena w h i c h o c c u r c y c l i c a l l y and a r e a s s o c i a t e d w i t h r e g u l a r c h a n g e s i n the endocrine glands. I t may o r may n o t be p o s s i b l e t o e x e r c i s e a c o n t r o l over migratory b e h a v i o u r b u t , i n any c a s e , i t i s e s s e n t i a l t o know s o m e t h i n g o f t h e i n t e r - r e l a t i o n s h i p s o f e n v i r o n m e n t a l and p h y s i o l o g i c a l c y c l e s i n o r d e r t o p r e d i c t the sequence o f b e h a v i o u r . A study of the p h y s i o l o g i c a l changes which a r e r e s p o n s i b l e f o r m i g r a t i o n demands p a r t i c u l a r a t t e n t i o n . I n a d d i t i o n , p h y s i o l o g y c a n make an i m p o r t a n t c o n t r i b u t i o n t o e t h o l o g y by e v a l u a t i n g t h e l i m i t s o f p e r c e p t i o n o f t h e r e c e p t o r mechanisms o f f i s h of a l l ages. P r e d i c t i o n o f many a c t i v i t i e s w h i c h depend upon c h a n g i n g l i g h t , t e m p e r a t u r e o r w a t e r c u r r e n t s demands s u c h i n f o r m a t i o n " ( H o a r , 1958) .  In summary, the main purpose  of t h i s i n v e s t i g a t i o n  i s t o f o l l o w a course of b a s i c r e s e a r c h concerning the e f f e c t s of p h o t o p e r i o d and temperature  on the  a c t i v i t y o f j u v e n i l e sockeye  The data presented  salmon.  locomotor  are a p p l i c a b l e to a v a r i e t y of problems which.consider or are dependent upon the d a i l y a c t i v i t y c y c l e s expressed teleosts.  by  6  2. The  MATERIALS AND  i n v e s t i g a t i o n was  environmental  conditions.  METHODS  conducted under The  l a b o r a t o r y was  i n a c o n c r e t e b a s e m e n t a r e a o f an where p e r i o d i c d i s t u r b a n c e s The  controlled  isolated  c o u l d be k e p t  located  building, a t a minimum.  o p e r a t i o n o f a i r pumps f o r a q u a r i a c r e a t e d a  n o i s e l e v e l , c o n f u s i n g any i n other p a r t s of the  incidental production.of  f l u c t u a t i o n s w h i c h may  that certain pervasive  environmental  have t h e c a p a c i t y t o e n t r a i n t h e  s u b j e c t s c o u l d n o t be m a n i p u l a t e d .  (For a review  s u c h n a t u r a l l y o s c i l l a t i n g f a c t o r s as  pressure  s e e B r o w n , 1960,  and  t h e i r e f f e c t s on c y c l i c  1965).  of  electromagnetic  c o s m i c r a y bombardment, c h a n g e s i n t h e  barometric  sound  building.  I t i s recognized  fields,  constant  At the present  local activity,  time, the  author  does not c o n s i d e r i t p r a c t i c a l t o a t t e m p t t h e c o n t r o l of these  subtle  2.1  Water  factors. Supply  C h l o r i n a t e d c i t y w a t e r was activities  and  as a s u p p l y  used f o r g e n e r a l  maintenance  f o r temperature regulated water  baths. A s y s t e m was a d j u s t t h e pH maintained  designed  to dechlorinate, purify  of the c i t y water.  a t f u l l w a t e r l e v e l by  and  This dechlorinator installing  valve d e l i v e r i n g a regulated water flow.  was  a control  The  unit  was  p l a c e d o u t s i d e t h e l a b o r a t o r y , i n a l o c a t i o n i n s u r i n g an  7  adequate water pressure  f o ra gravity flow  system.  The d e l i v e r y s y s t e m t o t h e a q u a r i a a n d a c t i v i t y c h a m b e r s was c o n s t r u c t e d polyethylene  pipe.  with the water supply  o f 1/2" (1.3 cm) i n s i d e d i a m e t e r  No m e t a l f i t t i n g s w e r e i n c o n t a c t a n d t h e c o m p l e t e s y s t e m was  i n s u l a t e d w i t h f i b e r g l a s s t o reduce v a r i a t i o n s i n the temperature. 2.2  Activity  Chambers  The e x t e r n a l d i m e n s i o n s o f e a c h a c t i v i t y  chamber  w e r e 48" x 48" x 8" (122 cm x 122 cm x 20.3 c m ) . chambers were c o n s t r u c t e d  o f 1/2" (1.3 cm) p l y w o o d a n d  t h e i n t e r i o r s were l i n e d w i t h f i b e r g l a s s c l o t h , with Estrex added  The  coated  #101 a l l p u r p o s e r e s i n f o r w a t e r p r o o f i n g  and  strength. E a c h chamber c o n t a i n e d  swimming c h a n n e l  a water bath  and a c i r c u l a r  m e a s u r i n g 7" w i d e a n d 7" (17.8 cm x  17.8 cm) d e e p w i t h a maximum d i a m e t e r o f 3 6 " (91.44 cm) (Fig. 1 ) .  The swimming c h a n n e l  was f o r m e d by two r i n g s  o f 1/16" x 8" (1.8 mm x 20.3 cm) p l e x i g l a s s w h i c h w e r e s e a l e d t o t h e b o t t o m o f t h e a c t i v i t y chamber w i t h f i b e r g l a s s resin.  The w a l l s w e r e p a i n t e d w i t h n o n - t o x i c  rust-oleum Illinois)  paint  (The R u s t - O l e u m C o r p o r a t i o n ,  flat  black  Evanston,  t o r e d u c e g l a r e a n d t o m i n i m i z e t h e number o f  l a t e r a l cues a v a i l a b l e t o t h e f i s h . All  d e c h l o r i n a t e d w a t e r was c o n t a i n e d  maximum c i r c u m f e r e n c e  within the  o f t h e swimming c h a n n e l .  The i n n e r  Diagram i l l u s t r a t i n g  top and s i d e  views of an a c t i v i t y  chamber. A l l  component s e c t i o n s are l a b e l l e d .  top view  \ TRANSDUCER  \ OUTER  WALL  J CABLE  TO  CIRCUIT WATER  LEVEL  1  side view  9  r i n g o f p l e x i g l a s s h a d s i x t e e n 1/8" (3.6.mm) p e r f o r a t i o n s to  a l l o w an exchange o f w a t e r i n t o and o u t o f t h e c h a n n e l .  These h o l e s level  a l s o a s s i s t e d i n keeping  b y a c t i n g a s a " s c o u r i n g s y s t e m " when w a t e r was  m o v i n g o u t o f t h e swimming A t o t a l o f nine for  d e t r i t u s a t a low  activity  theinvestigation.  vertical  chambers were  constructed  They w e r e mounted i n t h r e e  racks constructed  rack contained  channel.  o f #225 s t e e l d e x i o n .  Each  t h r e e a c t i v i t y chambers and measured  49" x 49" x 84" (124 cm x 124 cm x 213 c m ) . 2.3  Temperature  Controls  Water temperatures i n t h e a c t i v i t y c o n t r o l l e d b y Kodak t h e r m o s t a t i c m i x i n g Kodak C o . , R o c h e s t e r ,  N.Y.).  chambers were valves  (Eastman  These were c o n n e c t e d t o  the h o t and c o l d c i t y water o u t l e t s and s u p p l i e d t h e water baths surrounding The  temperatures o f these  allowed an  t h e o u t e r b o r d e r o f . t h e swimming two b o d i e s  channels.  o f w a t e r were a l w a y s  t o come i n t o e q u i l i b r i u m b e f o r e , p r o c e e d i n g  with  experiment. Low w a t e r t e m p e r a t u r e s w e r e m a i n t a i n e d  during the  summer m o n t h s b y c o o l i n g t h e c i t y w a t e r w i t h a p o r t a b l e refrigerating  unit.  A H o n e y w e l l t h e r m o g r a p h was u s e d t o r e c o r d t h e d a i l y t e m p e r a t u r e f l u c t u a t i o n s i n t h e swimming c h a n n e l s M a r c h , 1967. obtained  These d a t a were compared w i t h  during  readings  by hand-held thermometers and t h e d i a l i n d i c a t o r s  10  on t h e t h e r m o s t a t i c m i x i n g  valves.  A f t e r e q u i l i b r i u m was r e a c h e d b e t w e e n t h e swimming c h a n n e l s and t h e w a t e r b a t h s , t h e . r e c o r d i n d i c a t e d t h a n 0.3°C f l u c t u a t i o n i n t h e e n v i r o n m e n t a l temperature  d u r i n g a t w e n t y - f o u r hour p e r i o d  appendix). were adequate  less  water ( F i g . 1,  I t was assumed t h a t t h e t e m p e r a t u r e  controls  and t h e s l i g h t d a i l y v a r i a t i o n d i d n o t  c o n s t i t u t e a s u f f i c i e n t value t o e n t r a i n the locomotor a c t i v i t y o f j u v e n i l e sockeye  salmon.  The u s e o f t h e t h e r m o g r a p h  was d i s c o n t i n u e d a f t e r  e s t a b l i s h i n g that both the records obtained w i t h the m i x i n g v a l v e s and t h e h a n d - h e l d  thermometers were  sufficient  o v e r t h e c o u r s e o f a seven day e x p e r i m e n t a l p e r i o d . The e n v i r o n m e n t a l . t e m p e r a t u r e s  ranged  f r o m 5°C t o  15°C, w h i c h a r e w i t h i n t h e g e n e r a l l y a c c e p t e d l i m i t s o f t o l e r a n c e f o r j u v e n i l e sockeye 2.4  salmon  ( B r e t t , 1952, 1956).  Photoperiodic Controls I l l u m i n a t i o n was p r o v i d e d by i n c a n d e s c e n t  e x c e p t where s p e c i f i c a l l y  indicated.  was a c h i e v e d w i t h w h i t e p l a s t i c (30.5 cm) a b o v e t h e w a t e r  lamps,  Indirect  lighting  s h e e t i n g p o s i t i o n e d 12"  surface.  I t was assumed t h e  opaque s h e e t i n g a l s o r e d u c e d t h e number o f o v e r h e a d a v a i l a b l e to the f i s h s h i e l d e d each a c t i v i t y  for orientation. chamber t o p r e v e n t  Black  cues  plastic  interference  w i t h t h e e n v i r o n m e n t a l p h o t o p e r i o d by e x t e r n a l l i g h t Sudden c h a n g e s i n t h e room l i g h t i n t e n s i t y  were  sources.  prevented  by c o n s t r u c t i n g a " f a l s e e n t r y " t o t h e l a b o r a t o r y  and by m a i n t a i n i n g a c o n s t a n t l o w i l l u m i n a t i o n w i t h a s h i e l d e d 7 1/2 The  watt  environmental  m o d e l T-101  p h o t o p e r i o d s w e r e r e g u l a t e d by  I n t e r m a t i c time switches  C o o k s v i l l e , Ont.) and  lamp.  connected  (Marr E l e c t r i c L t d . ,  which were s y n c h r o n i z e d w i t h each o t h e r  to separate channels  on t h e e v e n t  recorder.  T h i s a r r a n g e m e n t i n d i c a t e d t h e e x a c t t i m e o f day l i g h t i n g c o n d i t i o n s w e r e c h a n g e d and  p r o v i d e d a measure o f  t h e p h a s e r e l a t i o n s h i p b e t w e e n p h o t o p e r i o d and termination of An  t h a t the  the onset  activity.  e l e c t r o n i c dimming system  e f f e c t to the a r t i f i c i a l  introduced a  environment.  The  twilight  circuitry  had  a 600 w a t t c a p a c i t y and was  a d j u s t e d t o produce a gradual  change i n . t h e e n v i r o n m e n t a l  l i g h t i n t e n s i t y over a  minute p e r i o d  or  ( F i g s . 2 & 3, a p p e n d i x ) .  These  twenty  "dimmers"  e l i m i n a t e d the d r a m a t i c changes i n l i g h t i n t e n s i t y a s s o c i a t e d w i t h i n t e r v a l t i m e r s and response  observed  avoided.the  i n j u v e n i l e sockeye  initial  "startle"  when t h e l i g h t s w e r e  t u r n e d on o r o f f . L i g h t i n t e n s i t y r e a d i n g s were t a k e n a t t h e s u r f a c e w i t h a Brockway i n c i d e n t l i g h t meter. w e r e c o n v e r t e d t o " l u x " w h e r e one  lux equals  water  A l l readings 0.0929  ,candlepower. 2.5  A c t i v i t y M o n i t o r i n g System The  e l e c t r o n i c instruments f o r recording  locomotor  12  a c t i v i t y were b u i l t  around.a compact s o n i c  (Enterprise Manufacturing The  plastic  Co., A k r o n ,  Ohio)  transducer (Fig.2).  t r a n s d u c e r head c o n t a i n e d t h e c i r c u i t r y i n  a sealed waterproof  unit. .  A l l electronic  components  w e r e s p r a y e d w i t h a r u b b e r i z e d , compound f o r a d d e d r e s i s t a n c e a g a i n s t shock and m o i s t u r e . Each t r a n s d u c e r e m i t t e d a continuous.wave o f 800 k i l o h e r t z This frequency  i n a 4° c o n e .  i s w e l l above t h e w a v e l e n g t h s h e a r d  f i s h i n general 1965;  (1 h e r t z = 1 c y c l e / s e c o n d )  signal  (Wodinski  and T a v o l g a ,  by  1963; M a r s h a l l ,  P r o t a s o v , 1967) a n d s o c k e y e s a l m o n i n . p a r t i c u l a r  ( B r e t t and Groot,  1 9 6 3 ; Van d e r W a l k e r , 1 9 6 6 ) .  beam was t r a n s m i t t e d t h r o u g h  When t h e  t h e w a t e r a n d came i n c o n t a c t  w i t h an o b j e c t , t h e s i g n a l was " r e f l e c t e d " b a c k t o t h e transducer. frequency  The o u t p u t  o f t h e i n s t r u m e n t was t h e a m p l i f i e d  d i f f e r e n c e between t h e e m i t t e d and r e t u r n i n g  signals.  I f an o b j e c t was t r a v e l l i n g  away f r o m t h e s o u r c e  o f sound e m i s s i o n , t h e w a v e l e n g t h o f t h e " r e f l e c t e d " w o u l d be of. a l o w e r f r e q u e n c y  signal  than i f t h e o b j e c t were  motionless.  I f t h e o b j e c t was m o v i n g t o w a r d s t h e t r a n s d u c e r ,  the frequency  o f t h e r e t u r n i n g w a v e l e n g t h w o u l d be g r e a t e r .  T h e r e f o r e , a . m o v i n g o b j e c t i n t h e "beam" r e s u l t e d i n a frequency event  change w h i c h could.be  a m p l i f i e d t o operate  an  recorder. Two u n i t s w e r e m o u n t e d i n o p p o s i t e c o r n e r s o f t h e  activity bath.  chamber  ( s e e F i g . 1) i n t h e a r e a o f t h e w a t e r  The beam p e n e t r a t e d  t h e 1/16" (1.3 mm)  outer  13  F i g u r e 2.  Photographs i l l u s t r a t i n g head and components.  the  transducer  (From l e f t t o  r i g h t , c a b l e , c a b l e s e c u r i n g screw, O-ring, waterproof body, end of c a b l e , p r i n t e d c i r c u i t board w i t h compound removed electronic  rubberized  t o r e v e a l the  components.  Located  above  i s the cap which houses both the sound emission  and d e t e c t i n g u n i t s ) .  14  p l e x i g l a s s w a l l and c r o s s e d t h e channel- a t a  tangent.  T h i s arrangement e l i m i n a t e d any . p o s s i b l e i n t e r f e r e n c e b e t w e e n t h e two u n i t s , a n d i n s u r e d t h a t an o b j e c t passing through  the f i e l d  o f sound would.be  recorded.  A c a b l e f r o m e a c h t r a n s d u c e r was c o u p l e d supporting electronic c i r c u i t r y . c o n s t r u c t i o n of these  instruments  with  The d e s i g n and were commissioned t o  N a t i o n a l E l e c t r o l a b A s s o c i a t e s , V a n c o u v e r , B.C. c i r c u i t diagrams a r e presented t h i s t h e s i s (Figs. The  4 t o 8,  The  i n the l a s t section of  appendix).  b a s i c o p e r a t i o n was a s f o l l o w s .  Each  activity  chamber was a s s o c i a t e d w i t h t w o t r a n s d u c e r s , a b i s t a b l e l o g i c c i r c u i t a n d a p e n o n a 20 c h a n n e l event recorder  (Fig. 3).  Esterline-Angus  The s y s t e m was a r r a n g e d  so t h a t  f i s h movement d e t e c t e d b y one t r a n s d u c e r w o u l d c a u s e t h e b i s t a b l e l o g i c c i r c u i t t o assume a " s e t " s t a t e .  Then,  f i s h movement d e t e c t e d b y t h e o t h e r t r a n s d u c e r w o u l d the l o g i c c i r c u i t  t o i t s "count"  release a pulse to the recorder. t o be d e t e c t e d  a g a i n by t h e f i r s t  state.  the next  count.could  T h i s . a c t i o n would  The f i s h movement h a d transducer  b i s t a b l e l o g i c c i r c u i t t o . r e v e r t back.to before  alter  and cause t h e  i t s "set" state  be r e l e a s e d .  T h i s c i r c u i t arrangement.was n e c e s s a r y  i n order t o  i n s u r e t h a t a n y c o u n t r e l e a s e d was n o t t h e r e s u l t o f f i s h movement i n t h e beam o f o n l y o n e . t r a n s d u c e r . .  This  prevented  r e c o r d i n g s w h i c h m i g h t be i n t e r p r e t e d , as. r e p r e s e n t i n g a h i g h l e v e l o f a c t i v i t y , b u t a c t u a l l y c o n s i s t e d o f movement w i t h i n  15  F i g u r e 3.  Block diagram i l l u s t r a t i n g  the  t r a n s d u c e r s and a s s o c i a t e d circuitry. 1  =  Transducer  2  =  Amplifier  3  =  Bistable logic  4  =  24 v o l t s  5  =  To  AC  Recorder  circuit  Block Diagram  16  a small area. is  The b e h a v i o u r  o f p i n k a n d chum s a l m o n f r y  s u c h t h a t c o n t i n u e d u n i d i r e c t i o n a l movement o c c u r s i n  a circular  swimming c h a n n e l  (Hoar,  1956).  The j u v e n i l e  sockeye salmon used i n these t e s t s e x h i b i t e d s i m i l a r movement p a t t e r n s . locomotor  The r e s u l t was a r e l i a b l e m e a s u r e o f  a c t i v i t y which,  i f needed, c o u l d a l s o  the approximate d i s t a n c e covered  indicate  d u r i n g any i n t e r v a l .  No v i s u a l o r t a c t i l e d i s t u r b a n c e c o u l d be - a t t r i b u t e d to the monitoring devices.  The t r a n s d u c e r s w e r e l o c a t e d  o u t s i d e o f t h e swimming, c h a n n e l , an a r r a n g e m e n t p e r m i t t i n g the i n v e s t i g a t i o n t o proceed  without  the a d d i t i o n o f another  v a r i a b l e t o t h e immediate environment. had  This  instrument  d e f i n i t e advantages over t h e use o f p h o t o c e l l s and  t h e i r ambient l i g h t i n t e n s i t y , o r mechanical i n t e r f e r e d w i t h f i s h movement Thines,  et a l . ,  The  g r i d s which  (Davis and B a r d a c h , 1965;  1 9 6 5 ; Bohun a n d W i n n , 1 9 6 6 ) .  t r a n s d u c e r s were t e s t e d f o r any i n t e r f e r e n c e w i t h  the a c t i v i t y o f t h e experimental  subjects.  i n t r o d u c e d s i n g l y i n t o a swimming c h a n n e l a c t i v i t i e s were r e c o r d e d t a l l y meter).  b y v i s u a l means  Ten f i s h and t h e i r  were motor  ( a i d e d by a hand  These measurements were conducted  fora  10 m i n u t e i n t e r v a l b e f o r e , d u r i n g a n d a f t e r t h e i n s t r u m e n t s were s e t i n t o o p e r a t i o n .  One mark was r e c o r d e d  r e v o l u t i o n o f t h e a c t i v i t y chamber.  f o r each  These d a t a were  s u b j e c t e d t o a n a l y s i s o f v a r i a n c e , i n d i c a t i n g t h a t no s i g n i f i c a n t a c t i v i t y changes r e s u l t e d from t h e h i g h s i g n a l p r o d u c e d by t h e t r a n s d u c e r s  (Table 1 ) .  frequency  17  T a b l e 1.  A c o m p a r i s o n o f a c t i v i t y p e r 10 m i n u t e  b e f o r e , d u r i n g and a f t e r  interval  i n s t r u m e n t o p e r a t i o n . (P<.01)  T e s t Number 1  2  3  4  5  6  7  8  9  10  Total  Before  "A"  6  1  4  3  2  5  4  1  1  7  34  During  "B"  7  0  4  3  3  7  4  0  . 2  5  35  After  "C"  6  1  3  1  5  6  5  0  1  4  32  T a b l e 2.  A comparison o f v i s u a l and i n s t r u m e n t a l  r e c o r d i n g t e c h n i q u e s . (P<.01)  T e s t Number 1.  2  3  4  5  6  7  8  9  10  Tally  0  7  4  0  2  1  1  4  6  1  26  Inst.  0  7  4  0  2  1  1  4  7  1  27  Total  18 A related  s e r i e s o f t e s t s c o m p a r e d v i s u a l and  automatic recording  methods t o e s t a b l i s h t h e a c c u r a c y  of  recording  the instrumental  system.  i n t r o d u c e d s i n g l y i n t o an a c t i v i t y simultaneous record  a reference point  b o t h methods.  each time t h e s u b j e c t  w h i c h was s i t u a t e d  passed  i n t h e beam o f  transducer t r i g g e r i n g the event recorder. facilitated  were  chamber a n d a  was o b t a i n e d w i t h  A c o u n t was r e g i s t e r e d  Ten f i s h  This  that  location  t h e c o m p a r a t i v e a s p e c t o f t h e two m e t h o d s .  Each t e s t l a s t e d t e n m i n u t e s and t e n r e p l i c a t i o n s were produced f o r the s e r i e s . The i n s t r u m e n t a l  t e c h n i q u e o f d a t a c o l l e c t i o n gave  records which w e r e . v i r t u a l l y by  v i s u a l observations  i d e n t i c a l to those  (Table 2 ) .  The i n s t r u m e n t s a n d c i r c u i t r y suited can  f o r recording  be a p p l i e d  obtained  a p p e a r t o be  t h e movements o f s m a l l  well  fish,  t o any s i t u a t i o n w h e r e i t m i g h t be  and desirable  t o i n d i c a t e a s u b j e c t has passed a c e r t a i n r e f e r e n c e i n an a c t i v i t y 2.6  Live All  chamber o r a q u a r i u m .  Material specimens o f j u v e n i l e  from t h e hatchery f a c i l i t i e s  sockeye salmon were o b t a i n e d  a t the F i s h e r i e s  o f C a n a d a , B i o l o g i c a l S t a t i o n , N a n a i m o , B.C. originated small  Research Board Most  fish  f r o m S k u l l y C r e e k a t L a k e l s e L a k e , B.C., b u t a  sample o f Kamchatka P e n i n s u l a p r e s m o l t s were  available  point  f o r comparative purposes.  Prior to a l l  also  19  e x p e r i m e n t s , the f i s h were m a i n t a i n e d food  (J.R. C l a r k , S a l t Lake C i t y , These f i s h were m a i n t a i n e d  provided  d i f f e r e n t photoperiodic  c o n d i t i o n s , semi-natural.and  on C l a r k ' s  dry  Utah). a t two and  s i t e s which  environmental  artificial.  The  semi-  n a t u r a l c o n d i t i o n s were o b t a i n e d by p l a c i n g f i s h i n a l a r g e o u t d o o r h o l d i n g tank (3,633 g a l l o n s ) . was  T h i s tank  s u b j e c t t o n a t u r a l f l u c t u a t i o n s i n the  photoperiod  and a l s o r e c e i v e d an unmeasurable i n f l u e n c e from artificial was  l i g h t sources i n nearby b u i l d i n g s .  d i v i d e d by. p l a s t i c  screens i n t o s i x equal  each of which c o n t a i n e d . i n d i v i d u a l s from one parental The achieved was  The  tank  compartments locality  and  stock. controlled a r t i f i c i a l w i t h two  h o l d i n g c o n d i t i o n s were  50 g a l l o n a q u a r i a .  The  photoperiod  r e g u l a t e d by an I n t e r m a t i c time s w i t c h and.. i n c a n d e s c e n t  lamps.  These a q u a r i a were s u p p l i e d w i t h a i r s t o n e s and  slow incoming, r a t e o f d e c h l o r i n a t e d w a t e r .  a  Temperature  v a r i a t i o n s were k e p t a t a minimum-by i n s u l a t i n g the water d e l i v e r y system. A separate  group of sockeye eggs f r o m . S k u l l y  were hatched and m a i n t a i n e d  Creek  i n the l a b o r a t o r y under  c o n t r o l l e d environmental.conditions.  The  f r y were  r e t a i n e d i n 10 g a l l o n a q u a r i a f o r p e r i o d i c t e s t i n g t o determine i f d e v e l o p m e n t a l changes e x i s t e d i n the photop e r i o d i c response.  2.7  Experimental Design All  t e s t s , e x c e p t where s p e c i f i c a l l y ,  were p e r f o r m e d on s i n g l e i n d i v i d u a l s . i n t r o d u c e d t o each a c t i v i t y  indicated,  A fish  was  chamber and a l l o w e d two  d a y s f o r a c c l i m a t i o n t o t h e new environment.-  The  a c c l i m a t i o n p e r i o d was d e t e r m i n e d f r o m t h e a n a l y s i s o f preliminary experiments.  The a c t u a l d a t a . r e c o r d i n g was  f o c u s e d upon t h e f o l l o w i n g s i x d a y s o f a c t i v i t y ,  but  o c c a s i o n a l l y i t was d e s i r a b l e t o p e r m i t an e x p e r i m e n t t o continue f o r longer periods of time. No f i s h was f e d . d u r i n g a n e x p e r i m e n t e x c e p t i n those t e s t s designed t o study the effects, of feeding upon t h e . a c t i v i t y  pattern.  The p r e l i m i n a r y e x p e r i m e n t s e x a m i n e d t h e b a s i c entrained response.  The c o m b i n e d t e m p e r a t u r e a n d p h o t o -  p e r i o d i c c o n d i t i o n s u s e d a r e l i s t e d i n T a b l e 3. tests i n this  Related  s e r i e s c o n s i d e r e d . t h e . r e l a t i o n s h i p o f age  t o - t h e b a s i c e n t r a i n e d response, and.the  synchronizing  e f f e c t s o f p h o t o p e r i o d u p o n b o t h i n d i v i d u a l s and g r o u p s of  fish. The e x p e r i m e n t s e x a m i n i n g . t h e e n d o g e n o u s component  ( T a b l e 4) w e r e c o n d u c t e d dark  i n constant l i g h t  (LL), constant  (DD) o r c o n s t a n t l i g h t w i t h , f e e d i n g - ( L L F ) .  T e m p e r a t u r e s w e r e m a i n t a i n e d c o n s t a n t a t 10°C a n d t h e light  i n t e n s i t y was h e l d c o n s t a n t a t 34.4 l u x ( e x c e p t  where s p e c i f i c a l l y  indicated).  21  Table 3.  Summary of temperature and photoperiod  conditions.  PHOTOPERIOD (°C) TEMPERATURE  8L 16D  12L 12D  1.6L 8D  5  +  +  +  10  +  +  +  15  +  +  +  T a b l e 4.  Summary o f c o n s t a n t  environmental.conditions.  ENVIRONMENTAL LIGHTING LL  DD  +  +  LL (F)  TEMPERATURE 10°C  +  22  S u r g i c a l t e c h n i q u e s used i n the f i n a l phase of the i n v e s t i g a t i o n are d e s c r i b e d i n the 2.8  Analytical  appendix.  Procedure  V i s u a l s c a n n i n g o f the event r e c o r d e r c h a r t s i n d i c a t e d the g e n e r a l performance o f each i n d i v i d u a l . The t o t a l number of events o c c u r r i n g i n each s i x t y minute i n t e r v a l were r e c o r d e d on d a t a s h e e t s and  then  t r a n s f e r r e d t o IBM punch c a r d s f o r a n a l y s i s . The d a i l y a c t i v i t y p a t t e r n o f each i n d i v i d u a l p l o t t e d by comparing.the h o u r l y a c t i v i t y a m p l i t u d e time.  was against  T h i s p r o v i d e d a survey of the o v e r a l l performance  f o r any i n d i v i d u a l o r group d u r i n g the e x p e r i m e n t a l p e r i o d . Mean h o u r l y v a l u e s were c a l c u l a t e d . t o condense these and t o c l a r i f y the g e n e r a l p a t t e r n s .  data  Mean v a l u e s were  a p p l i e d o n l y t o i n d i v i d u a l s . o r groups r e c e i v i n g the same t r e a t m e n t , such as the experiments  examining  e f f e c t s o f p h o t o p e r i o d and temperature rhythm.  the.combined  on the e n t r a i n e d  These d a t a were s u b j e c t e d t o . a n a l y s i s o f v a r i a n c e  and B a r t l e t t ' s  test.  Periodogram a n a l y s i s ( E n r i g h t , 1965a, b) was  used t o  i d e n t i f y any major p e r i o d i c i t y between 20.00 and 28.00 hours. T h i s was  a comparative  procedure  u s i n g the r o o t mean square  of the a m p l i t u d e . a s s o c i a t e d w i t h any e s t i m a t e d p e r i o d . Ap  ( r o o t mean square  amplitude)  E(avg.-grand avg.) e s t i m a t e of the period value  23  The completed  periodogram  provided.a  comparison  o f t h e a m p l i t u d e s . f o r s e l e c t e d p e r i o d s between 20.00 and 28.00 h o u r s . assumption  I t c o u l d i n d i c a t e whether t h e  o f a 24.0 hour p e r i o d , o r a 23.0  hour.period  e t c . , l e d t o an u n u s u a l l y l a r g e a m p l i t u d e , o r whether the a m p l i t u d e s were no g r e a t e r than background " n o i s e " (see F i g . 8b)..  The method d i d not a s s i g n any l e v e l o f  s i g n i f i c a n c e to the amplitude i n c r e a s e .  It.was  difficult  t o a s s i g n an o b j e c t i v e meaning t o c o n v e n t i o n a l s t a t i s t i c s such as a T - t e s t because t h e a c t i v i t y r e c o r d s were s e r i a l l y c o r r e l a t e d measurements and d i d n o t - r e p r e s e n t random independent  samples from t h e p o p u l a t i o n .  A t l e a s t f i v e r e p l i c a t i o n s o f a d a i l y c y c l e were recorded before attempting a n a l y s i s .  T h i s was  i n agreement  w i t h the g e n e r a l l y a c c e p t e d p o l i c y t o r e g i s t e r a minimum o f f i v e t o seven p e r i o d s b e f o r e examining rhythmicity  ( A s c h o f f , 1960).  Periodogram approach  an endogenous  a n a l y s i s provides a simple  t o c y c l i c b i o l o g i c a l phenomena.  The  statistical calculations  are e a s i l y d e t e r m i n e d , but a computer f a c i l i t a t e s the l a r g e number o f e s t i m a t e s r e q u i r e d .  3. 3.1  Determination  RESULTS  of a Basic Entrained  These e x p e r i m e n t s were f o c u s e d to photoperiod  as t h e p r i m a r y  I n d i v i d u a l s and introduced  upon e n t r a i n m e n t  environmental  factor.  o c c a s i o n a l l y groups of f i s h  t o an a c t i v i t y  m o t o r a c t i v i t y was analyzed  Response  chamber and  collected.  The  were  a r e c o r d of  chart output  the was  f o r t h e t o t a l number o f e v e n t s o c c u r r i n g  each s i x t y minute i n t e r v a l . information served  The  hourly  "bits"  during  of  as t h e b a s i c a c t i v i t y m e a s u r e  utilized  i n t h i s phase of the i n v e s t i g a t i o n . These t e s t s were d e s i g n e d following 1.  2.  to provide  answers t o  questions. Do  d i e l c y c l e s of locomotor a c t i v i t y  exist  under the a r t i f i c i a l  laboratory conditions?  Is the environmental  photoperiod  a major  synchronizer? 3.  Are  t h e o r g a n i s m s day  or crepuscular 4.  active, night active  in their activity  Does t h e o r g a n i s m n e e d t o a d a p t t o environmental  chambers b e f o r e  a consistent daily activity 5.  the  expressing pattern?  I s the r e s p o n s e s t a b l e o r does i t change during the course  6.  patterns?  of  development?  Is the e n t r a i n e d response expressed one  i n d i v i d u a l s i m i l a r to that  by a g r o u p o f i n d i v i d u a l s ?  by  expressed  the  25  N i n e L a k e l s e sockeye were i n t r o d u c e d s i n g l y the a c t i v i t y 8L 16D 5°C.  into  chambers i n a c o n t r o l l e d e n v i r o n m e n t o f  (8 h o u r s o f l i g h t and A continuous  s i x day  j u v e n i l e sockeye expressed the photoperiod  16 h o u r s o f d a r k n e s s )  at  record indicates that a  single  a d e f i n i t e c y c l i c response  to  This record i s considered  to  (Fig. 4).  be r e p r e s e n t a t i v e o f a l l i n d i v i d u a l s t e s t e d i n t h i s The  apparent  entrainment  to the photoperiod  series.  would tend  o m i t t h e p o s s i b i l i t y o f i n f l u e n c e by unknown f a c t o r s f u n c t i o n i n g as t h e e n t r a i n i n g a g e n t ,  to  environmental unless  these  f a c t o r s w e r e e x a c t l y 24.00 h o u r s i n t h e i r n a t u r a l periodicity. A t t h e o n s e t o f l i g h t t h e r e was activity  amplitude.  This increased a c t i v i t y  generally maintained t o day  d u r i n g the l i g h t phase, but  also an,increase  a s s o c i a t e d troughs  the f o u r t h , f i f t h  and  associated troughs  some  day  occurred. peaks  o c c u r r i n g d u r i n g the dark phase i n s i x t h days of the  test.  secondary  peaks  occur randomly, s i n c e the  o f mean v a l u e s f o r t h e s i x day  and  calculation  p e r i o d does not  c o n s i s t e n t a c t i v i t y peak i n t h e d a r k phase  indicate ( F i g . 5a).  There i s however a p o s i t i v e phase r e l a t i o n s h i p o f to the l i g h t c y c l e ( a c t i v i t y onset of l i g h t ) .  was  i n t h e number o f s e c o n d a r y  I t i s suggested, t h a t t h e s e  any  level  v a r i a t i o n i n the h o u r l y t o t a l s of events  T h e r e was and  an i n c r e a s e i n t h e  activity  l e v e l increases before  This i s a general c h a r a c t e r i s t i c  the of  26  Figure  4.  The e n t r a i n e d r e s p o n s e o f one to  8L 16D a t 5°C.  represented  The a m p l i t u d e i s  a s t h e t o t a l number o f  events o c c u r r i n g each hour. bars of  Figure  5.  a t t h e base r e p r e s e n t  The d a r k the periods  darkness.  (A) The mean d a i l y calculated presented  (B)  individual  activity  pattern  from t h e data i n Figure  4.  The mean d a i l y r e s p o n s e of a single  i n d i v i d u a l from t h e  Kamchatka P e n i n s u l a .  d i u r n a l l y a c t i v e organisms. Unfortunately,  grouping these data  omits  some  i n d i v i d u a l d a i l y c h a r a c t e r i s t i c s i n the a c t i v i t y but  the general  o v e r a l l pattern being  clearly defined.  cycle,  e x p r e s s e d i s more  The r e s u l t i n g p e r f o r m a n c e c u r v e  t o be s m o o t h e r t h a n t h e d a i l y  tends  record.  I t i s n o t known i f a s i m i l a r r e s p o n s e w o u l d be obtained  from g e n e t i c  of geographical  stocks o r i g i n a t i n g at a.variety  locations.  However, t h e s e  t e s t s d i d r e v e a l t h a t sockeye obtained Peninsula  expressed a s i m i l a r d i e l  will  from.the Kamchatka  response t o the photo-  p e r i o d i c environment i n t h i s l a b o r a t o r y These d a t a  ( F i g . 5b).  indicate that juvenile-sockeye  e x h i b i t a . c y c l i c a c t i v i t y pattern during  f o u r hour day under c o n t r o l l e d e n v i r o n m e n t a l A general the  preliminary  pattern of increased  period.  during  This  was  the dark phase.  This  pattern i s representative of the majority of  individuals The  tested.  photoperiodic  environment c o n s t i t u t e s a  e n t r a i n i n g a g e n t , and a t t h i s t e m p e r a t u r e i n the expression No g r o s s  conditions.  decrease i n the a c t i v i t y  amplitude occurring a t the onset.of general  a twenty  a c t i v i t y occurred  l i g h t phase o f t h e e x p e r i m e n t a l  a c c o m p a n i e d by a c o r r e s p o n d i n g  salmon  strong  (5°C) r e s u l t s  o f a b a s i c a l l y unimodal. a c t i v i t y  pattern  d i f f e r e n c e s i n t h e p a t t e r n c o u l d be i d e n t i f i e d a s  c h a r a c t e r i s t i c of e i t h e r Lakelse  o r Kamchatka s t o c k  tested  28  Further  analyses  t h a t a minimum two  of the d a i l y records  day  r e p e t i t i o n of the  indicated  cycle  was  necessary to determine the c h a r a c t e r i s t i c p a t t e r n ( i . e . w h e t h e r any active). day  i n d i v i d u a l was  a c t i v e or  In a d d i t i o n , a comparison of the  activity  p e r i o d was  i n d i c a t e d t h a t a minimum two  necessary before  o u t p u t c o u l d be  obtained.  a subject  activity  only  either light  day  t o an 3 plus  3.11  the  The  day  after  r e l a t i o n s h i p o f age  activity  introducing considered  This  to the b a s i c  s e r i e s was  conducted  valuable  diel  to  locomotor  p a t t e r n a t d i f f e r e n t stages of development.  i n v e s t i g a t i o n was  adaptation  chamber, d a t a a n a l y s i s  d e t e r m i n e i f t h e r e were changes i n the  was  to  f o l l o w i n g s i x days.  p a t t e r n of a c t i v i t y .  information  t o t a l day  a consistent daily Therefore,  dark  activity  This  i n determining.whether or not  examining a c o n s i s t e n t a c t i v i t y  the  pattern  i n the p r e - s m o l t s under i n v e s t i g a t i o n . A p p r o x i m a t e l y 900 two 10  of the  activity  gallon aquaria  for  one  year.  12L  12D  a t 5°C,  sockeye salmon eggs were h a t c h e d  chambers.  sac  r e s p o n s e was gravel.  f r y were m a i n t a i n e d  in  under c o n t r o l l e d e n v i r o n m e n t a l - c o n d i t i o n s  T e s t i n g was  conducted under c o n d i t i o n s  e x c e p t d a y s 1-14  V i s u a l observations with yolk  The  in  attached  14.5D.  i n d i c a t e d the newly hatched  were n e g a t i v e l y p h o t o t a c t i c .  gradually reversed  T h e r e was  w h i c h w e r e a t 9.5L  of  a f t e r emergence from  a p o s i t i v e r h e o t a c t i c response to  alevins This the the  29  w a t e r c u r r e n t i n t h e swimming c h a n n e l . ( p r o d u c e d b y an i n c o m i n g s u p p l y o f f r e s h w a t e r w h i c h was d i r e c t e d  into  t h e g r a v e l ) and..the f r y p r o c e e d e d i n . a u n i d i r e c t i o n a l column  around t h e a c t i v i t y  chamber.  This directional  movement was r e v e r s e d when t h e d i r e c t i o n o f t h e w a t e r f l o w was c h a n g e d  b y T80 d e g r e e s .  A l l unidirectional  movement b y t h e g r o u p t e r m i n a t e d when t h e . w a t e r ceased.  A sample  of this  s t o c k .was m a i n t a i n e d i n  s e v e r a l l a b o r a t o r y a q u a r i a for. f u r t h e r  testing.  A f t e r emergence from t h e g r a v e l t h e d a i l y was c h a n g e d  from a predominantly n o c t u r n a l ,  configuration to a diurnal activity.pattern b, c , d ) .  flow  pattern  bimodal ( F i g . 6 a,  T h i s r e s p o n s e t h e n remained unchanged i f  i n d i v i d u a l s w e r e s u b j e c t e d t o 12L 12D, 5°C e n v i r o n m e n t a l conditions.  A l l subjects maintained a synchrony  with  t h e p h o t o p e r i o d i c c y c l e a n d e x p r e s s e d more t h a n 50 p e r c e n t of t h e i r t o t a l a c t i v i t y d u r i n g t h e l i g h t phase  from t h e  t h i r d . m o n t h onward. 3.12  The s y n c h r o n i z i n g . e f f e c t o f . t h e e n v i r o n m e n t a l  l i g h t cycle a t a constant temperature.  Nine groups o f  L a k e l s e p r e - s m o l t s w e r e s u b j e c t e d t o an o s c i l l a t i n g p e r i o d i c environment. whether  photo-  The t e s t s w e r e d e s i g n e d t o d e t e r m i n e  or not the photoperiod could synchronize several  i n d i v i d u a l s t o t h e same a c t i v i t y  cycle.  Group s i z e ranged from f i v e t o t e n i n d i v i d u a l s w e r e i n t r o d u c e d t o a 12L 12D r e g i m e a t 5°C.  which  Over a p e r i o d  30  F i g u r e 6 ( a , b , c , d) .  The mean d a i l y  pattern at different life  activity  stages of the  cycle.  A  =  The f i r s t  9 days a f t e r  B  =  Days 10 t o 14.  C  =  3 months a f t e r  D  =  11 months a f t e r  emergence.  emergence. emergence.  Mean no. of events  31  of seven days t h i s group o f f i v e f i s h e x h i b i t e d a greater a c t i v i t y amplitude during the l i g h t period (Fig. 7a).  A decrease i n a c t i v i t y s i m i l a r t o t h a t  observed i n s o l i t a r y i n d i v i d u a l s occurred.during  the  dark. I t i s apparent from t h e s e grouped d a t a t h a t t h e a c t i v i t y p a t t e r n expressed i s s i m i l a r to.the response o f one i n d i v i d u a l .  C e r t a i n aspects  entrained of the  b e h a v i o u r w i l l . b e a l t e r e d due t o t h e s o c i a l e f f e c t s between s e v e r a l i n d i v i d u a l s , b u t t h e b a s i c p a t t e r n o f a day  a c t i v e r e l a t i o n s h i p t o t h e p h o t o p e r i o d i c c y c l e appears  t o be s i m i l a r . T h i s group i s e x p r e s s i n g a 24.00 hour p e r i o d i c i t y that i s apparently  synchronized  by t h e l i g h t c y c l e ( F i g . 7 b ) .  T h i s i s suggested because t h e p h o t o p e r i o d  a l s o has a 24 hour  component and i s t h e major o s c i l l a t i n g e n v i r o n m e n t a l f a c t o r involved. I t i s f u r t h e r suggested t h a t t h e l i g h t - d a r k c y c l e i s c a p a b l e o f s y n c h r o n i z i n g t h e i n d i v i d u a l - m e m b e r s i n a group to express s i m i l a r a c t i v i t y p a t t e r n s .  This,entrainment  t o a c o m m o n . s t i m u l u s . r e s u l t s i n a group e x p r e s s i o n o f a c t i v i t y w i t h a time p e r i o d s i m i l a r , t o t h a t o f t h e e n v i r o n mental cue.  The mean d a i l y a c t i v i t y  pattern  of f i v e i n d i v i d u a l s monitored s i m u l t a n e o u s l y f o r s i x days i n the same a c t i v i t y  chamber.  Periodogram a n a l y s i s of the s i x day r e c o r d i l l u s t r a t e d i n Fig.  7A.  Mean no. of events  rO O  to o rO 00  33  3.2  The E f f e c t s o f T e m p e r a t u r e a n d P h o t o p e r i o d - o n t h e  Daily Activity  P a t t e r n o f J u v e n i l e Sockeye Salmon.  These e x p e r i m e n t s of t h e temperature photoperiod.  were d e s i g n e d  e f f e c t on t h e . e n t r a i n e d response  Only temperature  and p h o t o p e r i o d  considered s i n c e they represented  t h e major  variables affecting the basic a c t i v i t y The  t o p r o v i d e a measure to  were  environmental  pattern.  t e s t s were p e r f o r m e d on s i n g l e L a k e l s e  pre-smolts  previously held i n the semi-natural photoperiodic conditions at  an average temperature  were r e c o r d e d day  o f 8°C.  The a c t i v i t y  f o r a d u r a t i o n . o f s i x days,  f o l l o w i n g a two  period of acclimation to the a r t i f i c i a l  environment.  N i n e i n d i v i d u a l s w e r e t o be e x a m i n e d a t e a c h and p h o t o p e r i o d  patterns  temperature  but several f a t a l i t i e s . a t the higher  t e m p e r a t u r e . v a l u e s , r e s u l t e d i n unequal sample s i z e s . total  A  o f 512 d a i l y a c t i v i t y . c y c l e s w e r e e x a m i n e d a n d t h e  mean d a i l y p a t t e r n s . f o r e a c h t e m p e r a t u r e . and., p h o t o p e r i o d i c value are presented. 3.21  The e n t r a i n e d . r e s p o n s e  photoperiods activity  a t 5°C:to.three d i f f e r e n t  (8L.16D, 12L 12D, 16L 8 D ) .  p a t t e r n s i n d i c a t e d . t h a t locomotor  The mean 24 h o u r activity  was  g r e a t e r d u r i n g t h e l i g h t phase o f t h e p h o t o p e r i o d i c c y c l e (Fig.  8 a, b, c ) . .  A t t h e onset o f l i g h t  three-fold increase i n the a c t i v i t y during a sixty  minute i n t e r v a l .  was  throughout  maintained  t h e r e was a  amplitude  occurring  This heightened  t h e l i g h t . p h a s e and-was  amplitude terminated  34  Figure  8 ( a , b, c ) .  Mean d a i l y a c t i v i t y  representing a t 5°C.  202 d a y s o f  The  standard  value.  activity  e r r o r s are  i n d i c a t e d by t h e v e r t i c a l a t each  patterns  brackets  MEAN NO. OF EVENTS  01  _»  O  _ i  01  PER  r\j o  HOUR  ro 01  w o  35  at  t h e o n s e t o f d a r k by = a c o r r e s p o n d i n g , d e c r e a s e i n  activity. A predawn i n c r e a s e i n a c t i v i t y decrease.in a c t i v i t y  are i l l u s t r a t e d  and. a  predusk  by.these  data,  and i n d i c a t e a p o s i t i v e p h a s e r e l a t i o n s h i p t o t h e photoperiodic conditions.(activity  increased or decreased  p r i o r t o the onset of the s y n c h r o n i z i n g stimulus).  This  p h a s e r e l a t i o n s h i p .was c i r c u m s t a n t i a l e v i d e n c e f o r a n endogenous r h y t h m i c i t y h a v i n g a c h a r a c t e r i s t i c p e r i o d o f l e s s t h a n 24 h o u r s . A comparison of  light  o f the amplitude.increases a t the onset  i n d i c a t e d a general.upper a c t i v i t y  photoperiod.  T h i s "dawn" p e a k was f o l l o w e d b y a  "midmorning" d e p r e s s i o n in. a c t i v i t y by d a y l e n g t h . b a s i c unimodal  l i m i t f o r each  The d e c r e a s e  t h a t was  i n activity  influenced  modified the  p a t t e r n . o b s e r v e d a t 8L 16D t o a b i m o d a l  p a t t e r n a t 16L. 8D. r e f e r r e d t o . a s t h e a l t e r n a n s ^ " t y p e ( A s c h o f f , 1966) . T h e r e was a n . i n c r e a s e i n t h e d u r a t i o n o f h e i g h t e n e d activity  when t h e a r t i f i c i a l  hours of l i g h t of  d a y was l e n g t h e n e d f r o m  to.twelve or sixteen hours.  these h i g h e r . a c t i v i t y  The  l e v e l s t o t h e l i g h t phase  t h a t a t 5°C t h e . l e n g t h o f t h e a r t i f i c i a l  day would  eight  limitation indicated not a l t e r  the b a s i c a l l y d i u r n a l pattern o f the entrained response. "^Alternans d e s c r i b e s a c h a r a c t e r i s t i c p a t t e r n where t h e major  peak f o l l o w s t h e m i n o r  peak.  36  3.22  The.entrained response  d i f f e r e n t photoperiods  (8L 16D  12L  f  a t 10°C 12D,  to three  16L  8D).  mean 24 h o u r a c t i v i t y p a t t e r n s e x a m i n e d a t t h i s s u p p o r t t h o s e d a t a o b t a i n e d a t 5°C  (Fig. 9 a ,  at  8L. 16D,  and  a g e n e r a l i n c r e a s e i n the  amplitude r e s u l t i n g from the h i g h e r  temperature  b, c ) .  The .major d i f f e r e n c e s o c c u r r i n g i n t h e s e include.an.apparent temperature-photoperiod  The  tests  interaction  activity  environmental  temperatures. A t 8L- 16D apparent  the i n c r e a s e d a c t i v i t y  i n the dark phase.  a c t i v i t y . l e v e l .decreased  a m p l i t u d e was  most  This heightened.nocturnal  the magnitude o f . t h e d i f f e r e n c e  between t h e . d i u r n a l . a n d n o c t u r n a l phases of. t h e  activity  pattern...  cyclic  However, t h e s e d a t a s t i l l .retained, a  r e l a t i o n s h i p t o the photoperiod.and differences in. a c t i v i t y  level  and d a r k p h a s e s o f t h e a c t i v i t y A t 12L  12D  and  significent  the a c t i v i t y  16L- 8D t h e r e . w a s  that observed  a t 5°C,  and  a definite The  day  magnitude  c o n s t i t u t e d a. l e v e l  d u r i n g the a r t i f i c i a l  Predawn.increases  and p r e d u s k  less twice  night. decreases  were i n d i c a t e d . b y t h e s e d a t a , s u p p o r t i n g t h e e v i d e n c e f o r an e n d o g e n o u s  light  cycle.  i n c r e a s e a t t h e o n s e t o f l i g h t was  than t h a t observed  <.01)  o c c u r r e d between the  active, r e l a t i o n s h i p to the photoperiod. of  (P  rhythmicity.  in activity available  gure  9  (a, b,  c).  Mean d a i l y  representing 10°C.  172  activity  days  of  patterns  activity  at  M E A N NO. O F E V E N T S  PER  HOUR  38  3.23  The e n t r a i n e d response a t 15"C  to three  d i f f e r e n t p h o t o p e r i o d s (8L 16D,  12L 12D,  major d i f f e r e n c e s between.these  t e s t s a t 15°C  a t 5°C and 10°C  16L 8D).  The  and t h o s e  include a continued.temperature-photoperiod  i n t e r a c t i o n at.8L.16D and h i g h e r a c t i v i t y  amplitudes  o c c u r r i n g a t twelve.and s i x t e e n hours o f l i g h t  ( F i g . 10 a,  b, c) . A t 8L 16D t h e r e was an apparent decrease., i n the mean a c t i v i t y a m p l i t u d e .during a l l hours o f t h e a r t i f i c i a l ( F i g . 10a).  The v a r i a t i o n s i n t h e s e grouped.data  differ.significantly  (P < . l ) .  day  d i d not  However, t h i s was due t o  t h e c o m b i n a t i o n o f b o t h . l i g h t a c t i v e and dark a c t i v e i n d i v i d u a l s . (see F i g . 15) which tended t o . c a n c e l each other. There was  s t i l l a l i g h t a c t i v e r e l a t i o n s h i p of  a c t i v i t y t o the p h o t o p e r i o d . a t . t w e l v e and s i x t e e n hours of  light.  However, the magnitude o f the i n c r e a s e a t the  o n s e t o f l i g h t was or 10°C,  and was  l e s s t h a n t h a t observed a t e i t h e r  5°C  f o l l o w e d by a s i g n i f i c a n t midmorning (P<.01)  d e p r e s s i o n o f a c t i v i t y r e s u l t i n g i n a bimodal p a t t e r n of t h e a l t e r n a n s t y p e . These, d a t a suggested t h a t the l o c o m o t o r . a c t i v i t y p a t t e r n was  i n f l u e n c e d by both the e n v i r o n m e n t a l . t e m p e r a t u r e  and p h o t o p e r i o d . .  These e x t r i n s i c . f a c t o r s produced  changes  i n b o t h the a m p l i t u d e of a c t i v i t y and t h e c o n t o u r o f the p a t t e r n e x p r e s s e d . i n a twenty f o u r hour p e r i o d .  39  Figure  10  (a, b,  c).  Mean  representing 15°C.  daily 138  activity  days  of  patterns  activity  at  M E A N NO. O F E V E N T S  PER  HOUR  40  3.24  The e f f e c t o f t e m p e r a t u r e  response. at  The mean h o u r l y v a l u e s r e c o r d e d  5°C, 10°C a n d 15°C i l l u s t r a t e d - t h a t  increased the a c t i v i t y (Fig.  on. t h e e n t r a i n e d  11).  f o r 12L 12D  temperature  a m p l i t u d e . i n both, l i g h t and dark  These f i g u r e s a l s o i n d i c a t e d . a development  o f t h e "midday" d e p r e s s i o n i n a c t i v i t y w h i c h  resulted  i n a bimodal  temperatures  a c t i v i t y pattern at.the higher  considered. The  mean a c t i v i t y values..were  temperature, f o r . e a c h .photoperiod.  plotted  The v a l u e s f o r  8L 16D a t 15°C i n d i c a t e d , a d e f i n i t e , d e c r e a s e number o f e v e n t s . d u r i n g  against  l i g h t and dark  i n . t h e mean  (Fig. 12).  H o w e v e r , t h e g e n e r a l t r e n d f r o m 5 ° C - 15°C- was one o f increasing activity  amplitudes.with  increasing  temperatures  ( s e e F i g . 1 2 , 12L 12D, 16L 8 D ) . The. d e c r e a s e d . a c t i v i t y a m p l i t u d e . t h a t , was a s s o c i a t e d w i t h . 8 L - 16D above.10°C i n d i c a t e d . t h a t optimum, v a l u e s o f photoperiod  and.temperature, might determine  of d i u r n a l or.nocturnal a c t i v i t y . temperature  "limit"  the expression  When t h e u p p e r  f o r a c e r t a i n d a y l e n g t h was e x c e e d e d ,  the organism, r e v e r s e d t h e p h o t o b e h a v i o u r a l response became d a r k 3.25 response. at  and  active. The, e f f e c t o f . p h o t o p e r i o d on t h e e n t r a i n e d Mean t o t a l a c t i v i t y was p l o t t e d a g a i n s t  each temperature  showed a v e r y s l i g h t  value  (Fig. 13).  A t 5°C, d a r k  daylength activity  i n c r e a s e b e t w e e n 8L a n d 1 6 L , w h e r e a s  41  F i g u r e 11.  Mean d a i l y a c t i v i t y 184 d a y s o f a c t i v i t y  patterns a t 12L  T e m p e r a t u r e s a r e i n °C.  representing 12D.  42  F i g u r e 12.  P l o t s o f a c t i v i t y on at three d i f f e r e n t  D  =  Dark;  L  =.  temperature  photoperiods.  Light  43  F i g u r e 13.  P l o t s o f a c t i v i t y on p h o t o p e r i o d three d i f f e r e n t  D  =  Dark;  temperatures.  L  =  Light  at  l i g h t a c t i v i t y i n d i c a t e d maximum a m p l i t u d e s a t 8L and 16L. A t 10°C b o t h l i g h t and dark phases i n d i c a t e d maximum a c t i v i t y a t 8L, whereas a t 15°C minimum a c t i v i t y a m p l i t u d e s were a s s o c i a t e d w i t h 8L. These d a t a p r o v i d e a d d i t i o n a l e v i d e n c e f o r an i n t e r a c t i o n between d a y l e n g t h and t e m p e r a t u r e .  It is  suggested t h a t a t 8L 16D a temperature above 10°C w i l l r e s u l t i n increased nocturnal a c t i v i t y .  When more t h a n  50% o f t h e t o t a l a c t i v i t y o c c u r s d u r i n g t h e d a r k , t h i s i s i n t e r p r e t e d as a r e v e r s a l i n t h e p h o t o b e h a v i o u r a l response and t h e organism i s c o n s i d e r e d t o be n o c t u r n a l l y a c t i v e . A t h r e e d i m e n s i o n a l p r e s e n t a t i o n o f mean t o t a l a c t i v i t y p l o t t e d a g a i n s t temperature and p h o t o p e r i o d indicated the general trend of the photobehavioural response  (Fig. 14).  A r i s e i n temperature r e s u l t e d i n  i n c r e a s e d mean t o t a l a c t i v i t y a t most d a y l e n g t h s . when s h o r t days (8L) and h i g h temperatures  However  (15°C) were  combined, d e c r e a s i n g a c t i v i t y o c c u r r e d . 3.26  The i n t e r a c t i o n o f temperature a n d . p h o t o p e r i o d  The mean d a i l y a c t i v i t y r e c o r d s f o r 8L 16D a t 10°C and 15° (see  F i g s . 9a and 10a) i n d i c a t e d an i n t e r a c t i o n between  temperature and p h o t o p e r i o d .  These r e c o r d s r e s u l t e d from  combining i n d i v i d u a l p a t t e r n s which tended t o c a n c e l each other  ( F i g . 1 5 ) , g i v i n g a mean d a i l y p a t t e r n t h a t d i d n o t  differ significantly  from a s t r a i g h t l i n e  of t h e p r e v i o u s s e c t i o n s  (P>.1).  Analysi  (2.4 and 2.5) i n d i c a t e d a g e n e r a l  45  F i g u r e 14.  Three dimensional i l l u s t r a t i o n of mean t o t a l a c t i v i t y amplitudes a t each temperature The broad l i n e s s u r f a c e contour.  and p h o t o p e r i o d . i n d i c a t e the  46  F i g u r e 15.  Mean d a i l y a c t i v i t y p a t t e r n s f o r two 8L  individuals 16D  at  15°C.  subjected  to  Mean no. of events  47  r e v e r s a l i n t h e a c t i v i t y response a t s h o r t d a y l e n g t h s and h i g h t e m p e r a t u r e s . A c o n s i d e r a t i o n of the percent frequency occurrence of n o c t u r n a l a c t i v i t y i n d i c a t e d a p r o g r e s s i v e a c t i v i t y i n c r e a s e which was emphasized where s h o r t p h o t o p e r i o d s and h i g h temperatures were combined ( F i g . 1 6 ) .  I t was  a l s o apparent t h a t t h e i n c r e a s e s were n o t p a r a l l e l a t a l l d a y l e n g t h and temperature c o m b i n a t i o n s . A t 8L 16D t h e e s t i m a t e d 50% d i s t r i b u t i o n  between  l i g h t and dark a c t i v i t y was a t 8°C ( F i g . 17, a, b, c ) . A t 12L 12D t h i s v a l u e was a t 15°C, and a t 16L 8D t h e e x t r a p o l a t e d v a l u e was a t 3 0°C. v a l u e was u n r e a l i s t i c  However, t h i s  latter  s i n c e i t exceeded t h e t o l e r a n c e  l i m i t s o f j u v e n i l e sockeye salmon.  I n each c a s e , a f o u r  hour i n c r e a s e i n d a y l e n g t h was s u f f i c i e n t  t o double t h e  e f f e c t i v e temperature where t h e 50% d a y / n i g h t a c t i v i t y d i s t r i b u t i o n occurred. I t i s hypothesized that f o r a c e r t a i n daylength there i s an upper temperature l i m i t which i f exceeded w i l l r e v e r s e the  a c t i v i t y response and j u v e n i l e sockeye salmon w i l l  e x p r e s s g r e a t e r a c t i v i t y a m p l i t u d e s d u r i n g dark than during l i g h t .  Lower temperature l i m i t s were n o t i n v e s t i -  g a t e d , b u t a r e assumed t o approximate 0°C i n j u v e n i l e sockeye.  48  F i g u r e 16.  Plot  of the percent occurrence  n o c t u r n a l a c t i v i t y a t each and  of  daylength  temperature.  0=  5°C  • =  10°C  A=  15°C  F i g u r e 1 7 . ( a , b, c ) .  Plots  illustrating activity  f o r each  photoperiod  the d i s t r i b u t i o n  f o r l i g h t and d a r k .  intersecting  of The  l i n e s r e p r e s e n t the  p o i n t s w h e r e a 50% distribution  activity  occurs.  O -  Light  • =  Dark  % Nocturnal activity  % Activity distribution Oi O  O O  -I  O  o  _L-  O  o  00  o  o o  Ol  o  _L_  o o  3.3  Endogenous. A c t i v i t y E x p r e s s e d , in.. A  Constant  Environment. An  i n v e s t i g a t i o n . c o n s i d e r i n g the p h o t o p e r i o d i c  responses  o f an o r g a n i s m . m i g h t  an e n d o g e n o u s . r h y t h m i c i t y by t h e l i g h t  cycle.  assume.the presence  which  of  i s being synchronized  T h e - d e s c r i p t i o n and a n a l y s i s  t h e endogenous r e s p o n s e . i n j u v e n i l e sockeye m i g h t b e . v a l u a b l e t o p a s t and  of  salmon  f u t u r e research which i s  f o c u s e d e i t h e r w h o l l y o r i n p a r t on t h e r e s p o n s e s t h e s e f i s h t o an e n v i r o n m e n t a l l i g h t  of  cycle.  Lakelse pre-smolts were.obtained  from  the.outdoor  h o l d i n g t a n k s and h e l d i n t h e l a b o r a t o r y , a q u a r i a f o r a minimum o f two w e e k s . i n t o the a c t i v i t y The  I n d i v i d u a l s were t h e n i n t r o d u c e d  c h a m b e r s u n d e r a 12L  t r a n s f e r from.the  12D. p h o t o p e r i o d .  controlled, environmental holding  a q u a r i a o c c u r r e d d u r i n g the l i g h t phase.of photoperiod.  the  artificial  At the end.of the l i g h t . p e r i o d the t i m e r s  were, p r e v e n t e d . f r o m  c o m p l e t i n g a r e v o l u t i o n and  the  e n v i r o n m e n t a l c o n d i t i o n s were m a i n t a i n e d i n . a c o n s t a n t state 10°C  (LL o r . D D ) . and  The  temperature  was  t h e e n v i r o n m e n t a l l i g h t i n g was  constant l e v e l  (34 . 4. l u x , e x c e p t i n DD,  held constant at maintained at a or where.specifica  indicated). T h e s e f i s h w e r e a l l o w e d 48 h o u r s t h e new  environmental conditions.  f o r adjustment  Locomotor  to  activities  w e r e r e c o r d e d f o r 5 o r 6 d a y s , b u t i n i s o l a t e d , c a s e s an  50  i n d i v i d u a l was a l l o w e d t o r e m a i n  i n the constant  environmental conditions f o r a longer period. These d a t a were a n a l y z e d a t t e n m i n u t e p e r i o d e s t i m a t e s b e t w e e n t h e 20.00 a n d 28.00 h o u r r a n g e o f the  periodogram. 3.31  The e n d o g e n o u s r e s p o n s e  (DP) a t a c o n s t a n t t e m p e r a t u r e f i s h r e p r e s e n t i n g 192 " d a y s " this series.  The r e s p o n s e  o f 1Q°C.  of a c t i v i t y t o DD w h i c h  subjects i n d i c a t e d that entrainment was a p p a r e n t  i n constant  during the f i r s t  darkness  A t o t a l o f 33 contributedto r e p r e s e n t e d most  t o the photoperiod  and l a s t t h r e e days o f t h e  t e s t , b u t t h e r e was no i n d i c a t i o n o f a n o b v i o u s component d u r i n g d a y s 4, 5, a n d 6 ( F i g . 18).  circadian Further  e x a m i n a t i o n o f t h e s e d a t a i n d i c a t e d a n e n d o g e n o u s component w i t h an approximate  5 hour  periodicity.  An e x c e p t i o n was n o t e d w h e r e a f r e e - r u n n i n g r h y t h m was m a i n t a i n e d w i t h a l o w a m p l i t u d e i n one i n d i v i d u a l . The p e r i o d o g r a m  a n a l y s i s o f t h e s e d a t a i n d i c a t e d a maximum  i n c r e a s e i n a m p l i t u d e a s s o c i a t e d w i t h a p e r i o d o f 22.90 hours  ( F i g . 19).  average  value  This i sa close approximation of the  (22.8 h o u r s )  a d e t e c t a b l e rhythm  calculated  for a l lfish  showing  (Table 5).  I t was c o n c l u d e d t h a t t h e e n d o g e n o u s c i r c a d i a n was n o t r e a d i l y e x p r e s s e d  i n constant darkness.  component  I n most  c a s e s w h e r e a d e t e c t a b l e i n c r e a s e i n a m p l i t u d e was o b s e r v e d , i t was e x p r e s s e d w i t h a p e r i o d v a l u e l e s s t h a n 24.00 h o u r s .  A continuous nine day r e c o r d f o r one i n d i v i d u a l i n 12L 12D constant dark a t 10°C.  and The  photoperiod i s i n d i c a t e d by the dark bars at the base of the graph.  Periodogram a n a l y s i s r e p r e s e n t i n g the response of one i n d i v i d u a l i n constant darkness.  4.0  Jo  _  3.0H  ° <b  aP o °  m  o ODD  2.0-| %  9xc  6>  1.<H  20  1  1  1  i  22  24  26  28  Period  T a b l e 5.  Periodicity  e x p r e s s e d by 16  i n constant darkness at  fish  10°C.  F i s h No.  Period  F i s h No.  1  20.8  9  21.6  2  20.0  10  25.4  3  21.7  11  24.7  4  20.8  12  25.0  5  20.8  13  23.1  6  24.0  14  23.8  7  24.7  15  24. 9  8  23 .2  16  21.0  Avg.  22.8  Period  -  54  This i s not t y p i c a l of d i u r n a l l y active which u s u a l l y express a spontaneous is  organisms  frequency that  l o n g e r t h a n 24.00 h o u r s i n c o n s t a n t d a r k n e s s . 3.32  The e n d o g e n o u s r e s p o n s e i n c o n s t a n t l i g h t  (LL) a t a c o n s t a n t t e m p e r a t u r e o f 10°C.  Thirty-five  s u b j e c t s r e p r e s e n t i n g 183 " d a y s " o f a c t i v i t y w e r e e x a m i n e d in this  series.  A p a r t i c u l a r l y good e x a m p l e o f t h e  e n d o g e n o u s component i s i l l u s t r a t e d by t h e t w e l v e d a y a c t i v i t y r e c o r d f o r a s i n g l e j u v e n i l e sockeye  (Fig. 20).  T h i s r e c o r d i n d i c a t e s t h a t some i n d i v i d u a l s c a n m a i n t a i n a r h y t h m i c a c t i v i t y p a t t e r n i n t h e absence environmental s t i m u l i .  of periodic  However, t h e r e s p o n s e  representative  o f most f i s h t e s t e d i n d i c a t e d a dampening o f t h e endogenous component a f t e r t h e f i r s t  t h r e e o r f o u r days  i n constant  light. P e r i o d o g r a m a n a l y s i s o f t h e above d a t a i n d i c a t e d a p e a k a m p l i t u d e i n c r e a s e a t 23.50 h o u r s f o r t h i s (Fig. 21).  The d a t a a n a l y s e s f o r a l l f i s h  individual  showing a  d e t e c t a b l e r h y t h m b e t w e e n 20.00 a n d 28.00 h o u r s r e v e a l e d an a v e r a g e e n d o g e n o u s p e r i o d i c i t y a p p r o a c h i n g 23.30 (Table 6 ) .  The e n d o g e n o u s component v a r i e d w i t h  hours each  i n d i v i d u a l a n d p e r i o d v a l u e s r a n g e d f r o m 20.00 h o u r s t o 26.40 h o u r s . I n c o n s t a n t l i g h t t h e mean p e r i o d i c i t y e x p r e s s e d was l e s s t h a n 24.00 h o u r s , w h i c h i s c h a r a c t e r i s t i c o f m o s t d i u r n a l l y a c t i v e organisms  ( A s c h o f f , 1960).  I t appeared  55  F i g u r e 20.  A continuous  12 day  r e c o r d of  i n d i v i d u a l i n constant (34.4  lux at  10°C).  light  one  No. of events per hour  56  F i g u r e 21.  Periodogram a n a l y s i s of the data presented i n F i g u r e 20.  •J  14-, 9 » ' oo o o  12J  o  o o  o o  o  10H  o  o  CL <  o  o o o  8H ~o -*—>  Q.  6H  o  E <  o o o  o o o CD o o<$> CD-  <*>o  o o o<Po o o  2^  —r 24 -  20  22  Period  I  26  28  T a b l e 6.  Periodicity  e x p r e s s e d by 30  i n constant l i g h t  at  fish  10°C.  F i s h No.  Period  F i s h No.  1  26.4  16  25.1  2  26.3  17  24.8  3  21.5  18  21.1  4  24.0  19  24.0  5  20.0  20  22.2  6  23.2  21  20.3  7  21.0  22  27.2  8  25.1  23  21.9  9  24.0  24  24.4  10  23.5  25  22.0  11  24.0  26  23.0  12  23.2  27  22.2  13  24.6  28  23.2  14  25.4  29  21.0  15  24.0  30  24.9  Avg.  23.3  Period  t h a t j u v e n i l e sockeye expressed more f r e e l y i n c o n s t a n t 3.33  a n e n d o g e n o u s r h y t h m much  l i g h t than i n constant  The r e s p o n s e o f t h e e n d o g e n o u s component t o  increasing light intensities. was m e a s u r e d i n s i x f i s h (<1 l u x a n d 34.4 l u x ) . maintained  dark.  constant  were a c h i e v e d  The e n d o g e n o u s  subjected  periodicity  t o two l i g h t  Environmental  intensities  t e m p e r a t u r e s were  a t 10°C a n d t h e l o w e r  light  by u s i n g a 1 w a t t neon lamp.  intensities  Periodogram  a n a l y s i s was u s e d t o d e t e r m i n e t h e c h a r a c t e r i s t i c f o r each  period  individual.  Table 7 illustrate!•. the values intensity.  The g e n e r a l  obtained  a t each  t r e n d i s f o r an i n c r e a s e i n p e r i o d  value with increasing l i g h t i n t e n s i t i e s .  This  i s noti n  a c c o r d a n c e w i t h p r e v i o u s l y o b s e r v e d r e s p o n s e s i n many day  a c t i v e species  been r e p o r t e d 3.34 constant  ( A s c h o f f , 1960), b u t e x c e p t i o n s  i n the l i t e r a t u r e  have  ( H o f f m a n n , 1965).  The e n t r a i n i n g e f f e c t s o f p e r i o d i c f e e d i n g i n  l i g h t a t 10°C.  Twenty f o u r f i s h  166 " d a y s " o f a c t i v i t y c o n t r i b u t e d t o t h i s was i n t r o d u c e d day.  other  t o the constant  representing series.  Food  e n v i r o n m e n t a t noon e a c h  The o b j e c t i v e was t o d e t e r m i n e w h e t h e r o r n o t  periodic feeding could entrain the a c t i v i t y The  mean v a l u e s  i n t r o d u c i n g food  f o ra l l tests indicated that  t o the constant  significant increase i n a c t i v i t y endogenous component.  pattern.  environment produced a and e n t r a i n e d t h e  Table 7.  Periodicity  (hours) expressed  sockeye s u b j e c t e d t o i n c r e a s i n g  Fish  1 lux  i n six juvenile  l i g h t i n t e n s i t y at  34.4  1  22.4  23.2  2  22.2  23.4  3  23.5  24.0  4  23.2  24.4  5  21.6  23.2  6  24.0  23.6  Avg.  22.8  23.6  10°C.  lux  60  The  mean d a i l y a c t i v i t y p a t t e r n f o r n i n e  individuals  i n d i c a t e d t h a t the increased a c t i v i t y amplitude  was  maintained  and  a t a peak f o r a p p r o x i m a t e l y  one  hour  then g r a d u a l l y returned to the background l e v e l Apparently the f i r s t with  t h e s y n c h r o n i z i n g e f f e c t s were s t r o n g e r t h r e e d a y s o f t h e t e s t , and  during  g r a d u a l l y weakened  concluded  t h a t the stimulus of p e r i o d i c  f e e d i n g c o u l d e n t r a i n t h e endogenous rhythm c o r r e s p o n d i n g l y a l t e r any present.  The  constant  only alternative for future  dark.  food a v a i l a b l e i n the  i n a random manner d u r i n g b o t h  Since the m a j o r i t y of experiments  here covered  a d u r a t i o n o f one  week, i t was  p r a c t i c a l not to i n t r o d u c e food to the  Mediation The  P r i n c i p a l Sensory Receptors  feeding light  reported  considered  Involved i n the  Response.  more o b v i o u s  accessible photoreceptive  i n c l u d e d t h e e y e s and  the p i n e a l body.  might i n v o l v e cutaneous photoreceptors  Less obvious or a  and  systems systems  general  s e n s i t i v i t y o f t h e n e r v o u s s y s t e m t o s t i m u l a t i o n by S i n c e t h e e y e s and  more  environment.  of the E n t r a i n e d and  was  long-term  s u p p l y , o r a t t e m p t t o mask t h e e f f e c t s o f  by p r e s e n t i n g f o o d  The  and  free-running rhythmicity that  e x p e r i m e n t s w o u l d be t o m a i n t a i n  3.4  22).  time. I t was  and  (Fig.  light.  t h e p i n e a l body a r e t h e more p r o m i n e n t  a c c e s s i b l e systems, a s e r i e s of e x p e r i m e n t s were  to i n v e s t i g a t e the r o l e of these  organs i n mediating  designed the  61  F i g u r e 22.  (A) The mean a c t i v i t y response o f 9 individuals  f o r 7 days.  The  grand means a r e presented a t the r i g h t (B).  i n d i c a t e s time of f e e d i n g  Mean no. of events  62  the e n t r a i n e d r e s p o n s e . These t e s t s a r e f o c u s e d upon t h e d a i l y e n t r a i n e d response o c c u r r i n g i n 12L 12D a t 10°C.  Longer  changes may o c c u r b u t t h e d a t a a r e n o t adequate a n a l y s i s o f s e a s o n a l o r annual c y c l e s . r e c o r d s a r e p r e s e n t e d and a r e adequate  term f o r the  Mean d a i l y to indicate  c o n s i s t e n t i n c r e a s e s o r d e c r e a s e s i n t h e locomotor activity  cycle.  3.41 fish.  The e n t r a i n e d response i n p i n e a l e c t o m i z e d  High a c t i v i t y l e v e l s a s s o c i a t e d w i t h t h e apparent  l o s s o f e n t r a i n m e n t were common i m m e d i a t e l y a f t e r p i n e a l ectomy.  F i g u r e 23 a, b i l l u s t r a t e s t h e response o f one  i n d i v i d u a l i m m e d i a t e l y a f t e r p i n e a l e c t o m y (a) and a g a i n , two weeks l a t e r  (b).  H y p e r a c t i v i t y and apparent l o s s o f  e n t r a i n m e n t o c c u r r e d on days one t h r o u g h f o u r .  The t o t a l  d a i l y a c t i v i t y was l e s s e n e d i n days 5, 6, and 7 w i t h t h e appearance  o f a d e f i n i t e response t o t h e e n v i r o n m e n t a l  light cycle.  Two weeks a f t e r p i n e a l e c t o m y (b) t h e response  had s t a b i l i z e d and e n t r a i n m e n t t o t h e l i g h t c y c l e was demonstrated.  A l l f u r t h e r experiments i n c l u d e d a two  week p e r i o d o f p o s t o p e r a t i v e r e c o v e r y i n o r d e r t o m a i n t a i n an a c c e p t a b l e degree o f comparison between each day o f t h e experiment and a l s o t o p r e v e n t masking o f t h e e n t r a i n e d response. The s i x t e e n f i s h t e s t e d f o r an i n t e r v a l o f s i x days each i n d i c a t e d t h a t a p i n e a l e c t o m i z e d f i s h would  respond  63  F i g u r e 23.  The  a c t i v i t y r e s p o n s e o f one  i n 12L  12D  a t 10°C  pinealectomy later  (B).  individual  immediately  (A) and  a g a i n two  after weeks  64  t o t h e l i g h t - d a r k c y c l e i n a manner s i m i l a r t o t h a t e x p r e s s e d b y sham o p e r a n t s  (Fig. 24).  The  average  periodicity calculated f o r a l lpinealectomized individuals was 23.97 h o u r s .  E i g h t i n d i v i d u a l s were i n e x a c t  w i t h t h e l i g h t c y c l e and t h e o t h e r e i g h t  expressed  p e r i o d i c i t i e s r a n g i n g f r o m 21.7 t o 26.8 h o u r s . suggested t h a t these l a t t e r  synchrony  Iti s  s u b j e c t s demonstrated  a scatter  a b o u t t h e 24.00 h o u r mean, w h i c h m i g h t be a t t r i b u t e d t o post-operative effects. t h e d i s t u r b a n c e was  However, t h e e x a c t n a t u r e o f  unknown.  E i g h t sham o p e r a n t f i s h w e r e t e s t e d i n t h e same manner a s p i n e a l e c t o m i z e d f i s h .  A l l eight  e x p r e s s e d a 24.00 h o u r mean p e r i o d i c i t y environment.  individuals  i n a light-dark  Two i n d i v i d u a l s i n t h i s g r o u p h a d a d d i t i o n a l  secondary peaks i n t h e periodogram  analysis, indicating  that  the r i g o u r s of the o p e r a t i o n might  r e s u l t i n some d i s t u r b a n c e  of the e n t r a i n e d response. I t was c o n c l u d e d t h a t p i n e a l e c t o m y a l t e r e d t h e d a i l y response.  A comparison  of the t o t a l a c t i v i t y  levels  e x p r e s s e d by p i n e a l e c t o m i z e d a n d sham o p e r a n t f i s h  indicated  that pinealectomy resulted i n s i g n i f i c a n t l y higher  (P<.01)  activity  levels  (see F i g . 2 4 ) .  However, even though t h e  mean d a i l y c y c l e was n o t w e l l e x p r e s s e d i n F i g . 24, periodogram  a n a l y s i s i n d i c a t e d t h a t most i n d i v i d u a l s were  entrained to the l i g h t  cycle.  65  F i g u r e 24.  The mean d a i l y a c t i v i t y p a t t e r n s f o r p i n e a l e c t o m i z e d and sham p i n e a l e c t o m i z e d fish.  Standard e r r o r s a r e i n d i c a t e d  by t h e v e r t i c a l  • O—-O  brackets.  Pinealectomized Sham P i n e a l e c t o m i z e d  Mean no. of events _L  o  ro o  66  3.42 fish.  The endogenous response i n p i n e a l e c t o m i z e d  A f t e r a two week p e r i o d o f p o s t - o p e r a t i v e r e c o v e r y ,  e i g h t p i n e a l e c t o m i z e d f i s h and f o u r sham o p e r a n t s subjected t o constant environmental 34.4 l u x and 10°C.  were  c o n d i t i o n s o f LL a t  Periodogram a n a l y s i s i n d i c a t e d t h a t  o n l y two o f t h e e i g h t p i n e a l e c t o m i z e d f i s h expressed peak i n a m p l i t u d e  t h a t c o u l d be v i s u a l l y s e p a r a t e d  the background.  The low a m p l i t u d e s  a  from  between 20 and 28  hours f o r a s i n g l e p i n e a l e c t o m i z e d f i s h made t h e assumption o f any p e r i o d i c element q u e s t i o n a b l e operant  f i s h expressed  (Fig. 25).  No sham  a p e r i o d i c i t y between 20 and 28 hours.  A p p a r e n t l y t h e r i g o u r s o f t h e o p e r a t i o n would u p s e t the endogenous component, s i n c e b o t h p i n e a l e c t o m i z e d and sham o p e r a n t s  expressed  similar disturbances.  i n v e s t i g a t i o n i s necessary  Continued  t o determine t h e r o l e o f t h e  p i n e a l i n t h e endogenous r e s p o n s e ,  and i t i s suggested  that  l o n g e r p e r i o d s o f p o s t - o p e r a t i v e r e c o v e r y might f a c i l i t a t e any such i n v e s t i g a t i o n . 3.43  The e n t r a i n e d response w i t h opaque o r  t r a n s p a r e n t p l a s t i c s h i e l d s over t h e s i t e o f t h e p i n e a l . S i n c e t h e p r e v i o u s e x p e r i m e n t s ( s e c t i o n s 3.41, 3.42) i n d i c a t e d t h a t p e n e t r a t i o n o f t h e cranium c o u l d a l t e r t h e e n t r a i n e d response,  p l a s t i c s h i e l d s were designed  e f f o r t t o simulate pinealectomy.  i n an  E i t h e r clear or black  p l a s t i c s h i e l d s were i n s e r t e d between t h e dermis and t h e o s s i f i e d c r a n i u m , beneath t h e deepest l a y e r o f c o n n e c t i v e  67  F i g u r e 25.  Periodogram a n a l y s i s of a s i n g l e pinealectomized  fish.  00  ODODJ,,  2.On  o o o o  00 o  Q. <  CD T3 •*—•  oco  o  6>o  OOD  c? c o o orf  oo<%  Q  °  o  o  °  °  °o°o  %  »  i.<H  Q.  E <  20  —i— 22  —I—  24  Period  — i — 26  —i 28  68  tissue.  T h i s p l a c e d t h e s h i e l d s between t h e p i n e a l body  and t h e s o u r c e o f i n d i r e c t o v e r h e a d i l l u m i n a t i o n .  No  l a t e r a l s o u r c e o f l i g h t was a v a i l a b l e t o t h e s u b j e c t s s i n c e t h e w a l l s o f t h e swimming c h a n n e l s w e r e c o a t e d w i t h non-reflective f l a t black paint. discount  H o w e v e r , one c a n n o t  the p o s s i b i l i t y that the pineal could  adequate l a t e r a l  illumination resulting  receive  from t h e d i f f r a c t i o n  of l i g h t by e i t h e r p a r t i c u l a t e m a t e r i a l i n t h e water o r t h e tissues surrounding All visible plastic  shields.  the skull.  l i g h t was a b l e t o p e n e t r a t e  the clear  The a b s o r p t i o n c u r v e o f t h i s m a t e r i a l  b e t w e e n 2 0 0 a n d 9 7 0 my i n d i c a t e d t h a t o n l y t h o s e w a v e l e n g t h s i n t h e u l t r a v i o l e t r e g i o n were n o t t r a n s m i t t e d . visible  spectrum  (38 0 t o 7 0 0 my) t r a n s m i t t a n c e  Inthe was a t a  maximum. (a)  Black  shield testss  Six f i s h representing to t h i s series. photoperiod  36 " d a y s " o f a c t i v i t y  contributed  The mean d a i l y r e s p o n s e t o a 12L 12D  a t 10°C i n d i c a t e d t h a t t h e p r e s e n c e o f t h e  black s h i e l d s over the p i n e a l region d i d not e f f e c t i v e l y a l t e r t h e e n t r a i n e d response t o t h e l i g h t - d a r k environment (Fig. 26).  H o w e v e r , i t was a p p a r e n t t h a t  preventing  i l l u m i n a t i o n of the p i n e a l resulted i n higher  levels of  activity. (b)  Clear shield  tests.  F o u r f i s h r e p r e s e n t i n g 24 " d a y s " o f a c t i v i t y  contributed  69  F i g u r e 26.  Mean d a i l y a c t i v i t y p a t t e r n s of b l a c k s h i e l d and c l e a r s h i e l d  9  • Black  shields  O—O Clear  shields  tests:  Mean no. of events  70  to t h i s s e r i e s .  The mean d a i l y r e s p o n s e i n d i c a t e d  that  c l e a r s h i e l d s d i d not i n t e r f e r e with the entrainment t o the photoperiodic significantly  cycle  (see F i g . 2 6 ) .  However,  l o w e r l e v e l s o f a c t i v i t y were o b t a i n e d  t h e s e d a t a were compared w i t h t h e group t e s t e d w i t h  when black  shields. T h e s e two e x p e r i m e n t s c o m p l i m e n t t h e d a t a with pinealectomized exerted  obtained  f i s h by i n d i c a t i n g t h e major d i f f e r e n c e  by t h e p i n e a l i s an a l t e r a t i o n i n t h e l e v e l o f  activity  rather than determining  period.  the entrainment t o photo-  These t e s t s a l s o p r o v i d e  penetration  further indication that  of the cranium a l t e r s the basic  24 h o u r  pattern  e x p r e s s e d by j u v e n i l e s o c k e y e . 3.44  The e n t r a i n e d  response i n blinded  experiments with pinealectomized of t h e e n t r a i n e d information the major blinded  fish  response occurred  route.  fish.  The  suggested that  mediation  v i a an e x t r a - p i n e a l  A t t e n t i o n was f o c u s e d u p o n t h e e y e s a s  (and p e r h a p s most o b v i o u s ) p h o t o r e c e p t o r .  i n d i v i d u a l s would c l a r i f y  a combined experiment w i t h b l i n d e d  Testing  t h e r o l e o f t h e e y e s , and and p i n e a l e c t o m i z e d  fish  m i g h t i n d i c a t e whether o r n o t any e x t r a - r e t i n a l , e x t r a p i n e a l p a t h w a y was  involved.  Four b l i n d i n g t e c h n i q u e s were used t o d e t e r m i n e t h e most s u i t a b l e a p p r o a c h . phemerol c h l o r i d e  These i n c l u d e d  the i n j e c t i o n of  (1:1,000, Parke D a v i s ) i n t o t h e p o s t e r i o r  chamber o f e a c h e y e , s e c t i o n i n g o f t h e o p t i c n e r v e ,  bilateral  71  e n u c l e a t i o n o f t h e eyes, and p l a c i n g b l a c k p l a s t i c d i s c s over the eyes.  Those i n d i v i d u a l s s u b j e c t e d t o s u r g i c a l  m a n i p u l a t i o n were a l l o w e d 2 weeks p o s t - o p e r a t i v e before t e s t i n g . to previous (a)  recovery  Data were c o l l e c t e d i n a manner s i m i l a r  tests. Chemical b l i n d i n g .  E i g h t f i s h were p r e p a r e d  f o r t h i s s e r i e s of t e s t s .  The a p p l i c a t i o n o f phemerol c h l o r i d e f o l l o w e d the p r e v i o u s l y described technique  (Hoar, 1955b; Gunning, 1959;  Hasler,  1966) . Gunning (1959) q u e s t i o n s the u s e f u l n e s s of t h i s c h e m i c a l , but bases h i s o b j e c t i o n s on a l t e r e d  behaviour  p a t t e r n s i n the b l u e g i l l s u n f i s h , and does not p r o v i d e a measure o f t h e organism's a b i l i t y t o d e t e c t  light.  The m a j o r i t y of j u v e n i l e sockeye t r e a t e d showed s i g n s of r e c o v e r y w i t h i n one week. c o l o r a t i o n was  I t was  noted t h a t the  s i m i l a r t o normal i n d i v i d u a l s and not  as  dark as those f i s h s u b j e c t e d t o b i l a t e r a l e n u c l e a t i o n . The  f i s h responded t o movements o r i g i n a t i n g o u t s i d e o f the  aquarium, and a p p a r e n t l y d e t e c t e d these movements by The  vision.  i n d i v i d u a l d a i l y r e c o r d s i n d i c a t e d t h a t most f i s h  responded t o the p h o t o p e r i o d and m a i n t a i n e d  a day a c t i v e  r e l a t i o n s h i p t o the l i g h t c y c l e . One  i n d i v i d u a l was  T h i s o b s e r v a t i o n was  a p p a r e n t l y b l i n d e d by t h i s  technique.  based upon c o l o r a t i o n and response t o  movement o u t s i d e o f the aquarium.  Subsequent t e s t i n g i n  72  the a c t i v i t y  chambers i n d i c a t e d a l o s s o f e n t r a i n m e n t t o  the environmental photoperiod. T h i s t e c h n i q u e was d i s c o n t i n u e d due t o t h e u n c e r t a i n results.  I t was c o n c l u d e d t h a t t h e c h e m i c a l i n d u c e d  b l i n d n e s s i n some, b u t n o t a l l i n d i v i d u a l s (b) the o p t i c  The e n t r a i n e d r e s p o n s e  treated.  i n fish after  sectioning  nerve.  E i g h t f i s h w e r e b l i n d e d by s e c t i o n i n g t h e o p t i c The mean d a i l y r e c o r d i n d i c a t e d a n a c t i v i t y the onset of l i g h t  ( F i g . 27) .  nerve.  increase after  A response t o photoperiod  was a p p a r e n t and t h e p r e d a w n a c t i v i t y  i n c r e a s e suggested  the. p r e s e n c e o f a n e n d o g e n o u s c o m p o n e n t .  Periodogram  analysis of the i n d i v i d u a l records indicated that s i x f i s h e x p r e s s e d a n e x a c t 24.00 h o u r p e r i o d i c i t y .  Slight  d e v i a t i o n s b y t h e o t h e r two i n d i v i d u a l s r e s u l t e d i n a n average  24.07 h o u r p e r i o d f o r a l l f i s h  tested.  These d a t a suggest t h a t t h e a c t i v i t y entrainment t o photoperiod are not mediated pathways such as t h e o p t i c (c)  and  v i a visual  nerve.  The e n t r a i n e d r e s p o n s e  pinealectomized  response  i n b l i n d e d and  individuals.  These e x p e r i m e n t s  investigated the influence of  p h o t o r e c e p t o r s o t h e r t h a n t h e eyes o r t h e p i n e a l body. B l i n d i n g was i n d u c e d by s e c t i o n i n g t h e o p t i c n e r v e a t l e a s t two weeks a f t e r p i n e a l e c t o m y .  The t e s t s  were  73  F i g u r e 27.  Mean d a i l y a c t i v i t y b l i n d e d and and  pinealectomized,  bilaterally  (Blinding  patterns for  =  enucleated  blinded,  groups.  s e v e r i n g of o p t i c  A—A  B l i n d e d and  O—O  Blinded Bilateral  nerve).  Pinealectomized  Enucleates  Mean no. of events  74  s t a r t e d a f t e r an a d d i t i o n a l two week p e r i o d o f p o s t operative recovery. The mean d a i l y a c t i v i t y p a t t e r n i n d i c a t e d t h a t t h e s e f i s h were e n t r a i n e d t o t h e e n v i r o n m e n t a l . p h o t o p e r i o d (Fig. 27).  A s i g n i f i c a n t a c t i v i t y i n c r e a s e (P<.01) o c c u r r e d  a t t h e o n s e t o f l i g h t and was t e r m i n a t e d by a c o r r e s p o n d i n g decrease a t t h e o n s e t o f d a r k n e s s .  These d a t a would tend  t o suggest t h e r o l e o f some e x t r a - r e t i n a l , e x t r a - p i n e a l source o f p h o t o r e c e p t i o n . These t e s t s were based upon t h e assumption  that  removal o f a s e c t i o n o f t h e o p t i c nerve would e f f e c t i v e l y b l i n d j u v e n i l e sockeye salmon.  As f a r as i s known, t h i s  p r o c e s s d i s r u p t s a l l v i s u a l c o n n e c t i o n between t h e r e t i n a and t h e b r a i n , b u t n o n - v i s u a l a f f e r e n t n e u r a l c o n n e c t i o n s from t h e r e t i n a t o t h e CNS might be p r e s e n t . Gunning (1959) c a u t i o n e d t h a t a l t h o u g h t h e o p t i c  nerve  was s e v e r e d , t h i s would n o t c o m p l e t e l y omit t h e p o s s i b i l i t y of a r e t i n a l response t o t h e changing l i g h t c y c l e . l e d t o an assumption  This  that r e t i n a l mediation of the l i g h t  c y c l e could s t i l l occur, but the t r a n s m i s s i o n of i n f o r m a t i o n was v i a pathways o t h e r than t h e o p t i c n e r v e . (d)  The e n t r a i n e d response w i t h b i l a t e r a l e n u c l e a t i o n  of t h e eyes. S i x f i s h were p r e p a r e d f o r t h i s s e r i e s o f t e s t s . s u r g i c a l t e c h n i q u e i s l i s t e d i n t h e appendix.  The  Activity  r e c o r d i n g s were o b t a i n e d two weeks a f t e r t h e o p e r a t i o n b u t  75  three f i s h f a i l e d  to s u r v i v e f o r the d u r a t i o n of  experiment.  remaining  for  11  The  t h r e e i n d i v i d u a l s were t e s t e d  d a y s e a c h , r e s u l t i n g i n 33  r e p r e s e n t i n g the These d a t a  indicated a very  t h e c h a r a c t e r i s t i c day  was  not present  the  s m a l l sample s i z e ) concluded the  light  The  photoperiod (due  of e l i c i t i n g the  entrained  apparent i n these  tests.  the f o l l o w i n g s e r i e s of t e s t s i n d i c a t e d  The  not  involved.  e n t r a i n e d r e s p o n s e w i t h b l a c k eye  f a t a l i t i e s associated with b i l a t e r a l  caps were d e s i g n e d  would prevent the eyes. curve  retinal  covers.  enucleation  s t i m u l a t i o n by  A total  which  l i g h t , without  c a p s w i t h t h e same  o f 21  f i s h were used i n t h e s e  o f 146  "days".of  light cycle  a  tests  activity.  ( F i g . 28).  t h e r e s p o n s e b e t w e e n n o r m a l f i s h and  removing  absorption  a p p l i c a t i o n of b l a c k caps a b o l i s h e d the  response to the  Black  f o r t h e p i n e a l s h i e l d s w e r e u s e d as  contributing a total The  as an a l t e r n a t e t e c h n i q u e  Clear plastic  described  control.  response  extra-retinal  i n d i c a t e d the b a s i c o b j e c t i o n to that technique. eye  to  eyes.  r e g i o n of  capable  activity  t h a t the e n t r a i n e d  t h a t t h e e x t r a - r e t i n a l e n t r a i n m e n t was (e)  of  tentatively  m e d i a t e d by t h e  r e s p o n s e t h e r e s u l t s w o u l d be and  low a m p l i t u d e  I t was  f a l l i n g on any  p h o t o s e n s i t i v i t y was  Both these  activity  a c t i v e response to  ( F i g . 27).  c y c l e was  If light  "days" o f  group.  and  to  the  The  entrained  similarity  the f i s h a f t e r  of  removal-  76  F i g u r e 28.  Mean d a i l y a c t i v i t y p a t t e r n s f o r b l a c k and c l e a r eye cap groups.  •  • B l a c k Eye Caps  O — O C l e a r Eye Caps  I  !  Mean no. of events  77  of the b l a c k caps i n d i c a t e d t h a t entrainment r e s u l t e d from r e t i n a l p h o t o s t i m u l a t i o n only eye  ( F i g . 29) .  caps were removed d u r i n g t h e c o u r s e  the s u b j e c t would respond immediately  o f an  to the  If  the  experiment, environmental  photoperiod. Before was  noted.  r e m o v a l an e n d o g e n o u s f o u r h o u r  T h i s was  environment 2 0.00  cap  an u l t r a d i a n r e s p o n s e t o t h e  ( l e s s t h a n a day,  h o u r s ) and  was  o b s e r v e d i n t h e DD  periodicity  but  ranging  s i m i l a r to the  tests.  No  short  constant  between 0  and  periodicity  endogenous p a t t e r n s  with  c i r c a d i a n c h a r a c t e r i s t i c s were o b s e r v e d i n f i s h w i t h caps i n p l a c e , a f a c t o r s u p p o r t i n g might i n h i b i t the e x p r e s s i o n  black  the assumption t h a t  o f an e n d o g e n o u s r h y t h m  DD  by  j u v e n i l e sockeye salmon. The  response obtained w i t h the c l e a r p l a s t i c  a p p e a r e d t o be  c h a r a c t e r i s t i c o f a day  s i n g l e i n d i v i d u a l s , but l e v e l s of a c t i v i t y Fig.  28).  The  and  shields  a c t i v e organism i n  the grouped data  indicated high  an a p p a r e n t l o s s o f e n t r a i n m e n t  c o n s t r u c t i o n of the p l a s t i c d i s c s f o r  (see these  t e s t s u n d o u b t e d l y c r e a t e d many v i s u a l a b e r r a t i o n s  which  i n t e r f e r e d w i t h the o p t i c s of the i n t a c t eye,  might  be  r e s p o n s i b l e f o r these These d a t a  variations in activity.  i n d i c a t e d a d e f i n i t e source  f o r the e n t r a i n e d response to photoperiod Since  the i n f o r m a t i o n about the  v i a the o p t i c nerve,  and  of  reception  in this  l i g h t c y c l e i s not  i t i s s u g g e s t e d t h a t t h e eye  species. transmitted might  78  F i g u r e 29.  12L 12D,5°C response i n normal  fish  and f i s h t e s t e d i m m e d i a t e l y a f t e r removal of t h e b l a c k eye caps.  O — O Normal  fish  B l a c k eye caps removed  Mean no. of events ai  _k oi  N> cn  79  s e c r e t e a b l o o d - b o r n agent w h i c h i s r e s p o n s i b l e f o r the c y c l i c a c t i v i t y response. 3.5  The E f f e c t s of M e l a t o n i n , S e r o t o n i n and  Teleost  S a l i n e on the E n t r a i n e d Response. The d a t a from the p r e c e d i n g  s e c t i o n s on  pinealectomized  and b l i n d e d f i s h suggested t h a t the p r o d u c t i o n of c e r t a i n c h e m i c a l agents may  be i n v o l v e d i n e n t r a i n m e n t .  i n d i v i d u a l s subjected to pinealectomy  or b l a c k s h i e l d s  over the p i n e a l r e g i o n e x h i b i t e d s i g n i f i c a n t l y activity levels.  The  Those  higher  e n t r a i n e d response and the  activity  l e v e l were b o t h . a l t e r e d by removal of the eyes and  the  a p p l i c a t i o n of b l a c k c a p s . The  o b j e c t i v e of t h i s s e r i e s was  i n j e c t i o n of melatonin  t o determine i f the  and s e r o t o n i n would induce  significant  changes i n the a c t i v i t y l e v e l s of j u v e n i l e sockeye salmon. S i n c e the p i n e a l i s g e n e r a l l y c o n s i d e r e d as the major s i t e of m e l a t o n i n  p r o d u c t i o n , i t i s assumed t h a t lower  l e v e l s w i l l be p r e s e n t  f o l l o w i n g pinealectomy  melatonin  (unless  compensatory i n c r e a s e s o c c u r i n the r e t i n a ) .  This decrease  i n the l e v e l of m e l a t o n i n might be r e s p o n s i b l e f o r the higher a c t i v i t y l e v e l s observed.  Alternatively,  i n c r e a s e d a c t i v i t y l e v e l might be due  the  to increased  of s e r o t o n i n , the p r e c u r s o r o f m e l a t o n i n .  This  c o u p l e d w i t h the f i n d i n g s of Wong and W h i t e s i d e  levels  information (1968) l e d  t o the f o r m u l a t i o n of a w o r k i n g h y p o t h e s i s p r e d i c t i n g an increase i n a c t i v i t y w i t h s e r o t o n i n , a decrease i n a c t i v i t y  80  w i t h m e l a t o n i n , and no change w i t h i n j e c t i o n s o f t e l e o s t saline. A t o t a l o f e i g h t e e n f i s h were t e s t e d i n t h r e e groups o f n i n e days each. upon t h e locomotor  The f i r s t t h r e e t e s t days were f o c u s e d a c t i v i t i e s i n a 12L 12D (10°C) e n v i r o n -  ment b e f o r e a d m i n i s t e r i n g i n t r a p e r i t o n e a l The  injections.  second t h r e e days o f t h e t e s t were f o c u s e d upon t h e  expression following  0.25 c c d a i l y i n j e c t i o n s  (lug per  gram body weight) o f m e l a t o n i n , s e r o t o n i n , o r t e l e o s t saline.  A l l i n j e c t i o n s were a d m i n i s t e r e d 1/2 hour b e f o r e  the dark phase o f t h e p h o t o p e r i o d i c c y c l e .  The f i n a l  days examined t h e a c t i v i t y r e c o r d s o f these  three  individuals  after the i n j e c t i o n s . S i g n i f i c a n t a c t i v i t y increases occurred a f t e r i n j e c t i o n of serotonin (Fig. 30). resulted  i n decreased  Melatonin  injections  a c t i v i t y , whereas no g r o s s changes  occurred i n the t e l e o s t s a l i n e treatment. i n d i c a t i o n t h a t s l i g h t a c t i v i t y decreases  There was an i n a l l tests  might be a t t r i b u t e d t o t h e method o f removing t h e f i s h from the environment and i n j e c t i n g by hand (see c o n t r o l ) . A comparison o f t h e mean d a i l y a c t i v i t y r e c o r d s f o r the c o n t r o l group and m e l a t o n i n t r e a t m e n t i n d i c a t e d the decrease  that  i n a c t i v i t y occurred mainly during the l i g h t  phase o f t h e p h o t o p e r i o d i c c y c l e ( F i g . 3 1 ) .  Conversely,  the major a r e a o f a c t i v i t y i n c r e a s e f o r t h e s e r o t o n i n t r e a t m e n t o c c u r r e d d u r i n g t h e dark phase o f t h e p h o t o p e r i o d .  81  F i g u r e 30.  Mean t o t a l d a i l y a c t i v i t y S e r o t o n i n , M e l a t o n i n and Saline injected  groups.  B  =  Before  Injection  D  =  During  Injection  A  =  After  Injection  for teleost  Mean no. of events co o  cn o  -NI  o  83  4.  DISCUSSION  The g e n e r a l importance o f b i o l o g i c a l r h y t h m i c i t y i s r e f l e c t e d by t h e amount o f i n t e r e s t t h e s u b j e c t has generated  i n the past f i f t e e n years.  i n c l u d e d examinations  Research has  o f d a i l y , t i d a l , l u n a r and annual  c y c l e s , and how t h e s e r e l a t e t o m i g r a t i o n , h i b e r n a t i o n , b r e e d i n g , c o l o r changes, m e t a b o l i c p r o c e s s e s , f e e d i n g and locomotor  a c t i v i t y patterns.  The m a j o r i t y o f i n t e r e s t has  f o c u s e d upon mammalian and a v i a n forms, a f a c t o r r e f l e c t i n g t h e i r a v a i l a b i l i t y and u s e f u l n e s s i n l o n g term l a b o r a t o r y projects. developed  As a r e s u l t , most r e c o r d i n g t e c h n i q u e s have been f o r a few t e r r e s t r i a l organisms.  The apparent l a c k o f i n t e r e s t i n t e l e o s t s has r e s u l t e d from t h e meagre s u p p l y o f adequate i n s t r u m e n t a t i o n f o r r e c o r d i n g locomotor  activity.  However, r e c e n t advances  i n t h e d e s i g n o f e l e c t r o n i c components and t h e i r  application  (Cummings, 1963; M u i r , e t a l . , 1965; M e f f e r t , 1968) have p r o v i d e d t h e o p p o r t u n i t y t o proceed w i t h i n v e s t i g a t i o n s i n a v a r i e t y o f a q u a t i c organisms. Environmental  c o n t r o l was o f paramount importance t o  t h i s i n v e s t i g a t i o n , and r e p r e s e n t e d a f a c t o r n o t a t t a i n a b l e i n the f i e l d .  However, t h e u l t i m a t e g o a l was t o a p p l y t h e  l a b o r a t o r y based hypotheses t o t h e f i e l d s i t u a t i o n ,  thus  i n d i c a t i n g the general e c o l o g i c a l s i g n i f i c a n c e of b i o l o g i c a l r h y t h m i c i t i e s i n j u v e n i l e sockeye salmon.  84  The adequate Bunning  c o n t r o l systems and f u l f i l l e d  used i n t h e l a b o r a t o r y were  t h e r e q u i r e m e n t s o u t l i n e d by  (1960) :  " P r o v i s i o n s f o r c o n s t a n t t e m p e r a t u r e and e x a c t c o n t r o l o f l i g h t and d a r k n e s s a r e p a r t i c u l a r l y i m p o r t a n t s i n c e we know t h a t i n some c a s e s t e m p e r a t u r e f l u c t u a t i o n s o f l e s s t h a n 1°C c a n s i g n i f i c a n t l y i n t e r f e r e with experiments, or t h a t an o b s e r v a t i o n l i g h t , e v e n i f a p p l i e d f o r o n l y a few seconds c a n a l s o u p s e t an e x p e r i m e n t " . The  use o f e l e c t r o n i c t r a n s d u c e r s f a c i l i t a t e d t h e  c o n s t r u c t i o n o f an a r t i f i c i a l  environment  and a c t i v i t y  m o n i t o r i n g system which enabled t h e i n v e s t i g a t i o n t o p r o c e e d w i t h o u t a d d i n g any i n s t r u m e n t s t o t h e c o n t r o l l e d environment.  The g e n e r a l a p p l i c a b i l i t y  was t e s t e d b y r e c o r d i n g a c t i v i t y instrument operation.  locomotor a c t i v i t y Spontaneous  b e f o r e , d u r i n g and a f t e r  These d a t a i n d i c a t e d t h a t use o f  h i g h f r e q u e n c y sound would of  of the transducer  f a c i l i t a t e a l o n g term  i n these  fish.  locomotor a c t i v i t y  i s an e a s i l y  b e h a v i o u r a l r e s p o n s e t o endogenous and exogenous which has been i n v e s t i g a t e d  measured factors  i n a v a r i e t y o f organisms  ( r e v i e w s : A s c h o f f , 1960, 1965a). of  study  This particular  measure  b e h a v i o u r was u s e d t o i n d i c a t e t h e e n t r a i n e d r e s p o n s e t o  p h o t o p e r i o d and a l s o s e r v e d as an e a s i l y o b t a i n e d a s s a y o f light  perception.  4.1  The B a s i c A c t i v i t y  Pattern  A d e f i n i t e e n t r a i n e d r e s p o n s e was o b t a i n e d i n an artificial  (LD) e n v i r o n m e n t .  The a c t i v i t y  rhythm  was  85  e x p r e s s e d as a l i g h t a c t i v e r e l a t i o n s h i p t o t h e e n v i r o n m e n t a l p h o t o p e r i o d w h i c h a l s o s e r v e d as t h e p r i m a r y e n t r a i n i n g agent of  fish.  The  f o r e i t h e r s i n g l e i n d i v i d u a l s or  response  at d i f f e r e n t developmental  i n d i c a t e d t h a t t h e a c t i v i t y p a t t e r n was at  an e a r l y age  groups  firmly  established  and m a i n t a i n e d f o r a t l e a s t one  S u f f i c i e n t i n f o r m a t i o n was  stages  year.  not a v a i l a b l e to determine  g e n e r a l a c t i v i t y p a t t e r n a f t e r one y e a r o f a g e .  the  Manzer  (1964) showed e v i d e n c e o f d i e l v e r t i c a l movements i n t h e G u l f o f A l a s k a , and G r o o t a b a s i c a c t i v i t y rhythm  (1965) s u g g e s t e d t h a t t h e r e  of sockeye  salmon,  was  young or o l d ,  migrating or not. F i s h o b t a i n e d from a d i f f e r e n t l o c a l i t y  (Kamchatka  Peninsula) expressed a s i m i l a r a c t i v i t y pattern at  5°C.  H o w e v e r , b o t h s t o c k s ( L a k e l s e and K a m c h a t k a ) w e r e r e a r e d under i d e n t i c a l  l a b o r a t o r y c o n d i t i o n s , a f a c t o r which  have c o n t r i b u t e d t o t h e e x p r e s s i o n o f s i m i l a r I t was  n o t known i f s i m i l a r d a t a w o u l d  identical  may  responses.  be o b t a i n e d i f  s t o c k s were r e a r e d a t t h e i r n a t i v e g e o g r a p h i c a l  locations. 4.11  The  Both temperature response.  i n f l u e n c e of temperature  and p h o t o p e r i o d a l t e r e d t h e e n t r a i n e d  I n c r e a s i n g temperatures  higher a c t i v i t y  and p h o t o p e r i o d .  usually resulted i n  a m p l i t u d e s d u r i n g b o t h l i g h t and  dark,  b u t when s h o r t d a y l e n g t h s w e r e c o m b i n e d w i t h h i g h decreased a c t i v i t y  occurred.  A unimodal  activity  temperatures, pattern  86 i  was e x p r e s s e d when s h o r t d a y l e n g t h s and low temperatures were used.  When e i t h e r d a y l e n g t h o r temperature  i n c r e a s e d , a bimodal p a t t e r n  appeared.  I t i s concluded t h a t c e r t a i n combinations of temperature and p h o t o p e r i o d can produce changes i n t h e c o n t o u r o f t h e a c t i v i t y response and can even i n i t i a t e a complete r e v e r s a l o f t h e p h o t o b e h a v i o u r a l r e s p o n s e . I t i s h y p o t h e s i z e d t h a t f o r each d a y l e n g t h t h e r e i s a c e r t a i n t e m p e r a t u r e , above which j u v e n i l e sockeye salmon w i l l r e v e r s e t h e c h a r a c t e r i s t i c l i g h t a c t i v e p a t t e r n and respond w i t h dark a c t i v i t y .  I t i s suggested t h a t  i n c r e a s i n g t e m p e r a t u r e s w i l l f u n c t i o n as t h e major " r e v e r s i n g " agent, whereas t h e p h o t o p e r i o d w i l l as a " c o n t r o l l i n g " agent.  I n t h i s way,  function  increasing  daylengths w i l l s t a b i l i z e the l i g h t a c t i v e response. The d a t a p r e s e n t e d r e f e r o n l y t o the l a b o r a t o r y i n v e s t i g a t i o n , but a r e s u p p o r t e d by the work o f Gibson and K e e n l e y s i d e (1966) who  suggested t h a t temperature  might be r e s p o n s i b l e f o r a r e v e r s e d p h o t o b e h a v i o u r a l response i n young A t l a n t i c  salmon.  P o i k i l o t h e r m s g e n e r a l l y respond t o i n c r e a s i n g temperatures  ( w i t h i n the l i m i t s o f t o l e r a n c e ) w i t h g r e a t e r  metabolic a c t i v i t y .  I n c r e a s e d locomotor a c t i v i t y  might  t h e n be i n t e r p r e t e d as a " r e f l e c t i o n " of the m e t a b o l i c s t a t e . The d a t a p r e s e n t e d i n s e c t i o n 3.24  clearly indicate this  g e n e r a l i z e d response i n young sockeye  salmon.  87  The a p p a r e n t t e m p e r a t u r e - d a y l e n g t h i n t e r a c t i o n complicates the interpretation of the data.  Hoar  (1965)  s u g g e s t s t h a t t h e t e m p e r a t u r e - p h o t o p e r i o d l i n k may be a s s o c i a t e d w i t h some p i t u i t a r y  factor, but the actual  mechanisms r e m a i n t o be d e t e r m i n e d . for  I t appears  that  e a c h d a y l e n g t h t h e r e i s a n optimum t e m p e r a t u r e f o r  c e r t a i n m e t a b o l i c p r o c e s s e s such as f a t d e p o s i t i o n o r resistance t o heat.  Seasonally changing photoperiods  might a d j u s t t h e metabolism of f i s h t o a n t i c i p a t e t e m p e r a t u r e c h a n g e s i n t h e s p r i n g a n d autumn. conflicting  environmental s t i m u l i  sharp  However,  ( s h o r t days and h i g h  temperatures) apparently r e s u l t i n a r e v e r s a l o f the photobehavioural Aschoff  response.  (1960)  suggested that a t d i f f e r e n t  i n t e n s i t i e s t h e r e was a n optimum l e v e l , organism would rest ratio.  light  above w h i c h an  seek t h e o p p o s i t e response i n t h e a c t i v i t y / A s i m i l a r mechanism might o c c u r h e r e , where  a manipulation of the temperature-daylength r e l a t i o n s h i p might r e g u l a t e t h e e x p r e s s i o n o f l i g h t o r dark These d i f f e r e n t a c t i v i t y  activity.  responses t o temperature  and p h o t o p e r i o d m i g h t a l s o a c c o u n t f o r some o f t h e conflicting  r e p o r t s on s a l m o n i d a c t i v i t y  w h i c h have  occurred a t separate geographical location Thompson a n d B u r g n e r ,  1967).  ( G r o o t , 1965;  88  4.2  The  Endogenous A c t i v i t y  Response  The  a n t i c i p a t o r y increase i n a c t i v i t y p r i o r to the  o n s e t o f l i g h t and a c o r r e s p o n d i n g d e c r e a s e i n a c t i v i t y b e f o r e d a r k s u g g e s t e d an e n d o g e n o u s c y c l e w i t h  a  c h a r a c t e r i s t i c p e r i o d t h a t was  hours.  Those e x p e r i m e n t s c o n d u c t e d t h a t t h e r e s p o n s e was  i n constant l i g h t  2 3 . 3 0  h o u r s was  The  of  periodic  identified. and  (1965)  w h i c h s u g g e s t e d t h e p r e s e n c e o f an  clock i n this  indicated  A f r e e - r u n n i n g rhythm w i t h  data supported the hypothesis of Groot (1965)  2 4 . 0 0  m a i n t a i n e d i n absence  environmental s t i m u l i . period approximating  l e s s than  a These  Hoar  endogenous  species.  response to i n c r e a s i n g l i g h t i n t e n s i t i e s  not i n accordance w i t h A s c h o f f ' s general r u l e  was  (1960)  which  states: "With i n c r e a s i n g l i g h t i n t e n s i t i e s the p e r i o d of a l i g h t a c t i v e organism w i l l decrease". H o w e v e r , t h i s i s a g r o s s g e n e r a l i z a t i o n and  several  e x c e p t i o n s have been n o t e d i n t h e l i t e r a t u r e Hoffmann, The  (review:  1 9 6 5 ) .  i n c r e a s i n g period i n these j u v e n i l e  sockeye  m i g h t be a s s o c i a t e d w i t h t h e i r c h a r a c t e r i s t i c  ability  t o e x p r e s s b o t h d i u r n a l and n o c t u r n a l a c t i v i t y p a t t e r n s at d i f f e r e n t stages i n the l i f e i n t e r e s t i n g t o determine whether  cycle.  I t would  be  o r n o t any c h a n g e s o c c u r  i n t h e e n d o g e n o u s component d u r i n g t h e c o u r s e o f  development,  89  especially  d u r i n g the  with nocturnal Periodic cycle  present  feeding  could also constant  or continuously  entrainment  sectioning response, response  of•the  i n image  the  (possibly consider  optic  nerve  induced  removal of  suppressed  the  experiments.  a b o l i s h the  "blindness",  different  However,  possibility  indicated  of  of  the light  than the is  entrained visual intensity  processes.  It  i n f o r m a t i o n might  it  Since  optic  equally  is  be  nerve  feasible  some unknown a f f e r e n t  to  nervous  retina.  The h y p o t h e s i s First,  d i d not  some s y s t e m o t h e r  p a t h w a y s from t h e  continuously  term  f o r m a t i o n and d e t e c t i o n  vascular). the  is  e y e s and p i n e a l body  that photoperiodic by  short  It  occurred through r e t i n a l mediation.  m i g h t be b a s e d upon e n t i r e l y  transmitted  endogenous  Entrainment  but presumably  hypothesized  the  environment.  absent for  A consideration of that  affect  f o o d w i l l have t o be e i t h e r  Mechanism o f  4.3  coincide  activity.  i n an o t h e r w i s e  concluded that  p e r i o d s when m i g r a t i o n s  the  was b a s e d upon t h e eye,  following  or placing black  e n t r a i n e d response  (i.e.  discs  apparently  information. over the  it fish  c o u l d not d e t e c t the  photoperiodic  environment).  Secondly,  sectioning  nerve  result  of  entrainment This  the  optic  (i.e.  the  fish  information indicated  d i d not  could detect that  i n loss  the  some change  light  cycle).  occurred within  90  the eye, and t h e r e s u l t i n g a c t i v i t y response was mediated  by pathways w i t h i n the o p t i c n e r v e .  not The  p o s s i b i l i t y o f e n t r a i n m e n t by o t h e r p h o t o r e c e p t o r s as t h e p i n e a l body was  r e j e c t e d s i n c e e n t r a i n m e n t d i d not  occur i n b i l a t e r a l l y enucleated f i s h w i t h t h i s intact.  such  organ  However, Menaker (1968) and G a s t e r and Menaker  (1968) demonstrated  t h a t i l l u m i n a t i o n o f the p i n e a l c o u l d  s e r v e t o e n t r a i n b l i n d e d sparrows.  C o n v e r s e l y , Quay  (1968) f a i l e d t o demonstrate any p i n e a l m e d i a t i o n i n the a c t i v i t y response o f t h e l a b o r a t o r y r a t , but d i d r e v e a l t h a t e n t r a i n m e n t was  a response t o i l l u m i n a t i o n of the  retina. My i n v e s t i g a t i o n i n d i c a t e d t h a t t h e p i n e a l body i n j u v e n i l e sockeye response.  salmon was not i n v o l v e d i n the e n t r a i n e d  However, removal o f t h e p i n e a l o r s h a d i n g i t  r e s u l t e d w i t h increased a c t i v i t y l e v e l s during both and d a r k .  light  S i n c e e n t r a i n m e n t i s p o s s i b l e i n some forms  v i a p i n e a l i l l u m i n a t i o n , and i n o t h e r s by r e t i n a l  mediation,  one might f i r s t l o o k f o r s i m i l a r mechanisms w i t h i n the  two  organs. Quay (1965) demonstrated  the presence of p i n e a l  and  r e t i n a l HIOMT ( h y d r o x y i n d o l e - o - m e t h y l t r a n s f e r a s e ) i n lower v e r t e b r a t e s , i n c l u d i n g f i s h .  This i s the m e t h y l a t i n g  enzyme r e s p o n s i b l e f o r the f o r m a t i o n o f m e l a t o n i n ( N - a c e t y l •5-methoxy-tryptamine) from i t s p r e c u r s o r N - a c e t y l s e r o t o n i n (N-acetyl-5-hydroxy-tryptamine).  Fenwick ( i n p r e s s )  has  91  demonstrated the presence o f both r e t i n a l and p i n e a l melatonin and s e r o t o n i n i n two c l o s e l y r e l a t e d s p e c i e s of P a c i f i c salmon.  Bagnara  (I960), Wurtman, e t a l . ,  (1963) and Quay (1964) have shown t h a t an apparent c o r r e l a t i o n e x i s t s between the environmental l i g h t c y c l e and  v a r i a t i o n s i n the l e v e l o f p i n e a l melatonin and  s e r o t o n i n i n s e v e r a l higher  vertebrates.  Quay s experiments with the l a b o r a t o r y r a t i n d i c a t e d 1  low  s e r o t o n i n l e v e l s d u r i n g dark, with a 900% i n c r e a s e  during l i g h t .  R e i s s , e t a l . , (1963) and Wong and Whiteside  (1968) have demonstrated t h a t i n j e c t i o n s o f p i n e a l e x t r a c t s or melatonin w i l l s i g n i f i c a n t l y reduce the t o t a l wheelrunning  a c t i v i t y of laboratory  I t was hypothesized  rats.  that i n t r a p e r i t o n e a l i n j e c t i o n s  of melatonin might reduce locomotor a c t i v i t y i n j u v e n i l e sockeye salmon i f s i m i l a r c o r r e l a t i o n s e x i s t e d between the production cycle.  o f t h i s chemical  and the environmental  light  I f an opposing response c o u l d be a t t r i b u t e d t o  s e r o t o n i n , an i n c r e a s e d amplitude of a c t i v i t y might be expected.  The l a b o r a t o r y t e s t s i n d i c a t e d t h a t  serotonin  i n c r e a s e d the a c t i v i t y l e v e l d u r i n g the dark phase of the photoperiod  and melatonin decreased the a c t i v i t y  level  d u r i n g the l i g h t phase. Since the p i n e a l i s g e n e r a l l y considered major s i t e o f melatonin p r o d u c t i o n ,  t o be the  the t r i g g e r e d r e l e a s e  by e i t h e r r e t i n a l i n f o r m a t i o n or d i r e c t i l l u m i n a t i o n may  92  involved.  H o w e v e r , t h e m e c h a n i s m s by w h i c h t h i s  o c c u r s r e m a i n t o be The  determined.  onset of darkness might  s y n t h e s i s from s e r o t o n i n . l e v e l s may  release  The  f a c i l i t a t e melatonin  increased melatonin  be c o r r e l a t e d w i t h t h e d e c r e a s e d  activity  l e v e l s d u r i n g d a r k n e s s , however, t h e a c t u a l  function  w i t h i n t h e o r g a n i s m r e m a i n s t o be d e t e r m i n e d . n o t known w h e t h e r  the m e l a t o n i n formed  a hormone, o r where t h e s p e c i f i c  It is  i s s e c r e t e d as  s i t e s of a c t i o n  are  located. Serotonin  (5-HT) o r i g i n a t e s as a p r o d u c t o f  tryptophan metabolism.  The  b r a i n are s m a l l , but appear  a b s o l u t e amounts i n t h e t o have g r e a t i m p o r t a n c e  (Quay, 1965).  I t s d i s t r i b u t i o n i s not c o n f i n e d t o the  c e n t r a l nervous  system.  identified  I n mammals, 5-HT  i n many t i s s u e s and  found i n the blood  has  been  l a r g e amounts h a v e b e e n  platelets.  C i r c u m s t a n t i a l e v i d e n c e i n d i c a t e s t h a t 5-HT as a t r a n s m i t t e r s u b s t a n c e  ( R o c h e , 1964).  may  act  However,  c u r r e n t r e s e a r c h on t h e o v e r a l l f u n c t i o n o f 5-HT  is  c o m p l i c a t e d by i t s n e a r l y u b i q u i t o u s d i s t r i b u t i o n . In j u v e n i l e sockeye salmon, o f 5-HT  increased dark a c t i v i t y .  known w h e t h e r  intraperitoneal  injections  Here a g a i n , i t i s n o t  t h e s i t e s o f a c t i o n were i n t h e b r a i n o r a t  peripheral areas.  A d m i n i s t r a t i o n of  5-hydroxytryptophan  (5-HTP), t h e p r e c u r s o r o f s e r o t o n i n , w i l l  increase  the  93  amounts o f 5-HT t h a t d o g s and somatic, those  p r o d u c e d by  5-HT  changes  levels  with  resembling  w i t h l o s s o f r e f l e x e s , a p p a r e n t f e a r , sham  data  research.  disorientation).  s u g g e s t a p r o f i t a b l e avenue o f  future  A c o l l a b o r a t i v e approach p r o v i d i n g a  o f i n f o r m a t i o n on chemical  behavioural  (1964) i n d i c a t e s  lysergic acid diethylamide ( i . e .  b l i n d n e s s and My  Roche  cats respond to increased  a u t o n o m i c and  excitement rage,  i n most t i s s u e s .  and  synthesis  the o v e r t e n t r a i n e d response w i t h  the  c e l l u l a r changes o c c u r r i n g i n the c e n t r a l  n e r v o u s s y s t e m w o u l d be m o s t v a l u a b l e .  4.4  but  The  Adaptive  S i g n i f i c a n c e of a D a i l y A c t i v i t y  The  e x p r e s s i o n o f an a c t i v i t y r h y t h m i s n o t  unique,  the f u n c t i o n a l s i g n i f i c a n c e of such a response i s  evasive.  One  o b v i o u s a d v a n t a g e m i g h t be  the v a r i o u s p h y s i o l o g i c a l processes and  Cycle.  to  synchronize  with daily,  seasonal  y e a r l y c y c l e s , thus e n a b l i n g the organism to  fully  e x p l o i t i t s environment. I f c e r t a i n l e v e l s of s e n s i t i v i t y are w i t h the environmental  photoperiod  o r g a n i s m c o u l d make f u l l u s e i n performing  (Aschoff, 1965b),  of i t s sensory  such d i v e r s e a c t i v i t i e s  m i g r a t i n g or evading  predators.  b e t w e e n e n d o g e n o u s and t h e s i t u a t i o n and  However, t h e  simple  an  information  as f e e d i n g ,  exogenous components  o b s c u r e s any  synchronized  schooling,  interaction  complicates  answer t o t h e  general  94  s i g n i f i c a n c e of b i o l o g i c a l The  rhythmicity.  i n t e r a c t i o n o b s e r v e d between temperature  and  photoperiod supported the hypothesis that thermal r e s i s t a n c e c o u l d be m o d i f i e d by t h e l i g h t c y c l e 1965b)  (i.e.  long photoperiods could increase  (Hoar,  resistance  to heat w h i l e short photoperiods could increase to  chilling). My  study indicated that long photoperiods increased  t h e t e m p e r a t u r e v a l u e a t w h i c h t h e r e was of l i g h t / d a r k a c t i v i t y . if  resistance  I t was  a 50%  f u r t h e r demonstrated  the e n v i r o n m e n t a l temperature exceeded  t h e r e was  distribution  a certain  limit,  a r e v e r s a l of the photobehavioural response  j u v e n i l e s o c k e y e s a l m o n became d a r k a c t i v e .  that  and  The  c h a r a c t e r i s t i c n o c t u r n a l smolt o u t m i g r a t i o n might represent a s i m i l a r response t o the r a p i d l y  increasing  water temperatures i n s p r i n g . 4.41  E m e r g e n c e and m i g r a t i o n .  Under  natural  c o n d i t i o n s sockeye f r y are n e g a t i v e l y p h o t o t a c t i c remain h i d d e n i n t h e g r a v e l d u r i n g t h e day. r e a d y t o m i g r a t e emerge a t d u s k , e x h i b i t an  and  Those f r y apparent  n e g a t i v e r h e o t a x i s and move d o w n s t r e a m t o n u r s e r y l a k e s . The m i g r a n t s e x p r e s s a b i m o d a l a c t i v i t y p a t t e r n and not school.  A f t e r reaching the lake the f i s h  form  s c h o o l s , e x h i b i t a p o s i t i v e r h e o t a x i s and r e m a i n and f e e d i n g d u r i n g t h e d a y 1960;  Hartman, e t a l . ,  ( H o a r , 1954,  1962;  Heard,  1958;  1965).  do  swimming  McDonald,  95  My l a b o r a t o r y t e s t s o f f e r e d p a r a l l e l e v i d e n c e f o r a bimodal a c t i v i t y p a t t e r n i m m e d i a t e l y a f t e r h a t c h i n g . A s m a l l morning a c t i v i t y peak o c c u r r e d a t t h e o n s e t o f l i g h t , and a major e v e n i n g a c t i v i t y peak o c c u r r e d a f t e r t h e o n s e t of d a r k n e s s .  These peaks were s e p a r a t e d by  a midday d e p r e s s i o n of a c t i v i t y which p e r s i s t e d f o r a p p r o x i m a t e l y t e n days.  Newly emerged f r y i n a s w i f t l y  f l o w i n g s t r e a m , unable t o p e r f o r m v i s u a l  landmark  o r i e n t a t i o n , might be s u b j e c t e d t o a p a s s i v e downstream displacement.  The h i g h a m p l i t u d e s of n o c t u r n a l a c t i v i t y  i n the newly emerged f r y , c o u p l e d w i t h reduced acuity  ( A l i , 1959)  visual  suggest a mechanism f o r downstream  displacement. Upstream m i g r a n t s (a s i t u a t i o n where the  spawning  grounds a r e i n t r i b u t a r i e s f l o w i n g out o f t h e n u r s e r y l a k e ) e x p r e s s a p a t t e r n of day a c t i v e m i g r a t i o n (McCart, 1967; C a l a p r i c e , p e r s . comm.).  Andrew and Geen (1960)  r e p o r t t h a t i n t h e C h i l c o l a k e system, t h e newly emerged f r y move downstream u n t i l . r e a c h i n g areas of low v e l o c i t y a l o n g t h e r i v e r edge.  The f r y e v i d e n t l y h o l d i n t h e s e  a r e a s and move upstream seven t o t e n days after'.emergence. My i n v e s t i g a t i o n i n d i c a t e d t h a t a f t e r t e n d a y s , downstream m i g r a n t f r y became l i g h t a c t i v e .  The i n c r e a s e d  l e v e l s o f day a c t i v i t y c o u p l e d w i t h a s t r o n g r h e o t a x i s would be o f paramount importance t o upstream m i g r a t i o n d u r i n g t h e day.  T h i s study would f a v o u r the h y p o t h e s i s  96  t h a t u p s t r e a m m i g r a t i o n s a r e f a c i l i t a t e d by changes i n behaviour  c o u p l e d w i t h an i n c r e a s e i n s i z e  (7 t o 10 d a y s  a d d i t i o n a l d e v e l o p m e n t ) a n d swimming a b i l i t y  (Hoar,  1958).  H o w e v e r , C a l a p r i c e ( p e r s . comm.) a l s o c o n s i d e r s e q u a l l y f e a s i b l e a g e n e t i c a l l y r e g u l a t e d l i g h t and c u r r e n t f o r u p s t r e a m and downstream m i g r a n t s  response  (see a l s o R a l e i g h ,  1967; B r a n n o n , 1967). 4.42  P e l a g i c stage.  After entering the nursery  l a k e s , f r y d i s p e r s e along shore  entirely  pelagic.  This study  maintained  i n t h o s e age g r o u p s n o r m a l l y r e s i d e n t i n t h e  nursery age  lakes.  suggested  a n d l a t e r become  that light activity  The e x p e r i m e n t s  on t h e e n t r a i n e d r e s p o n s e ,  examining  was  the effect of  a n d t h e two y e a r a n a l y s i s  of r e l a t e d data i n d i c a t e d the general a p p l i c a b i l i t y of a l i g h t a c t i v e p a t t e r n t o t h i s age g r o u p . E i t h e r s i n g l e i n d i v i d u a l s o r g r o u p s c o u l d be e n t r a i n e d t o t h e same e n v i r o n m e n t a l  cycle.  This  provided  a m e c h a n i s m e n a b l i n g i n d i v i d u a l s t o become c o r r e s p o n d i n g l y a c t i v e and i n a c t i v e a t a p p r o x i m a t e l y  t h e same t i m e s o f d a y .  T h i s m e c h a n i s m m i g h t be v a l u a b l e i n m a i n t a i n i n g t h e cohesiveness accuracy The  o f a s c h o o l a n d may a l s o p r o v i d e  i n t i m i n g the onset of d a i l y  activity.  i n v e s t i g a t i o n a t B a b i n e L a k e , B.C. w i t h " s e a  s c a n a r " echo soundings i n d i c a t e s a r e l a t i v e and  absence o f dense s c h o o l s a t n i g h t  Marshall  greater  (1965) a n d D e n t o n a n d N i c o l  inactivity  (Narver,  unpublished).  (1966) e m p h a s i z e d t h e  97  r o l e of v i s i o n i n schooling species, but i n d i c a t e d that o t h e r sensory systems were undoubtedly i n v o l v e d . i s a p p a r e n t l y a v i s u a l response i n t h i s s p e c i e s  Schooling (Hoar, 1958;  A l i , 1959), a f a c t o r s u g g e s t i n g t h e g e n e r a l importance o f light  activity. 4.43  Feeding.  The importance o f v i s i o n i n t h e  l o c a t i o n and c a p t u r e o f food i s a c h a r a c t e r i s t i c o f Pacific 1963).  salmon (Hoar, 1958; A l i , 1959; B r e t t and G r o o t , F i e l d observations i n d i c a t e d that feeding occurred  d u r i n g t h e day i n upstream m i g r a n t Babine l a k e p r e - s m o l t s  f r y (McCart, 1967) and  (Narver, p e r s . comm.).  The Babine  lake i n v e s t i g a t i o n i n d i c a t e d that feeding a c t i v i t y  occurred  when t h e f i s h were 150 t o 200 f e e t deep, however, some f e e d i n g a l s o o c c u r r e d a t dawn and dusk b e f o r e and a f t e r the d i e l v e r t i c a l m i g r a t i o n ( F o e r s t e r , 1968).  Groot  (1965) demonstrates dawn and dusk peaks i n s u r f a c e f e e d i n g a c t i v i t y f o r m i g r a t i n g smolts  ( a t 14°C avg. t e m p e r a t u r e ) .  The h i g h l e v e l s o f a c t i v i t y d u r i n g t h e day, coupled w i t h i n c r e a s e d v i s u a l a c u i t y might f a c i l i t a t e t h e l o c a t i o n and c a p t u r e o f p r e y .  The f e e d i n g response i s a p p a r e n t l y  e s t a b l i s h e d a t an e a r l y age a f t e r c l o s u r e o f t h e y o l k s a c , when t h e f i s h have developed C e r t a i n combinations  a light active pattern.  o f temperature and p h o t o p e r i o d  may  determine a b i m o d a l p a t t e r n o f f e e d i n g a c t i v i t y i n t h e summer, and a unimodal p a t t e r n o f f e e d i n g a c t i v i t y i n t h e winter.  98  4.44  Diel v e r t i c a l migrations.  The v e r t i c a l  movements o f t h i s s p e c i e s h a v e b e e n w e l l d o c u m e n t e d (Johnson, 1956, 1961; B u r g n e r , 1 9 6 2 ) , however t h e p a t t e r n s o f movement a n d d i s t r i b u t i o n a r e n o t i d e n t i c a l i n a l l sockeye l a k e s .  I n B a b i n e L a k e , B.C. t h e m i g r a t i o n  c a n be d i v i d e d i n t o f o u r p h a s e s 1. 150  (Narver, pers.  Dawn, v e r t i c a l d e s c e n t t o d e p t h s  pattern  comm.).  approximating  t o 200 f e e t . 2.  Midday, h o l d i n g a t depth.  3.  Dusk, v e r t i c a l a s c e n t  activity  with a short period of  at the surface.  4.  N i g h t , d e s c e n t t o 10 t o 25 f e e t a n d r e l a t i v e  inactivity. The  responses obtained  i n the laboratory  parallel  the v a r i o u s a c t i v i t i e s observed i n the f i e l d .  I t appeared  t h a t l i g h t a c t i v i t y was a s s o c i a t e d w i t h f e e d i n g , and  schooling  d i e l v e r t i c a l m i g r a t i o n , w h i l e dark i n a c t i v i t y  was  a s s o c i a t e d w i t h the h o l d i n g response i n the l a k e , the c e s s a t i o n o f f e e d i n g , and t h e n o c t u r n a l d i s p e r s a l o f individuals  ( i . e . t h e breakdown o f s c h o o l s ) .  The a v a i l -  a b i l i t y o f j u v e n i l e sockeye salmon t o s u r f a c e techniques support  at night  (Burgner,  sampling  1962) p r o v i d e d a d d i t i o n a l  f o r t h e i r presence i n t h i s l a y e r .  H o w e v e r , my  i n v e s t i g a t i o n s u g g e s t e d t h a t i n d i v i d u a l s were i n a c t i v e a t night while present  relatively  a t o r near t h e surface.  99  4.5  Seasonal Regulation of the Entrained  Response  This investigation indicated that short photoperiods and l o w t e m p e r a t u r e s activity  pattern.  (16L 8D,  15°C)  (8L 16D,  5°C)  resulted  in a  unimodal  L o n g p h o t o p e r i o d s and h i g h t e m p e r a t u r e s  r e s u l t e d i n a bimodal pattern with  amplitudes of a c t i v i t y  during both l i g h t  and  increased  dark.  Combining s h o r t photoperiods w i t h h i g h temperatures (8L  16D,  15°C)  r e s u l t e d i n an a p p a r e n t r e v e r s a l o f t h e  photobehavioural response. A t L a k e l s e L a k e , B.C. 15th  to A p r i l  1 5 t h a p p r o x i m a t e 3.5°C b e t w e e n 20 f e e t  80 f e e t d e p t h s .  May  and J u n e a r e c h a r a c t e r i z e d by  r a p i d temperature increase forty feet.  t h e t e m p e r a t u r e s f r o m December  (10°C  t o 15°C)  and a  i n the upper  The w a t e r t e m p e r a t u r e s d r o p t o t h e w i n t e r  l e v e l s f r o m S e p t e m b e r t o November  ( B r e t t , 1950).  The  l a b o r a t o r y t e m p e r a t u r e s approximated t h e range measured at  L a k e l s e Lake, a f a c t o r suggesting the g e n e r a l a p p l i c -  ability  of t h i s  study to f i e l d  conditions.  Assuming  that  the  l a b o r a t o r y study approximated the general p a t t e r n i n  the  f i e l d , we  activity  can s p e c u l a t e upon t h e f o l l o w i n g s e a s o n a l  response.  Winter:  S h o r t p h o t o p e r i o d s and l o w t e m p e r a t u r e s i n  m o s t s o c k e y e l a k e s may  r e s u l t i n a unimodal a c t i v i t y  L i g h t i n t e n s i t y c a n be f u r t h e r d e c r e a s e d by i c e and cover.  A unimodal a c t i v i t y  sockeye t o u t i l i z e  p a t t e r n would enable  pattern. snow  resident  a l la v a i l a b l e d a y l i g h t f o r the capture  100  of f o o d .  Growth and energy e x p e n d i t u r e  lowest annual l e v e l , responding and d e c r e a s e d food Spring:  are a t t h e i r  t o t h e low t e m p e r a t u r e s  supply.  Increasing photoperiod  and temperature  might r e s u l t i n h i g h e r l e v e l s and i n c r e a s e d d u r a t i o n s o f activity.  I c e breakup precedes t h e smolt m i g r a t i o n s t o  a marine environment.  Foerster  (1937) p r o v i d e d a  s i g n i f i c a n t c o r r e l a t i o n between temperature c o n d i t i o n s and seaward m i g r a t i o n s from C u l t u s Lake, B.C.  The  i n c r e a s e d n o c t u r n a l a c t i v i t y may be a response t o t h e r a p i d t e m p e r a t u r e i n c r e a s e s i n s p r i n g and c o u l d  impart  a c h a r a c t e r i s t i c dark a c t i v e p a t t e r n o f movement t o many smolt m i g r a t i o n s .  Further increases i n photoperiod  c o u l d a c t t o s t a b i l i z e t h e day a c t i v e r e l a t i o n s h i p o f t h e a c t i v i t y p a t t e r n , thus b r i n g i n g a c e s s a t i o n t o t h e outmigration. Summer:  The response t o l o n g p h o t o p e r i o d s  temperatures i n d i c a t e s t h a t the a c t i v i t y p a t t e r n  and h i g h expressed  by new r e s i d e n t s and r e s i d u a l s might be day a c t i v e , and b i m o d a l , w i t h a c t i v i t y peaks o c c u r r i n g a t dawn and a t dusk. Fall:  Decreasing  photoperiod  and temperature c o u l d  regulated a r e v e r s a l of the process, r e s u l t i n g i n the e x p r e s s i o n o f a unimodal a c t i v i t y p a t t e r n d u r i n g t h e w i n t e r . To s t a t e t h a t t h e organism i s a complete s u b j e c t o f i t s environment i s an o v e r s i m p l i f i c a t i o n o f t h e problem. E n t r a i n m e n t i s a p p a r e n t l y t h e response t o an endogenous  101  p a t t e r n of p h y s i o l o g i c a l e x c i t a b i l i t y t h a t i s r e f l e c t e d by i n c r e a s e s and decreases i n the locomotor The  activity.  e n t r a i n e d response i s a s y n c h r o n i z a t i o n of  this  endogenous p a t t e r n with c e r t a i n environmental cues a c t i n g as s t i m u l i f o r the onset and cycle  t e r m i n a t i o n of the  (Aschoff, e t a l . , 1965).  activity  102  5. 1.  SUMMARY AND CONCLUSIONS  The o b j e c t i v e o f t h i s  s t u d y was t o p r o v i d e a  d e s c r i p t i o n and a n a l y s i s o f t h e endogenous and exogenous f a c t o r s c o n t r i b u t i n g t o a d a i l y pattern of locomotor activity 2.  i n j u v e n i l e sockeye  salmon.  The b a s i c m e a s u r e e m p l o y e d t h r o u g h o u t t h e i n v e s t i g a t i o n  was s p o n t a n e o u s  locomotor a c t i v i t y .  High frequency  was u s e d t o c o n t i n u o u s l y m o n i t o r a c t i v i t y d i a m e t e r swimming c h a n n e l s .  sound  i n 36" (91.4 cm)  The a p p a r a t u s p r o v i d e d a  m o n i t o r i n g system w i t h o u t any a p p a r e n t d i s t u r b a n c e t o e i t h e r t h e c o n t r o l l e d environment  or the experimental  subjects. 3.  The i n f l u e n c e o f p h o t o p e r i o d a n d t e m p e r a t u r e o n t h e  entrained a c t i v i t y artificial  p a t t e r n was i n v e s t i g a t e d .  p h o t o p e r i o d f u n c t i o n e d as t h e p r i m a r y e n t r a i n i n g  agent f o r s i n g l e i n d i v i d u a l s o r groups o f f i s h . activity  The  The  r e c o r d s i n d i c a t e d t h a t sockeye f r y were d a r k  i m m e d i a t e l y a f t e r emergence.  A light  active  a c t i v e p a t t e r n was  d e v e l o p e d two weeks a f t e r e m e r g e n c e a n d was m a i n t a i n e d f o r at 4.  l e a s t one y e a r . B o t h p h o t o p e r i o d a n d t e m p e r a t u r e i n f l u e n c e d t h e 24 h o u r  activity  pattern i n yearling fish.  facilitated  light activity  high temperatures  Low t e m p e r a t u r e s  (5°C)  a t a l l p h o t o p e r i o d s , whereas  (15°C) c o m b i n e d w i t h s h o r t p h o t o p e r i o d s  (8L 16D) i n c r e a s e d d a r k  activity.  103  5.  The  f r y was  d a i l y e n t r a i n e d a c t i v i t y p a t t e r n of bimodal  responded  i m m e d i a t e l y a f t e r emergence.  t o low temperatures  a unimodal  T o t a l a c t i v i t y was  used.  The  active pattern at The  i n a bimodal  activity  (16L 8D)  interaction  facilitated a  The  intensity.  entraining 8.  The  pattern. temperatures  indicated light  15°C. examined i n a c o n s t a n t  p e r i o d v a l u e was  23.30 h o u r s  c o n s t a n t l i g h t , b u t c o u l d be l e n g t h e n e d by light  temperature  i n c r e a s e d a t the h i g h e r  e n d o g e n o u s r h y t h m was  environment.  Yearlings  short photoperiods with  temperature-photoperiod  that longer photoperiods  7.  and  a c t i v i t y p a t t e r n , but i n c r e a s i n g  and/or p h o t o p e r i o d r e s u l t e d 6.  sockeye  in  increasing  P e r i o d i c f e e d i n g c o u l d f u n c t i o n as  an  agent.  eyes were i d e n t i f i e d  as t h e p r i m a r y  sensory  r e c e p t o r s m e d i a t i n g i n f o r m a t i o n about the environmental ' photoperiod.  T r a n s f e r o f i n f o r m a t i o n was  o p t i c nerve.  This suggested  a f f e r e n t nervous by a b l o o d - b o r n 9.  The  not v i a the  the p o s s i b i l i t y  of other  pathways from the r e t i n a , o r m e d i a t i o n agent.  p i n e a l b o d y had  no e n t r a i n i n g r o l e i n t h i s s p e c i e s .  H o w e v e r , b o t h p i n e a l e c t o m y and  inserting  p i n e a l region increased the t o t a l d a i l y  s h i e l d s over activity.  the  104  10.  I n t r a p e r i t o n e a l melatonin i n j e c t i o n s decreased  a c t i v i t y d u r i n g l i g h t , whereas s i m i l a r i n j e c t i o n s of s e r o t o n i n i n c r e a s e d a c t i v i t y d u r i n g dark. supported the hypothesis i n other  based upon an apparent c o r r e l a t i o n  s p e c i e s t h a t melatonin and s e r o t o n i n may be  i n v o l v e d i n the r e g u l a t i o n of a c t i v i t y 11.  levels.  The l a b o r a t o r y f i n d i n g s were d i s c u s s e d  to p u b l i s h e d 12.  T h i s experiment  f i e l d observations  in relation  on j u v e n i l e salmonids.  The f u n c t i o n a l s i g n i f i c a n c e of the a c t i v i t y c y c l e  was d i s c u s s e d  i n r e l a t i o n to migration, o r i e n t a t i o n ,  f e e d i n g , and s c h o o l i n g behaviour.  105  6. Ali,  LITERATURE  CITED  M.A. 1 9 5 9 . The o c u l a r s t r u c t u r e , r e t i n o m o t o r a n d photobehavioral responses of j u v e n i l e P a c i f i c s a l m o n . C a n . J . Z o o l . , 3 7 : 965-996.  A n d r e w , L . J . , a n d G.H. Geen. 1 9 6 0 . S o c k e y e a n d p i n k s a l m o n p r o d u c t i o n i n r e l a t i o n t o p r o p o s e d dams i n t h e F r a s e r R i v e r System. I n t e r n . P a c . Salmon F i s h . Comm., B u l l . , No.11. 259p. A s c h o f f , J . 1960. Exogenous and endogenous components i n c i r c a d i a n r h y t h m s . C o l d S p r i n g s H a r b o r Symp. Quant. B i o l . , 25: 11-27. 1965a. Response c u r v e s i n c i r c a d i a n p e r i o d i c i t y , I n : C i r c a d i a n C l o c k s (ed. J . 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DeGroot. 1967. D i u r n a l a c t i v i t y p a t t e r n o f p l a i c e and f l o u n d e r (Pleuronectidae) in aquaria. N e t h . J . S e a . R e s . , 3: 383-390. W o d i n s k y , J . , a n d W.N. T a v o l g a . 1 9 6 3 . Sound d e t e c t i o n in teleost fishes. I n : Marine B i o a c o u s t i c s . (Ed. W.N. T a v o l g a ) , p . 2 6 9 - 2 8 0 . New Y o r k : Pergamon P r e s s . Wong, R., a n d C.B.C. W h i t e s i d e . 1 9 6 8 . The e f f e c t o f m e l a t o n i n on t h e w h e e l - r u n n i n g a c t i v i t y o f r a t s - deprived of food. J . Endocrinol., 40: 383-384. Wurtman, R . J . , J . A x e l r o d , a n d L . S . P h i l l i p s . 1 9 6 3 . Melatonin synthesis i n the p i n e a l gland: c o n t r o l by l i g h t . S c i e n c e , 142: 1071-1073.  APPENDIX  114  F i g u r e 1.  A continuous 24 hour r e c o r d of room temperature and water  temperature  i n the swimming channels d u r i n g March, 1967.  Temperature (°C) Cl  _l  (>• 1  N 1  :  "O 1-  115  F i g u r e 2.  C i r c u i t diagram o f 600 w a t t lamp controller. Key: RI - 470K l/2w  C2 - 1000/25v  R2 - 1. 5M  C3 - 0.1/lOOv  R3 - 1. 0M  Dl,D2,D3 - IN681  R4 - 2. 2K R5 -  "  68K  D4 - Z4XL20 D5,D6,D7,D8 - IN3571  R6 - 220  Ql - 2N2925  R7 -  Q2 - 2N2646  47  R8 - 4700 C l - l/25v  2w  SCR1 - C20C T l - 12.6v Hammond 166F12  U n l e s s o t h e r w i s e n o t e d , a l l cap. i n mf d. , r e s i s t o r s i n ohms l/2w and 2w, 10%  R8  load  line LAMP CONTROLLER  116  F i g u r e 3.  P l o t of l i g h t i n t e n s i t y a g a i n s t time, i n d i c a t i n g the d u r a t i o n of the  twilight  p e r i o d r e g u l a t e d by the lamp c o n t r o l l e r .  gures 4 - 8 .  C i r c u i t diagrams o f t h e f i s h movement c o u n t e r .  LOGIC & OUTPUT  POWER SUPPLY  SYSTEM BLOCK DIAGRAM  OUT  118  RD I  WH  2 **  BK 3 SHLD 4 - *  AMPLIFIER  U n l e s s otherwise noted, a l l c a p . i n mfd., r e s i s t o r s i n ohms -gw 10%. Front panel a d j . p o t ^ c w r o t a t i o n .  119  LOGIC & OUTPUT DRIVER U n l e s s o t h e r w i s e noted a l l c a p s , i n mfd. A l l r e s i s t o r s -|w 10% L e t t e r s i n d i c a t e c i r c u i t board  terminal  120  -25v A  POWER SUPPLY Unless otherwise noted, c a p . i n mfd. Resistors 10%  2N13U  TYPICAL OPERATE CIRCUIT \ RELAY coto.ACTS  HAMMOND'  167K25  c C  1 2  3  19 20  ¥  ?  ?  i  A  A  ?  2 4 vAG  r v. A  20  5~ i PEN i  TYPICAL RECORDER CIRCUIT  JUNCTION BOX  122  Surgical  techniques.  All in  fish  subjected  a s o l u t i o n o f M.S.  to operations  222  anaesthesia  was  Sulfonate;  The amount u s e d  0.2 gram p e r g a l l o n , w h i c h  a c o n c e n t r a t i o n o f 1:19,230. g e n e r a l l y t o l e r a t e d by t h i s General  anaesthetized  ( T r i c a i n e Methane  K e n t C h e m i c a l C o . , Vancouver., B . C . ) . for  were  anaesthesia  represented  T h i s was w i t h i n t h e r a n g e species  (Bell,  1967).  was assumed when t h e s u b j e c t  c e a s e d movement and f l o a t e d v e n t r a l s i d e u p .  I f no  r e f l e x a c t i o n was o b s e r v e d when t h e t a i l was g r a s p e d  with  a p a i r o f f o r c e p s , t h e f i s h was removed f r o m t h e s o l u t i o n and  p l a c e d on a p o r t a b l e o p e r a t i n g p l a t f o r m .  A glass  t u b e was c o n n e c t e d t o t h e d e c h l o r i n a t e d w a t e r s u p p l y  and  i n s e r t e d i n t o t h e mouth o r o p e r c u l a r chamber t o i r r i g a t e the  gills. A.  Pinealectomy  The p i n e a l o f t h e s e  fish  i s a small structure located  on t h e r o o f o f t h e b r a i n between t h e t e l e n c e p h a l o n optic  lobes  (Fig. 9).  The l o c a t i o n was e a s i l y  through the top of the s k u l l  and t h e  visible  (Hoar, 1 9 5 5 ) , and a f l a p o f  t i s s u e was l a i d b a c k i n t h i s r e g i o n t o e x p o s e t h e c r a n i u m . The s k u l l was p e n e t r a t e d  w i t h a s h a r p g l a s s t u b e and a  c y l i n d e r o f o s s i f i e d and c a r t i l a g i n o u s m a t e r i a l was removed and  discarded.  A f i n e , blunt, hollow  g l a s s tube t h a t  c o n n e c t e d t o a n a s p i r a t o r was t o u c h e d t o t h e t o p o f t h e exposed p i n e a l .  The o r g a n was d r a w n i n t o t h e t u b e a n d  was  123  Figure  9.  Dorsal  view  illustrating  of  the  the  exposed  location  general  appearance  of  body.  Magnification  the  brain, and pineal  6.5X.  124  separation i n v a r i a b l y occurred  a t the base of the s t a l k .  The s i t e was s e a l e d w i t h S q u i b b o r a b a s e medication epidermal  (a d e n t a l  t h a t c o n t a i n s no a n t i b i o t i c s )  and t h e  f l a p was r e p l a c e d i n p o s i t i o n .  The  incision  was t h e n s e a l e d w i t h a n o t h e r l a y e r o f S q u i b b o r a b a s e . Sham o p e r a t i o n s  consisted of f o l l o w i n g s i m i l a r  p r o c e d u r e s f o r removing t h e p i n e a l body, however t h e o r g a n was n o t r e m o v e d . immediately  Penetration of the cranium  p o s t e r i o r to the p i n e a l .  was n e c e s s a r y  t o prevent  was  This modification  damage t o t h e o r g a n .  The  i n c i s i o n was s e a l e d i n a manner i d e n t i c a l t o n o r m a l pinealectomy. The s u b j e c t s w e r e r e v i v e d a n d p l a c e d holding aquaria  i n postoperative  (10 g a l l o n s ) w h i c h w e r e m a i n t a i n e d  controlled photoperiodic  conditions.  under  H e a l i n g was  completed  w i t h i n two weeks and t h e s u b j e c t s w e r e t h e n i n t r o d u c e d t o t h e a c t i v i t y chambers f o r e x p e r i m e n t a t i o n . S u c c e s s o f r e m o v a l was v e r i f i e d by o b s e r v a t i o n s  with  a d i s s e c t i n g m i c r o s c o p e d u r i n g t h e o p e r a t i o n and by histological the extent  s e c t i o n s w h i c h were p r e p a r e d  of pinealectomy.  were p r e s e r v e d  t o demonstrate  A l l m a t e r i a l f o r these  slides  i n Weber's s o l u t i o n a n d s t a i n e d w i t h hema-  t o x y l i n and e o s i n . B.  Plastic  screens  A similar preparatory placement of transparent  p r o c e d u r e was f o l l o w e d f o r t h e  and opaque p l a s t i c  screens  over  125  the r e g i o n of the p i n e a l .  I n t h i s case,  only the skin  t i s s u e was l a i d b a c k a n d t h e p l a s t i c  screen  immediately  The t i s s u e s w e r e  superior to the s k u l l .  then sutured and  allowed C.  inserted  back i n t o p o s i t i o n and t h e f i s h were r e v i v e d t o recover  i n the holding  aquaria.  Blinding  S u r g i c a l techniques  f o r b l i n d i n g were as f o l l o w s .  The f i s h w e r e a n a e s t h e t i z e d  a n d p l a c e d on t h e o p e r a t i n g  p l a t f o r m w i t h t h e v e n t r a l s i d e up.  The l o w e r  j a w was  h e l d o p e n b y a b l u n t hook a n d a s m a l l i n c i s i o n was made in the o r a l epithelium.  T h i s exposed t h e o p t i c nerve  w h i c h was l i f t e d s l i g h t l y  w i t h a f i n e g l a s s hook.  was s e v e r e d  medially with a p a i r of scissors.  was s e v e r e d  again  laterally  procedure described  The n e r v e  and a s e c t i o n e s t i m a t e d  1/16 t o 1/8 i n c h was r e m o v e d .  It  at  R e c o v e r y f o l l o w e d t h e same  above.  A l i m i t e d number o f i n d i v i d u a l s w e r e s u b j e c t e d t o bilateral  e n u c l e a t i o n of the eyes.  A l l f l u i d was removed  from t h e p o s t e r i o r chamber, t h e m u s c u l a t u r e and t h e o p t i c nerve severed, socket  a n d t h e c o m p l e t e e y e was l i f t e d f r o m t h e  

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