THE EFFECTS OF PHOTOPERIOD AND TEMPERATURE ON THE DAILY PATTERN OF LOCOMOTOR ACTIVITY IN JUVENILE SOCKEYE SALMON, ONCORHYNCHUS NERKA (WALBAUM). by JOHN EDWARD BYRNE B.A., U n i v e r s i t y o f H a w a i i , 1962 M . S c , U n i v e r s i t y o f H a w a i i , 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f ZOOLOGY We a c c e p t t h i s t h e s i s as co n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA December, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I ag r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my wr i t t e n . p e r m i ss i on. John E. Byrne Department of Zoology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 16 December, 1968 i S u p e r v i s o r : N. R. L i l e y ABSTRACT The 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 i e l rhythm of l o c o m o t o r a c t i v i t y i n j u v e n i l e sockeye salmon Oncorhynchus n e r k a (Walbaum) were examined i n t h e l a b o r a t o r y . The b a s i c measure employed i n t h e i n v e s t i g a t i o n was spontaneous locomotor a c t i v i t y . H i g h f r e q u e n c y sound (800 kHz) was used as a m o n i t o r i n g t e c h n i q u e t o c o l l e c t c o n t i n u o u s a c t i v i t y r e c o r d s . Three major a r e a s r e c e i v e d a t t e n t i o n . F i r s t , a d e s c r i p t i o n and a n a l y s i s were p r o v i d e d f o r t h e e n t r a i n e d d i e l a c t i v i t y p a t t e r n under t h r e e d i f f e r e n t t e m p e r a t u r e s (5°, 10°, and 15°C) and 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 (8L 16D, 12L 12D, and 16L 8D). The combined e f f e c t s o f t e m p e r a t u r e and p h o t o p e r i o d upon th e b a s i c 24 hour response were r e c o r d e d and a n a l y z e d . J u v e n i l e sockeye salmon were n o c t u r n a l l y a c t i v e 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 . A d i u r n a l a c t i v i t y p a t t e r n was g r a d u a l l y a c q u i r e d d u r i n g t h e f o l l o w i n g 14 days and was m a i n t a i n e d f o r 12 months. P h o t o p e r i o d was t h e p r i m a r y e n v i r o n m e n t a l s y n c h r o n i z e r f o r e i t h e r d i u r n a l o r n o c t u r n a l a c t i v i t y . The endogenous component o f t h e a c t i v i t y rhythm was 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 . C o n s t a n t l i g h t (34.4 l u x a t 10°C) f a c i l i t a t e d t h e f r e e - r u n n i n g response w h i l e c o n s t a n t dark i n h i b i t e d i t . The spontaneous 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 23.30 hours b u t t h i s c o u l d be a l t e r e d by l i g h t i n t e n s i t y o r p e r i o d i c f e e d i n g . The f i n a l e x p e r i m e n t s f o c u s e d upon t h e r e l a t i o n s h i p b etween t h e e n v i r o n m e n t a l s t i m u l u s ( p h o t o p e r i o d ) and t h e p h y s i o l o g i c a l 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 e y e s were t h e p r i m a r y p h o t o 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 a b o u t t h e l i g h t - d a r k e n v i r o n m e n t . The e n t r a i n e d a c t i v i t y r e s p o n s e d i s a p p e a r e d when t h e r e t i n a was n o t i l l u m i n a t e d . When t h e p i n e a l body was removed o r s h a d e d , j u v e n i l e s o c k e y e r e s p o n d e d w i t h i n c r e a s e d a c t i v i t y . 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 o f m e l a t o n i n ( N - a c e t y l - 5 - h y d r o x y - t r y p t a m i n e ) o r s e r o t o n i n ( 5 - h y d r o x y - t r y p t a m i n e ) s e l e c t i v e l y a l t e r e d t h e a c t i v i t y a m p l i t u d e i n e i t h e r t h e l i g h t o r d a r k r e s p e c t i v e l y . J u v e n i l e s o c k e y e s a l m o n p o s s e s s an endogenous c i r c a d i a n a c t i v i t y r h y t h m w h i c h i s s y n c h r o n i z e d by t h e p h o t o p e r i o d i c c y c l e . The f i s h a r e g e n e r a l l y l i g h t a c t i v e , e x c e p t f o r t h e p e r i o d i m m e d i a t e l y a f t e r emergence. However, i n t e r a c t i o n s b etween d a y l e n g t h and t e m p e r a t u r e c a n r e s u l t i n t e m p o r a r y d a r k a c t i v e r e s p o n s e s . M e d i a t i o n o f t h e p h o t o p e r i o d i c i n f o r m a t i o n o c c u r s v i a t h e r e t i n a , b u t w i t h o u t t r a n s m i s s i o n by o p t i c n e r v e p a t h w a y s . C h e m i c a l a g e n t s ( m e l a t o n i n and s e r o t o n i n ) p r o d u c e d by t h e r e t i n a a n d / o r p i n e a l m i g h t c o n t r o l t h e a c t i v i t y a m p l i t u d e i n l i g h t and d a r k , t h u s r e s u l t i n g i n t h e c h a r a c t e r i s t i c e n t r a i n e d p a t t e r n . ERRATA Bunning s h o u l d r e a d Blinning D a n i e l e v s k i i s h o u l d r e a d D a n i l e v s k i i p. 36, l i n e 15, s i g n i f i c a n t p. 87, l i n e 23, l o c a t i o n s p. 105, l i n e 22, Baggerman, B. 19 57 p. 106, l i n e 24, p r i n c i p l e s p. 10 7, l i n e 1, rhythms p. 112, l i n e 30, d i v i s i o n i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS. x 1. INTRODUCTION 1 2. MATERIALS AND METHODS 6 2.1 WATER SUPPLY 6 2.2 ACTIVITY CHAMBERS 7 2.3 TEMPERATURE CONTROLS 9 2.4 PHOTOPERIOD CONTROLS 10 2.5 ACTIVITY MONITORING SYSTEM 11 2.6 LIVE MATERIAL 18 2.7 EXPERIMENTAL DESIGN 20 2.8 ANALYTICAL PROCEDURE 22 3. RESULTS 24 3.1 DETERMINATION OF A BASIC ENTRAINED RESPONSE. 24 3.11 The 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 28 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 C y c l e a t a Co n s t a n t Temperature 29 3.2 THE EFFECTS OF TEMPERATURE AND PHOTOPERIOD ON THE DAILY ACTIVITY PATTERN OF JUVENILE SOCKEYE SALMON 33 i v Page 3.21 The E n t r a i n e d Response a t 5°C t o 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, 16L 8D) 33 3.22 The E n t r a i n e d Response a t 10°C t o 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, 16L 8D) 36 3.23 The E n t r a i n e d Response a t 15°C t o 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, 16L 8D) 38 3.24 The E f f e c t o f Temperature on the 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 4 0 3.26 The I n t e r a c t i o n o f Temperature and P h o t o p e r i o d 44 3.3 ENDOGENOUS ACTIVITY EXPRESSED IN A CONSTANT ENVIRONMENT 49 3.31 The Endogenous Response i n C o n s t a n t Darkness (DD) a t a C o n s t a n t Temperature o f 10°C 50 3.32 The Endogenous Response 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 Temperature o f 10°C 54 3.33 The Response o f t h e Endogenous Component t o 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 ; 58 3.34 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 C o n s t a n t L i g h t a t 10°C 58 3.4 THE PRINCIPAL SENSORY RECEPTORS INVOLVED IN THE MEDIATION OF THE ENTRAINED RESPONSE. 6 0 3.41 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 F i s h 62 V Page 3.42 The Endogenous Response i n P i n e a l e c t o m i z e d F i s h 66 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 o v e r t h e s i t e o f t h e P i n e a l . 66 3.44 The E n t r a i n e d Response i n B l i n d e d F i s h 69 3.5 THE EFFECTS OF MELATONIN, SEROTONIN AND TELEOST SALINE ON THE ENTRAINED RESPONSE 7 9 4. DISCUSSION ; 83 4.1 THE BASIC ACTIVITY PATTERN 84 4.11 The I n f l u e n c e o f Temperature and P h o t o p e r i o d 85 4.2 THE ENDOGENOUS ACTIVITY RESPONSE 88 4.3 MECHANISMS OF ENTRAINMENT 8 9 4.4 THE ADAPTIVE SIGNIFICANCE OF A DAILY ACTIVITY 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 F e e d i n g 97 4.44 D i e l V e r t i c a l M i g r a t i o n s 98 4.5 SEASONAL REGULATION OF THE ENTRAINED RESPONSE '99 5. SUMMARY AND CONCLUSIONS 102 6. LITERATURE CITED. 105 APPENDIX - C i r c u i t Diagrams and S u r g i c a l T e c h n i q u e s . . . 114 v i LIST OF TABLES T a b l e Page I A comparison o f a c t i v i t y 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 a f t e r i n s t r u m e n t o p e r a t i o n 17 I I 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 17 I I I Summary o f te m p e r a t u r e and p h o t o p e r i o d c o n d i t i o n s 21 IV Summary o f 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 21 V P e r i o d i c i t y e x p r e s s e d i n c o n s t a n t d a r k n e s s a t 10°C 53 VI P e r i o d i c i t y e x p r e s s e d i n c o n s t a n t l i g h t a t 10°C 57 V I I P e r i o d i c i t y e x p r e s s e d w i t h 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 a t 10°C 59 v i i LIST OF FIGURES Figure Page 1 Top and side views of a c t i v i t y chambers 8 2 Sonic transducer and components 13 3 Block diagram of monitoring instruments and c i r c u i t r y 15 4 The entrained response of one i n d i v i d u a l at 8L 16D, 5°C 26 5 The mean d a i l y a c t i v i t y patterns of j u v e n i l e Lakelse and Kamchatka sockeye 26 6 The mean d a i l y a c t i v i t y patterns at d i f f e r e n t stages of development 3 0 7 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 of f i v e i n d i v i d u a l s recorded simultaneously 32 8 The mean d a i l y a c t i v i t y patterns f o r three photoperiods at 5°C 34 9 The mean d a i l y a c t i v i t y patterns f o r three photoperiods at 10°C 37 10 The mean d a i l y a c t i v i t y patterns f o r three photoperiods at 15°C 39 11 The mean d a i l y a c t i v i t y patterns at 12L 12D f o r 5°C, 10°C, and 15°C 41 v i i i F i gure Page 12 P l o t s of a c t i v i t y and temperature at three d i f f e r e n t photoperiods 42 13 P l o t s of a c t i v i t y and photoperiod at three d i f f e r e n t temperatures 43 14 Three dimensional i l l u s t r a t i o n of the mean t o t a l a c t i v i t y at each temperature and photoperiod 45 15 Mean d a i l y a c t i v i t y patterns f o r two i n d i v i d u a l s at 8L 16D, 15°C 46 16 Percent occurrence of noctu r n a l a c t i v i t y at 5°C, 10°C, and 1 5 ° C .. 48 17 Percent a c t i v i t y d uring l i g h t and dark at three photoperiods 48 18 The endogenous response i n constant dark at 10°C 51 19 Periodogram a n a l y s i s of one i n d i v i d u a l i n constant dark ;. 52 20 A continuous 12 day record of one i n d i v i d u a l i n constant l i g h t a t 10°C 55 21 Periodogram a n a l y s i s of the data presented i n Figure 20 56 22 The mean a c t i v i t y p a t t e r n i n constant l i g h t to p e r i o d i c feeding 61 i x F i g u r e Page 23 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 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 f i s h 65 25 P e r iodogram a n a l y s i s o f 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 i n c o n s t a n t l i g h t . . 67 26 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 27 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 , and b i l a t e r a l l y e n u c l e a t e d groups 73 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 76 29 12L 12D, 5°C response i n normal 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 3 0 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 31 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 by t h e F i s h e r i e s R e s e a r c h Board o f Canada t h r o u g h g r a n t s made t o Dr. N.R. L i l e y and th e Department o f Zoo l o g y , U n i v e r s i t y o f B r i t i s h C olumbia. I o f f e r my s i n c e r e thanks t o Dr. C. Groot f o r t h e i n i t i a l 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 the Nanaimo B i o l o g i c a l S t a t i o n . Dr. J.R. B r e t t p r o v i d e d many h e l p f u l comments on t h e work and 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 work under t h e gui d a n c e o f Dr. N.R. L i l e y , my major 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 s t u d y . Drs. W.S. Hoar, N.J. W i l i m o v s k y , N.R. L i l e y > T.G. N o r t h c o t e and J.R. B r e t t c r i t i c a l l y r e a d t h e t h e s i s and 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 . Dr. H.C. Nordan p r o v i d e d l a b o r a t o r y space on 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 l a b o r a t o r y environment. P e r s o n a l funds were 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 from t h e F i s h e r i e s R esearch Board 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 a s s i s t a n c e i n c o u n t i n g t h e " e n d l e s s m i l e s " o f event r e c o r d e r c h a r t s 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 Zoology 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. INTRODUCTION The p r i m a r y o b j e c t i v e o f t h i s t h e s i s i s t o p r o v i d e an 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 t o t h e e x p r e s s i o n o f 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 sockeye salmon, Oncorhynctius n e r k a (Walbaum). The term " j u v e n i l e " r e f e r s t o t h e i n t e r v a l i n t h e l i f e c y c l e w hich t h e s e f i s h spend as r e s i d e n t s i n f r e s h -w a ter l a k e s b e f o r e m i g r a t i n g t o a marine environment. The d u r a t i o n o f t h i s p e r i o d i s u s u a l l y one, f r e q u e n t l y two, and o c c a s i o n a l l y 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 l a k e s o f t h e i r g e o g r a p h i c a l d i s t r i b u t i o n the l o n g e r terms of r e s i d e n c e a r e more common ( F o e r s t e r , 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 a r e u b i q u i t o u s i n t h e a n i m a l kingdom, and can 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 p r o c e s s e s . The d i s p l a y o f p e r i o d i c phenomena a t b o t h the c e l l u l a r 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 o f 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 i n r e c e n t y e a r s ( r e v i e w s : Cloudsley-Thompson, 1960, 1961; D a n i e l e v s k i i , 1965; H a r k e r , 1964; F a r n e r , 1965; Bunning, 1967). C i r c a d i a n and l u n a r 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, 1965), and a t the s u b - c e l l u l a r l e v e l t h e r h y t h m i c p r o d u c t i o n o f n u c l e i c a c i d s has r e c e i v e d l i m i t e d a t t e n t i o n ( E l i n g , 1967; J e r u s a l e m , 1967). 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 o r g a n i s m l e v e l i n a number o f s p e c i e s r a n g i n g from t h e p r o t o z o a t o t h e h i g h e r v e r t e b r a t e s . The u n i c e l l u l a r d i n o f l a g e l l a t e Gonyaulax p o l y e d r a e x h i b i t s a d i e l rhythm i n b i o l u m i n e s c e n c e and c e l l d i v i s i o n (Sweeney and H a s t i n g s , 1957, 1958), 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 , r o d e n t s and man have been demonstrated 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 a r e s y n c h r o n i z e d by s p e c i f i c e n v i r o n m e n t a l s t i m u l i (DeCoursey, 1960; Hauty and Adams, 1965; Menaker, 1965; Menaker and E s k i n , 1967). 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 c o n s i d e r -a t i o n i n t h o s e 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 t h e f i e l d s o f o r i e n t a t i o n and n a v i g a t i o n . Here, an endogenous r h y t h m i c i w i t h a c h a r a c t e r i s t i c 24 hour p e r i o d i s suggested t o p l a y an i m p o r t a n t r o l e i n t h e d i r e c t i o n f i n d i n g o f a v a r i e t y o f o r g a n i s m s , i n c l u d i n g f i s h (Schwassmann, 196 0; Schwassmann and Braemar, 1961; G r o o t , 1965; H a s l e r , 1967) . A s u r v e y o f t h e l i t e r a t u r e r e v e a l s t h a t o n l y l i m i t e d a t t e n t i o n has been f o c u s e d upon p h o t o p e r i o d i c mechanisms i n t e l e o s t s . Most e f f o r t s have c o n s i d e r e d t h e a d a p t i v e s i g n i f i c a n c e o f t h e r e s p o n s e t o d a y l e n g t h and t e m p e r a t u r e , and 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 m i g r a t i o n s o r t h e c o n t r o l o f r e p r o d u c t i v e b e h a v i o u r ( r e v i e w s : Baggerman, 1957, 1959, 1960a, b; F a r n e r , 1961a, 1965; H a r r i n g t o n , 1959; Hoar, 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 ( N o r t h c o t e , 1962; Blahm, 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; G r o o t , 1965; T h i n e s , e t a l . , 1965; Bohun and Winn, 1966; L i c h t e n h e l d , 1967; V e r h e i j e n and DeGroot, 1967; C h a s t o n , 1968). The e v i d e n c e a v a i l a b l e t o d a t e s u g g e s t s t h a t a r h y t h m i c d i e l a c t i v i t y p a t t e r n i s e x p r e s s e d by sockeye 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 c y c l e (Hoar, 1958; Johnson, 1961; G r o o t , 1965). In t h i s i n v e s t i g a t i o n t h e l o c o m o t o r a c t i v i t i e s o f j u v e n i l e sockeye 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 former 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 o r o t h e r a q u a t i c o r g a n i s m s . 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. S u p p o r t i n g e l e c t r o n i c i n s t r u m e n t s t r a n s f o r m e d t h e o u t p u t from t h e s e u n i t s t o an e l e c t r i c a l i m p u l s e 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 f i r s t e x p e r i m e n t s 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 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 t e m p e r a t u r e s . The d e s i g n o f t h e e x p e r i m e n t s e n a b l e d t h e i n v e s t i g a t o r t o r e c o r d and a n a l y z e t h e combined e f f e c t s o f t e m p e r a t u r e and p h o t o p e r i o d upon t h e . b a s i c t w e n t y - f o u r hour c y c l e . The second phase f o c u s e s upon an a n a l y s i s o f the endogenous component, 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 o f 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 c o n d i t i o n s . 4 T h i s i s c o n s i d e r e d . n e c e s s a r y s i n c e i t i s t h i s component whi c h i s e n t r a i n e d by the e n v i r o n m e n t a l s t i m u l i . E n t r a i n -ment r e s u l t s i n the c h a r a c t e r i s t i c d i e l p e r i o d i c i t y a p p a r e n t under normal o s c i l l a t i n g e n v i r o n m e n t a l c o n d i t i o n s . The t h i r d and f i n a l phase 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 between th 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 p h y s i o l o g i c a l 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 most s t r i k i n g changes i n motor 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 t h e o n s e t and t e r m i n a t i o n o f l i g h t . Emphasis i s f o c u s e d upon t h e r o l e o f t h e eyes and/or p i n e a l body 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 . B oth 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 were u t i l i z e d i n t h i s phase o f t h e i n v e s t i g a t i o n . The 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 t o t h e problems a t hand. " I f any attempt 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 o f f i s h t h r o u g h a m a n i p u l a t i o n o f 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 t h e b e h a v i o u r . T h i s i s p a r t i c u l a r l y t r u e w i t h r e s p e c t t o m i g r a t o r y phenomena whi 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 changes i n t h e e n d o c r i n e g l a n d s . I t may o r may not 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 o v e r m i g r a t o r y 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 something 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 t h e sequence o f b e h a v i o u r . A s t u d y of t h e p h y s i o l o g i c a l changes wh i c h 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 can 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 o f 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 water c u r r e n t s demands such i n f o r m a t i o n " (Hoar, 1958) . In summary, the main purpose of t h i s investigation i s to follow a course of basic research concerning the ef f e c t s of photoperiod and temperature on the locomotor a c t i v i t y of juvenile sockeye salmon. The data presented are applicable to a variety of problems which.consider or are dependent upon the d a i l y a c t i v i t y cycles expressed by te l e o s t s . 6 2. MATERIALS AND METHODS The i n v e s t i g a t i o n was conducted 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 . The l a b o r a t o r y was l o c a t e d i n a c o n c r e t e basement a r e a o f an i s o l a t e d b u i l d i n g , where p e r i o d i c d i s t u r b a n c e s c o u l d be k e p t a t a minimum. The 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 c o n s t a n t n o i s e l e v e l , c o n f u s i n g any i n c i d e n t a l p r o d u c t i o n . o f sound i n o t h e r p a r t s o f t h e b u i l d i n g . I t i s r e c o g n i z e d t h a t c e r t a i n p e r v a s i v e e n v i r o n m e n t a l f l u c t u a t i o n s w h i c h may have th 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 r e v i e w o f such 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 e l e c t r o m a g n e t i c f i e l d s , cosmic r a y bombardment, changes i n the l o c a l b a r o m e t r i c p r e s s u r e and t h e i r e f f e c t s on c y c l i c a c t i v i t y , see Brown, 1960, 1965). A t the p r e s e n t t i m e , t h e a u t h o r 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 empt t h e c o n t r o l o f t h e s e s u b t l e f a c t o r s . 2.1 Water Supply C h l o r i n a t e d c i t y w a t e r was used f o r g e n e r a l maintenance a c t i v i t i e s and as a s u p p l y f o r t e m p e r a t u r e r e g u l a t e d w a t e r b a t h s . A system was d e s i g n e d t o d e c h l o r i n a t e , p u r i f y and a d j u s t t h e pH o f t h e c i t y w a t e r . T h i s d e c h l o r i n a t o r was m a i n t a i n e d a t f u l l w a ter l e v e l by i n s t a l l i n g a c o n t r o l v a l v e d e l i v e r i n g a r e g u l a t e d w a t e r f l o w . The u n i t 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 p r e s s u r e f o r a g r a v i t y f l o w system. The d e l i v e r y system t o t h e a q u a r i a and a c t i v i t y chambers was c o n s t r u c t e d o f 1/2" (1.3 cm) i n s i d e d i a m e t e r p o l y e t h y l e n e p i p e . No m e t a l f i t t i n g s were i n c o n t a c t w i t h t h e wa t e r s u p p l y and t h e complete system 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 te m p e r a t u r e . 2.2 A c t i v i t y Chambers The e x t e r n a l d i m e n s i o n s o f each a c t i v i t y chamber were 48" x 48" x 8" (122 cm x 122 cm x 20.3 cm). The chambers were c o n s t r u c t e d o f 1/2" (1.3 cm) plywood and the 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 , c o a t e d w i t h E s t r e x #101 a l l purpose r e s i n f o r w a t e r p r o o f i n g and added s t r e n g t h . Each chamber c o n t a i n e d a water b a t h and a c i r c u l a r swimming c h a n n e l measuring 7" wide and 7" (17.8 cm x 17.8 cm) deep w i t h a maximum d i a m e t e r o f 36" (91.44 cm) ( F i g . 1 ) . The swimming c h a n n e l was formed 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 were s e a l e d t o t h e bottom o f the a c t i v i t y chamber w i t h f i b e r g l a s s r e s i n . The w a l l s were p a i n t e d w i t h n o n - t o x i c f l a t b l a c k r u s t - o l e u m p a i n t (The Rust-Oleum C o r p o r a t i o n , E v a n s t o n , I l l i n o i s ) t o reduce g l a r e and 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 . A l l 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 w i t h i n t h e maximum c i r c u m f e r e n c e 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 side views of an a c t i v i t y chamber. A l l component sections are l a b e l l e d . top view CABLE TO CIRCUIT WATER LEVEL \ TRANSDUCER \ OUTER WALL J 1 side view 9 r i n g o f p l e x i g l a s s had s i x t e e n 1/8" (3.6.mm) p e r f o r a t i o n s t o a l l o w an exchange of wat e r i n t o and out o f t h e c h a n n e l . These h o l e s a l s o a s s i s t e d i n k e e p i n g d e t r i t u s a t a low l e v e l by a c t i n g as a " s c o u r i n g system" when water was moving o u t o f t h e swimming c h a n n e l . A t o t a l o f n i n e a c t i v i t y chambers were c o n s t r u c t e d f o r t h e i n v e s t i g a t i o n . They were mounted i n t h r e e v e r t i c a l r a c k s c o n s t r u c t e d o f #225 s t e e l d e x i o n . Each r a c k c o n t a i n e d 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 cm). 2.3 Temperature C o n t r o l s Water t e m p e r a t u r e s i n th e a c t i v i t y chambers were c o n t r o l l e d by Kodak t h e r m o s t a t i c m i x i n g v a l v e s (Eastman Kodak Co., R o c h e s t e r , N.Y.). These were connected t o the h o t and c o l d c i t y w ater o u t l e t s and s u p p l i e d t h e water b a t h s s u r r o u n d i n g t h e o u t e r b o r d e r o f . t h e swimming c h a n n e l s . The t e m p e r a t u r e s o f t h e s e two b o d i e s o f water were always a l l o w e d t o come i n t o e q u i l i b r i u m before, p r o c e e d i n g w i t h an e x p e r i m e n t . Low wa t e r t e m p e r a t u r e s were m a i n t a i n e d d u r i n g t he summer months by c o o l i n g t h e c i t y water w i t h a p o r t a b l e r e f r i g e r a t i n g u n i t . A Honeywell thermograph was used 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 d u r i n g March, 1967. These d a t a were compared w i t h r e a d i n g s o b t a i n e d by hand-held thermometers and th 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 v a l v e s . A f t e r e q u i l i b r i u m was reached between t h e swimming c h a n n e l s and t h e water b a t h s , t h e . r e c o r d i n d i c a t e d l e s s 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 w a t e r t e m p e r a t u r e d u r i n g a t w e n t y - f o u r hour p e r i o d ( F i g . 1, a p p e n d i x ) . I t was assumed t h a t t h e tem p e r a t u r e c o n t r o l s were adequate and the s l i g h t d a i l y v a r i a t i o n d i d not c o n s t i t u t e a s u f f i c i e n t v a l u e t o e n t r a i n t h e l o c o m o t o r a c t i v i t y o f j u v e n i l e sockeye salmon. The use o f t h e thermograph 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 t h a t b o t h t h e r e c o r d s o b t a i n e d w i t h t h e m i x i n g v a l v e s and t h e hand-held thermometers were s u f f i c i e n t 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 from 5°C t o 15°C, wh 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 salmon ( B r e t t , 1952, 1956). 2.4 P h o t o p e r i o d i c C o n t r o l s 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 lamps, e x c e p t where s p e c i f i c a l l y i n d i c a t e d . I n d i r e c t l i g h t i n g 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 s h e e t i n g p o s i t i o n e d 12" (30.5 cm) above t h e wa t e r s u r f a c e . I t was assumed the opaque s h e e t i n g a l s o reduced t h e number o f overhead cues a v a i l a b l e t o t h e f i s h f o r o r i e n t a t i o n . B l a c k p l a s t i c s h i e l d e d each a c t i v i t y chamber t o p r e v e n t i n t e r f e r e n c e 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 s o u r c e s . Sudden changes i n t h e room l i g h t i n t e n s i t y were p r e v e n t e d 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 low 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 w a t t lamp. The e n v i r o n m e n t a l p h o t o p e r i o d s were r e g u l a t e d by model T-101 I n t e r m a t i c time s w i t c h e s (Marr E l e c t r i c L t d . , C o o k s v i l l e , Ont.) w h i c h were s y n c h r o n i z e d w i t h each o t h e r and c o n n e c t e d t o s e p a r a t e c h a n n e l s on t h e event r e c o r d e r . T h i s arrangement i n d i c a t e d t h e e x a c t time o f day t h a t the l i g h t i n g c o n d i t i o n s were changed and p r o v i d e d a measure o f t h e phase r e l a t i o n s h i p between p h o t o p e r i o d and t h e o n s e t o r t e r m i n a t i o n o f a c t i v i t y . An e l e c t r o n i c dimming system i n t r o d u c e d a t w i l i g h t e f f e c t t o t h e a r t i f i c i a l e nvironment. The c i r c u i t r y 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 g r a d u a l 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 o v e r a twenty minute p e r i o d ( F i g s . 2 & 3, a p p e n d i x ) . These "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 a v o i d e d . t h e i n i t i a l " s t a r t l e " r e s p onse o b s e r v e d i n j u v e n i l e sockeye when the l i g h t s were 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 water 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. A l l r e a d i n g s were c o n v e r t e d t o " l u x " where one l u x e q u a l s 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 i n s t r u m e n t s f o r r e c o r d i n g l o c o m o t o r 12 a c t i v i t y were b u i l t around.a compact s o n i c t r a n s d u c e r ( E n t e r p r i s e M a n u f a c t u r i n g Co., A k r o n , Ohio) ( F i g . 2 ) . The p l a s t i c 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 s e a l e d w a t e r p r o o f u n i t . . A l l e l e c t r o n i c components were 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 added 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 s i g n a l o f 800 k i l o h e r t z (1 h e r t z = 1 c y c l e / s e c o n d ) i n a 4° cone. T h i s f r e q u e n c y i s w e l l above t h e wavelengths h e a r d by f i s h i n g e n e r a l (Wodinski and T a v o l g a , 1963; M a r s h a l l , 1965; P r o t a s o v , 1967) and sockeye salmon i n . p a r t i c u l a r ( B r e t t and G r o o t , 1963; Van der Walker, 1966). When the beam was t r a n s m i t t e d t h r o u g h t h e water and came i n c o n t a c t w i t h an o b j e c t , the s i g n a l was " r e f l e c t e d " back t o t h e t r a n s d u c e r . 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 f r e q u e n c y d i f f e r e n c e between the e m i t t e d and r e t u r n i n g s i g n a l s . I f an o b j e c t was t r a v e l l i n g away from t h e so 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 the " r e f l e c t e d " s i g n a l would be of. a l o w e r f r e q u e n c y t h a n i f t h e o b j e c t were m o t i o n l e s s . I f t h e o b j e c t was moving towards t h e t r a n s d u c e r , t h e f r e q u e n c y o f t h e r e t u r n i n g w a v e l e n g t h would be g r e a t e r . T h e r e f o r e , a.moving o b j e c t i n t h e "beam" r e s u l t e d i n a f r e q u e n c y change w h i c h c o u l d . b e a m p l i f i e d t o o p e r a t e an event r e c o r d e r . Two u n i t s were mounted i n o p p o s i t e c o r n e r s o f t h e a c t i v i t y chamber (see F i g . 1) i n t h e a r e a o f the water b a t h . The beam p e n e t r a t e d t h e 1/16" (1.3 mm) o u t e r 13 Figure 2. Photographs i l l u s t r a t i n g the transducer head and components. (From l e f t to r i g h t , cable, cable securing screw, O-ring, waterproof body, end of cable, printed c i r c u i t board with rubberized compound removed to reveal the electronic components. Located above i s the cap which houses both the sound emission and detecting 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 t a n g e n t . 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 between t h e two u n i t s , and i n s u r e d t h a t an o b j e c t p a s s i n g t h r o u g h t h e f i e l d o f sound would.be r e c o r d e d . A c a b l e from each t r a n s d u c e r was c o u p l e d w i t h s u p p o r t i n g e l e c t r o n i c c i r c u i t r y . The d e s i g n and c o n s t r u c t i o n o f t h e s e i n s t r u m e n t s 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 , Vancouver, B.C. The c i r c u i t diagrams a r e p r e s e n t e d i n t h e l a s t s e c t i o n o f t h i s t h e s i s ( F i g s . 4 t o 8, a p p e n d i x ) . The b a s i c o p e r a t i o n was as f o l l o w s . Each a c t i v i t y chamber was a s s o c i a t e d w i t h two 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 and a pen on a 20 c h a n n e l E s t e r l i n e - A n g u s e v e n t r e c o r d e r ( F i g . 3 ) . The system 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 by one t r a n s d u c e r would cause 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 by the o t h e r t r a n s d u c e r would a l t e r t h e l o g i c c i r c u i t t o i t s "count" s t a t e . T h i s . a c t i o n would r e l e a s e a p u l s e t o the r e c o r d e r . The f i s h movement had 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 t r a n s d u c e r and cause 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 . r e v e r t b a c k . t o i t s " s e t " s t a t e b e f o r e t h e n e x t c o u n t . c o u l d 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 o r d e r t o i n s u r e t h a t any count r e l e a s e d was not 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 . . T h i s p r e v e n t e d r e c o r d i n g s w h i c h might 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 , but 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. B l o c k diagram i l l u s t r a t i n g t h e t r a n s d u c e r s and a s s o c i a t e d c i r c u i t r y . 1 = T r a n s d u c e r 2 = A m p l i f i e r 3 = B i s t a b l e l o g i c c i r c u i t 4 = 24 v o l t s AC 5 = To Recorder Block Diagram 16 a s m a l l a r e a . The b e h a v i o u r o f p i n k and chum salmon f r y i s such 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 c i r c u l a r swimming c h a n n e l (Hoar, 1956). The j u v e n i l e sockeye salmon used i n t h e s e 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 . The r e s u l t was a r e l i a b l e measure o f loco m o t o r a c t i v i t y w h i c h , i f needed, c o u l d a l s o i n d i c a t e t h e approximate d i s t a n c e c o v e r e d 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 t o t h e m o n i t o r i n g d e v i c e s . The t r a n s d u c e r s were 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 arrangement p e r m i t t i n g 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 t h e a d d i t i o n o f a n o t h e r v a r i a b l e t o the immediate environment. T h i s i n s t r u m e n t had d e f i n i t e advantages o v e r the 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 m e c h a n i c a l g r i d s which i n t e r f e r e d w i t h f i s h movement (Davis and Ba r d a c h , 1965; T h i n e s , e t a l . , 1965; Bohun and Winn, 1966). The 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 t h e a c t i v i t y o f t h e e x p e r i m e n t a l s u b j e c t s . Ten f i s h were 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 and t h e i r motor a c t i v i t i e s were r e c o r d e d by v i s u a l means ( a i d e d by a hand t a l l y m e t e r ) . These measurements were conducted f o r a 10 minute i n t e r v a l b e f o r e , d u r i n g and 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 f o r each r e v o l u t i o n o f t h e a c t i v i t y chamber. 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 f r e q u e n c y s i g n a l produced by t h e t r a n s d u c e r s (Table 1 ) . 17 Ta b l e 1. A comparison o f a c t i v i t y 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 a f t e r 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 T o t a l B e f o r e "A" 6 1 4 3 2 5 4 1 1 7 34 D u r i n g "B" 7 0 4 3 3 7 4 0 . 2 5 35 A f t e r "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) Te s t Number 1 . 2 3 4 5 6 7 8 9 10 T o t a l T a l l y 0 7 4 0 2 1 1 4 6 1 26 I n s t . 0 7 4 0 2 1 1 4 7 1 27 18 A r e l a t e d s e r i e s o f t e s t s compared v i s u a l and a u t o m a t i c r e c o r d i n g methods t o e s t a b l i s h t h e a c c u r a c y of t h e i n s t r u m e n t a l r e c o r d i n g system. Ten f i s h were 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 chamber and a s i m u l t a n e o u s r e c o r d was o b t a i n e d w i t h b o t h methods. A count was r e g i s t e r e d each time t h e s u b j e c t passed a r e f e r e n c e p o i n t w h i c h was s i t u a t e d i n t h e beam o f t h a t t r a n s d u c e r t r i g g e r i n g t h e event r e c o r d e r . T h i s l o c a t i o n f a c i l i t a t e d t h e c o m p a r a t i v e a s p e c t o f the two methods. Each t e s t l a s t e d t e n minutes 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 r e c o r d s w h i c h w e r e . v i r t u a l l y i d e n t i c a l t o t h o s e o b t a i n e d by v i s u a l o b s e r v a t i o n s (Table 2 ) . The i n s t r u m e n t s and c i r c u i t r y appear t o be w e l l s u i t e d 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 , and can be a p p l i e d t o any s i t u a t i o n where i t might be d e s i r a b l e t o i n d i c a t e a s u b j e c t has pass e d a c e r t a i n r e f e r e n c e p o i n t i n an a c t i v i t y chamber o r aquarium. 2.6 L i v e M a t e r i a l A l l specimens o f j u v e n i l e sockeye salmon were o b t a i n e d from t h e h a t c h e r y f a c i l i t i e s a t the F i s h e r i e s R e search Board of Canada, B i o l o g i c a l S t a t i o n , Nanaimo, B.C. Most f i s h o r i g i n a t e d from S k u l l y Creek a t L a k e l s e Lake, B.C., but a s m a l l sample o f Kamchatka P e n i n s u l a p r e s m o l t s were a l s o a v a i l a b l e f o r c o m p a r a t i v e p u r p o s e s . P r i o r t o a l l 19 experiments, the f i s h were maintained on C l a r k ' s dry food (J.R. C l a r k , S a l t Lake C i t y , Utah). These f i s h were maintained at two s i t e s which provided d i f f e r e n t photoperiodic and environmental c o n d i t i o n s , semi-natural.and a r t i f i c i a l . The semi-n a t u r a l c o n d i t i o n s were obtained by p l a c i n g f i s h i n a l a r g e outdoor h o l d i n g tank (3,633 g a l l o n s ) . This tank was subject to 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 a r t i f i c i a l l i g h t sources i n nearby b u i l d i n g s . The tank was d i v i d e d by. p l a s t i c screens i n t o s i x equal compartments 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 l o c a l i t y and p a r e n t a l stock. The c o n t r o l l e d a r t i f i c i a l h o l d i n g c o n d i t i o n s were achieved w i t h two 50 g a l l o n aquaria. The photoperiod was regulated by an Intermatic time switch and.. incandescent lamps. These aquaria were suppli e d w i t h a i r s t o n e s and a slow incoming, r a t e of d e c h l o r i n a t e d water. Temperature v a r i a t i o n s were kept at 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 from.Skully Creek were hatched and maintained 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 aquaria f o r p e r i o d i c t e s t i n g to determine i f developmental changes e x i s t e d i n the photo-p e r i o d i c response. 2.7 E x p e r i m e n t a l Design A l l t e s t s , e x c e p t where s p e c i f i c a l l y , i n d i c a t e d , were performed on s i n g l e i n d i v i d u a l s . A f i s h was i n t r o d u c e d t o each a c t i v i t y chamber and a l l o w e d two days f o r a c c l i m a t i o n t o the 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 from t h e a n a l y s i s o f p r e l i m i n a r y e x p e r i m e n t s . 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 days 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 experiment t o c o n t i n u e f o r l o n g e r p e r i o d s o f t i m e . No f i s h was f e d . d u r i n g an experiment e x c e p t i n t h o s e t e s t s d e s i g n e d t o s t u d y t h e e f f e c t s , o f f e e d i n g upon t h e . a c t i v i t y p a t t e r n . The p r e l i m i n a r y e x p e r i m e n t s examined the b a s i c e n t r a i n e d r e s p o n s e . The combined temperature and ph o t o -p e r i o d i c c o n d i t i o n s used a r e l i s t e d i n Table 3. R e l a t e d t e s t s i n t h i s 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 r e s p o n s e , and.the s y n c h r o n i z i n g e f f e c t s o f p h o t o p e r i o d upon b o t h i n d i v i d u a l s and groups of f i s h . The e x p e r i m e n t s e x a m i n i n g . t h e endogenous component (Table 4) were conducted i n c o n s t a n t l i g h t ( L L ) , c o n s t a n t dark (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 ) . Temperatures were m a i n t a i n e d c o n s t a n t a t 10°C and the l i g h t 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 (except where s p e c i f i c a l l y i n d i c a t e d ) . 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 of 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 . ENVIRONMENTAL LIGHTING TEMPERATURE 10°C LL + DD + LL (F) + 22 S u r g i c a l techniques 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 described i n the appendix. 2.8 A n a l y t i c a l Procedure V i s u a l scanning of the event recorder charts i n d i c a t e d the general performance of 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 recorded on data sheets and then t r a n s f e r r e d to IBM punch cards 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 of each i n d i v i d u a l was p l o t t e d by comparing.the hourly a c t i v i t y amplitude against time. This provided 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 or group during the experimental p e r i o d . Mean hourly values were c a l c u l a t e d . t o condense these data and to c l a r i f y the general p a t t e r n s . Mean values were a p p l i e d only to 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 treatment, such as the experiments examining the.combined e f f e c t s of photoperiod and temperature on the entrained rhythm. These data were subjected t o . a n a l y s i s of variance and B a r t l e t t ' s t e s t . Periodogram a n a l y s i s (Enright, 1965a, b) was used to 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. This was a comparative procedure using the root mean square of the amplitude.associated w i t h any estimated p e r i o d . Ap (root mean square amplitude) E(avg.-grand avg.) estimate of the p e r i o d value 23 The completed periodogram provided.a comparison of the amplitudes.for s e l e c t e d periods between 20.00 and 28.00 hours. I t could i n d i c a t e whether the assumption of a 24.0 hour p e r i o d , or a 23.0 hour.period e t c . , l e d to an unusually l a r g e amplitude, or whether the amplitudes were no greater than background "noise" (see F i g . 8b).. The method d i d not assign any l e v e l of 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 d i f f i c u l t to a s s i g n an o b j e c t i v e meaning to conventional s t a t i s t i c s such as a T-test because the a c t i v i t y records were s e r i a l l y c o r r e l a t e d measurements and d i d not-represent random independent samples from the p o p u l a t i o n . At l e a s t f i v e r e p l i c a t i o n s of a d a i l y c y c l e were recorded before attempting a n a l y s i s . This was i n agreement w i t h the g e n e r a l l y accepted p o l i c y to r e g i s t e r a minimum of f i v e to seven periods before examining an endogenous r h y t h m i c i t y (Aschoff, 1960). Periodogram a n a l y s i s provides a simple s t a t i s t i c a l approach to c y c l i c b i o l o g i c a l phenomena. The c a l c u l a t i o n s are e a s i l y determined, but a computer f a c i l i t a t e s the l a r g e number of estimates r e q u i r e d . 3. RESULTS 3.1 D e t e r m i n a t i o n o f a B a s i c E n t r a i n e d Response These e x p e r i m e n t s were f o c u s e d upon e n t r a i n m e n t t o p h o t o p e r i o d as t h e p r i m a r y e n v i r o n m e n t a l f a c t o r . I n d i v i d u a l s and o c c a s i o n a l l y groups of f i s h were i n t r o d u c e d t o an a c t i v i t y chamber and a r e c o r d o f t h e motor a c t i v i t y was c o l l e c t e d . The c h a r t o u t p u t was a n a l y z e d 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 d u r i n g each s i x t y minute i n t e r v a l . The h o u r l y " b i t s " o f i n f o r m a t i o n s e r v e d as the b a s i c a c t i v i t y measure u t i l i z e d i n t h i s phase of t h e 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 t o p r o v i d e answers t o t h e f o l l o w i n g q u e s t i o n s . 1. Do d i e l c y c l e s o f l o c o m o t o r a c t i v i t y e x i s t under th e a r t i f i c i a l l a b o r a t o r y c o n d i t i o n s ? 2. I s 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 a major s y n c h r o n i z e r ? 3. Are t h e organisms day a c t i v e , n i g h t a c t i v e o r c r e p u s c u l a r i n t h e i r a c t i v i t y p a t t e r n s ? 4. Does the o r g a n i s m need t o adapt t o the e n v i r o n m e n t a l chambers b e f o r e e x p r e s s i n g a c o n s i s t e n t d a i l y a c t i v i t y p a t t e r n ? 5. I s the r e s p o n s e s t a b l e o r does i t change d u r i n g t h e c o u r s e o f development? 6. I s t h e e n t r a i n e d response e x p r e s s e d by one i n d i v i d u a l s i m i l a r t o t h a t e x p r e s s e d by a group o f i n d i v i d u a l s ? 25 Nine 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 i n t o t h e a c t i v i t y chambers i n a c o n t r o l l e d environment of 8L 16D (8 hours of l i g h t and 16 hours of darkness) a t 5°C. A c o n t i n u o u s s i x day r e c o r d i n d i c a t e s t h a t a s i n g l e j u v e n i l e sockeye e x p r e s s e d a d e f i n i t e c y c l i c response t o the p h o t o p e r i o d ( F i g . 4 ) . T h i s r e c o r d i s c o n s i d e r e d t o 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 s e r i e s . The a p p a r e n t e n t r a i n m e n t t o the p h o t o p e r i o d would t e n d t o 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 e n v i r o n m e n t a l 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 agent, u n l e s s t h e s e f a c t o r s were e x a c t l y 24.00 hours i n t h e i r n a t u r a l p e r i o d i c i t y . A t t h e o n s e t o f l i g h t t h e r e was an i n c r e a s e i n the a c t i v i t y a m p l i t u d e . T h i s i n c r e a s e d a c t i v i t y l e v e l was g e n e r a l l y m a i n t a i n e d d u r i n g the l i g h t phase, but some day t o day v a r i a t i o n i n t h e h o u r l y t o t a l s o f e v e n t s o c c u r r e d . There was a l s o a n , i n c r e a s e i n t h e number of secondary peaks and a s s o c i a t e d t r o u g h s o c c u r r i n g d u r i n g t h e dark phase i n the f o u r t h , f i f t h and s i x t h days of the t e s t . I t i s suggested, t h a t t h e s e secondary peaks and a s s o c i a t e d t r o u g h s o c c u r randomly, s i n c e t h e c a l c u l a t i o n of mean v a l u e s f o r t h e s i x day p e r i o d does not i n d i c a t e any 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 ( F i g . 5 a ) . 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 a c t i v i t y t o t h e l i g h t c y c l e ( a c t i v i t y l e v e l i n c r e a s e s b e f o r e t h e o n s e t o f l i g h t ) . T h i s i s a g e n e r a l c h a r a c t e r i s t i c o f 26 F i g u r e 4. The e n t r a i n e d response o f one i n d i v i d u a l t o 8L 16D a t 5°C. The a m p l i t u d e i s r e p r e s e n t e d as the t o t a l number o f e v e n t s o c c u r r i n g each hour. The dark b a r s a t t h e base r e p r e s e n t t h e p e r i o d s o f d a r k n e s s . F i g u r e 5. (A) The mean d a i l y a c t i v i t y p a t t e r n c a l c u l a t e d from t h e d a t a p r e s e n t e d i n F i g u r e 4. (B) The mean d a i l y response of a s i n g l e i n d i v i d u a l from the Kamchatka Peninsula. d i u r n a l l y a c t i v e o r g anisms. U n f o r t u n a t e l y , g r o u p i n g t h e s e d a t a o m i t s 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 t h e a c t i v i t y c y c l e , but t h e g e n e r a l o v e r a l l p a t t e r n b e i n g e x p r e s s e d i s more c l e a r l y d e f i n e d . The r e s u l t i n g performance c u r v e tends t o be smoother t h a n t h e d a i l y r e c o r d . I t i s not known i f a s i m i l a r r e s ponse would be o b t a i n e d from g e n e t i c s t o c k s o r i g i n a t i n g a t a . v a r i e t y o f g e o g r a p h i c a l l o c a t i o n s . However, t h e s e p r e l i m i n a r y t e s t s d i d r e v e a l t h a t sockeye o b t a i n e d from.the Kamchatka P e n i n s u l a e x p r e s s e d a s i m i l a r d i e l r esponse t o t h e 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 ( F i g . 5b). These d a t a i n d i c a t e t h a t j u v e n i l e - s o c k e y e salmon w i l l e x h i b i t a . c y c l i c a c t i v i t y p a t t e r n d u r i n g a twenty 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 c o n d i t i o n s . A g e n e r a l p a t t e r n o f i n c r e a s e d a c t i v i t y o c c u r r e d d u r i n g t h e l i g h t phase o f t h e e x p e r i m e n t a l p e r i o d . T h i s was accompanied 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 t h e a c t i v i t y a m p l i t u d e o c c u r r i n g a t t h e o n s e t . o f the dark phase. T h i s g e n e r a l p a t t e r n i s r e p r e s e n t a t i v e o f the m a j o r i t y o f i n d i v i d u a l s t e s t e d . The p h o t o p e r i o d i c environment c o n s t i t u t e s a s t r o n g 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 (5°C) r e s u l t s i n t h e e x p r e s s i o n o f a b a s i c a l l y unimodal. a c t i v i t y p a t t e r n No g r o s s 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 as c h a r a c t e r i s t i c o f e i t h e r L a k e l s e o r Kamchatka s t o c k t e s t e d 28 F u r t h e r a n a l y s e s o f t h e 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 a minimum two day r e p e t i t i o n o f the c y c l e was n e c e s s a r y t o d e t e r m i n e th e c h a r a c t e r i s t i c p a t t e r n ( i . e . whether any i n d i v i d u a l was e i t h e r l i g h t a c t i v e o r dark a c t i v e ) . I n a d d i t i o n , a comparison o f t h e t o t a l day t o day a c t i v i t y i n d i c a t e d t h a t a minimum two day a d a p t a t i o n p e r i o d was n e c e s s a r y b e f o r e a c o n s i s t e n t d a i l y a c t i v i t y o u t p u t c o u l d be o b t a i n e d . T h e r e f o r e , a f t e r i n t r o d u c i n g a s u b j e c t t o an a c t i v i t y chamber, d a t a a n a l y s i s c o n s i d e r e d o n l y day 3 p l u s t h e f o l l o w i n g s i x days. 3.11 The 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 d i e l  p a t t e r n o f a c t i v i t y . T h i s s e r i e s was conducted t o d e t e r m i n e i f t h e r e were changes i n t h e l o c o m o t o r a c t i v i t y p a t t e r n a t d i f f e r e n t s t a g e s o f development. T h i s i n f o r m a t i o n was v a l u a b l e i n d e t e r m i n i n g . w h e t h e r o r n o t t h e i n v e s t i g a t i o n was e x a m i n i n g a c o n s i s t e n t a c t i v i t y p a t t e r n i n t h e 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 sockeye salmon eggs were hat c h e d i n two o f t h e a c t i v i t y chambers. The f r y were m a i n t a i n e d i n 10 g a l l o n a q u a r i a 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 f o r one y e a r . T e s t i n g was conducted under c o n d i t i o n s o f 12L 12D a t 5°C, e x c e p t days 1-14 which were a t 9.5L 14.5D. V i s u a l o b s e r v a t i o n s i n d i c a t e d t h e newly h a t c h e d a l e v i n s w i t h y o l k sac a t t a c h e d were n e g a t i v e l y p h o t o t a c t i c . T h i s r e s p o n s e was g r a d u a l l y r e v e r s e d a f t e r emergence from the g r a v e l . There was a p o s i t i v e r h e o t a c t i c response t o the 29 water c u r r e n t i n t h e swimming c h a n n e l . (produced by an inc 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 i n t o t h e g r a v e l ) and..the f r y proceeded 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. T h i s d i r e c t i o n a l movement was r e v e r s e d when the d i r e c t i o n o f the w a t e r f l o w was changed by T80 d e g r e e s . A l l u n i d i r e c t i o n a l movement by t h e group t e r m i n a t e d when t h e . w a t e r f l o w ceased. A sample o f t h i s 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 f o r . f u r t h e r t e s t i n g . A f t e r emergence from t h e g r a v e l t h e d a i l y p a t t e r n was changed from a p r e d o m i n a n t l y n o c t u r n a l , b i m o d a l c o n f i g u r a t i o n t o a d i u r n a l a c t i v i t y . p a t t e r n ( F i g . 6 a, b, c, d ) . T h i s response t h e n remained unchanged i f i n d i v i d u a l s were 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 c o n d i t i o n s . A l l s u b j e c t s m a i n t a i n e d a synchrony w i t h t h e p h o t o p e r i o d i c c y c l e and 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 c y c l e a t a c o n s t a n t t e m p e r a t u r e . Nine groups o f L a k e l s e p r e - s m o l t s were s u b j e c t e d t o an o s c i l l a t i n g p h oto-p e r i o d i c environment. The t e s t s were d e s i g n e d t o det e r m i n e whether o r not t h e p h o t o p e r i o d c o u l d s y n c h r o n i z e s e v e r a l 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 c y c l e . 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 hich were i n t r o d u c e d t o a 12L 12D regime a t 5°C. 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 a c t i v i t y p a t t e r n a t d i f f e r e n t s t a g e s o f t h e l i f e c y c l e . A = The f i r s t 9 days a f t e r emergence. B = Days 10 t o 14. C = 3 months a f t e r emergence. D = 11 months a f t e r emergence. Mean no. of events 31 of seven days t h i s group of 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 p e r i o d ( F i g . 7a). A decrease i n a c t i v i t y s i m i l a r to th 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 these grouped data t h a t the 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 entrained response of one i n d i v i d u a l . C e r t a i n aspects of the behaviour w i l l . b e a l t e r e d due to the 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 , but the b a s i c p a t t e r n of a day a c t i v e r e l a t i o n s h i p t o the photoperiodic c y c l e appears to be s i m i l a r . This group i s expressing a 24.00 hour p e r i o d i c i t y t h a t i s apparently synchronized by the l i g h t c y c l e ( F i g . 7b). This i s suggested because the photoperiod a l s o has a 24 hour component and i s the major o s c i l l a t i n g environmental f a c t o r i n v o l v e d . I t i s f u r t h e r suggested t h a t the l i g h t - d a r k c y c l e i s capable of synchronizing the individual-members 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 to a common.stimulus.results i n a group expression of 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 , to th a t of the environ-mental cue. The mean d a i l y a c t i v i t y pattern of f i v e individuals monitored simultaneously for six days i n the same a c t i v i t y chamber. Periodogram analysis of the six day record i l l u s t r a t e d i n Fig . 7 A . rO O to o rO 00 Mean no. of events 33 3.2 The E f f e c t s o f Temperature and P h o t o p e r i o d - on t h e  D a i l y A c t i v i t y 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 were d e s i g n e d t o p r o v i d e a measure of t h e temp e r a t u r e e f f e c t on t h e . e n t r a i n e d response t o p h o t o p e r i o d . Only temperature and p h o t o p e r i o d were c o n s i d e r e d s i n c e they r e p r e s e n t e d t h e major e n v i r o n m e n t a l v a r i a b l e s a f f e c t i n g t h e b a s i c a c t i v i t y p a t t e r n . The t e s t s were performed on s i n g l e L a k e l s e p r e - s m o l t s p r e v i o u s l y h e l d i n t h e s e m i - n a t u r a l p h o t o p e r i o d i c c o n d i t i o n s a t an average te m p e r a t u r e o f 8°C. The a c t i v i t y p a t t e r n s were r e c o r d e d 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 day p e r i o d o f a c c l i m a t i o n t o the a r t i f i c i a l e nvironment. Nine i n d i v i d u a l s were t o be examined a t each temperature and p h o t o p e r i o d b u t s e v e r a l f a t a l i t i e s . a t t h e h i g h e r 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 . A t o t a l o f 512 d a i l y a c t i v i t y . c y c l e s were examined and t h e mean d a i l y p a t t e r n s . f o r each temperature . and., p h o t o p e r i o d i c v a l u e a r e p r e s e n t e d . 3.21 The e n t r a i n e d . r e s p o n s e a t 5°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, 16L 8D). The mean 24 hour a c t i v i t y p a t t e r n s i n d i c a t e d . t h a t l o c o m o t o r a c t i v i t y 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 the p h o t o p e r i o d i c c y c l e ( F i g . 8 a, b, c ) . . A t t h e o n s e t o f l i g h t t h e r e was a t h r e e - f o l d i n c r e a s e i n the a c t i v i t y a m p l i t u d e o c c u r r i n g d u r i n g a s i x t y minute i n t e r v a l . T h i s h e i g h t e n e d a m p l i t u d e was m a i n t a i n e d t h r o u g h o u t t h e l i g h t . p h a s e and-was t e r m i n a t e d 34 F i g u r e 8 ( a , b, c ) . Mean d a i l y a c t i v i t y p a t t e r n s r e p r e s e n t i n g 202 days o f a c t i v i t y a t 5°C. The s t a n d a r d 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 b r a c k e t s a t each v a l u e . MEAN NO. OF EVENTS PER HOUR _» _ i r\j ro w 01 O 01 o 01 o 35 a t t h e o n s e t o f dark 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 a c t i v i t y . A predawn i n c r e a s e i n a c t i v i t y and. a predusk d e c r e a s e . i n a c t i v i t y a r e i l l u s t r a t e d b y . t h e s e d a t a , and i n d i c a t e a p o s i t i v e phase 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 o n d i t i o n s . ( a c t i v i t y i n c r e a s e d o r d e c r e a s e d p r i o r t o t h e o n s e t o f the s y n c h r o n i z i n g s t i m u l u s ) . T h i s phase 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 an 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 o f t h e a m p l i t u d e . i n c r e a s e s a t t h e o n s e t of l i g h t i n d i c a t e d a g e n e r a l . u p p e r a c t i v i t y l i m i t f o r each p h o t o p e r i o d . T h i s "dawn" peak was f o l l o w e d by a "midmorning" d e p r e s s i o n i n . a c t i v i t y t h a t was i n f l u e n c e d by d a y l e n g t h . The d e c r e a s e i n a c t i v i t y m o d i f i e d t h e b a s i c unimodal 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 . as the a l t e r n a n s ^ " t y p e ( A s c h o f f , 1966) . There was a n . i n c r e a s e i n the d u r a t i o n o f h e i g h t e n e d a c t i v i t y when t h e a r t i f i c i a l day was l e n g t h e n e d from e i g h t hours o f l i g h t t o . t w e l v e o r s i x t e e n h o u r s . The l i m i t a t i o n o f t h e s e h i g h e r . a c t i v i t y l e v e l s t o t h e l i g h t phase i n d i c a t e d 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 n ot a l t e r t h e b a s i c a l l y d i u r n a l p a t t e r n o f the e n t r a i n e d r e s p o n s e . "^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 the major peak f o l l o w s t h e minor peak. 36 3.22 T h e . e n t r a i n e d response a t 10°C t o 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 (8L 16D f 12L 12D, 16L 8D). The mean 24 hour a c t i v i t y p a t t e r n s examined a t t h i s t e m p e r a t u r e 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 ( F i g . 9 a , 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 t e s t s i n c l u d e . a n . a p p a r e n t t e m p e r a t u r e - p h o t o p e r i o d i n t e r a c t i o n a t 8L. 16D, and a g e n e r a l i n c r e a s e i n the a c t i v i t y a m p l i t u d e r e s u l t i n g from t h e h i g h e r 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 . A t 8L- 16D t h e i n c r e a s e d a c t i v i t y a m p l i t u d e was most app a r e n t i n t h e dark phase. T h i s h e i g h t e n e d . n o c t u r n a l a c t i v i t y . l e v e l .decreased 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 a c t i v i t y pattern... However, t h e s e d a t a s t i l l . r e tained, a c y c l i c r e l a t i o n s h i p t o the p h o t o p e r i o d . a n d s i g n i f i c e n t (P <.01) d i f f e r e n c e s i n . a c t i v i t y l e v e l o c c u r r e d between th e l i g h t and dark phases of t h e a c t i v i t y c y c l e . A t 12L 12D and 16L- 8D there.was a d e f i n i t e 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 . The magnitude of t h e a c t i v i t y i n c r e a s e a t the o n s e t o f l i g h t was l e s s t h a n t h a t o b s e r v e d a t 5°C, and c o n s t i t u t e d a. l e v e l t w i c e t h a t o b s e r v e d d u r i n g t h e a r t i f i c i a l n i g h t . P r e d a w n . i n c r e a s e s and predusk d e c r e a s e s i n a c t i v i t y 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 a v a i l a b l e e v i d e n c e f o r an endogenous r h y t h m i c i t y . g u r e 9 ( a , b , c ) . Mean d a i l y a c t i v i t y p a t t e r n s r e p r e s e n t i n g 172 d a y s o f a c t i v i t y a t 10°C. MEAN NO. OF EVENTS PER HOUR 38 3.23 The entrained response at 15"C to three  d i f f e r e n t photoperiods (8L 16D, 12L 12D, 16L 8D). The major d i f f e r e n c e s between.these t e s t s at 15°C and those at 5°C and 10°C i n c l u d e a continued.temperature-photoperiod i n t e r a c t i o n at.8L.16D and higher a c t i v i t y amplitudes o c c u r r i n g at twelve.and s i x t e e n hours of l i g h t ( F i g . 10 a, b, c) . At 8L 16D there was an apparent decrease., i n the mean a c t i v i t y amplitude .during a l l hours of the a r t i f i c i a l day ( F i g . 10a). The v a r i a t i o n s i n these grouped.data d i d not d i f f e r . s i g n i f i c a n t l y (P < . l ) . However, t h i s was due to the combination of 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 to the photoperiod.at.twelve and s i x t e e n hours of l i g h t . However, the magnitude of the increase at the onset of l i g h t was l e s s than t h a t observed at e i t h e r 5°C or 10°C, and was followed by a s i g n i f i c a n t midmorning (P<.01) depression of 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 the alternans type. These, data 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 environmental.temperature and photoperiod.. These e x t r i n s i c . f a c t o r s produced changes i n both the amplitude of a c t i v i t y and the contour of the p a t t e r n expressed.in a twenty four hour p e r i o d . 3 9 F i g u r e 10 ( a , b, c ) . Mean d a i l y a c t i v i t y p a t t e r n s r e p r e s e n t i n g 138 d a y s o f a c t i v i t y a t 1 5 ° C . MEAN NO. OF EVENTS PER HOUR 40 3.24 The e f f e c t o f temp e r a t u r e on. t h e e n t r a i n e d  r e s p o n s e . The mean h o u r l y v a l u e s r e c o r d e d f o r 12L 12D a t 5°C, 10°C and 15°C i l l u s t r a t e d - t h a t t e m p e r a t u r e i n c r e a s e d t h e a c t i v i t y a m p l i t u d e . i n both, l i g h t and dark ( F i g . 1 1 ) . 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 r e s u l t e d i n a b i m o d a l a c t i v i t y p a t t e r n a t . t h e h i g h e r t e m p e r a t u r e s c o n s i d e r e d . The mean a c t i v i t y values..were p l o t t e d a g a i n s t temperature, f o r . e a c h .photoperiod. 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 i n . t h e mean number o f e v e n t s . d u r i n g l i g h t and dark ( F i g . 1 2 ) . However, t h e g e n e r a l t r e n d from 5°C - 15°C- was one o f i n c r e a s i n g a c t i v i t y a m p l i t u d e s . w i t h i n c r e a s i n g t e m p e r a t u r e s (see F i g . 12, 12L 12D, 16L 8D). 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 with.8L- 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 p h o t o p e r i o d and.temperature, might determine the e x p r e s s i o n o f d i u r n a l o r . n o c t u r n a l a c t i v i t y . When t h e upper temp e r a t u r e " l i m i t " f o r a c e r t a i n d a y l e n g t h was exceeded, 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 and became dark a c t i v e . 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  r e s p o n s e . 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 d a y l e n g t h a t each te m p e r a t u r e v a l u e ( F i g . 1 3 ) . A t 5°C, dark a c t i v i t y showed a v e r y s l i g h t i n c r e a s e between 8L and 16L, whereas 41 F i g u r e 11. Mean d a i l y a c t i v i t y p a t t e r n s r e p r e s e n t i n g 184 days o f a c t i v i t y a t 12L 12D. Temperatures a r e i n °C. 42 F i g u r e 12. P l o t s o f a c t i v i t y on temperature a t 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 . D = Dark; L =. L i g h t 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 a t t h r e e d i f f e r e n t t e m p e r a t u r e s . D = Dark; L = L i g h t l i g h t a c t i v i t y i n d i c a t e d maximum amplitudes at 8L and 16L. At 10°C both 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 at 15°C minimum a c t i v i t y amplitudes were a s s o c i a t e d w i t h 8L. These data provide a d d i t i o n a l evidence f o r an i n t e r a c t i o n between daylength and temperature. I t i s suggested t h a t at 8L 16D a temperature above 10°C w i l l r e s u l t i n increased n o c t u r n a l a c t i v i t y . When more than 50% of the t o t a l a c t i v i t y occurs during the dark, 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 the photobehavioural response and the organism i s considered to be n o c t u r n a l l y a c t i v e . A three dimensional p r e s e n t a t i o n of mean t o t a l a c t i v i t y p l o t t e d against temperature and photoperiod i n d i c a t e d the general trend of the photobehavioural response ( F i g . 14). A r i s e i n temperature r e s u l t e d i n increased mean t o t a l a c t i v i t y at most daylengths. However when short days (8L) and high temperatures (15°C) were combined, decreasing a c t i v i t y occurred. 3.26 The i n t e r a c t i o n of temperature and.photoperiod The mean d a i l y a c t i v i t y records f o r 8L 16D at 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 photoperiod. These records r e s u l t e d from combining i n d i v i d u a l patterns which tended to cancel each other ( F i g . 15), g i v i n g a mean d a i l y p a t t e r n that d i d not d i f f e r s i g n i f i c a n t l y from a s t r a i g h t l i n e (P>.1). A n a l y s i of the previous s e c t i o n s (2.4 and 2.5) i n d i c a t e d a general 45 Figure 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 at each temperature and photoperiod. The broad l i n e s indicate the surface contour. 46 Figure 15. Mean d a i l y a c t i v i t y patterns f o r two i n d i v i d u a l s subjected to 8L 16D at 15°C. Mean no. of events 47 r e v e r s a l i n the a c t i v i t y response at short daylengths and high temperatures. 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 progressive a c t i v i t y i ncrease which was emphasized where short photoperiods and high temperatures were combined ( F i g . 16). I t was als o apparent t h a t the increases were not p a r a l l e l at a l l daylength and temperature combinations. At 8L 16D the estimated 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 at 8°C ( F i g . 17, a, b, c ) . At 12L 12D t h i s value was at 15°C, and at 16L 8D the extrapolated value was at 3 0°C. However, t h i s l a t t e r value was u n r e a l i s t i c since i t exceeded the to l e r a n c e l i m i t s of j u v e n i l e sockeye salmon. In each case, a four hour increase i n daylength was s u f f i c i e n t to double the e f f e c t i v e temperature where the 50% day/night 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 t h a t 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 reverse 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 express greater a c t i v i t y amplitudes during dark than during l i g h t . Lower temperature l i m i t s were not i n v e s t i -gated, but are assumed to approximate 0°C i n j u v e n i l e sockeye. 48 F i g u r e 16. P l o t o f t h e p e r c e n t o c c u r r e n c e o f n o c t u r n a l a c t i v i t y a t each d a y l e n g t h and t e m p e r a t u r e . 0 = 5°C • = 10°C A = 15°C F i g u r e 1 7 . ( a , b, c ) . P l o t s f o r each p h o t o p e r i o d i l l u s t r a t i n g t h e d i s t r i b u t i o n o f a c t i v i t y f o r l i g h t and d a r k . The i n t e r s e c t i n g l i n e s r e p r e s e n t the p o i n t s where a 50% a c t i v i t y d i s t r i b u t i o n o c c u r s . O -• = L i g h t Dark % Activity distribution % Nocturnal activity 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 Expressed, in.. A C o n s t a n t  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 t h e p h o t o p e r i o d i c r e s p o n s e s o f an organism.might assume.the presence o f an e n d o g e n o u s . r h y t h m i c i t y w h i c h i s b e i n g s y n c h r o n i z e d by t h e l i g h t c y c l e . T h 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 e endogenous r e s p o n s e . i n j u v e n i l e sockeye salmon might b e . v a l u a b l e t o p a s t and f u t u r e r e s e a r c h w h i c h 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 o f 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 c y c l e . L a k e l s e p r e - s m o l t s w e r e . o b t a i n e d from t h e . o u t d o o r h o l d i n g t a n k s and h e l d i n the l a b o r a t o r y , a q u a r i a f o r a minimum of two weeks. I n d i v i d u a l s were th e n i n t r o d u c e d i n t o t h e a c t i v i t y chambers under a 12L 12D. p h o t o p e r i o d . The t r a n s f e r from.the c o n t r o l l e d , e n v i r o n m e n t a l h o l d i n g a q u a r i a o c c u r r e d d u r i n g t h e l i g h t phase.of t h e a r t i f i c i a l p h o t o p e r i o d . A t t h e end.of t h e l i g h t . p e r i o d t h e 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 t h e 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 s t a t e (LL or.DD). The t e m p e r a t u r e was h e l d c o n s t a n t a t 10°C and 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 m a i n t a i n e d a t a c o n s t a n t l e v e l (34 . 4. l u x , e x c e p t i n DD, o r w h e r e . s p e c i f i c a i n d i c a t e d ) . These f i s h were a l l o w e d 48 hours f o r adjustment t o t h e new e n v i r o n m e n t a l c o n d i t i o n s . Locomotor a c t i v i t i e s were 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 remain i n t h e 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 f o r a l o n g e r p e r i o d . These d a t a were a n a l y z e d a t t e n minute p e r i o d e s t i m a t e s between t h e 20.00 and 28.00 hour range o f th e periodogram. 3.31 The endogenous response i n c o n s t a n t d a r k n e s s  (DP) a t a c o n s t a n t t e m p e r a t u r e of 1Q°C. A t o t a l o f 33 f i s h r e p r e s e n t i n g 192 "days" of 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 s e r i e s . The response t o DD whi c h r e p r e s e n t e d most s u b j e c t s i n d i c a t e d t h a t e n t r a i n m e n t t o t h e p h o t o p e r i o d was a p p a r e n t d u r i n g t h e f i r s t 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 an o b v i o u s c i r c a d i a n component d u r i n g days 4, 5, and 6 ( F i g . 18). F u r t h e r 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 an endogenous component w i t h an approximate 5 hour p e r i o d i c i t y . An e x c e p t i o n was noted where a f r e e - r u n n i n g rhythm was m a i n t a i n e d w i t h a low a m p l i t u d e i n one i n d i v i d u a l . The periodogram 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). T h i s i s a c l o s e a p p r o x i m a t i o n o f t h e average v a l u e (22.8 hours) c a l c u l a t e d f o r a l l f i s h showing a d e t e c t a b l e rhythm (Table 5). I t was c o n c l u d e d t h a t the endogenous c i r c a d i a n component was n ot r e a d i l y e x p r e s s e d i n c o n s t a n t d a r k n e s s . I n most cases where 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 record for one i n d i v i d u a l i n 12L 12D and constant dark at 10°C. The photoperiod i s indicated by the dark bars at the base of the graph. Periodogram analysis representing the response of one i n d i v i d u a l i n constant darkness. 4.0 _ J o aP 3.0H ° o m <b ° o ODD 2.0-| % 9xc 6> 1.<H 1 1 1 i 20 22 24 26 28 Period T a b l e 5. P e r i o d i c i t y e x p r e s s e d by 16 f i s h i n c o n s t a n t d a r k n e s s a t 10°C. F i s h No. P e r i o d F i s h No. P e r i o d 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 54 T h i s i s not t y p i c a l o f d i u r n a l l y a c t i v e organisms w h i c h u s u a l l y e x p r e s s a spontaneous f r e q u e n c y t h a t i s l o n g e r t h a n 24.00 hours i n c o n s t a n t d a r k n e s s . 3.32 The endogenous 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. T h i r t y - f i v e s u b j e c t s r e p r e s e n t i n g 183 "days" o f a c t i v i t y were examined i n t h i s s e r i e s . A p a r t i c u l a r l y good example o f t h e endogenous component i s i l l u s t r a t e d by t h e t w e l v e day 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 ( F i g . 2 0 ) . 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 can 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 o f p e r i o d i c e n v i r o n m e n t a l s t i m u l i . However, t h e response r e p r e s e n t a t i v e 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 c o n s t a n t l i g h t . P e r iodogram 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 peak a m p l i t u d e i n c r e a s e a t 23.50 hours f o r t h i s i n d i v i d u a l ( F i g . 2 1 ) . The d a t a a n a l y s e s f o r a l l f i s h showing a d e t e c t a b l e rhythm between 20.00 and 28.00 hours r e v e a l e d an average endogenous p e r i o d i c i t y a p p r o a c h i n g 23.30 hours (Table 6 ) . The endogenous component v a r i e d w i t h each i n d i v i d u a l and p e r i o d v a l u e s ranged from 20.00 hours 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 , which i s c h a r a c t e r i s t i c o f most 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 Figure 20. A continuous 12 day record of one i n d i v i d u a l i n constant l i g h t (34.4 lux at 10°C). No. of events per hour 56 Figure 21. Periodogram analysis of the data presented i n Figure 20. •J CL < ~o -*—> Q. E < 14-, 12J 10H 8H 6H 2^ 9» ' o oo o o o o o o o o o o o o o o o o o o o o o<$> o CD o o <*>o CD- o<Po o o 20 22 — r-24 I 2 6 28 Period T a b l e 6. P e r i o d i c i t y e x p r e s s e d by 30 f i s h i n c o n s t a n t l i g h t a t 10°C. F i s h No. P e r i o d F i s h No. P e r i o d 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 t h a t j u v e n i l e sockeye e x p r e s s e d an endogenous rhythm much more f r e e l y i n c o n s t a n t l i g h t t h a n i n c o n s t a n t d a r k . 3.33 The response o f t h e endogenous component t o  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 endogenous p e r i o d i c i t y was measured i n s i x f i s h s u b j e c t e d t o two l i g h t i n t e n s i t i e s (<1 l u x and 34.4 l u x ) . 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 were m a i n t a i n e d c o n s t a n t a t 10°C and t h e lower l i g h t i n t e n s i t i e s were a c h i e v e d by u s i n g a 1 w a t t neon lamp. Periodogram a n a l y s i s was used t o det e r m i n e the c h a r a c t e r i s t i c p e r i o d f o r each i n d i v i d u a l . T a b l e 7 i l l u s t r a t e ! • . t h e v a l u e s o b t a i n e d a t each i n t e n s i t y . The g e n e r a l 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 v a l u e w i t h 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 . T h i s i s n o t i n accordance 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 o t h e r day a c t i v e s p e c i e s ( A s c h o f f , 1960), b u t e x c e p t i o n s have been r e p o r t e d i n t h e l i t e r a t u r e (Hoffmann, 1965). 3.34 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  c o n s t a n t l i g h t a t 10°C. Twenty f o u r f i s h r e p r e s e n t i n g 166 "days" 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 s e r i e s . Food was i n t r o d u c e d t o t h e c o n s t a n t environment a t noon each day. The o b j e c t i v e was t o det e r m i n e whether o r not 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 a c t i v i t y p a t t e r n . The mean v a l u e s f o r a l l t e s t s i n d i c a t e d t h a t i n t r o d u c i n g f o o d t o t h e c o n s t a n t environment produced a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y and e n t r a i n e d t h e endogenous component. Table 7. P e r i o d i c i t y (hours) expressed i n six juvenile sockeye subjected to increasing l i g h t i n t e n s i t y at 10°C. Fish 1 lux 34.4 lux 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 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 i n d i v i d u a l s i n d i c a t e d t h a t t h e i n c r e a s e d a c t i v i t y a m p l i t u d e was m a i n t a i n e d a t a peak f o r a p p r o x i m a t e l y one hour and t h e n g r a d u a l l y r e t u r n e d t o t h e background l e v e l ( F i g . 22). A p p a r e n t l y 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 d u r i n g t h e f i r s t t h r e e days o f t h e t e s t , and g r a d u a l l y weakened w i t h t i m e . I t was c o n c l u d e d t h a t t h e s t i m u l u s o f 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 and c o r r e s p o n d i n g l y a l t e r any f r e e - r u n n i n g r h y t h m i c i t y t h a t was p r e s e n t . The o n l y a l t e r n a t i v e f o r f u t u r e l o n g - t e r m e x p e r i m e n t s would be t o m a i n t a i n f o o d a v a i l a b l e i n the c o n s t a n t s u p p l y , o r a t t empt t o mask t h e e f f e c t s o f f e e d i n g by p r e s e n t i n g f o o d i n a random manner d u r i n g b o t h l i g h t and d a r k . S i n c e t h e m a j o r i t y o f e x p e r i m e n t s r e p o r t e d here c o v e r e d a d u r a t i o n o f one week, i t was c o n s i d e r e d more p r a c t i c a l n o t t o i n t r o d u c e f o o d t o t h e environment. 3.4 The P r i n c i p a l Sensory R e c e p t o r s I n v o l v e d i n t h e  M e d i a t i o n o f t h e E n t r a i n e d Response. The more o b v i o u s and a c c e s s i b l e p h o t o r e c e p t i v e systems i n c l u d e d t h e eyes and t h e p i n e a l body. Less o b v i o u s systems might i n v o l v e cutaneous p h o t o r e c e p t o r s o r a g e n e r a l s e n s i t i v i t y o f the nervous system t o s t i m u l a t i o n by l i g h t . S i n c e t h e eyes and t h e p i n e a l body a r e t h e more prominent and a c c e s s i b l e systems, a s e r i e s o f e x p e r i m e n t s were d e s i g n e d t o i n v e s t i g a t e t h e r o l e o f t h e s e organs i n m e d i a t i n g t h e 61 Figure 22. (A) The mean a c t i v i t y response of 9 individuals for 7 days. The grand means are presented at the ri g h t (B). indicates time of feeding Mean no. of events 62 the en t r a i n e d response. These t e s t s are focused upon the d a i l y entrained response o c c u r r i n g i n 12L 12D at 10°C. Longer term changes may occur but the data are not adequate f o r the a n a l y s i s of seasonal or annual c y c l e s . Mean d a i l y records are presented and are adequate to i n d i c a t e c o n s i s t e n t increases or decreases i n the locomotor a c t i v i t y c y c l e . 3.41 The entrained response i n pinealectomized f i s h . 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 the apparent l o s s of entrainment were common immediately a f t e r p i n e a l -ectomy. Figure 23 a, b i l l u s t r a t e s the response of one i n d i v i d u a l immediately a f t e r pinealectomy (a) and again, 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 of entrainment occurred on days one through fou r . The t o t a l d a i l y a c t i v i t y was lessened i n days 5, 6, and 7 w i t h the appearance of a d e f i n i t e response to the environmental l i g h t c y c l e . Two weeks a f t e r pinealectomy (b) the response had s t a b i l i z e d and entrainment to the l i g h t c y c l e was demonstrated. A l l f u r t h e r experiments included a two week per i o d of postoperative recovery i n order to maintain an acceptable degree of comparison between each day of the experiment and a l s o to prevent masking of the entrained 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 of s i x days each i n d i c a t e d t h a t a pinealectomized 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 of one i n d i v i d u a l i n 12L 12D a t 10°C 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 ) . 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 by sham o p e r a n t s ( F i g . 2 4 ) . The average p e r i o d i c i t y c a l c u l a t e d f o r a l l p i n e a l e c t o m i z e d i n d i v i d u a l s 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 synchrony 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 e x p r e s s e d p e r i o d i c i t i e s r a n g i n g from 21.7 t o 26.8 h o u r s . I t i s suggested t h a t t h e s e l a t t e r s u b j e c t s demonstrated a s c a t t e r about t h e 24.00 hour mean, whi c h might be a t t r i b u t e d t o p o s t - o p e r a t i v e e f f e c t s . However, t h e e x a c t n a t u r e o f the d i s t u r b a n c e was unknown. E i g h t sham o p e r a n t f i s h were t e s t e d i n t h e same manner as p i n e a l e c t o m i z e d f i s h . A l l e i g h t i n d i v i d u a l s e x p r e s s e d a 24.00 hour mean p e r i o d i c i t y i n a l i g h t - d a r k e nvironment. Two i n d i v i d u a l s i n t h i s group had a d d i t i o n a l s econdary peaks i n t h e pe r i o d o g r a m a n a l y s i s , i n d i c a t i n g t h a t t h e r i g o u r s o f t h e 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 o f t h e e n t r a i n e d r e s p o n s e . 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 r e s p o n s e . A comparison o f t h e t o t a l a c t i v i t y l e v e l s 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 and sham o p e r a n t f i s h i n d i c a t e d t h a t p i n e a l e c t o m y r e s u l t e d i n s i g n i f i c a n t l y h i g h e r (P<.01) a c t i v i t y l e v e l s (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 e n t r a i n e d t o t h e l i g h t c y c l e . 65 Figure 24. The mean d a i l y a c t i v i t y patterns f o r pinealectomized and sham pinealectomized f i s h . Standard e r r o r s are i n d i c a t e d by the v e r t i c a l brackets. • Pinealectomized O—-O Sham Pinealectomized Mean no. of events _L ro o o 66 3.42 The endogenous response i n pinealectomized  f i s h . A f t e r a two week p e r i o d of post-operative recovery, e i g h t pinealectomized f i s h and four sham operants were subjected to constant environmental c o n d i t i o n s of LL at 34.4 l u x and 10°C. Periodogram a n a l y s i s i n d i c a t e d t h a t only two of the e i g h t pinealectomized f i s h expressed a peak i n amplitude t h a t could be v i s u a l l y separated from the background. The low amplitudes between 20 and 28 hours f o r a s i n g l e pinealectomized f i s h made the assumption of any p e r i o d i c element questionable ( F i g . 25). No sham operant f i s h expressed a p e r i o d i c i t y between 20 and 28 hours. Apparently the r i g o u r s of the operation would upset the endogenous component, since both pinealectomized and sham operants expressed s i m i l a r disturbances. Continued i n v e s t i g a t i o n i s necessary to determine the r o l e of the p i n e a l i n the endogenous response, and i t i s suggested that longer periods of post-operative recovery 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 entrained response w i t h opaque or  transparent p l a s t i c s h i e l d s over the s i t e of the p i n e a l . Since the previous experiments (sections 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 of the cranium could a l t e r the entrained response, p l a s t i c s h i e l d s were designed i n an e f f o r t to simulate pinealectomy. E i t h e r c l e a r 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 the dermis and the o s s i f i e d cranium, beneath the deepest l a y e r of connective 67 Figure 25. Periodogram analysis of a single pinealectomized f i s h . 2.On Q. < CD T3 •*—• Q. E < ° o ° o i.<H 00 o o o o o OOD 00 ODODJ,, o 20 — i — 22 oco 6>o c? Q coo orf oo<% ° °o°o % » — I — 24 — i — 26 — i 28 Period 68 t i s s u e . 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 overhead 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 were c o a t e d w i t h n o n - r e f l e c t i v e f l a t b l a c k p a i n t . However, one cannot d i s c o u n t t h e p o s s i b i l i t y t h a t t h e p i n e a l c o u l d r e c e i v e adequate l a t e r a l i l l u m i n a t i o n r e s u l t i n g from t h e d i f f r a c t i o n o f 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 t i s s u e s s u r r o u n d i n g t h e s k u l l . A l l v i s i b l e l i g h t was a b l e t o p e n e t r a t e t h e c l e a r p l a s t i c s h i e l d s . 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 between 200 and 970 my i n d i c a t e d t h a t o n l y t h o s e wavelengths i n t h e u l t r a v i o l e t r e g i o n were n ot t r a n s m i t t e d . I n t h e v i s i b l e spectrum (38 0 t o 7 0 0 my) t r a n s m i t t a n c e was a t a maximum. (a) B l a c k s h i e l d t e s t s s S i x f i s h r e p r e s e n t i n g 36 "days" 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 s e r i e s . The mean d a i l y r e s ponse t o a 12L 12D p h o t o p e r i o d a t 10°C i n d i c a t e d t h a t t h e presence o f t h e b l a c k s h i e l d s o ver t h e p i n e a l r e g i o n 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 r e sponse t o t h e l i g h t - d a r k environment ( F i g . 2 6 ) . However, i t was apparent t h a t p r e v e n t i n g i l l u m i n a t i o n o f t h e p i n e a l r e s u l t e d i n h i g h e r l e v e l s o f a c t i v i t y . (b) C l e a r s h i e l d t e s t s . Four f i s h r e p r e s e n t i n g 24 "days" o f a c t i v i t y c o n t r i b u t e d 69 Figure 26. Mean d a i l y a c t i v i t y patterns of black s h i e l d and c l e a r s h i e l d t e s t s : 9 • Black s h i e l d s O — O C l e a r s h i e l d s Mean no. of events 70 t o t h i s s e r i e s . The mean d a i l y r esponse i n d i c a t e d t h a t 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 w i t h t h e e n t r a i n m e n t t o th e p h o t o p e r i o d i c c y c l e (see F i g . 2 6 ) . However, s i g n i f i c a n t l y 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 when 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 b l a c k s h i e l d s . These two e x p e r i m e n t s compliment t h e d a t a o b t a i n e d w i t h p i n e a l e c t o m i z e d 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 e x e r t e d 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 a c t i v i t y r a t h e r t h a n d e t e r m i n i n g t h e e n t r a i n m e n t t o ph o t o -p e r i o d . These t e s t s a l s o p r o v i d e f u r t h e r i n d i c a t i o n t h a t p e n e t r a t i o n o f t h e c r a n i u m a l t e r s the b a s i c 24 hour p a t t e r n e x p r e s s e d by j u v e n i l e sockeye. 3.44 The e n t r a i n e d r e s ponse i n b l i n d e d f i s h . The ex p e r i m e n t s w i t h p i n e a l e c t o m i z e d f i s h s uggested t h a t m e d i a t i o n of t h e e n t r a i n e d r e s ponse o c c u r r e d v i a an e x t r a - p i n e a l i n f o r m a t i o n r o u t e . A t t e n t i o n was f o c u s e d upon t h e eyes as th e major (and perhaps most o b v i o u s ) p h o t o r e c e p t o r . T e s t i n g b l i n d e d i n d i v i d u a l s would c l a r i f y t h e r o l e o f t h e e y e s , and a combined e x p e r i m e n t w i t h b l i n d e d and p i n e a l e c t o m i z e d f i s h m ight i n d i c a t e whether o r not any e x t r a - r e t i n a l , e x t r a -p i n e a l pathway was i n v o l v e d . 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 approach. These i n c l u d e d t h e i n j e c t i o n o f phemerol c h l o r i d e (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 each eye, 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 , b i l a t e r a l 71 e n u c l e a t i o n of the eyes, and p l a c i n g black 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 subjected t o s u r g i c a l manipulation were allowed 2 weeks post-operative recovery before t e s t i n g . Data were c o l l e c t e d i n a manner s i m i l a r to previous t e s t s . (a) Chemical b l i n d i n g . E i g h t f i s h were prepared 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 of phemerol c h l o r i d e followed the p r e v i o u s l y described technique (Hoar, 1955b; Gunning, 1959; H a s l e r , 1966) . Gunning (1959) questions the usefulness of t h i s chemical, but bases h i s o b j e c t i o n s on a l t e r e d behaviour patterns i n the b l u e g i l l s u n f i s h , and does not provide a measure of the organism's a b i l i t y to detect l i g h t . 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 signs of recovery w i t h i n one week. I t was noted t h a t the c o l o r a t i o n was s i m i l a r to normal i n d i v i d u a l s and not as dark as those f i s h subjected to 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 to movements o r i g i n a t i n g o utside of the aquarium, and apparently detected these movements by v i s i o n . The i n d i v i d u a l d a i l y records i n d i c a t e d t h a t most f i s h responded to the photoperiod and maintained a day a c t i v e r e l a t i o n s h i p to the l i g h t c y c l e . One i n d i v i d u a l was apparently b l i n d e d by t h i s technique. This observation was based upon c o l o r a t i o n and response to movement outside of 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 e n v i r o n m e n t a l p h o t o p e r i o d . 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 r e s u l t s . 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, bu t not a l l i n d i v i d u a l s t r e a t e d . (b) The e n t r a i n e d response i n f i s h a f t e r s e c t i o n i n g t h e o p t i c n e r v e . E i g h t f i s h were 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 n e r v e . The mean d a i l y r e c o r d i n d i c a t e d an a c t i v i t y i n c r e a s e a f t e r t h e o n s e t o f l i g h t ( F i g . 27) . A response t o p h o t o p e r i o d was a p p a r e n t and t h e predawn 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 an endogenous component. Periodogram a n a l y s i s o f t h e i n d i v i d u a l r e c o r d s i n d i c a t e d t h a t s i x f i s h e x p r e s s e d an e x a c t 24.00 hour p e r i o d i c i t y . S l i g h t d e v i a t i o n s by 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 an average 24.07 hour p e r i o d f o r a l l f i s h t e s t e d . These d a t a s u g g e s t t h a t t h e a c t i v i t y r e s p onse and e n t r a i n m e n t t o p h o t o p e r i o d a r e n o t mediated v i a v i s u a l pathways such as t h e o p t i c n e r v e . (c) The e n t r a i n e d response i n b l i n d e d and p i n e a l e c t o m i z e d i n d i v i d u a l s . These e x p e r i m e n t s i n v e s t i g a t e d t h e i n f l u e n c e o f 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 erve 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 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 , and b i l a t e r a l l y e n u c l e a t e d groups. ( B l i n d i n g = s e v e r i n g o f o p t i c n e r v e ) . A — A B l i n d e d and P i n e a l e c t o m i z e d O — O B l i n d e d B i l a t e r a l E n u c l e a t e s 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 of post-op e r a t i v e 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 these f i s h were entrained to the environmental.photoperiod ( F i g . 27). A s i g n i f i c a n t a c t i v i t y i ncrease (P<.01) occurred at the onset of l i g h t and was terminated by a corresponding decrease at the onset of darkness. These data would tend to suggest the r o l e of 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 of photoreception. These t e s t s were based upon the assumption t h a t removal of a s e c t i o n of the 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 process d i s r u p t s a l l v i s u a l connection between the r e t i n a and the b r a i n , but non-vi s u a l a f f e r e n t neural connections from the r e t i n a to the CNS might be present. Gunning (1959) cautioned t h a t although the o p t i c nerve was severed, t h i s would not completely omit the p o s s i b i l i t y of a r e t i n a l response to the changing l i g h t c y c l e . This l e d to an assumption t h a t 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 transmi s s i o n of informa t i o n was v i a pathways other than the o p t i c nerve. (d) The entrained 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 the eyes. S i x f i s h were prepared f o r t h i s s e r i e s of t e s t s . The s u r g i c a l technique i s l i s t e d i n the appendix. A c t i v i t y recordings were obtained two weeks a f t e r the operation but 75 t h r e e f i s h f a i l e d t o s u r v i v e f o r t h e d u r a t i o n o f t h e e x p e r i m e n t . The r e m a i n i n g t h r e e i n d i v i d u a l s were t e s t e d f o r 11 days eac h , r e s u l t i n g i n 33 "days" o f a c t i v i t y r e p r e s e n t i n g t h e group. These d a t a i n d i c a t e d a v e r y low a m p l i t u d e o f a c t i v i t y and t h e c h a r a c t e r i s t i c day a c t i v e response t o p h o t o p e r i o d was n o t p r e s e n t ( F i g . 27). I t was t e n t a t i v e l y (due t o t h e s m a l l sample s i z e ) c o n c l u d e d t h a t t h e e n t r a i n e d r e s p o n s e t o t h e l i g h t c y c l e was mediated by t h e eyes. I f l i g h t f a l l i n g on any r e g i o n o f e x t r a - r e t i n a l p h o t o s e n s i t i v i t y was c a p a b l e o f e l i c i t i n g t h e e n t r a i n e d r e s ponse t h e r e s u l t s would be a p p a r e n t i n t h e s e t e s t s . B o t h t h e s e and t h e f o l l o w i n g s e r i e s o f t e s t s i n d i c a t 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 n o t i n v o l v e d . (e) The e n t r a i n e d r e sponse w i t h b l a c k eye c o v e r s . The f a t a l i t i e s a s s o c i a t e d w i t h b i l a t e r a l e n u c l e a t i o n i n d i c a t e d t h e b a s i c o b j e c t i o n t o t h a t t e c h n i q u e . B l a c k eye caps were d e s i g n e d as an a l t e r n a t e t e c h n i q u e w h i c h would p r e v e n t r e t i n a l s t i m u l a t i o n by l i g h t , w i t h o u t removing the eyes. C l e a r p l a s t i c caps w i t h t h e same a b s o r p t i o n c u r v e d e s c r i b e d f o r t h e p i n e a l s h i e l d s were used as a c o n t r o l . A t o t a l o f 21 f i s h were used i n t h e s e t e s t s c o n t r i b u t i n g a t o t a l o f 146 "days".of a c t i v i t y . The a p p l i c a t i o n o f b l a c k caps a b o l i s h e d t h e e n t r a i n e d r e s ponse t o t h e l i g h t c y c l e ( F i g . 28). The s i m i l a r i t y o f t h e r e sponse between normal f i s h and t h e f i s h a f t e r removal-76 Figure 28. Mean d a i l y a c t i v i t y patterns f o r black and c l e a r eye cap groups. • • Black Eye Caps O — O C l e a r Eye Caps I ! Mean no. of events 77 o f t h e b l a c k caps i n d i c a t e d t h a t e n t r a i n m e n t 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 o n l y ( F i g . 29) . I f t h e eye caps were removed d u r i n g t h e c o u r s e o f an e x p e r i m e n t , the s u b j e c t would respond i m m e d i a t e l y 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 . B e f o r e cap removal an endogenous f o u r hour p e r i o d i c i t y was n o t e d . T h i s was an u l t r a d i a n response t o t h e c o n s t a n t environment ( l e s s t h a n a day, b u t r a n g i n g between 0 and 2 0.00 hours) and was s i m i l a r t o t h e s h o r t p e r i o d i c i t y o b s e r v e d i n t h e DD t e s t s . No endogenous p a t t e r n s w i t h 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 obse r v e d i n f i s h w i t h b l a c k 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 t h e a ssumption t h a t DD might i n h i b i t t h e e x p r e s s i o n o f an endogenous rhythm by j u v e n i l e sockeye salmon. The r e sponse o b t a i n e d w i t h t h e c l e a r p l a s t i c s h i e l d s appeared t o be c h a r a c t e r i s t i c o f a day a c t i v e o r g a n i s m i n s i n g l e i n d i v i d u a l s , b u t t h e grouped d a t a i n d i c a t e d h i g h l e v e l s o f a c t i v i t y and 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 (see F i g . 2 8 ) . The c o n s t r u c t i o n o f t h e p l a s t i c d i s c s f o r t h e s e 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 w h i c h i n t e r f e r e d w i t h t h e o p t i c s o f t h e i n t a c t eye, and might be r e s p o n s i b l e f o r t h e s e v a r i a t i o n s i n a c t i v i t y . These d a t a i n d i c a t e d a d e f i n i t e s o u r c e o f r e c e p t i o n f o r t h e e n t r a i n e d r e sponse t o p h o t o p e r i o d i n t h i s s p e c i e s . S i n c e t h e i n f o r m a t i o n about t h e l i g h t c y c l e i s n o t t r a n s m i t t e d v i a t h e o p t i c n e r v e , i t i s s u g g ested t h a t t h e eye might 78 Figure 29. 12L 12D,5°C response i n normal f i s h and f i s h t e s t e d immediately a f t e r removal of the black eye caps. O — O Normal f i s h Black eye caps removed Mean no. of events _k N> a i o i cn 79 secrete a blood-born agent which 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 Melatonin, Serotonin and Teleost  S a l i n e on the Entrained Response. The data from the preceding 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 production of c e r t a i n chemical agents may be i n v o l v e d i n entrainment. Those i n d i v i d u a l s subjected to pinealectomy or black s h i e l d s over the p i n e a l region e x h i b i t e d s i g n i f i c a n t l y higher a c t i v i t y l e v e l s . The entrained response and the a c t i v i t y l e v e l were both .altered by removal of the eyes and the a p p l i c a t i o n of black caps. The o b j e c t i v e of t h i s s e r i e s was to determine i f the i n j e c t i o n of melatonin and s e r o t o n i n would induce s i g n i f i c a n t 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. Since the p i n e a l i s g e n e r a l l y considered as the major s i t e of melatonin production, i t i s assumed th a t lower melatonin l e v e l s w i l l be present f o l l o w i n g pinealectomy (unless compensatory increases occur i n the r e t i n a ) . This decrease i n the l e v e l of melatonin 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. A l t e r n a t i v e l y , the increased a c t i v i t y l e v e l might be due to increased l e v e l s of s e r o t o n i n , the precursor of melatonin. This information coupled w i t h the f i n d i n g s of Wong and Whiteside (1968) l e d to the f o r m u l a t i o n of a working hypothesis 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 melatonin, and no change wi t h i n j e c t i o n s of t e l e o s t s a l i n e . A t o t a l of eighteen f i s h were t e s t e d i n three groups of nine days each. The f i r s t three t e s t days were focused upon the locomotor a c t i v i t i e s i n a 12L 12D (10°C) environ-ment before 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 i n j e c t i o n s . The second three days of the t e s t were focused upon the expression f o l l o w i n g 0.25 cc d a i l y i n j e c t i o n s (lug per gram body weight) of melatonin, s e r o t o n i n , or t e l e o s t s a l i n e . A l l i n j e c t i o n s were administered 1/2 hour before the dark phase of the photoperiodic c y c l e . The f i n a l three days examined the a c t i v i t y records of these i n d i v i d u a l s a f t e r 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 s e r o t o n i n ( F i g . 30). Melatonin i n j e c t i o n s r e s u l t e d i n decreased a c t i v i t y , whereas no gross changes occurred i n the t e l e o s t s a l i n e treatment. There was an 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 i n a l l t e s t s might be a t t r i b u t e d to the method of removing the 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 of the mean d a i l y a c t i v i t y records f o r the c o n t r o l group and melatonin treatment i n d i c a t e d t h a t the decrease i n a c t i v i t y occurred mainly during the l i g h t phase of the photoperiodic c y c l e ( F i g . 31). Conversely, the major area of a c t i v i t y i ncrease f o r the serot o n i n treatment occurred during the dark phase of the photoperiod. 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 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 i n j e c t e d groups. B = B e f o r e I n j e c t i o n D = D u r i n g I n j e c t i o n A = A f t e r I n j e c t i o n Mean no. of events co cn -NI o o o 83 4. DISCUSSION The general importance of 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 the amount of i n t e r e s t the subject has generated i n the past f i f t e e n years. Research has included examinations of d a i l y , t i d a l , lunar and annual c y c l e s , and how these r e l a t e to m i g r a t i o n , h i b e r n a t i o n , breeding, c o l o r changes, metabolic processes, feeding and locomotor a c t i v i t y p a t t e r n s . The m a j o r i t y of i n t e r e s t has focused upon mammalian and avian 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 usefulness i n long term l a b o r a t o r y p r o j e c t s . As a r e s u l t , most recording techniques have been developed f o r a few t e r r e s t r i a l organisms. The apparent l a c k of 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 the meagre supply of adequate instrumentation f o r reco r d i n g locomotor a c t i v i t y . However, recent advances i n the design of e l e c t r o n i c components and t h e i r a p p l i c a t i o n (Cummings, 1963; Muir, et a l . , 1965; M e f f e r t , 1968) have provided the opportunity to 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 of aquatic organisms. Environmental c o n t r o l was of paramount importance to t h i s i n v e s t i g a t i o n , and represented a f a c t o r not a t t a i n a b l e i n the f i e l d . However, the u l t i m a t e goal was to apply the la b o r a t o r y based hypotheses to the 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 c o n t r o l systems used i n t h e l a b o r a t o r y were adequate and f u l f i l l e d t h e r e q u i r e m e n t s o u t l i n e d by Bunning (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 ca 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 can s i g n i f i c a n t l y i n t e r f e r e w i t h e x p e r i m e n t s , o r t h a t an o b s e r v a t i o n l i g h t , even i f a p p l i e d f o r o n l y a few seconds can 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 w h i c h e n a b l e d t h e i n v e s t i g a t i o n t o pro 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 the c o n t r o l l e d e nvironment. The g e n e r a l a p p l i c a b i l i t y o f t h e t r a n s d u c e r was t e s t e d by r e c o r d i n g a c t i v i t y b e f o r e , d u r i n g and a f t e r i n s t r u m e n t o p e r a t i o n . 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 f a c i l i t a t e a l o n g term s t u d y o f l o c o m o t o r a c t i v i t y i n t h e s e f i s h . Spontaneous l o c o m o t o r a c t i v i t y i s an e a s i l y measured b e h a v i o u r a l r e sponse t o endogenous and exogenous f a c t o r s w h i c h has been i n v e s t i g a t e d 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). T h i s p a r t i c u l a r measure of b e h a v i o u r was used t o i n d i c a t e the e n t r a i n e d r e sponse 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 assay o f l i g h t p e r c e p t i o n . 4.1 The B a s i c A c t i v i t y P a t t e r n A d e f i n i t e e n t r a i n e d r e sponse was o b t a i n e d i n an a r t i f i c i a l (LD) environment. 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 the p r i m a r y e n t r a i n i n g agent 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 o r groups of f i s h . The response a t d i f f e r e n t d e v e l o p m e n t a l s t a g e s 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 f i r m l y e s t a b l i s h e d a t an e a r l y age and m a i n t a i n e d f o r a t l e a s t one y e a r . S u f f i c i e n t i n f o r m a t i o n was not a v a i l a b l e t o d e t e r m i n e t h e 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 age. 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 Groot (1965) suggested t h a t t h e r e was a b a s i c a c t i v i t y rhythm o f sockeye salmon, young or o l d , m i g r a t i n g o r n o t . 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 P e n i n s u l a ) e x p r e s s e d a s i m i l a r a c t i v i t y p a t t e r n a t 5°C. However, b o t h s t o c k s ( L a k e l s e and Kamchatka) were 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 may 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 r e s p o n s e s . I t was not known i f s i m i l a r d a t a would be o b t a i n e d i f i d e n t i c a l 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 l o c a t i o n s . 4.11 The i n f l u e n c e o f t e m p e r a t u r e and p h o t o p e r i o d . Both t e m p e r a t u r e 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 r e s p o n s e . I n c r e a s i n g t e m p e r a t u r e s u s u a l l y r e s u l t e d i n h i g h 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 b o t h l i g h t and d a r k , but when s h o r t d a y l e n g t h s were combined w i t h h i g h t e m p e r a t u r e s , d e c r e a s e d a c t i v i t y o c c u r r e d . A unimodal a c t i v i t y p a t t e r n 86 i was expressed when short daylengths and low temperatures were used. When e i t h e r daylength or temperature in 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 photoperiod can produce changes i n the contour of the 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 of the photobehavioural response. I t i s hypothesized t h a t f o r each daylength there i s a c e r t a i n temperature, above which j u v e n i l e sockeye salmon w i l l reverse the 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 temperatures w i l l f u n c t i o n as the major " r e v e r s i n g " agent, whereas the photoperiod w i l l f u n c t i o n as a " c o n t r o l l i n g " agent. In t h i s way, i n c r e a s i n g 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 data presented r e f e r only to 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 are supported by the work of Gibson and Keenleyside (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 reversed photobehavioural response i n young A t l a n t i c salmon. Po i k i l o t h e r m s g e n e r a l l y respond to i n c r e a s i n g temperatures ( w i t h i n the l i m i t s of tolerance) w i t h greater metabolic a c t i v i t y . Increased locomotor a c t i v i t y might then 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 metabolic s t a t e . The data presented i n s e c t i o n 3.24 c l e a r l y i n d i c a t e t h i s 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 c o m p l i c a t e s t h e i n t e r p r e t a t i o n o f t h e d a t a . 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 f a c t o r , but t h e a c t u a l mechanisms remai n t o be d e t e r m i n e d . I t appears t h a t f o r each d a y l e n g t h t h e r e i s an optimum tem 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 r e s i s t a n c e t o h e a t . S e a s o n a l l y c h a n g i n g p h o t o p e r i o d s might a d j u s t t h e m e t a b o l i s m o f f i s h t o a n t i c i p a t e s harp t e m p e r a t u r e changes i n t h e s p r i n g and autumn. However, c o n f l i c t i n g e n v i r o n m e n t a l s t i m u l i ( s h o r t days and h i g h t e m p e r a t u r e s ) a p p a r e n t l y r e s u l t i n a r e v e r s a l o f the p h o t o b e h a v i o u r a l r e s p o n s e . A s c h o f f (1960) suggested t h a t a t d i f f e r e n t l i g h t i n t e n s i t i e s t h e r e was an optimum l e v e l , above which an organis m would seek the o p p o s i t e response i n t h e a c t i v i t y / r e s t r a t i o . A s i m i l a r mechanism might o c c u r h e r e , where a m a n i p u l a t i o n o f t h e t e m p e r a t u r e - d a y l e n g t h 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 a c t i v i t y . These d i f f e r e n t a c t i v i t y r e s p o n s e s t o te m p e r a t u r e and p h o t o p e r i o d might a l s o account f o r some o f t h e c o n f l i c t i n g 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 o c c u r r e d a t s e p a r a t e g e o g r a p h i c a l l o c a t i o n ( G r o o t , 1965; Thompson and B u r g n e r , 1967). 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 i n c r e a s e i n a c t i v i t y p r i o r t o t h e 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 suggested an endogenous 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 l e s s t h a n 2 4 . 0 0 h o u r s . Those e x p e r i m e n t s conducted i n c o n s t a n t l i g h t i n d i c a t e d t h a t t h e response was m a i n t a i n e d i n absence o f p e r i o d i c e n v i r o n m e n t a l s t i m u l i . A f r e e - r u n n i n g rhythm w i t h a p e r i o d a p p r o x i m a t i n g 2 3 . 3 0 hours was i d e n t i f i e d . These d a t a s u p p o r t e d t h e h y p o t h e s i s o f Groot ( 1 9 6 5 ) and Hoar ( 1 9 6 5 ) w h i c h suggested t h e p r e s e n c e o f an endogenous c l o c k i n t h i s s p e c i e s . The r e s p o n s e t o 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 was n o t i n a c c o r d a n c e w i t h A s c h o f f ' s g e n e r a l r u l e ( 1 9 6 0 ) w h i c h s t a t e s : "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 t h e p e r i o d o f a l i g h t a c t i v e o r g a n i s m w i l l d e c r e a s e " . However, 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 s e v e r a l e x c e p t i o n s have been noted i n t h e l i t e r a t u r e ( r e v i e w : Hoffmann, 1 9 6 5 ) . The i n c r e a s i n g p e r i o d i n t h e s e j u v e n i l e sockeye might 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 a b i l i t y 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 a t d i f f e r e n t s t a g e s i n t h e l i f e c y c l e . I t would be i n t e r e s t i n g t o d e t e r m i n e whether o r not any changes o c c u r i n t h e endogenous component d u r i n g t h e c o u r s e o f development, 89 e s p e c i a l l y d u r i n g t h e p e r i o d s when m i g r a t i o n s c o i n c i d e w i t h n o c t u r n a l a c t i v i t y . P e r i o d i c f e e d i n g c o u l d a l s o a f f e c t t h e endogenous c y c l e i n an o t h e r w i s e c o n s t a n t e n v i r o n m e n t . I t i s c o n c l u d e d t h a t f o o d w i l l have t o be e i t h e r c o n t i n u o u s l y p r e s e n t o r c o n t i n u o u s l y a b s e n t f o r s h o r t term e x p e r i m e n t s . 4.3 Mechanism o f E n t r a i n m e n t A c o n s i d e r a t i o n o f t h e eyes and p i n e a l body i n d i c a t e d t h a t e n t r a i n m e n t o c c u r r e d t h r o u g h r e t i n a l m e d i a t i o n . S i n c 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 d i d n o t a b o l i s h t h e e n t r a i n e d r e s p o n s e , b u t p r e s u m a b l y i n d u c e d " b l i n d n e s s " , t h e v i s u a l r e s p o n s e i n image f o r m a t i o n and d e t e c t i o n o f l i g h t i n t e n s i t y m i g h t be b a s e d upon e n t i r e l y d i f f e r e n t p r o c e s s e s . I t i s h y p o t h e s i z e d t h a t p h o t o p e r i o d i c i n f o r m a t i o n m i g h t be t r a n s m i t t e d by some system o t h e r t h a n t h e o p t i c n e r v e ( p o s s i b l y v a s c u l a r ) . However, i t i s e q u a l l y f e a s i b l e t o c o n s i d e r t h e p o s s i b i l i t y o f some unknown a f f e r e n t n e r v o u s pathways from t h e r e t i n a . The h y p o t h e s i s was b a s e d upon t h e f o l l o w i n g i n f o r m a t i o n . F i r s t , r e m o v a l o f t h e e y e , o r p l a c i n g b l a c k d i s c s o v e r i t s u p p r e s s e d t h e e n t r a i n e d r e s p o n s e ( i . e . a p p a r e n t l y t h e f i s h c o u l d n o t d e t e c t t h e p h o t o p e r i o d i c e n v i r o n m e n t ) . S e c o n d l y , s e c t i o n i n g t h e o p t i c n e r v e d i d n o t r e s u l t i n l o s s o f e n t r a i n m e n t ( i . e . t h e f i s h c o u l d d e t e c t t h e l i g h t c y c l e ) . T h i s i n f o r m a t i o n i n d i c a t e d t h a t some change o c c u r r e d w i t h i n 90 the eye, and the r e s u l t i n g a c t i v i t y response was not mediated by pathways w i t h i n the o p t i c nerve. The p o s s i b i l i t y of entrainment by other photoreceptors such as the p i n e a l body was r e j e c t e d since entrainment 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 organ i n t a c t . However, Menaker (1968) and Gaster and Menaker (1968) demonstrated t h a t i l l u m i n a t i o n of the p i n e a l could serve to e n t r a i n b l i n d e d sparrows. Conversely, Quay (1968) f a i l e d to demonstrate any p i n e a l mediation i n the a c t i v i t y response of the 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 entrainment was a response to i l l u m i n a t i o n of the r e t i n a . 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 the p i n e a l body i n j u v e n i l e sockeye salmon was not i n v o l v e d i n the e n t r a i n e d response. However, removal of the p i n e a l or shading 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 l i g h t and dark. Since entrainment 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 others by r e t i n a l mediation, one might f i r s t look 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 (hydroxyindole-o-methyl transferase) 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 methylating enzyme r e s p o n s i b l e f o r the formation of melatonin (N-acetyl-•5-methoxy-tryptamine) from i t s precursor N-acetyl s e r o t o n i n (N-acetyl-5-hydroxy-tryptamine). Fenwick ( i n press) has 91 demonstrated the presence of both r e t i n a l and pineal melatonin and serotonin i n two cl o s e l y related species of P a c i f i c salmon. Bagnara (I960), Wurtman, et a l . , (1963) and Quay (1964) have shown that an apparent c o r r e l a t i o n exists between the environmental l i g h t cycle and variations i n the l e v e l of pineal melatonin and serotonin i n several higher vertebrates. Quay 1s experiments with the laboratory r a t indicated low serotonin levels during dark, with a 900% increase during l i g h t . Reiss, et a l . , (1963) and Wong and Whiteside (1968) have demonstrated that injections of pineal extracts 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 wheel-running a c t i v i t y of laboratory r a t s . I t was hypothesized that intraperitoneal injections of melatonin might reduce locomotor a c t i v i t y i n juvenile sockeye salmon i f sim i l a r correlations existed between the production of t h i s chemical and the environmental l i g h t cycle. If an opposing response could be attributed to serotonin, an increased amplitude of a c t i v i t y might be expected. The laboratory tests indicated that serotonin increased the a c t i v i t y l e v e l during the dark phase of the photoperiod and melatonin decreased the a c t i v i t y l e v e l during the l i g h t phase. Since the pineal i s generally considered to be the major s i t e of melatonin production, the triggered release by either r e t i n a l information or d i r e c t illumination may 92 i n v o l v e d . However, th e mechanisms by w h i c h t h i s r e l e a s e o c c u r s remain t o be d e t e r m i n e d . The o n s e t o f d a r k n e s s might f a c i l i t a t e m e l a t o n i n s y n t h e s i s from s e r o t o n i n . The i n c r e a s e d m e l a t o n i n l e v e l s may 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 a c t i v i t y 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 f u n c t i o n w i t h i n t h e o r g a n i s m remains t o be d e t e r m i n e d . I t i s n o t known whether t h e m e l a t o n i n formed i s s e c r e t e d as a hormone, o r where th e s p e c i f i c s i t e s o f a c t i o n a r e l o c a t e d . S e r o t o n i n (5-HT) o r i g i n a t e s as a p r o d u c t o f t r y p t o p h a n m e t a b o l i s m . The a b s o l u t e amounts i n t h e b r a i n a r e s m a l l , b u t appear 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 t h e c e n t r a l nervous system. I n mammals, 5-HT has been i d e n t i f i e d i n many t i s s u e s and l a r g e amounts have been found i n t h e b l o o d p l a t e l e t s . 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 may a c t as a t r a n s m i t t e r s u b s t a n c e (Roche, 1964). 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 i s 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 . I n j u v e n i l e sockeye salmon, 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 o f 5-HT i n c r e a s e d d a r k a c t i v i t y . Here a g a i n , i t i s not known whether 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 p e r i p h e r a l a r e a s . A d m i n i s t r a t i o n o f 5 - h y d r o x y t r y p t o p h a n (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 i n c r e a s e t h e 93 amounts o f 5-HT i n most t i s s u e s . Roche (1964) i n d i c a t e s t h a t dogs and c a t s respond t o i n c r e a s e d 5-HT l e v e l s w i t h s o m a t i c , autonomic and b e h a v i o u r a l changes r e s e m b l i n g t h o s e produced by l y s e r g i c a c i d d i e t h y l a m i d e ( i . e . e x c i t e m e n t 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 r a g e , b l i n d n e s s and d i s o r i e n t a t i o n ) . My d a t a s uggest a p r o f i t a b l e avenue of f u t u r e r e s e a r c h . 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 s y n t h e s i s o f i n f o r m a t i o n on t h e o v e r t e n t r a i n e d r e sponse w i t h t h e c h e m i c a l and c e l l u l a r changes o c c u r r i n g i n t h e c e n t r a l nervous system would be most v a l u a b l e . 4.4 The A d a p t i v e S i g n i f i c a n c e o f a D a i l y A c t i v i t y C y c l e . The e x p r e s s i o n o f an a c t i v i t y rhythm i s not u n i q u e , but t h e f u n c t i o n a l s i g n i f i c a n c e o f such a r e s p o n s e i s e v a s i v e . One o b v i o u s advantage might be t o s y n c h r o n i z e t h e v a r i o u s p h y s i o l o g i c a l p r o c e s s e s w i t h d a i l y , s e a s o n a l and y e a r l y c y c l e s , t h u s e n a b l i n g t h e o r g a n i s m t o f u l l y 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 o f s e n s i t i v i t y a r e s y n c h r o n i z e d 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 ( A s c h o f f , 1965b), an o r g a n i s m c o u l d make f u l l use o f i t s s e n s o r y i n f o r m a t i o n i n p e r f o r m i n g such d i v e r s e a c t i v i t i e s as f e e d i n g , s c h o o l i n g , m i g r a t i n g o r e v a d i n g p r e d a t o r s . However, the i n t e r a c t i o n between endogenous and exogenous components c o m p l i c a t e s t h e s i t u a t i o n and o b s c u r e s any s i m p l e answer t o t h e g e n e r a l 94 s i g n i f i c a n c e o f b i o l o g i c a l r h y t h m i c i t y . The i n t e r a c t i o n o b s e r v e d between te m p e r a t u r e and p h o t o p e r i o d s u p p o r t e d t h e h y p o t h e s i s t h a t t h e r m a l 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 (Hoar, 1965b) ( i . e . l o n g p h o t o p e r i o d s c o u l d i n c r e a s e r e s i s t a n c e t o h e a t w h i l e s h o r t p h o t o p e r i o d s c o u l d i n c r e a s e r e s i s t a n c e t o c h i l l i n g ) . My s t u d y i n d i c a t e d t h a t l o n g p h o t o p e r i o d s i n c r e a s e d 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 a 50% d i s t r i b u t i o n o f l i g h t / d a r k a c t i v i t y . I t was f u r t h e r demonstrated t h a t i f t h e e n v i r o n m e n t a l t e m p e r a t u r e exceeded a c e r t a i n l i m i t , t h e r e was a 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 and j u v e n i l e sockeye salmon became dark a c t i v e . The c h a r a c t e r i s t i c n o c t u r n a l s m o l t o u t m i g r a t i o n might r e p r e s e n t a s i m i l a r r e s ponse t o t h e r a p i d l y i n c r e a s i n g w ater t e m p e r a t u r e s i n s p r i n g . 4.41 Emergence and m i g r a t i o n . Under n a t u r a l c o n d i t i o n s sockeye f r y a r e n e g a t i v e l y p h o t o t a c t i c and 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. Those f r y r e a d y t o m i g r a t e emerge a t dusk, e x h i b i t an a p p a r e n t n e g a t i v e r h e o t a x i s and move downstream 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 do n o t s c h o o l . A f t e r r e a c h i n g t h e l a k e t h e 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 remain swimming and f e e d i n g d u r i n g t h e day (Hoar, 1954, 1958; McDonald, 1960; Hartman, e t a l . , 1962; Heard, 1965). 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 evidence f o r a bimodal a c t i v i t y p a t t e r n immediately a f t e r hatching. A small morning a c t i v i t y peak occurred at the onset of l i g h t , and a major evening a c t i v i t y peak occurred a f t e r the onset of darkness. These peaks were separated by a midday depression of a c t i v i t y which p e r s i s t e d f o r approximately ten days. Newly emerged f r y i n a s w i f t l y f l o w i n g stream, unable to perform v i s u a l landmark o r i e n t a t i o n , might be subjected to a passive downstream displacement. The high amplitudes 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 , coupled w i t h reduced v i s u a l a c u i t y ( A l i , 1959) suggest a mechanism f o r downstream displacement. Upstream migrants (a s i t u a t i o n where the spawning grounds are i n t r i b u t a r i e s f l o w i n g out of the nursery lake) express 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 , pers. comm.). Andrew and Geen (1960) re p o r t t h a t i n the C h i l c o lake system, the 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 along the r i v e r edge. The f r y e v i d e n t l y hold i n these areas and move upstream seven to ten 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 ten days, downstream migrant f r y became l i g h t a c t i v e . The increased l e v e l s of day a c t i v i t y coupled w i t h a strong r h e o t a x i s would be of paramount importance to upstream m i g r a t i o n during the day. This study would favour the hypothesis 96 t h a t upstream 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 b e h a v i o u r 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 days a d d i t i o n a l development) and swimming a b i l i t y (Hoar, 1958). However, 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 response f o r upstream and downstream m i g r a n t s (see a l s o R a l e i g h , 1967; Brannon, 1967). 4.42 P e l a g i c s t a g e . A f t e r e n t e r i n g t h e n u r s e r y l a k e s , f r y d i s p e r s e a l o n g shore and l a t e r become e n t i r e l y p e l a g i c . T h i s s t u d y suggested t h a t l i g h t a c t i v i t y was m a i n t a i n e d i n t h o s e age groups n o r m a l l y r e s i d e n t i n t h e n u r s e r y l a k e s . The e x p e r i m e n t s examining t h e e f f e c t o f age on t h e e n t r a i n e d r e s p o n s e , and t h e two y e a r a n a l y s i s o f r e l a t e d d a t a i n d i c a t e d t h e g e n e r a l a p p l i c a b i l i t y o f 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 group. 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 groups 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 c y c l e . T h i s p r o v i d e d a mechanism 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 day. T h i s mechanism might 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 c o h e s i v e n e s s o f a s c h o o l and may a l s o p r o v i d e g r e a t e r a c c u r a c y i n t i m i n g t h e o n s e t o f d a i l y a c t i v i t y . The i n v e s t i g a t i o n a t Babine Lake, B.C. w i t h "sea 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 i n a c t i v i t y and absence o f dense s c h o o l s a t n i g h t ( N a r v e r , u n p u b l i s h e d ) . M a r s h a l l (1965) and Denton and N i c o l (1966) emphasized t h e 97 r o l e of v i s i o n i n school i n g s p e c i e s , but i n d i c a t e d t h a t other sensory systems were undoubtedly i n v o l v e d . Schooling i s apparently a v i s u a l response i n t h i s species (Hoar, 1958; A l i , 1959), a f a c t o r suggesting the general importance of l i g h t a c t i v i t y . 4.43 Feeding. The importance of v i s i o n i n the l o c a t i o n and capture of food i s a c h a r a c t e r i s t i c of P a c i f i c salmon (Hoar, 1958; A l i , 1959; B r e t t and Groot, 1963). F i e l d observations i n d i c a t e d t h a t feeding occurred during the day i n upstream migrant f r y (McCart, 1967) and Babine lake pre-smolts (Narver, pers. 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 t h a t feeding a c t i v i t y occurred when the f i s h were 150 to 200 f e e t deep, however, some feeding a l s o occurred at dawn and dusk before 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 (Foerster, 1968). Groot (1965) demonstrates dawn and dusk peaks i n surface feeding a c t i v i t y f o r m i g r a t i n g smolts (at 14°C avg. temperature). The high l e v e l s of a c t i v i t y during the day, coupled w i t h increased v i s u a l a c u i t y might f a c i l i t a t e the l o c a t i o n and capture of prey. The feeding response i s apparently e s t a b l i s h e d at an e a r l y age a f t e r c l o s u r e of the yol k sac, when the f i s h have developed a l i g h t a c t i v e p a t t e r n . C e r t a i n combinations of temperature and photoperiod may determine a bimodal p a t t e r n of feeding a c t i v i t y i n the summer, and a unimodal p a t t e r n of feeding a c t i v i t y i n the win t e r . 98 4.44 D i e l v e r t i c a l m i g r a t i o n s . The v e r t i c a l movements o f t h i s s p e c i e s have been w e l l documented (Johnson, 1956, 1961; B u r g n e r , 1962), however t h e p a t t e r n s o f movement and d i s t r i b u t i o n a r e not i d e n t i c a l i n a l l sockeye l a k e s . I n Babi n e Lake, B.C. t h e m i g r a t i o n p a t t e r n can be d i v i d e d i n t o f o u r phases ( N a r v e r , p e r s . comm.). 1. Dawn, v e r t i c a l d e s c e n t t o depths a p p r o x i m a t i n g 150 t o 200 f e e t . 2. Midday, h o l d i n g a t d e p t h . 3. Dusk, v e r t i c a l a s c e n t w i t h a s h o r t p e r i o d o f a c t i v i t y a t t h e s u r f a c e . 4. N i g h t , d e s c e n t t o 10 t o 25 f e e t and r e l a t i v e i n a c t i v i t y . The r e s p o n s e s o b t a i n e d i n t h e l a b o r a t o r y p a r a l l e l t h e v a r i o u s a c t i v i t i e s o b s e r v e d 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 , s c h o o l i n g and 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 t h e h o l d i n g r e sponse i n t h e l a k e , t h e 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 i n d i v i d u a l s ( i . e . the 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 s a m p l i n g t e c h n i q u e s a t n i g h t (Burgner, 1962) p r o v i d e d a d d i t i o n a l s u p p o r t f o r t h e i r p r e s e n c e i n t h i s l a y e r . However, my i n v e s t i g a t i o n s u g g e sted t h a t i n d i v i d u a l s were r e l a t i v e l y i n a c t i v e a t n i g h t w h i l e p r e s e n t a t o r near t h e s u r f a c e . 99 4.5 S e a s o n a l R e g u l a t i o n o f t h e E n t r a i n e d Response T h i s 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 s h o r t p h o t o p e r i o d s and low t e m p e r a t u r e s (8L 16D, 5°C) r e s u l t e d i n a unimodal a c t i v i t y p a t t e r n . Long 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 (16L 8D, 15°C) r e s u l t e d i n a b i m o d a l p a t t e r n w i t h i n c r e a s e d a m p l i t u d e s o f a c t i v i t y d u r i n g b o t h l i g h t and d a r k . Combining s h o r t p h o t o p e r i o d s w i t h h i g h t e m p e r a t u r e s (8L 16D, 15°C) r e s u l t e d i n an apparent 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 . A t L a k e l s e Lake, B.C. t h e t e m p e r a t u r e s from December 15th t o A p r i l 1 5 t h approximate 3.5°C between 20 f e e t and 80 f e e t d e p t h s . May and June a r e c h a r a c t e r i z e d by a r a p i d t e m p e r a t u r e i n c r e a s e (10°C t o 15°C) i n t h e upper f o r t y f e e t . The water t e m p e r a t u r e s drop t o t h e w i n t e r l e v e l s from September 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 a t L a k e l s e Lake, a f a c t o r s u g g e s t i n g the g e n e r a l a p p l i c -a b i l i t y o f t h i s s t u d y t o f i e l d c o n d i t i o n s . Assuming t h a t the l a b o r a t o r y s t u d y approximated t h e g e n e r a l p a t t e r n i n th e f i e l d , we 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 a c t i v i t y r e s p o n s e . W i n t e r : S h o r t p h o t o p e r i o d s and low t e m p e r a t u r e s i n most sockeye l a k e s may r e s u l t i n a unimodal a c t i v i t y p a t t e r n . L i g h t i n t e n s i t y can be f u r t h e r d e c r e a s e d by i c e and snow c o v e r . A unimodal a c t i v i t y p a t t e r n would e n a b l e r e s i d e n t sockeye t o u t i l i z e a l l a v a i l a b l e d a y l i g h t f o r t h e c a p t u r e 100 of food. Growth and energy expenditure are at t h e i r lowest annual l e v e l , responding to the low temperatures and decreased food supply. Spring: I n c r e a s i n g photoperiod and temperature might r e s u l t i n higher l e v e l s and increased durations of a c t i v i t y . Ice breakup precedes the smolt migrations to a marine environment. Foerster (1937) provided 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 migrations from Cultus Lake, B.C. The increased n o c t u r n a l a c t i v i t y may be a response to the r a p i d temperature increases i n s p r i n g and could 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 of movement to many smolt m i g r a t i o n s . Further increases i n photoperiod could a ct to s t a b i l i z e the day a c t i v e r e l a t i o n s h i p of the 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 to the outmigration. Summer: The response to long photoperiods and high 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 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 bimodal, w i t h a c t i v i t y peaks o c c u r r i n g at dawn and at dusk. F a l l : Decreasing photoperiod and temperature could r e g u l a t e d a r e v e r s a l of the process, r e s u l t i n g i n the expression of a unimodal a c t i v i t y p a t t e r n during the win t e r . To s t a t e t h a t the organism i s a complete subject of 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 of the problem. Entrainment i s apparently the response to an endogenous 101 pattern of physiological e x c i t a b i l i t y that i s r e f l e c t e d by increases and decreases i n the locomotor a c t i v i t y . The entrained response i s a synchronization of t h i s endogenous pattern with c e r t a i n environmental cues acting as s t i m u l i for the onset and termination of the a c t i v i t y cycle (Aschoff, et a l . , 1965). 102 5. SUMMARY AND CONCLUSIONS 1. 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 the 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 p a t t e r n o f l o c o m o t o r a c t i v i t y i n j u v e n i l e sockeye salmon. 2. The b a s i c measure employed 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 spontaneous l o c o m o t o r a c t i v i t y . H i g h f r e q u e n c y sound was used 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 i n 36" (91.4 cm) d i a m e t e r swimming c h a n n e l s . 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 ap 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 o r t h e e x p e r i m e n t a l s u b j e c t s . 3. The i n f l u e n c e o f p h o t o p e r i o d and temp e r a t u r e on t h e e n t r a i n e d a c t i v i t y p a t t e r n was i n v e s t i g a t e d . The a r t i f i c i a l 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 . The a c t i v i t y 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 dark a c t i v e i m m e d i a t e l y a f t e r emergence. A l i g h t a c t i v e p a t t e r n was de v e l o p e d two weeks a f t e r emergence and was m a i n t a i n e d f o r a t l e a s t one y e a r . 4. Both p h o t o p e r i o d and te m p e r a t u r e i n f l u e n c e d t h e 24 hour a c t i v i t y p a t t e r n i n y e a r l i n g f i s h . Low t e m p e r a t u r e s (5°C) f a c i l i t a t e d l i g h t a c t i v i t y a t a l l p h o t o p e r i o d s , whereas h i g h t e m p e r a t u r e s (15°C) combined 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 dark a c t i v i t y . 103 5. The 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 o f sockeye f r y was b i m o d a l i m m e d i a t e l y a f t e r emergence. Y e a r l i n g s responded t o low t e m p e r a t u r e s and s h o r t p h o t o p e r i o d s w i t h a unimodal 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 t e m p e r a t u r e and/or p h o t o p e r i o d r e s u l t e d i n a b i m o d a l a c t i v i t y p a t t e r n . 6. T o t a l a c t i v i t y was i n c r e a s e d a t the h i g h e r t e m p e r a t u r e s used. The t e m p e r a t u r e - p h o t o p e r i o d i n t e r a c t i o n i n d i c a t e d t h a t l o n g e r p h o t o p e r i o d s (16L 8D) f a c i l i t a t e d a l i g h t a c t i v e p a t t e r n a t 15°C. 7. The endogenous rhythm was examined i n a c o n s t a n t environment. The p e r i o d v a l u e was 23.30 hours i n 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 i n c r e a s i n g l i g h t i n t e n s i t y . 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 e n t r a i n i n g agent. 8. The eyes were i d e n t i f i e d as t h e p r i m a r y s e n s o r y 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 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 . T r a n s f e r o f i n f o r m a t i o n was not v i a t h e o p t i c n e r v e . T h i s suggested t h e p o s s i b i l i t y o f o t h e r a f f e r e n t nervous pathways from t h e r e t i n a , o r m e d i a t i o n by a b l o o d - b o r n agent. 9. The p i n e a l body 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 . However, b o t h p i n e a l e c t o m y and i n s e r t i n g s h i e l d s o v er t h e p i n e a l r e g i o n i n c r e a s e d t h e t o t a l d a i l y a c t i v i t y . 104 10. Intraperitoneal melatonin injections decreased a c t i v i t y during l i g h t , whereas similar injections of serotonin increased a c t i v i t y during dark. This experiment supported the hypothesis based upon an apparent c o r r e l a t i o n i n other species that melatonin and serotonin may be involved i n the regulation of a c t i v i t y l e v e l s . 11. The laboratory findings were discussed i n r e l a t i o n to published f i e l d observations on juvenile salmonids. 12. The functional significance of the a c t i v i t y cycle was discussed i n r e l a t i o n to migration, orientation, feeding, and schooling behaviour. 105 6. LITERATURE CITED A l i , M.A. 1959. 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 and p h o t o b e h a v i o r a l r e s p o n s e s o f j u v e n i l e P a c i f i c salmon. Can. J . Z o o l . , 37: 965-996. Andrew, L . J . , and G.H. Geen. 1960. Sockeye and p i n k salmon p r o d u c t i o n i n r e l a t i o n t o proposed dams i n t h e F r a s e r R i v e r System. I n t e r n . Pac. Salmon F i s h . Comm., B u l l . , No.11. 259p. A s c h o f f , J . 1960. 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Etude c o m p a r a t i v e de l ' a c t i v i t e du p o i s s o n c a v e r n i c o l e A n o p t i c h t h y s a n t r o b i u s A l v a r e z , e t de son a n c e t r e e p i g e A s t y n a x mexicanus ( F i l i p p i ) . Ann. Soc. Roy. Z o o l . B e l g . , 96: 61-116. 113 Thompson, R.B., and R.L. Burgner. 1967. Review: On the o r i e n t a t i o n o f young sockeye salmon (Oncorhynchus nerka) d u r i n g t h e i r seaward m i g r a t i o n out o f l a k e s . T r a n s . Amer. F i s h . S o c , 96: p.431. Van D e r w a l k e r , J.G. 1967. Response o f s a l m o n i d s t o low f r e q u e n c y sound. I n : Ma r i n e B i o a c o u s t i c s . (Ed. W.N. T a v o l g a ) , p.45-58. New York: Pergamon P r e s s . V e r h e i j e n , F . J . , and S.J. 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 ( P l e u r o n e c t i d a e ) i n a q u a r i a . Neth. J . Sea. Res., 3: 383-390. Wodinsky, J . , and W.N. T a v o l g a . 1963. Sound d e t e c t i o n i n t e l e o s t f i s h e s . I n : Ma r i n e B i o a c o u s t i c s . (Ed. W.N. T a v o l g a ) , p.269-280. New York: Pergamon P r e s s . Wong, R., and C.B.C. W h i t e s i d e . 1968. 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 - d e p r i v e d o f f o o d . J . E n d o c r i n o l . , 40: 383-384. Wurtman, R . J . , J . A x e l r o d , and L.S. P h i l l i p s . 1963. M e l a t o n i n s y n t h e s i s i n t h e p i n e a l g l a n d : 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 Figure 1. A continuous 24 hour record of room temperature and water temperature i n the swimming channels during March, 1967. Temperature (°C) Cl (>• N "O _ l 1 1 : 1-115 Figure 2. C i r c u i t diagram of 600 watt lamp c o n t r o l l e r . 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 " D4 - Z4XL20 R5 - 68K D5,D6,D7,D8 - IN3571 R6 - 220 Ql - 2N2925 R7 - 47 Q2 - 2N2646 R8 - 4700 2w SCR1 - C20C C l - l/25v T l - 12.6v Hammond 166F12 Unless otherwise noted, 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 1 1 6 Figure 3. Plot of l i g h t i n t e n s i t y against time, in d i c a t i n g the duration of the t w i l i g h t period regulated by the lamp c o n t r o l l e r . gures 4 - 8 . C i r c u i t diagrams of the f i s h movement counter. POWER SUPPLY LOGIC & OUTPUT OUT SYSTEM BLOCK DIAGRAM 118 RD I WH 2 ** BK 3 SHLD 4 -* AMPLIFIER Unless otherwise noted, a l l cap. 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 Unless otherwise noted a l l caps, 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 2N13U POWER SUPPLY Unless otherwise noted, cap. i n mfd. R e s i s t o r s 1 0 % TYPICAL OPERATE CIRCUIT \ RELAY coto. HAMMOND' 167K25 c C ACTS 1 2 3 19 20 ¥ ? ? i ? 24 vAG r v. A A A 20 5~ i PEN i TYPICAL RECORDER CIRCUIT JUNCTION BOX 122 S u r g i c a l t e c h n i q u e s . A l l f i s h s u b j e c t e d t o o p e r a t i o n s were a n a e s t h e t i z e d i n a s o l u t i o n o f M.S. 222 ( T r i c a i n e Methane S u l f o n a t e ; Kent C h e m i c a l Co., Vancouver., B.C.). The amount used f o r a n a e s t h e s i a was 0.2 gram per g a l l o n , w h i c h r e p r e s e n t e d a c o n c e n t r a t i o n o f 1:19,230. T h i s was w i t h i n t h e range g e n e r a l l y t o l e r a t e d by t h i s s p e c i e s ( B e l l , 1967). G e n e r a l a n a e s t h e s i a was assumed when th e s u b j e c t ceased movement and f l o a t e d v e n t r a l s i d e up. 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 the t a i l was g r a s p e d w i t h a p a i r o f f o r c e p s , t h e f i s h was removed from 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 g l a s s tube was co 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 water 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 t h e g i l l s . A. P i n e a l e c t o m y The p i n e a l o f t h e s e f i s h i s a s m a l l s t r u c t u r e l o c a t e d 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 and the o p t i c l o b e s ( F i g . 9 ) . The l o c a t i o n was e a s i l y v i s i b l e t h r o u g h t h e t o p o f t h e s k u l l (Hoar, 1955), and a f l a p o f t i s s u e was l a i d back i n t h i s r e g i o n t o expose t h e cranium. The s k u l l was p e n e t r a t e d w i t h a sharp g l a s s tube 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 d i s c a r d e d . A f i n e , b l u n t , h o l l o w g l a s s tube t h a t was con n e c t e d t o an a s p i r a t o r was touched t o t h e t o p o f t h e exposed p i n e a l . The organ was drawn i n t o t h e tube and 123 F i g u r e 9. D o r s a l v i e w o f t h e e x p o s e d b r a i n , i l l u s t r a t i n g t h e l o c a t i o n and g e n e r a l a p p e a r a n c e o f t h e p i n e a l body. M a g n i f i c a t i o n 6.5X. 124 s e p a r a t i o n i n v a r i a b l y o c c u r r e d a t t h e base o f t h e s t a l k . The s i t e was s e a l e d w i t h Squibb orabase (a d e n t a l m e d i c a t i o n 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 e p i d e r m a l f l a p was r e p l a c e d i n p o s i t i o n . The i n c i s i o n 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 Squibb o r a b a s e . Sham o p e r a t i o n s c o n s i s t e d o f 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 organ was not removed. P e n e t r a t i o n o f t h e cra n i u m was i m m e d i a t e l y p o s t e r i o r t o t h e p i n e a l . T h i s m o d i f i c a t i o n was n e c e s s a r y t o p r e v e n t damage t o t h e or 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 normal p i n e a l e c t o m y . The s u b j e c t s were r e v i v e d and p l a c e d i n p o s t o p e r a t i v e h o l d i n g a q u a r i a (10 g a l l o n s ) which were m a i n t a i n e d under c o n t r o l l e d p h o t o p e r i o d i c c o n d i t i o n s . H e a l i n g was completed w i t h i n two weeks and the s u b j e c t s were t h e n i n t r o d u c e d t o the 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 . Success o f removal was v e r i f i e d by o b s e r v a t i o n s w i t h 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 h i s t o l o g i c a l s e c t i o n s w h i c h were p r e p a r e d t o demonstrate the e x t e n t o f p i n e a l e c t o m y . A l l m a t e r i a l f o r t h e s e s l i d e s were p r e s e r v e d i n Weber's s o l u t i o n and 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. P l a s t i c s c r e e n s A s i m i l a r p r e p a r a t o r y p r o c e d u r e was f o l l o w e d f o r t h e placement o f t r a n s p a r e n t and opaque p l a s t i c s c r e e n s o v e r 125 the r e g i o n o f t h e p i n e a l . I n t h i s c a s e , o n l y t h e s k i n t i s s u e was l a i d back and t h e p l a s t i c s c r e e n i n s e r t e d i m m e d i a t e l y s u p e r i o r t o t h e s k u l l . The t i s s u e s were the n s u t u r e d back i n t o p o s i t i o n and the f i s h were r e v i v e d and a l l o w e d t o r e c o v e r i n t h e h o l d i n g a q u a r i a . C. B l i n d i n g S u r g i c a l t e c h n i q u e s 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 were a n a e s t h e t i z e d and 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 lo w e r jaw was h e l d open by a b l u n t hook and a s m a l l i n c i s i o n was made i n t h e o r a l e p i t h e l i u m . T h i s exposed t h e o p t i c nerve which 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. I t was s e v e r e d m e d i a l l y w i t h a p a i r o f s c i s s o r s . The nerve was s e v e r e d a g a i n l a t e r a l l y and a s e c t i o n e s t i m a t e d a t 1/16 t o 1/8 i n c h was removed. Recovery f o l l o w e d t h e same pr o c e d u r e d e s c r i b e d above. A l i m i t e d number o f i n d i v i d u a l s were 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 o f t h e 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 s e v e r e d , and t h e complete eye was l i f t e d from the s o c k e t