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Factors affecting the production of daily growth increments in the otoliths of fishes Campana, Steven E. 1983

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FACTORS AFFECTING THE PRODUCTION OF DAILY GROWTH INCREMENTS IN THE OTOLITHS OF FISHES by STEVEN E. CAMPANA B . S c , Dalhousie U n i v e r s i t y , H a l i f a x , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May 1983 © Steven E. Campana, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of f The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT O t o l i t h growth in young f i s h e s occurs with a c i r c a d i a n p e r i o d i c i t y , r e s u l t i n g i n a c o n c e n t r i c growth r e c o r d of c a l c a r e o u s m a t e r i a l . The r e s u l t a n t sequence of d a i l y growth increments i s o f t e n punctuated by prominent checks ( d i s c o n t i n u i t i e s ) . Yet l i t t l e i s known of those v a r i a b l e s that may c o n t r o l or modify the p r o d u c t i o n of one increment per day and/or checks. The o b j e c t i v e of t h i s t h e s i s was t h r e e f o l d : 1) to assess the i n f l u e n c e of some environmental v a r i a b l e s on d a i l y increment p r o d u c t i o n 2) to develop a mechanism and e x p l a n a t i o n f o r check formation 3) to i n t e r p r e t the o t o l i t h m i c r o s t r u c t u r e of w i l d f i s h e s i n terms of knowledge gained from the f i r s t two o b j e c t i v e s . The i n f l u e n c e of l i g h t and temperature on d a i l y increment formation was the s u b j e c t of the f i r s t experiment. T e t r a c y c l i n e i n j e c t e d i n t o j u v e n i l e s t a r r y f l o u n d e r s ( P l a t i c h t h y s s t e l l a t u s ) was i n c o r p o r a t e d i n t o the p e r i p h e r y of the s a g i t t a l o t o l i t h s w i t h i n 24 h. The r e s u l t i n g band, v i s i b l e under u l t r a v i o l e t l i g h t , was used as a dated mark on the o t o l i t h growth increments. T h i s technique was used to v e r i f y that increments were l a i d down on a d a i l y b a s i s , both i n f i e l d and l a b o r a t o r y environments. S u b - d a i l y increments were v i s i b l e i n o t o l i t h s of f i s h e s reared under most environmental c o n d i t i o n s . The p r o d u c t i o n of d a i l y increments i n j u v e n i l e s t a r r y f l o u n d e r s p r e c o n d i t i o n e d to a n a t u r a l environmental regime was u n a f f e c t e d by photoperiod or temperature f l u c t u a t i o n , suggesting the presence of an i n t e r n a l c i r c a d i a n rhythm. J u v e n i l e s t e e l h e a d t r o u t (Salmo g a i r d n e r i ) and s t a r r y f l o u n d e r s were reared 64-76 d under v a r i o u s experimental feeding regimes to determine i f fe e d i n g p e r i o d i c i t y i n f l u e n c e d the p r o d u c t i o n of d a i l y growth increments on the o t o l i t h s . Both s p e c i e s produced d a i l y increments when fed t h r i c e d a i l y , d a i l y or once every three days, as w e l l as through 26-32 d of s t a r v a t i o n . D a i l y growth increments were a l s o d e p o s i t e d i n v a t e r i t i c ( " c r y s t a l l i n e " ) o t o l i t h s , which comprised 27% of the t r o u t s a g i t t a e sampled. Feeding frequency a f f e c t e d increment appearance and the i n c i d e n c e of s u b d a i l y increments i n t r o u t , but not i n f l o u n d e r s . The d i f f e r e n c e i n e f f e c t was probably due to the higher metabolic r a t e of t r o u t . Increment d e p o s i t i o n i n a l l f l o u n d e r s was f a r more v a r i a b l e than i n t r o u t , and some f l o u n d e r s a p p a r e n t l y ceased increment formation a l t o g e t h e r . Since the r e s u l t s of the f i r s t two experiments were a p p a r e n t l y c o n t r a r y to some p r e v i o u s s t u d i e s , I suspected that age may i n t e r a c t with l i g h t and/or temperature to i n f l u e n c e o t o l i t h growth. P l a i n f i n midshipman, P o r i c h t h y s notatus, were reared i n the l a b o r a t o r y under three environmental regimes to determine the i n f l u e n c e of c e r t a i n v a r i a b l e s upon o t o l i t h growth increment formation. Both l a r v a l and j u v e n i l e midshipman were used to t e s t d i e l c y c l e s and constant c o n d i t i o n s of l i g h t and temperature. In the absence of a d i e l p hotoperiod, d a i l y growth increments were not formed upon hatch. However, a f t e r an a c c l i m a t i o n p e r i o d of 2-3 wk, an endogenous c i r c a d i a n rhythm became e v i d e n t , r e s u l t i n g i n d a i l y increment p r o d u c t i o n . With i v i n c r e a s i n g age, the i n f l u e n c e of l i g h t as a z e i t g e b e r decreased, while d a i l y increments became more prominent i n a l l environments. Temperature f l u c t u a t i o n a f f e c t e d increment appearance, but d i d not e n t r a i n increment d e p o s i t i o n . P e r i o d s of s t r e s s can d i s r u p t d a i l y growth increment formation on a f i s h o t o l i t h , producing a check ( d i s c o n t i n u i t y ) . Calcium-45 was used to monitor c a l c i u m d e p o s i t i o n on the s a g i t t a e of coho salmon, Oncorhynchus k i s u t c h , d u r i n g p e r i o d s of check formation. " 5Ca d e p o s i t i o n on the o t o l i t h c o ntinued f o r 12 hr a f t e r t r a n s f e r from a 5 C a water. When s t r e s s was a p p l i e d d u r i n g fl5Ca immersion, tt5Ca d e p o s i t i o n was reduced. However, s t r e s s a p p l i e d immediately a f t e r t r a n s f e r from a 5 C a water had no e f f e c t on U 5 C a d e p o s i t i o n . S t r e s s i n d i r e c t l y d i s r u p t e d * 5Ca d e p o s i t i o n on the o t o l i t h through a r e d u c t i o n i n b r a n c h i a l uptake of c a l c i u m . Check formation was not a s s o c i a t e d with r e s o r p t i o n of o t o l i t h c a l c i u m . "Lunar" growth p a t t e r n s have been observed i n the o t o l i t h s of many marine f i s h e s . I examined the o t o l i t h m i c r o s t r u c t u r e of j u v e n i l e s t a r r y f l o u n d e r s sampled from a monitored environment f o r evidence of luna r p e r i o d i c i t y . Three types of biweekly c y c l e s were observed i n a l l of the f l o u n d e r o t o l i t h s ; two of the c y c l e s were c o r r e l a t e d with a t i d a l modulation of the environment. Through a m u l t i p l e r e g r e s s i o n model, much of the day-to-day v a r i a b i l i t y i n d a i l y increment width c o u l d be e x p l a i n e d by d a i l y v a r i a t i o n s i n temperature, s a l i n i t y and t i d a l mixing. My r e s u l t s suggest that a 15-d increment width c y c l e was e n t r a i n e d by the i n t e r a c t i o n of a 15-d t i d a l c y c l e with V temperature and s a l i n i t y . The same t i d a l cycle/temperature i n t e r a c t i o n probably produced a semi-lunar p a t t e r n of increment c o n t r a s t . However, the presence of o t o l i t h checks formed at weekly and/or biweekly i n t e r v a l s c o u l d not be so e x p l a i n e d , although checks were c o n s i s t e n t l y formed on the new and f u l l moons. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES x ACKNOWLEDGEMENTS x i i General I n t r o d u c t i o n 1 Chapter 1. L i g h t and temperature e f f e c t s on d a i l y increment p r o d u c t i o n i n s t a r r y f l o u n d e r s 9 I n t r o d u c t i o n 9 M a t e r i a l s and Methods 10 Re s u l t s and D i s c u s s i o n 14 Chapter 2. Feeding p e r i o d i c i t y and the pr o d u c t i o n of d a i l y growth increments i n o t o l i t h s 29 I n t r o d u c t i o n 29 M a t e r i a l s and Methods 30 Trout 30 Flounders 32 ' O t o l i t h P r e p a r a t i o n 32 Re s u l t s 34 Trout 34 Flounders 40 D i s c u s s i o n 46 Chapter 3. Age-environment i n t e r a c t i o n s i n the pr o d u c t i o n of d a i l y growth increments i n midshipman 50 I n t r o d u c t i o n 50 v i i M a t e r i a l s and Methods 52 R e s u l t s 57 D i e l L i g h t Cycle 62 D i e l Temperature C y c l e 73 Constant Environment 77 D i s c u s s i o n 81 Chapter 4. Calcium d e p o s i t i o n and o t o l i t h check formation d u r i n g p e r i o d s of s t r e s s i n coho salmon 89 I n t r o d u c t i o n 89 M a t e r i a l s and Methods 91 Ca-45 Immersion 91 Ca-45 I n j e c t i o n 95 S t r e s s 96 O t o l i t h p r e p a r a t i o n 96 R e s u l t s 97 D i s c u s s i o n 106 Chapter 5. Lunar c y c l e s of o t o l i t h growth i n j u v e n i l e s t a r r y f l o u n d e r s 112 I n t r o d u c t i o n 112 M a t e r i a l s and Methods 113 R e s u l t s 115 Lunar Checks 115 Lunar Pa t t e r n s 120 Lunar C o r r e l a t e s of Feeding 123 D a i l y Increment Width 123 D i s c u s s i o n i 128 General C o n c l u s i o n s 137 L i t e r a t u r e C i t e d LIST OF TABLES Table 1. Increment counts under v a r i o u s environmental c o n d i t i o n s 16 Table 2. Mean number of d a i l y growth increments produced under v a r i o u s experimental f e e d i n g regimes 35 Table 3. Growth increment counts i n r e l a t i o n to e l a p s e d time f o r v a r i o u s environmental regimes 74 Table 4. Age e f f e c t s on growth increment p r o d u c t i o n i n midshipman reared under three a r t i f i c i a l environments. . 83 Table 5. Experiments i n which coho salmon were immersed f o r v a r i o u s lengths of time i n Ca-45 water 92 Table 6. Mean a c t i v i t y of Ca-45 d e p o s i t e d onto o t o l i t h s of f i s h kept under v a r i o u s experimental c o n d i t i o n s . 103 Table 7. M u l t i p l e r e g r e s s i o n equation f o r d a i l y increment width 1 27 Table 8. C o r r e l a t i o n and p a r t i a l c o r r e l a t i o n v a l u e s f o r the v a r i a b l e s i n the m u l t i p l e r e g r e s s i o n equation 131 X LIST OF FIGURES F i g . 1. A s e c t i o n through an i d e a l i z e d s a g i t t a l o t o l i t h . .. 3 F i g . 2. O t o l i t h m i c r o s t r u c t u r e of a s t a r r y f l o u n d e r under b r i g h t f i e l d i l l u m i n a t i o n and UV 17 F i g . 3. Increment count vs. elapsed time f o r a l l environments 20 F i g . 4. S u b d a i l y increments i n the ground s a g i t t a e of a s t a r r y f l o u n d e r 25 F i g . 5. O t o l i t h increment counts as a f u n c t i o n of time f o r s t a r v e d s t e e l h e a d t r o u t (A) and s t a r r y f l o u n d e r (B). ... 37 F i g . 6. Ground and p o l i s h e d v a t e r i t i c s a g i t t a from a s t e e l h e a d t r o u t showing d a i l y growth increments 41 F i g . 7. S a g i t t a of a s t a r r y f l o u n d e r fed once d a i l y f o r 76 < d 44 F i g . 8. Summary of experimental environmental regimes through time 54 F i g . 9. T o t a l increment count as a f u n c t i o n of time f o r f i s h sampled from a l l experimental environments 59 F i g . 10. D a i l y increment width as a f u n c t i o n of age f o r o t o l i t h samples from each of the three experimental environments 63 F i g . 11. Growth increments on the p o l i s h e d s a g i t t a e of l a r v a l midshipman 65 F i g . 12. S a g i t t a l growth increments produced before and a f t e r t r a n s f e r to a constant environment 69 F i g . 13. D a i l y growth increments produced on the s a g i t t a e of midshipman a f t e r 15-25 d of r e a r i n g under a d i e l temperature c y c l e 75 F i g . 14. Index of d a i l y increment width r e g u l a r i t y as a f u n c t i o n of age f o r o t o l i t h samples from each of the three experimental environments 78 F i g . 15. Mean a c t i v i t y of 4 5 - C a d e p o s i t e d onto o t o l i t h s through time 98 F i g . 16. Prepared s a g i t t a of experimental coho salmon. Checks due to c o l l e c t i o n (A), experimental s t r e s s (B) and water temperature change (C) are i n d i c a t e d . Bar = 10 um 101 F i g . 17. O t o l i t h m i c r o s t r u c t u r e of a w i l d s t a r r y f l o u n d e r . 116 F i g . 18. Histogram d e p i c t i n g the time i n t e r v a l between checks i n o t o l i t h s of j u v e n i l e f l o u n d e r s (N = 19). ..'...118 F i g . 19. Date of check formation i n o t o l i t h s of j u v e n i l e f l o u n d e r s sampled at c a . 5 d i n t e r v a l s between 2 Sept and 2 Oct 121 F i g . 20. Food consumption as a f u n c t i o n . o f time f o r s t a r r y f l o u n d e r s sampled between 2 Sept and 2 Oct 124 F i g . 21. Mean d a i l y increment width ( s o l i d l i n e ) as a f u n c t i o n of time. 129 x i i ACKNOWLEDGMENTS Dr. Arthur Tautz of the F i s h and W i l d l i f e Branch k i n d l y p r o v i d e d the s t e e l h e a d t r o u t used i n the feeding experiments, while Mr. Jim McNutt of Ayerst L a b o r a t o r i e s donated the a n t i b i o t i c s used i n r e a r i n g the midshipman l a r v a e . Dr. John Steeves p r o v i d e d an i n t r o d u c t i o n to the f l u o r e s e n c e microscope. Mr. L a s z l o Veto donated much of h i s time to i n s t r u c t i n g me i n the o p e r a t i o n of the SEM. I thank Mr. Dave Z i t t i n f o r h i s a s s i s t a n c e with computer a n a l y s i s and programming. The f i e l d a s s i s t a n c e of Paul Bentzen and Doug Walton was g r e a t l y a p p r e c i a t e d . Doug Begle was e s p e c i a l l y h e l p f u l i n t h i s regard, both i n the f i e l d and i n the l a b o r a t o r y . The members of my re s e a r c h committee were h e l p f u l i n g u i d i n g me through t h i s t h e s i s . My thanks to Drs. A. B. Acton, H. D. F i s h e r , J . M. G o s l i n e , A. G. Lewis and N. J . Wilimovsky. Manuscript reviews by Drs. G. H. Geen, P. A. L a r k i n , N. J . Wilimovsky and Mr. K. L o f t u s c o n t r i b u t e d to an improved t h e s i s p r e s e n t a t i o n . I e s p e c i a l l y acknowledge the comments and c r i t i c i s m s of John D. N e i l s o n of Simon F r a s e r U n i v e r s i t y ; discussion's with John always p r o v i d e d me with' new i n s i g h t s . S p e c i a l thanks go to my s u p e r v i s o r , Norman J . Wilimovsky, f o r h i s support through every phase of t h i s study. F i n a l l y , I'd l i k e to thank my wife Jane, who aid e d me i n a l l a s pects of my r e s e a r c h , and p r o v i d e d i n v a l u a b l e moral support throughout. 1 GENERAL INTRODUCTION Age d e t e r m i n a t i o n of f i s h e s i s an i n t e g r a l p a r t of f i s h e r i e s b i o l o g y . Age data are r e q u i r e d f o r c a l c u l a t i o n s as simple as that of growth r a t e , or as complex as the c u r r e n t age-s t r u c t u r e d f i s h e r i e s y i e l d models. The accuracy of such data i s oft e n assumed, yet recent s t u d i e s i n d i c a t e that such assumptions are o f t e n unwarranted (Bennett et a l . 1982; C h i l t o n and Beamish 1982). C l a s s i c a l l y , counts of a n n u l i on s c a l e s have been used f o r aging temperate f i s h e s . Although u s e f u l f o r fast-growing young f i s h , the r e l i a b i l i t y of s c a l e readings i s l i m i t e d by the p o t e n t i a l f o r s c a l e r e s o r p t i o n ( B i l t o n 1974) and the poor d i f f e r e n t i a t i o n of a n n u l i i n o l d f i s h (Bagenal and Tesch 1978). Other c a l c i f i e d s t r u c t u r e s may be aged - a n n u l i have been counted i n ve r t e b r a e (Munekiyo et a l . 1982), o p e r c u l a (Bagenal and Tesch 1978), f i n rays (Beamish 1981) and c l e i t h r a (Casselman 1974). However, the o t o l i t h s , or ear bones, have become the s t r u c t u r e of ch o i c e f o r age det e r m i n a t i o n of many f i s h e s . O t o l i t h s grow through the c o n c e n t r i c d e p o s i t i o n of m a t e r i a l around a nucleus. The primary o t o l i t h c o n s t i t u e n t s are a min e r a l form of c a l c i u m carbonate, a r a g o n i t e , and a hig h molecular weight p r o t e i n , o t o l i n (Degens et a l . 1969). The p r o p o r t i o n s of these components vary s e a s o n a l l y , r e s u l t i n g i n the formation of a t r a n s l u c e n t , h y a l i n e zone (annulus) dur i n g the winter months. Annulus formation i s g e n e r a l l y c o n s i s t e n t and r e l i a b l e ; however, o t o l i t h growth i s asymmetric ( F i g . 1A), a f a c t that a f f e c t s reading accuracy, and which only recent aging methodologies take 2 i n t o account ( C h r i s t e n s e n 1964; C h i l t o n and Beamish 1982). L a r v a l , j u v e n i l e (< 1 yr old) and t r o p i c a l f i s h cannot be aged by annular counts, s i n c e a n n u l i are not present. I n t e r p r e t a t i o n of length-frequency analyses i s o f t e n confounded by s i z e - s e l e c t i v e m o r t a l i t y and extended h a t c h i n g p e r i o d s . T h e r e f o r e , the d i s c o v e r y of d a i l y growth r i n g s ( F i g . 1B) i n t r o p i c a l f i s h o t o l i t h s was of c o n s i d e r a b l e s i g n i f i c a n c e to f i s h e r i e s b i o l o g i s t s ( P a n n e l l a 1971) 1. D a i l y growth increments 2 have now been observed i n hundreds of s p e c i e s of f i s h e s (Pannella 1971, 1974, 1980; Brothers et a l . 1976; Brothers 1980; Wilson and L a r k i n 1980), although the d a i l y nature of the growth r e c o r d has been v e r i f i e d f o r only a few. Since the d a i l y increment sequence p r o v i d e s a c h r o n o l o g i c a l r e c o r d of past f i s h growth, i t has been used f o r the de t e r m i n a t i o n of hat c h i n g date (Rosenberg and Haugen 1982), instantaneous growth r a t e (Methot 1981), recruitment p a t t e r n s ( V i c t o r 1983) and date of l i f e h i s t o r y t r a n s i t i o n s (Brothers and McFarland 1981). D e s p i t e i t s s u c c e s s f u l a p p l i c a t i o n s , the d a i l y growth rec o r d may be d i s c o n t i n u o u s at p o s t - j u v e n i l e ages (Pannella 1971), and i n some s p e c i e s (Geffen 1982; Laroche et a l . 1982; Lough et a l . 1982), p a r t i c u l a r l y i n those f i s h reared i n an a r t i f i c i a l environment. For these reasons, v a l i d a t i o n of d a i l y 1 - H i c k l i n g (1931) observed d a i l y r i n g s at a much e a r l i e r date, but P a n n e l l a was the f i r s t to r e a l i z e t h e i r t rue s i g n i f i c a n c e . 2 - Although the term " r i n g " i s used by some workers, the term "increment" more p r o p e r l y i n t e r p r e t s the three-dimensional nature of d a i l y o t o l i t h growth. 1. A s e c t i o n through an i d e a l i z e d s a g i t t a l o t o l i t h . . (A) Schematic diagram of a macroscopic view. (B) Showing t y p i c a l m i c r o s t r u c t u r a l f e a t u r e s . C = check D = d a i l y increments SD = s u b d a i l y increments < V e n t r a l 6 increment production in the species under study i s mandatory. More importantly, information i s needed concerning the variables that influence the circadian rhythm of o t o l i t h deposition. Laboratory environments are seldom representative of natural habitats, suggesting that the reports of non-daily increment production are a r t i f a c t s of the laboratory milieu. Several environmental variables cycle with a d i e l p e r i o d i c i t y , and each has been implicated as an entrainment factor for rhythmic o t o l i t h growth. A 24-h light-dark cycle appeared to entrain an endogenous circadian rhythm in l a r v a l Lepomis (Taubert and Coble 1977). But in a study of temperate stream fishes, photoperiod e f f e c t s on increment deposition were secondary to those of d i e l temperature fluctuation (Brothers 1981). In a t h i r d study, d a i l y increment production in chinook salmon, Oncorhynchu-s tshawytscha, continued in the presence of constant conditions of both environmental variables (Neilson and Geen 1982). The above results were further confused by suggestions that feeding influenced (Neilson and Geen 1982) or did not influence (Marshall and Parker 1982) increment p e r i o d i c i t y . In l i g h t of these apparently c o n f l i c t i n g r e s u l t s , a major objective of my thesis was the determination of those variables that influence the formation of one increment per 24 h. Otoli t h s of wild f i s h are characterized by sequences of incremental growth punctuated by prominent structures known as checks (or d i s c o n t i n u i t i e s ) . Checks may occur at apparently random locations in the growth record, or they may delimit 7 incremental p a t t e r n s of 14-15 d ( P a n n e l l a 1971, 1980; Brothers 1980). In both cases, check l o c a t i o n can be a s s o c i a t e d with a date of formation by enumerating the d a i l y increments between the check and the o t o l i t h p e r i p h e r y (=date of sampling). Use of t h i s technique has demonstrated t h a t n o n - p e r i o d i c check formation i s g e n e r a l l y a s s o c i a t e d with p e r i o d s of s t r e s s ( P a n n e l l a 1980). Through an unknown mechanism, s t r e s s must i n t e r f e r e with the d e p o s i t i o n of a t , l e a s t one of the o t o l i t h ' s two major components to r e s u l t i n the formation of such a v i s u a l l y - d i s t i n c t i v e s t r u c t u r e . As a second o b j e c t i v e of t h i s t h e s i s , a r a d i o i s o t o p e of c a l c i u m was used to monitor c a l c i u m d e p o s i t i o n on the o t o l i t h through s t r e s s f u l p e r i o d s , i n an attempt to develop a p l a u s i b l e mechanism f o r check formation. The p e r i o d i c i t y of the 14-15 d incremental p a t t e r n s i n marine f i s h o t o l i t h s suggests a lu n a r or t i d a l l y - m o d u l a t e d cause. Lunar p a t t e r n s have been observed i n a l a r g e number of s p e c i e s ( P a n n e l l a 1971, 1974, 1980; Brothers et a l . 1976; Rosenberg 1982), but are as yet u n s t u d i e d . Since o t o l i t h growth i s g e n e r a l l y p r o p o r t i o n a l to f i s h growth (Wilson and L a r k i n 1982), lunar p a t t e r n s of o t o l i t h growth may r e f l e c t s i m i l a r c y c l e s of f i s h growth. The f i n a l o b j e c t i v e of t h i s t h e s i s was the f o r m u l a t i o n of a r e l a t i o n s h i p between lunar p a t t e r n s i n o t o l i t h s and environmental v a r i a b l e s known to i n f l u e n c e o t o l i t h growth. T h i s and the other t h e s i s o b j e c t i v e s c o n s t i t u t e d i s t i n c t but r e l a t e d q u e s t i o n s , and have been p u b l i s h e d s e p a r a t e l y . T h e r e f o r e , the t h e s i s format i s one of s e c t i o n s , with each s e c t i o n s e l f - c o n t a i n e d i n j o u r n a l form. A general c o n c l u s i o n at 8 the end of the t h e s i s attempts to i n t e r - r e l a t e the v a r i o u s s e c t i o n a l c o n c l u s i o n s . 9 CHAPTER 1. LIGHT AND TEMPERATURE EFFECTS ON DAILY INCREMENT PRODUCTION IN STARRY FLOUNDERS Int r o d u c t ion The e x i s t e n c e of d a i l y growth increments on o t o l i t h s of marine and freshwater f i s h e s has been both suggested (Schmidt and F a b r i z i o 1980; S t e f f e n s e n 1980; Townsend 1980) and confirmed ( P a n n e l l a 1971; Brothers et a l . 1976; Struhsaker and Uchiyama 1976; Taubert and Coble 1977; Radtke 1978; Wild and Foreman 1980; Wilson and L a r k i n 1980). These c o n c e n t r i c a l l y formed increments have c o n s i d e r a b l e p o t e n t i a l f o r the exact d e t e r m i n a t i o n of hat c h i n g dates and e a r l y l i f e h i s t o r y growth r a t e s of w i l d f i s h p o p u l a t i o n s . However, d a i l y increments are not always produced i n some s p e c i e s (Wild and Foreman 1980) and at some ages (Pannella 1971), making the v e r i f i c a t i o n of the increment to age r e l a t i o n s h i p e s s e n t i a l before i t s a p p l i c a t i o n to the aging of w i l d p o p u l a t i o n s . Most d a i l y increment v e r i f i c a t i o n s t u d i e s to date have employed l a b o r a t o r y - r e a r e d f i s h of known hat c h i n g date. However, many s p e c i e s , i n c l u d i n g the s t a r r y f l o u n d e r ( P l a t i c h t h y s  s t e l l a t u s ) , are not e a s i l y r e ared from ha t c h i n g i n a l a b o r a t o r y environment. In a d d i t i o n , c y c l i c a l l a b o r a t o r y events may i n f l u e n c e growth increment formation. As a r e s u l t , l a b o r a t o r y r e s u l t s may not be a p p l i c a b l e to w i l d p o p u l a t i o n s . In a s e r i e s of experiments, Taubert and Coble (1977) concluded that an i n t e r n a l b i o l o g i c a l c l o c k e n t r a i n e d by a 24 h l i g h t - d a r k c y c l e was r e s p o n s i b l e f o r d a i l y increment formation i n young 10 mouthbrooders. In c o n t r a s t , Brothers (1978) regarded temperature f l u c t u a t i o n as the key f a c t o r to the t i m i n g of increment d e p o s i t i o n i n temperate stream f i s h e s . Photoperiod and f e e d i n g were c o n s i d e r e d to be l e s s s i g n i f i c a n t as increment i n d u c e r s . The p o s s i b i l i t y of an endogenous r h y t h m i c i t y was not d i s c u s s e d . T e t r a c y c l i n e a p p l i c a t i o n to a f i s h at a known date can be used in age v a l i d a t i o n s t u d i e s (Holden and Vince 1973) and as a temporal mark i n growth s t u d i e s (Wild and Foreman 1980). T e t r a c y c l i n e i s known to be i n c o r p o r a t e d i n t o c a l c i f y i n g t i s s u e s i n a f i s h d u r i n g growth, d e p o s i t i n g a band f l u o r e s c e n t under u l t r a v i o l e t l i g h t (Weber and Ridgway 1967; Meunier and B o i v i n 1974). We used the t e t r a c y c l i n e band as a temporal mark, f i r s t of a l l to v e r i f y the e x i s t e n c e of d a i l y growth increments i n j u v e n i l e s t a r r y f l o u n d e r s , both i n the l a b and in s i t u , and secondly, to t e s t the c u r r e n t suggestions on the r o l e of p o s s i b l e environmental m o d i f i e r s on the d e p o s i t i o n of o t o l i t h d a i l y growth increments. M a t e r i a l s and Methods I n t r a - p e r i t o n e a l i n j e c t i o n of o x y t e t r a c y c l i n e h y d r o c h l o r i d e (100 mg t e t r a c y c l i n e / k g f i s h ) r e s u l t e d i n d e p o s i t i o n of a UV-f l u o r e s c e n t band on the s a g i t t a e of the f l o u n d e r s . I n j e c t e d volume was 0.025 mL. Although s i m i l a r r e s u l t s were achieved by immersing the f i s h i n a 0.02% o x y t e t r a c y c l i n e / s a l i n e s o l u t i o n f o r 1 d, the experiments d e s c r i b e d h e r e i n were c a r r i e d out by means of an i n j e c t i o n . A l l experimental f i s h i n t h i s study were 4-8 cm in standard l e n g t h and approximately 8 mo o l d . 11 To be e f f e c t i v e as an a c c u r a t e l y dated marker, the t e t r a c y c l i n e must be i n c o r p o r a t e d i n t o the o t o l i t h soon a f t e r a p p l i c a t i o n . To determine how q u i c k l y t e t r a c y c l i n e was i n c o r p o r a t e d i n t o the o t o l i t h , 12 f l o u n d e r s were i n j e c t e d and s a c r i f i c e d a f t e r 0,6,10,24 and 48 h. L e f t and r i g h t hand s i d e s a g i t t a e were removed immediately and processed (see f o l l o w i n g p aragraphs). For the in s i t u experiment, 25 f l o u n d e r s were c o l l e c t e d at the mouth of the Nooksak River i n Bellingham Bay, Washington on Oct. 10,1980. The f i s h were i n j e c t e d and p l a c e d immediately i n a 3.1 X 3.1 X 1 m mesh e n c l o s u r e i n a t i d a l channel of the e s t u a r y . F i v e c o n t r o l f i s h were c o l l e c t e d by seine and e i g h t f i s h were sampled from the e n c l o s u r e on October 24, 1980; f u r t h e r sampling was impossible due to f l o o d waters. Unmarked c o n t r o l f i s h served to check f o r n a t u r a l l y o c c u r r i n g f l u o r e s c e n c e not induced by t e t r a c y c l i n e . L e f t and r i g h t s a g i t t a e were removed from the f i s h the same day, brushed c l e a r of t i s s u e and attached s u l c u s - s i d e down with i n s t a n t glue onto a standard microscope s l i d e . S l i d e s were s t o r e d i n darkness u n t i l p r o c e s s i n g . Dark storage f o r up to four months r e s u l t e d i n no r e d u c t i o n of f l u o r e s c e n c e i n t e n s i t y . For the l a b o r a t o r y experiments, w i l d - c o l l e c t e d f l o u n d e r s were f i r s t r e a red i n the l a b f o r 2 mo under a n a t u r a l l i g h t - d a r k c y c l e . F i v e aquaria i n separate, l i g h t - p r o o f c u b i c l e s were stocked with 20 f l o u n d e r s each. Experimental c o n d i t i o n s f o r each aquarium were designed to t e s t the e f f e c t of photoperiod and temperature f l u c t u a t i o n upon increment d e p o s i t i o n . Temperatures 1 2 were s e l e c t e d to approximate those measured in s i t u . Twenty four and 36 h photoperiods were t e s t e d at constant temperature, with the l a t t e r a l s o t e s t e d i n c o n j u n c t i o n with a 36 h temperature c y c l e . Constant l i g h t c o n d i t i o n s with a 36 h temperature c y c l e were used t o examine the e f f e c t of temperature a l o n e . I f the p e r i o d i c i t y of e i t h e r v a r i a b l e i s r e l a t e d to increment t i m i n g , the 36 h c y c l e should r e s u l t i n f i s h with 67% of the increments v i s i b l e under a 24 h c y c l e . F i s h were a l s o exposed to constant c o n d i t i o n s f o r each v a r i a b l e , i n the event that f l u c t u a t i o n of e i t h e r environmental v a r i a b l e e n t r a i n e d an endogenous c i r c a d i a n rhythm. A l l experimental c o n d i t i o n s are d e t a i l e d below: 1) Photoperiod of 13 h l i g h t : 1 1 h dark ( i e . , a n a t u r a l l i g h t regime); constant temperature at 15°C (=13L:11D/CT) 2) 24 h l i g h t ; constant temperature (=24L/CT) 3) 24 h l i g h t ; 24 h at 15°C:12 h at 18°C (=24L/24T1:12T2) 4) 24 h l i g h t : 1 2 h dark; constant temperature (=24L:12D/CT) 5) 24 h l i g h t : 1 2 h dark; 24 h at 15°C:12 h at 18°C (=24L:12D/24T1:12T2) Temperatures were monitored with a continuous temperature recorder a c c u r a t e to 0.25°C. During temperature changes, the f i n a l temperature was reached l e s s than 2 h a f t e r i n i t i a t i o n . A l l l i g h t i n g was f l u o r e s c e n t . One s a t i a t i o n f e e d i n g with l i v e T u b ifex and frozen zooplankton was given at a random time d u r i n g a 24 h p e r i o d , with some p e r i o d s omitted t o t a l l y to a v o i d any entrainment of d a i l y growth p a t t e r n s due to feed i n g p e r i o d i c i t y . A l l f i s h were a c c l i m a t e d to experimental c o n d i t i o n s f o r 2 wk p r i o r to t e t r a c y c l i n e i n j e c t i o n . F i v e f i s h from each aquarium 1 3 were sampled w i t h i n 1 d of i n j e c t i o n , with the remainder of the f i s h sampled 25-26 d and 43-47 d a f t e r i n j e c t i o n . Mounted s a g i t t a e were ground to a plane where the p e r i p h e r a l growth increments were most v i s i b l e . O t o l i t h s l i d e s were mounted on a g r i n d i n g apparatus ( N e i l s o n and Geen 1981) coupled to an automated r o t a t e r and ground on m e t a l l u r g i c a l l a p p i n g f i l m (3 jam) . F i n a l p r e p a r a t i o n s were s u p e r i o r to those made by hand on s i n t e r e d g l a s s with aluminum oxide, as the g r i n d was f i n e r and on a more even plane. A l l prepared o t o l i t h s were examined and photographed at 500X-1250X under both white and u l t r a v i o l e t l i g h t on a L e i t z Orthoplan f l u o r e s c e n c e microscope with a 35mm camera attachment. We used e x c i t a t i o n f i l t e r bands from 450-490 nm and b a r r i e r f i l t e r s at 510 and 515 nm . To determine the p o s i t i o n of the f l u o r e s c e n t band r e l a t i v e to the growth increments v i s i b l e under white l i g h t , each o t o l i t h was photographed i n a p a i r e d sequence: once under b r i g h t f i e l d i l l u m i n a t i o n and once under UV l i g h t . The o t o l i t h p o s i t i o n and focus were not a d j u s t e d w i t h i n a photograph p a i r sequence. Developed negatives were mounted d i r e c t l y i n t o p r o j e c t o r s l i d e s . Increment counts were made from the p r o j e c t e d image. We d e f i n e d an increment as a b i p a r t i t e s t r u c t u r e . Under t r a n s m i t t e d l i g h t , i t c o n s i s t e d of a narrow, opaque band and an adjacent wider, t r a n s l u c e n t r e g i o n . Both major and t o t a l increment counts were made along the long a x i s of the s a g i t t a e . Major increments were g e n e r a l l y e a s i l y d i s t i n g u i s h e d on the b a s i s of t h e i r c o n t i n u i t y , i n t e n s i t y and r e g u l a r i t y of s p a c i n g . Minor increments d i f f e r e d , 1 4 i n t h a t they o f t e n merged with other increments or were s i g n i f i c a n t l y l i g h t e r and t h i n n e r i n appearance than the surrounding increments. The presence and spacing of increments i n some o t o l i t h s was v e r i f i e d with a P e r k i n Elmer Autoscan scanning e l e c t r o n microscope a f t e r e t c h i n g the o t o l i t h s with 1% HC1 f o r 90 s and c o a t i n g with g o l d . A l l counts were r e p l i c a t e d at l e a s t three times i n a random sequence by each author i n d i v i d u a l l y . Agreement w i t h i n 10% between readers was c o n s i d e r e d a c c e p t a b l e and the mean taken; a l l q u e s t i o n a b l e counts and/or p r e p a r a t i o n s were removed from f u r t h e r a n a l y s i s . These r e j e c t i o n s comprised 3% and 9%, r e s p e c t i v e l y , of the t o t a l number of o t o l i t h s . O t o l i t h anomalies that c o u l d c o n c e i v a b l y a f f e c t the f i n a l increment count were n o t i c e a b l e i n 10-20% of the l i g h t and e l e c t r o n microscopy photographs. These anomalies i n c l u d e d the e x i s t e n c e of m u l t i p l e growth f o c i l e a d i n g to growth increments that merged with others and d i s c o n t i n u o u s increments. In two cases, these anomalies c o u l d have s i g n i f i c a n t l y a l t e r e d the e v e n t u a l increment count; these o t o l i t h s were r e j e c t e d . R e s u l t s and D i s c u s s i o n T e t r a c y c l i n e i n c o r p o r a t i o n i n t o the o t o l i t h was evident i n 97% of the f l o u n d e r s i n j e c t e d . In each of those l e f t a l i v e at l e a s t a month a f t e r i n j e c t i o n , the r e s u l t i n g f l u o r e s c e n t band spanned a minimum of f i v e major increments and g e n e r a l l y 14-20. I n c o r p o r a t i o n i n t o the o t o l i t h began i n l e s s than a day. A f l u o r e s c e n t band was v i s i b l e i n 50% of the f i s h , 10 h a f t e r 1 5 i n j e c t i o n , and i n 100% of the f i s h , 24 h a f t e r i n j e c t i o n . T h e r e f o r e , the proximal edge of the band was c o n s i d e r e d to be l o c a t e d w i t h i n one or two increments of the o t o l i t h p e r i p h e r y at the time of i n j e c t i o n . A t h i n f l u o r e s c e n t l i n e o f t e n a s s o c i a t e d with the ground o t o l i t h / g l u e i n t e r f a c e was e a s i l y d i s t i n g u i s h a b l e from the f l u o r e s c e n c e due to the t e t r a c y c l i n e . Both major and t o t a l increment counts were made from the proximal edge of the t e t r a c y c l i n e band to the p e r i p h e r y of the o t o l i t h . Growth increments i n l e f t and r i g h t s a g i t t a e were counted s e p a r a t e l y f o r each f i s h . However, the mean of the two s a g i t t a e was used i n the data a n a l y s i s , as the two s i d e s were not s i g n i f i c a n t l y d i f f e r e n t (95% co n f i d e n c e l e v e l ) . The width of the major increments v a r i e d between 0.9 /am and 3.4 >um, depending on the i n d i v i d u a l f i s h and l o c a t i o n on the o t o l i t h . Minor increments numbered up to three between adjacent major increments; scanning e l e c t r o n microscope measurements i n d i c a t e d that these c o u l d be as narrow as 0.25 /um. It the major growth increments on the o t o l i t h are l a i d down on a d a i l y b a s i s , a slope (b) of one should be obtained i n the r e g r e s s i o n of major increment count on the number of days elapsed s i n c e t e t r a c y c l i n e i n j e c t i o n . A l l e x p e r i m e n t a l l y c o n d i t i o n e d f i s h , as w e l l as the f i s h maintained in s i t u , produced o t o l i t h s with a day:major increment slope not s i g n i f i c a n t l y d i f f e r e n t from one at the 95% co n f i d e n c e l e v e l (Table 1). We conclude that d a i l y increments were l a i d down both i n s i t u and i n the experimental s i t u a t i o n s s t u d i e d . D a i l y increments can be seen i n the s a g i t t a of F i g . 2. The major T A B L E I. Regression coefficient (b). standard error (SK). and corre-sponding R2 value for the regression of major increment count on the number of days elapsed, for each of the experimental and in situ environments. Expt.a No. of fish b SE R2 In situ 13 1.06 0.06 0.96 13L:11D/CT 10 1.04 0.07 0.96 24L/CT 20 0.90 0.08 0.89 24L/24T,: 12T, 16 0.90 0.06 0.93 24L:12D/CT 15 0.99 0.09 0.90 24L: I2D/24T,: I2T\ 10 0.96 0.06 0.97 'L - light hours; D = dark hours; CT = constant temperature; 7", !5°C; T2 = 18°C. 2. O t o l i t h micro-structure of a s t a r r y flounder under b r i g h t f i e l d i l l u m i n a t i o n and UV. (Top). Under b r i g h t f i e l d i l l u m i n a t i o n . (Bottom). Under UV l i g h t . The f l u o r e s c e n t band i s due to i n c o r p o r a t e d t e t r a c y c l i n e , whose l o c a t i o n can now be a s s o c i a t e d with s p e c i f i c increments v i s i b l e i n (top) view. The flounder was sampled 45 d a f t e r t e t r a c y c l i n e i n j e c t i o n . Bar = 20 ^im. IS 19 increments are d a i l y , making the minor increments s u b - d a i l y i n nature. The l a b o r a t o r y data were then pooled as the slopes were not s i g n i f i c a n t l y d i f f e r e n t and B a r t l e t t ' s t e s t i n d i c a t e d homogeneity of v a r i a n c e between the experiments. F i g . 3 shows the pooled l a b o r a t o r y d a i l y increment data. The f i e l d data c o u l d not be pooled with the l a b r e s u l t s , d e s p i t e t h e i r s i m i l a r s l o p e s , due to the s i g n i f i c a n t l y lower v a r i a n c e of the former. The decreased v a r i a n c e was at l e a s t p a r t i a l l y due to the r e l a t i v e l y short time p e r i o d between i n j e c t i o n and sampling i n  s i t u . Both the f i e l d and l a b r e s u l t s c o n f i r m a p r e v i o u s l y u n v e r i f i e d o b s e r v a t i o n of d a i l y growth increments in s t a r r y f l o u n d e r s (Wilson and L a r k i n 1980). Our r e s u l t s i n d i c a t e that d a i l y increments are formed i n the s a g i t t a e of j u v e n i l e s t a r r y f l o u n d e r s under a v a r i e t y of experimental c o n d i t i o n s , as w e l l as under a n a t u r a l environment. T h i s study a l s o supports the view that u n n a t u r a l l i g h t or temperature s t i m u l i are not l i k e l y to induce a s i g n i f i c a n t d e v i a t i o n from a n a t u r a l d a i l y increment p a t t e r n i n p o s t - l a r v a l f i s h p r e c o n d i t i o n e d to a n a t u r a l environmental regime. In s t a r r y f l o u n d e r s at l e a s t , l a b o r a t o r y o b s e r v a t i o n s of d a i l y increment p a t t e r n s i n j u v e n i l e s can probably be e x t r a p o l a t e d to the n a t u r a l s i t u a t i o n . The v a r i a n c e a s s o c i a t e d with the increment counts i s somewhat higher than that r e p o r t e d i n most pre v i o u s p u b l i c a t i o n s . Although some of t h i s may be a t t r i b u t a b l e to poor o t o l i t h p r e p a r a t i o n , there appears to be a l a r g e s p e c i e s e f f e c t present i n the presence and/or c l a r i t y of increment d e p o s i t i o n . 20 F i g . 3. Increment count vs. elapsed time f o r a l l environments. 22 I d e n t i c a l p r e p a r a t o r y and reader counts made on s t i c k l e b a c k (Gasterosteus a c u l e a t u s ) s a g i t t a e r e s u l t e d i n a much lower l e v e l of i n t r a - s p e c i f i c v a r i a n c e (Campana, unpublished d a t a ) . Flounder increment p r o d u c t i o n r a t e s may vary among i n d i v i d u a l s , or d i f f e r e n t i a t i o n of d a i l y and s u b d a i l y increments may be d i f f i c u l t i n t h i s s p e c i e s . T h i s phenomenon does not appear to be common to many s p e c i e s . Suggestions that temperature f l u c t u a t i o n (Brothers 1978) or photoperiod (Taubert and Coble 1977; Radtke 1978) might be the major f a c t o r s c o n t r o l l i n g increment p e r i o d i c i t y were not supported by our r e s u l t s . T h i r t y - s i x h "days" of the two c o n d i t i o n s , even i n c o n j u n c t i o n , were i n e f f e c t i v e i n a l t e r i n g the p r o d u c t i o n of one major o t o l i t h growth increment per 24 h, averaged over a number of i n d i v i d u a l s . S i m i l a r i l y , constant temperatures and photoperiods had no v i s i b l e e f f e c t on d a i l y increment p r o d u c t i o n i n d i c a t i n g t h a t n e i t h e r environmental stimulus i s mandatory f o r the maintenance of a 24 h p e r i o d i c i t y i n o t o l i t h growth, at l e a s t i n f l o u n d e r s of s e v e r a l months age. T h i s i s not to say that l i g h t or temperature can have no e f f e c t on increment d e p o s i t i o n , but that a c i r c a d i a n rhythm i s maintained d e s p i t e t h e i r i n f l u e n c e . I t i s p o s s i b l e that l a r v a l f i s h would be more s u s c e p t i b l e to abnormal photoperiod or temperature c y c l e s , as a l l of the f l o u n d e r s s t u d i e d here were j u v e n i l e s and t h e r e f o r e p r e c o n d i t i o n e d to a n a t u r a l l i g h t - d a r k c y c l e . I f such were the case, these r e s u l t s would not be i n c o n s i s t e n t with Taubert and Coble's (1977) o b s e r v a t i o n s . However, N e i l s o n and Geen (1982) have reared chinook salmon 23 (Oncorhynchus tshawytscha) eggs and a l e v i n s i n t o t a l darkness and observed c l e a r d a i l y increment p r o d u c t i o n i n a l l cases. The p o s s i b i l i t y t h a t f i s h possess an i n t e r n a l , d i e l c l o c k has been suggested before (Gibson et a l . 1978; E r i k s s o n and van Veen 1980). In mammals, endogenous 24 h rhythms are o f t e n e n t r a i n e d by a c y c l i c photoperiod, although a c i r c a d i a n rhythm p e r s i s t s under constant l i g h t or dark (Kramm 1980; Lynch et a l . 1980). Taubert and Coble (1977) noted that l a r v a l s u n f i s h (Lepomis) responded to an u n n a t u r a l photoperiod by the p r o d u c t i o n of n o n - d a i l y growth increments that were not c o r r e l a t e d with the r e l a t i v e "day" l e n g t h . T h e r e f o r e , they concluded t h a t an endogenous b i o l o g i c a l c l o c k , u sing photoperiod as a z e i t g e b e r , was i n o p e r a t i o n . The r e s u l t s of , our study d i f f e r , i n that a 24 h p e r i o d i c i t y i n increment p r o d u c t i o n was continued d e s p i t e a 36 h photoperiod or constant l i g h t c o n d i t i o n s . T h i s i n c o n s i s t e n c y may be due to the d i f f e r e n t ages of the f i s h e s used. However, our evidence does support the hypothesis that c e r t a i n f i s h ' s p e c i e s possess an i n t e r n a l l y -r e g u l a t e d c i r c a d i a n rhythm. The d i u r n a l f e e d i n g and locomotory behaviour i n s t a r r y f l o u n d e r s supports t h i s suggestion (Campana, unpublished d a t a ) . In a d d i t i o n , an endogenous p e r i o d i c i t y i s not c o n t r a r y t o the concept of v a r i a n c e a s s o c i a t e d with increment counts. Large i n d i v i d u a l v a r i a t i o n s i n the e x p r e s s i o n of endogenous rhythms have been demonstrated i n other f i s h s p e c i e s (Godin 1981). A measure of the i n c i d e n c e of s u b - d a i l y increments was given by t o t a l increment counts. A high degree of v a r i a b i l i t y 24 was a s s o c i a t e d with t o t a l counts , p o s s i b l y due to the d i f f i c u l t y of viewing the s u b - d a i l y increments and p o t e n t i a l c o n f u s i o n with o p t i c a l a r t i f a c t s ( F i g . 4). The slopes f o r the v a r i o u s experimental c o n d i t i o n s using t o t a l counts were not s i g n i f i c a n t l y d i f f e r e n t (95% c o n f i d e n c e l e v e l ) . However, the r e s u l t s d i d seem to i n d i c a t e a high i n c i d e n c e of s u b - d a i l y increments under a n a t u r a l photoperiod regime (13L:11D/CT) (b =1.80: SE=0.24) and v i r t u a l l y none under c o n d i t i o n s of constant l i g h t and temperature (24L/CT) (b=0.99: SE=0.17). The i n s i t u and other experimental f i s h were intermediate i n t h e i r i n c i d e n c e of s u b - d a i l y increments. Growth r a t e s d i d not vary between experiments and t h e r e f o r e c o u l d not be i m p l i c a t e d i n the i n c i d e n c e of s u b - d a i l y increments. The v a r i a t i o n i n number of s u b - d a i l y increments between experimental treatments was o-f i n t e r e s t . Although the d i f f e r e n c e was not s i g n i f i c a n t by t - t e s t (0 .05<P<0.10), constant l i g h t c o n d i t i o n s produced only 13% of the s u b - d a i l y increments produced under a normal photoperiod. S u b - d a i l y increments were a l s o observed i n the f i s h kept i n the outdoor e n c l o s u r e . Sub-d a i l y p a t t e r n s have been noted by other r e s e a r c h e r s (Taubert and Coble 1977; Brothers 1978), but the reasons behind t h e i r formation remain obscure. Both temperature cues (Brothers 1978) and f e e d i n g frequency ( N e i l s o n and Geen, i n p r e p a r a t i o n ) have been i m p l i c a t e d i n t h e i r f o rmation. The value of t e t r a c y c l i n e f o r growth increment s t u d i e s i s enhanced by i t s r a p i d i n c o r p o r a t i o n i n t o the o t o l i t h , as r e p o r t e d here and elsewhere (Meunier and B o i v i n 1974). However, 25 F i g . 4. S u b d a i l y increments in the ground s a g i t t a e of a s t a r r y f l o u n d e r . Bar = 10 um. 27 our r e s u l t s were not c o n s i s t e n t with other o b s e r v a t i o n s of t o t a l i n c o r p o r a t i o n over a s i n g l e day i n tuna (Wild and Foreman 1980). T e t r a c y c l i n e bands were g e n e r a l l y 14-20 increments wide i n t h i s study; as s a g i t t a l growth occurs c o n c e n t r i c a l l y with increments added through time, t e t r a c y c l i n e i n c o r p o r a t i o n must be o c c u r r i n g over a 14-20 increment time p e r i o d . Kobayashi et a l . ( l 9 6 4 ) a l s o noted t h a t dual t e t r a c y c l i n e i n j e c t i o n s remained d i s t i n c t only when a month or more was a l l o t e d between a p p l i c a t i o n s . The d i s c r e p a n c y may be due to the higher metabolic r a t e of tuna r e l a t i v e t o f l o u n d e r s and g o l d f i s h . N e v e r t h e l e s s , i f the proximal edge of the band i s accepted as corresponding to the date of i n j e c t i o n , the width of the band i s i r r e l e v a n t , as the o r i g i n i s a c c u r a t e l y dated. The t e t r a c y c l i n e technique we used i s one approach to c o n f i r m i n g d a i l y increment e x i s t e n c e i n w i l d f i s h , or i n f i s h of unknown age. D a i l y growth increments have been v e r i f i e d i n only a few f i s h s p e c i e s . However, t h e i r occurrence has been suggested in numerous other s p e c i e s ( P a n n e l l a 1971,1974; Brothers et a l . 1976; Wilson and L a r k i n 1980). The method o u t l i n e d here p r o v i d e s a means of c o r r o b o r a t i n g the' occurrence of d a i l y growth increments. In those f i s h s p e c i e s where o t o l i t h d a i l y increments are shown to e x i s t , counts should prove u s e f u l i n c o n f i r m i n g the nature of a n n u l i or spawning checks on s c a l e s or o t o l i t h s . S i m i l a r l y , they can be used to determine the h a t c h i n g dates of n a t u r a l p o p u l a t i o n s . Such i n f o r m a t i o n i s v a l u a b l e f o r the study of many s p e c i e s where spawning grounds or times are unknown. A 28 p r e c a u t i o n i s necessary i n that d a i l y increment p r o d u c t i o n may not p e r s i s t i n o t o l i t h s of o l d e r f i s h ( P a n n e l l a 1974; Campana, unpublished d a t a ) . A l s o , the time of f i r s t increment formation i n r e l a t i o n to the hatching date would have to be determined. Perhaps more imp o r t a n t l y , the c l o s e correspondence between o t o l i t h l e n g t h and f i s h l e n g t h (Struhsaker and Uchiyama 197.6; Taubert and Coble 1977; Wild and Foreman 1980) suggests that the d i s t a n c e between increments may be an a c c u r a t e measure of d a i l y growth i n some ca s e s . Such a measure would prove i n v a l u a b l e to the study of l a r v a l f i s h p o p u l a t i o n s and i s a s u i t a b l e t o p i c f o r f u t u r e r e s e a r c h . 29 CHAPTER 2. FEEDING PERIODICITY AND THE PRODUCTION OF DAILY GROWTH INCREMENTS IN OTOLITHS I n t r o d u c t i o n Since P a n n e l l a (1971) f i r s t d e s c r i b e d d a i l y growth increments i n the o t o l i t h s of some f i s h e s , numerous i n v e s t i g a t i o n s have confirmed h i s o b s e r v a t i o n s on other s p e c i e s (Brothers et a l . 1976; Struhsaker and Uchiyama 1976; Taubert and Coble 1977; Wilson and L a r k i n 1980; and o t h e r s ) . The apparent u n i v e r s a l i t y of d a i l y increments in young f i s h has l e d to t h e i r a p p l i c a t i o n i n many f i s h e r i e s problems. D a i l y increments are now being used to age l a r v a l f i s h e s (Townsend and Graham 1981; K e n d a l l and Gordon 1981), determine d a i l y growth r a t e s (Methot 1981) and assess l i f e h i s t o r y changes i n i n d i v i d u a l f i s h ( P a n n e l l a 1980; Brothers and McFarland 1981). L i t t l e i s known of the f a c t o r s t h a t i n f l u e n c e d a i l y increment p r o d u c t i o n , and consequently, t h e i r r e l i a b i l i t y . Taubert and Coble (1977) suggested that the d e p o s i t i o n of d a i l y growth increments was r e g u l a t e d by an i n t e r n a l c i r c a d i a n rhythm, e n t r a i n e d by a 24 h l i g h t - d a r k c y c l e . S t u d i e s supporting (Tanaka et a l . 1981) and r e j e c t i n g (Campana and N e i l s o n 1982; N e i l s o n and Geen 1982) the r o l e of photoperiod as a z e i t g e b e r i n d i c a t e that age or s p e c i e s e f f e c t s may be important. D i e l temperature f l u c t u a t i o n has a l s o been i m p l i c a t e d as a f a c t o r behind increment p e r i o d i c i t y (Brothers 1981). The r o l e of f e e d i n g p e r i o d i c i t y i n o t o l i t h d e p o s i t i o n has only r e c e n t l y been examined. F i s h given m u l t i p l e d a i l y feedings 30 have been r e p o r t e d to produce more increments than would be expected of d a i l y p r o d u c t i o n ( P a n n e l l a 1980; N e i l s o n and Geen 1982), although M a r s h a l l and Parker (1982) r e p o r t e d continued d a i l y increment p r o d u c t i o n under c o n d i t i o n s of s t a r v a t i o n . Time of f e e d i n g a p p a r e n t l y does not i n f l u e n c e the t i m i n g of increment d e p o s i t i o n (Tanaka et a l . 1981). None of the s t u d i e s c i t e d s u b j e c t e d f i s h to more than one n o n - d a i l y f e e d i n g regime. The o b j e c t of t h i s study was to examine the r o l e of f e e d i n g p e r i o d i c i t y and s t a r v a t i o n i n the p r o d u c t i o n of d a i l y growth increments i n o t o l i t h s . Both s t e e l h e a d t r o u t (Salmo g a i r d n e r i ) and s t a r r y f l o u n d e r ( P l a t i c h t h y s s t e l l a t u s ) were s t u d i e d to t e s t f o r any i n f l u e n c e of widely d i f f e r e n t metabolic r a t e s . In a d d i t i o n , the c o n s i s t e n c y of increment d e p o s i t i o n was compared i n the two s p e c i e s . M a t e r i a l s and Methods Trout Young-of-the-year s t e e l h e a d t r o u t were obtained from a hatchery on 19 Oct 1981 and h e l d i n flow-through tanks f o r 7 d. On 26 Oct 1981, 111 t r o u t (standard l e n g t h = 7.58 cm; SD=0.66 cm) were i n j e c t e d IP with 0.05 ml of 20 mg/ml o x y t e t r a c y c l i n e h y d r o c h l o r i d e . T e t r a c y c l i n e i s known to be i n c o r p o r a t e d i n t o f l o u n d e r o t o l i t h s w i t h i n a day of i n j e c t i o n (Campana and N e i l s o n 1982). A s i m i l a r response to i n j e c t i o n has been noted i n t r o u t (Campana un p u b l i s h e d ) . Since the i n c o r p o r a t e d compound f l u o r e s c e s under u l t r a v i o l e t (UV) l i g h t , the r e s u l t a n t band can 31 be used as a temporal mark on the o t o l i t h , with the medial aspect of the band corresponding to the d a i l y growth increment d e p o s i t e d on the day of i n j e c t i o n . The i n j e c t e d t r o u t were d i v i d e d i n t o 4 200-1 tanks (30 f i s h i n each of Tanks 2-4 and 18 i n Tank 1). A semi-natural photoperiod was maintained through the use of an o u t s i d e p h o t o c e l l , and i n i t i a l water temperature was 10°C. By the end of the experiment, water temperature had dropped to 4.5°C. Rations c o n s i s t e d of a commercial t r o u t food. Each aquarium was maintained under a d i f f e r e n t feeding regime as f o l l o w s : Tank 1 - Once d a i l y f o r 13 d, f o l l o w e d by s t a r v a t i o n f o r up to 32 d more. Tank 2 - Once every 3 d f o r 64 d. Tank 3 - Once d a i l y f o r 64 d. Tank 4 - T h r i c e d a i l y (at 8 h i n t e r v a l s ) f o r 64 d. Feeding was c a r r i e d out at 1400 h f o r Tanks 1-3, and at 0800, 1600 and 2400 h f o r Tank 4 ( v i a an automatic f e e d e r ) . Excess food was removed 4 h a f t e r each f e e d i n g (once d a i l y i n the case of Tank 4). The q u a n t i t y of food given to each tank through the course of the experiment was roughly e q u a l . F i s h from Tank 1 were sampled at 6 d i n t e r v a l s ; f i s h from the other tanks were sampled 64 d a f t e r t e t r a c y c l i n e i n j e c t i o n . S a g i t t a l o t o l i t h s were removed and prepared as d e s c r i b e d below. 32 Flounders Young-of-the-year s t a r r y f l o u n d e r s were c o l l e c t e d from Bellingham Bay, Washington on 12 Sept 1981 and h e l d i n l a b o r a t o r y a q u a r i a f o r 6-7 d. Age of these f i s h was roughly 7 mo and t h e i r mean standard l e n g t h was 5.36 cm (SD=0.79 cm). D a i l y meals of l i v e t u b i f i c i d worms were p r o v i d e d . On 18 Sept 1981, 90 f l o u n d e r s were i n j e c t e d IP with 20 mg/ml of o x y t e t r a c y c l i n e h y d r o c h l o r i d e at a t i t r e of 0.025 ml/5 g f i s h . Experimental design was s i m i l a r to that of the t r o u t : 30 f i s h i n each of 3 50-1 a q u a r i a (Tanks 2-4). A f u r t h e r 25 f l o u n d e r s were i n j e c t e d and p l a c e d i n Tank 1 on 19 Sept 1981. A l l a q u a r i a were maintained under a 14L:10D photoperiod at i a temperature of 16°C. By the end of the experiment, water temperature i n a l l a q u a r i a had g r a d u a l l y decreased to 10°C, although no d i e l f l u c t u a t i o n s were observed. S a l i n i t y was <5 O/oo, as per the c o l l e c t i o n s i t e . A l l f i s h from Tanks 2-4 were k i l l e d a f t e r 74-76 d. F i s h from Tank 1 were sampled s e q u e n t i a l l y at 4-10 d i n t e r v a l s once s t a r v a t i o n had s t a r t e d . O t o l i t h P r e p a r a t i o n A f t e r removal, l e f t and r i g h t s a g i t t a l o t o l i t h s were brushed f r e e of t i s s u e and embedded s e p a r a t e l y , s u l c u s - s i d e down, i n Krazy Glue on a microscope s l i d e . M e t a l l u r g i c a l l a p p i n g f i l m ( g r i t s i z e 30 um to 0.3 um) and a g r i n d i n g j i g (N e i l s o n and Geen 1981) were used to g r i n d and p o l i s h the o t o l i t h to the mid-33 plane, or u n t i l the d a i l y growth increments on the postrostrum were most v i s i b l e . Increment counts were made on the p o s t r o s t r a l t i p ( f l o u n d e r s ) or the l a t e r a l aspect of the postrostrum ( t r o u t ) s i n c e increments were most d i s t i n c t i n t h i s region of the o t o l i t h . V i s u a l counts were made from the medial aspect of the f l u o r e s c e n t t e t r a c y c l i n e band (Campana and N e i l s o n 1982) at 1250X on a L e i t z Orthoplan f l u o r e s c e n c e microscope. Counts were repeated at a l a t e r date. Scanning e l e c t r o n microscope (SEM) photographs were made of some o t o l i t h s , p a r t i c u l a r l y when higher r e s o l u t i o n was r e q u i r e d . Flounder o t o l i t h s f o r SEM were prepared by e t c h i n g with 0.1 M EDTA ( e t h y l e n e d i n i t r i l o t e t r a a c e t i c a c i d ) f o r 10 min or with 2% HC1 f o r 16 min. Trout o t o l i t h s were etched i n e i t h e r 0.1 M EDTA f o r 4 min or i n 2% HC1 f o r 2 min. Etched o t o l i t h s were coated with g o l d and viewed at 10 kV on a Cambridge Stereoscan 250 scanning e l e c t r o n microscope. As a check f o r v i s u a l c o u n t i n g accuracy, p a i r e d UV-bright l i g h t micrographs were prepared of some v i s u a l l y - c o u n t e d r e g i o n s , and counts were made o f f the p r o j e c t e d image (Campana and N e i l s o n 1982). V i s u a l , photographic and SEM counts were then compared. In wel l - p r e p a r e d s a g i t t a e , increment counts d i d not d i f f e r s i g n i f i c a n t l y among the techniques (P>0.05). However, regions of narrow increments were d i f f i c u l t to r e s o l v e v i s u a l l y , optimal e t c h i n g f o r SEM was imp o s s i b l e when o t o l i t h c u r v a t u r e was present ( e s p e c i a l l y i n f l o u n d e r s ) , and photographs introduced d a i l y / s u b d a i l y c o n f u s i o n due to the f i x e d f o c a l l e n g t h . 34 The mean of the l e f t and r i g h t s a g i t t a l increment counts from each f i s h was used i n the data a n a l y s i s . L e f t and r i g h t s a g i t t a l counts do not d i f f e r s y s t e m a t i c a l l y i n f l o u n d e r s (Campana and N e i l s o n 1982) or t r o u t (Campana unpu b l i s h e d ) . In in s t a n c e s where one o t o l i t h had a s u b s t a n t i a l l y lower increment count than i t s p a i r e d c o u n t e r p a r t (> 30% d i f f e r e n c e ) , the lower value was assumed to be anomalous and excluded from f u r t h e r a n a l y s i s . Anomalous values were uncommon i n t r o u t , but occur r e d more f r e q u e n t l y i n f l o u n d e r s where increment d e p o s i t i o n i s v a r i a b l e (Campana and N e i l s o n 1982). C e r t a i n t r o u t o t o l i t h s appeared aberrant i n both appearance and morphology r e l a t i v e to " t y p i c a l " s a g i t t a e ; aberrant o t o l i t h s were l o b u l a t e d and i r r e g u l a r i n shape, and g l a s s y (as opposed to white) i n c o l o u r . On the b a s i s of X-ray d i f f r a c t i o n s t u d i e s of s i m i l a r - a p p e a r i n g o t o l i t h s i n rainbow t r o u t (Mugiya 1972), the aberrant o t o l i t h s l i k e l y had a v a t e r i t i c c r y s t a l l i n e s t r u c t u r e . R e s u l t s Trout D a i l y growth p a t t e r n s i n t r o u t o t o l i t h s were r e g u l a r i n c o n t r a s t , with d e c l i n i n g .widths a p p a r e n t l y due to de c r e a s i n g temperatures. Increment counts d i d not d i f f e r s i g n i f i c a n t l y among the v a r i o u s feeding regimes, suggesting that f e e d i n g p e r i o d i c i t y d i d not i n f l u e n c e the d a i l y p r o d u c t i o n of growth increments (P>0.05)(Table 2). However, f i s h fed once/3 d (Tank 2) d e p o s i t e d s i g n i f i c a n t l y fewer increments than would be I Table 2. Mean number of daily growth increments produced under various experimental feeding regimes. • Feeding Regime Increment Count SE Days Elapsed N Flounder Tank 2(once per 3 d) 73.5 3.7 76 20 Tank 3(once daily) 72.2 2.1 75 27 Tank 4(thrice daily) 67.6 6.5 74 11 Trout Tank 2(once per 3 d) 60.6 1.1 64 29 Tank 3(once daily) 62.8 0.6 64 28 Tank 4(thrice daily) 62.8 1.0 64 28 36 expected of d a i l y p r o d u c t i o n . Since the d i f f e r e n c e was minimal (< 6%), the " l o s t " increments were l i k e l y due to poor r e s o l u t i o n r a t h e r than absence. Increments i n the o t o l i t h s of f i s h f ed once every 3 d tended to be narrower than those of f i s h f ed more f r e q u e n t l y ( i e - as narrow as 0.85 /am); s i n c e o t o l i t h growth o f t e n r e f l e c t s . f i s h growth (Wilson and L a r k i n 1982), a s i g n i f i c a n t l y slower growth r a t e was probably r e s p o n s i b l e ( S c h e f f e ' s t e s t , P<0.05). F i s h s t a r v e d f o r p e r i o d s of up to 32 d d i d not cease p r o d u c t i o n of d a i l y increments ( F i g . 5). Subdaily increments were not counted, but v i s u a l o b s e r v a t i o n s suggested t h a t t h e i r frequency i n c r e a s e d with the fe e d i n g frequency of the f i s h . S u b d a i l y increments were not observed i n f i s h that had been s t a r v e d . Although s t a r v a t i o n and fe e d i n g p e r i o d i c i t y had no e f f e c t on increment number, o t o l i t h m i c r o s t r u c t u r e d i f f e r e d among the experimental treatments. Increments d e p o s i t e d i n s t a r v e d f i s h were narrower than those produced before the s t a r t of the s t a r v a t i o n p e r i o d . The change was gradual but g e n e r a l l y became n o t i c e a b l e 0-4 d a f t e r s t a r v a t i o n s t a r t e d . A s s o c i a t e d with the r e d u c t i o n i n increment width was a decrease i n the v i s u a l c o n t r a s t of adjacent increments ( i e - i n c r e m e n t s became f a i n t e r ) . In a d d i t i o n , I observed a s u b t l e 3-d p a t t e r n i n many of the o t o l i t h s of f i s h fed once per 3 d. The p a t t e r n c o n s i s t e d of a dark increment f o l l o w e d by two f a i n t e r increments, but d i d not become evident u n t i l 15-30 d a f t e r the experiment began. Increment width d i d not vary w i t h i n the p a t t e r n . 5. O t o l i t h increment counts as a f u n c t i o n of time f o r s t a r v e d s t e e l h e a d t r o u t (A) and s t a r r y ' f l o u n d e r (B). S t a r v a t i o n began on Day 10 f o r f l o u n d e r s and Day 13 f o r t r o u t . N e i t h e r r e g r e s s i o n slope i s s i g n i f i c a n t l y d i f f e r e n t from 1.0 at the 95% confidence l e v e l . Numbers beside data p o i n t s r e f e r to c o i n c i d e n t v a l u e s . TIME (DAYS) B T I M E ( D A Y S ) 40 Twenty seven percent of the s a g i t t a e removed from the t r o u t were v a t e r i t i c ( " c r y s t a l l i n e " ) , as opposed to a r a g o n i t i c ( v a t e r i t e i s an a l t e r n a t e m i n e r a l form of c a l c i u m carbonate; a r a g o n i t e i s the mineral that g e n e r a l l y makes up an o t o l i t h ) . D e spite the a l t e r e d s t r u c t u r e and g l a s s - l i k e appearance, d a i l y increments were present ( F i g . 6). In s a g i t t a e which -were p a r t i a l l y a r a g o n i t i c and p a r t i a l l y v a t e r i t i c , increment counts were s i m i l a r i n the two m i n e r a l forms. Both regions had an o t o l i t h check a s s o c i a t e d with the t e t r a c y c l i n e band. However, l i g h t t r a n s m i t t e d through v a t e r i t e was h i g h l y r e f r a c t e d , r e s u l t i n g i n a high p r o p o r t i o n of v i s u a l a r t i f a c t s a s s o c i a t e d with the incremental s t r u c t u r e . I t was not d i f f i c u l t to d i s t i n g u i s h d a i l y increments from v i s u a l a r t i f a c t s , but counts of s u b d a i l y increments were u n r e l i a b l e . Flounders D a i l y growth increments i n f l o u n d e r s were f a r more d i f f i c u l t to count than were those of t r o u t . Increment sequences were o f t e n i n t e r r u p t e d by one or more o t o l i t h checks ( d i s c o n t i n u i t i e s ) . Increments a s s o c i a t e d with checks were d i f f i c u l t to read, s i n c e t h e i r i d e n t i t y as d a i l y , s u b d a i l y or v i s u a l a r t i f a c t was g e n e r a l l y confused. Increment width was of t e n i r r e g u l a r , p a r t i c u l a r l y i n the p r o x i m i t y of checks. Standard e r r o r s of mean increment counts were much higher i n the flounder experiments than i n the t r o u t experiments ( T a b l e 2), demonstrating the g r e a t e r i r r e g u l a r i t y of the floun d e r o t o l i t h m i c r o s t r u c t u r e . S u b d a i l y increments c o n t r i b u t e d to the floun d e r 6. Ground and p o l i s h e d v a t e r i t i c s a g i t t a from a s t e e l h e a d t r o u t showing d a i l y growth increments. Bar=lO pjm. M X 43 data v a r i a n c e much more than was the case with the t r o u t . Feeding frequency had no e f f e c t on the mean number of increments produced i n experimental f l o u n d e r s . D a i l y increments were produced i n f i s h f ed three times per day, d a i l y or once every three days (Table 2). Nor d i d s t a r v a t i o n f o r p e r i o d s up to 26 d r e s u l t i n c e s s a t i o n of increment d e p o s i t i o n ( F i g . 5). However, 1-6 f i s h from a l l feeding regimes d e p o s i t e d f a r l e s s than one increment per day on one or both of t h e i r s a g i t t a e ( F i g . 7 ). These i n s t a n c e s , though i n f r e q u e n t , c o u l d not be a t t r i b u t e d to counting e r r o r or o t o l i t h c u r v a t u r e . Although s u b d a i l y increments on flounder o t o l i t h s were not counted, q u a l i t a t i v e assessments i n d i c a t e d that they were more frequent on f l o u n d e r s than on s t e e l h e a d , with no apparent c o r r e l a t i o n to f e e d i n g frequency. The a b i l i t y to d i s c e r n s u b d a i l y p a t t e r n s was r e l a t e d to the width of the growth zone; r e s o l u t i o n of very narrow increments was d i f f i c u l t . Feeding p e r i o d i c i t y had no observable e f f e c t on the o t o l i t h m i c r o s t r u c t u r e of f l o u n d e r s . Although some of the s t a r v e d f l o u n d e r s produced narrow increments, other i n d i v i d u a l s were not so a f f e c t e d . P o s t - s t a r v a t i o n increments d i d not appear to be f a i n t e r than those d e p o s i t e d p r e v i o u s l y . No three-day p a t t e r n was observed i n f i s h f ed once every 3 d. If a p a t t e r n as s u b t l e as that d e s c r i b e d i n Tank 2 t r o u t s a g i t t a e had been present, i t may have been obscured by the g e n e r a l i r r e g u l a r i t y of flounder d e p o s i t i o n . 7. S a g i t t a of a s t a r r y f l o u n d e r fed once d a i l y f o r 76 d. Far fewer than 76 growth increments are v i s i b l e , s uggesting c e s s a t i o n of s a g i t t a l growth at some p o i n t d u r i n g the experiment. Arrow i n d i c a t e s increment formed on day of experiment o r i g i n . Bar=lO um. 46 D i s c u s s i o n D a i l y growth increments were d e p o s i t e d on the o t o l i t h s of s t e e l h e a d t r o u t and s t a r r y f l o u n d e r under both d a i l y and non-d a i l y f e e d i n g regimes. In t r o u t , there was some i n d i c a t i o n that the number of s u b d a i l y increments i n c r e a s e d with f e e d i n g p e r i o d i c i t y . No such r e l a t i o n s h i p was evident i n the flounder o t o l i t h s . These r e s u l t s are c o n s i s t e n t with those of Taubert and Coble (1977), who r e p o r t e d that young mouthbrooders ( T i l a p i a  mossambica) given m u l t i p l e d a i l y feedings under a 15L:10D photop e r i o d , continued to produce d a i l y growth zones. In c o n t r a s t , N e i l s o n and Geen (1982) found that f i s h fed 4 times per day produced a higher number of narrow increments than those f i s h fed d a i l y , although increment number was not d i r e c t l y p r o p o r t i o n a l to the number of meals p r o v i d e d . My r e s u l t s and those of N e i l s o n and Geen (1982) are not n e c e s s a r i l y c o n t r a d i c t o r y i f one assumes that the i n c i d e n c e of s u b d a i l y increments i s r e l a t e d to f e e d i n g frequency. Such a r e l a t i o n s h i p was suggested by t h i s study; what appear to be broad d a i l y and numerous i n t e r v e n i n g s u b d a i l y increments were a l s o v i s i b l e i n the o t o l i t h s of chinook salmon (Oncorhynchus tshawytscha) fed 4 times per day ( N e i l s o n and Geen 1982 - F i g . 2). Trout and f l o u n d e r s s t a r v e d f o r p e r i o d s of 32 and 26 d, r e s p e c t i v e l y , continued to d e p o s i t d a i l y growth increments on t h e i r o t o l i t h s . S i m i l a r r e s u l t s have been r e p o r t e d f o r sockeye salmon (Oncorhynchus nerka) ( M a r s h a l l and Parker 1982). The body f a t r e s e r v e s present i n most f i s h e s may provide s u f f i c i e n t energy f o r l i m i t e d s k e l e t a l and o t o l i t h growth duri n g p e r i o d s of 47 s t a r v a t i o n (Buckley 1980; J o b l i n g 1980; M a r s h a l l and Parker 1982). T h e r e f o r e , d a i l y increment d e p o s i t i o n d u r i n g s t a r v a t i o n i s p l a u s i b l e . However, f i r s t - f e e d i n g f i s h l a r v a e would not have such a reserve and may cease increment d e p o s i t i o n as a r e s u l t . Examination of the o t o l i t h s of s t a r v e d anchovy ( E n g r a u l i s mordax) l a r v a e support t h i s suggestion (Methot and Kramer 1979); thus, the age d e t e r m i n a t i o n of s t a r v e d e a r l y - s t a g e l a r v a e by means of d a i l y growth increments may be u n r e l i a b l e . Although f l o u n d e r s were not so a f f e c t e d , the o t o l i t h m i c r o s t r u c t u r e of t r o u t was i n f l u e n c e d by f e e d i n g p e r i o d i c i t y . Many s t a r v e d t r o u t d e p o s i t e d narrow increments, while a s u b t l e t r i d a i l y p a t t e r n was produced i n f i s h fed once every 3 d. The absence of these c h a r a c t e r s i n f l o u n d e r s i s probably r e l a t e d to the d i f f e r e n t metabolic r a t e s of the two s p e c i e s . Salmonids such as the sockeye salmon have a standard metabolic r a t e of approximately 75 ml 02/kg-h at 10°C ( B r e t t and Glass 1973), while p l e u r o n e c t i d s such as the flounder P l a t i c h t h y s f l e s u s , are c l o s e r to 23 ml 02/kg-h at 10°C (Jorgensen and Mustafa 1980). (Metabolic r a t e s have not been p u b l i s h e d f o r the s p e c i e s and/or weights of f i s h used in my study, but the values mentioned are r e p r e s e n t a t i v e of the taxa (Altman and Dittmer 1974).) Consequently, food i s i n c o r p o r a t e d i n t o t i s s u e s f a r more q u i c k l y i n t r o u t than i n f l o u n d e r s ; a r a p i d response to food a v a i l a b i l i t y / f e e d i n g would t h e r e f o r e be expected i n t r o u t , and i s a p p a r e n t l y r e f l e c t e d i n the p a t t e r n and width of d a i l y growth zones, as w e l l as the i n c i d e n c e of s u b d a i l y increments i n the o t o l i t h s . M e t a b o l i c response to food intake i n f l o u n d e r s may be 48 too slow to a f f e c t the appearance of d a i l y increments. Trout and f l o u n d e r s were reared a t d i f f e r e n t temperatures i n t h i s study. However, the lower temperature at which the t r o u t were reared would tend to mask, not enhance, short-term metabolic responses. Few f i s h e s feed only once per day. Rainbow t r o u t (Salmo  q a i r d n e r i ) o f t e n feed s e v e r a l times a day (Grove et a l . 1978), while young f l o u n d e r s feed i n t e r t i d a l l y , 1-2 times per day, depending on the t i d e s ( T h i j s s e n et a l . 1974; Campana un p u b l i s h e d ) . P e r i o d s of s t a r v a t i o n probably occur i n most i n d i v i d u a l s at one time or another. If increment number was p r o p o r t i o n a l to f e e d i n g frequency, o t o l i t h growth increments c o u l d not be used r e l i a b l y i n age or growth s t u d i e s of w i l d f i s h . T h i s study demonstrates that such concern i s probably unwarranted, although the r e s u l t s cannot be e x t r a p o l a t e d to e a r l y - s t a g e l a r v a l f i s h e s . Tanaka et a l . (1981) a l s o r e p o r t e d that f e e d i n g d i d not e n t r a i n the p r o d u c t i o n of d a i l y increments. To my knowledge, d a i l y growth increments have not been p r e v i o u s l y r e p o r t e d i n v a t e r i t i c o t o l i t h s . V a t e r i t i c o t o l i t h s are uncommon among most w i l d f i s h e s , but appear f r e q u e n t l y i n h a t c h e r y - r e a r e d salmonids (Campana u n p u b l i s h e d ) . Although v a t e r i t e i s a d i f f e r e n t mineral form than i s a r a g o n i t e , the f a c t t h a t d a i l y growth increments are present i n the former i m p l i e s that the d e p o s i t i o n a l processes are s i m i l a r i n the two cases. T e t r a c y c l i n e i n c o r p o r a t i o n d i d not d i f f e r a p p r e c i a b l y between the two m i n e r a l s . A small number of f l o u n d e r s possessed o t o l i t h s that had a p p a r e n t l y ceased increment d e p o s i t i o n at v a r i o u s times a f t e r 49 t e t r a c y c l i n e i n j e c t i o n . SEM o b s e r v a t i o n of r e p r e s e n t a t i v e o t o l i t h s i n d i c a t e d that increments too narrow to be viewed by l i g h t microscopy were not r e s p o n s i b l e f o r the low increment counts. Curvature of the o t o l i t h p e r i p h e r y accounted f o r some " l o s t " increments, as was demonstrated when o t o l i t h s were ground from both s i d e s to a t h i n s e c t i o n . However, I can only conclude that increment p r o d u c t i o n ceased i n a number of i n d i v i d u a l s , and in some f i s h , on only one of the s a g i t t a l p a i r . These anomalies remain unexplained, although they have been noted p r e v i o u s l y i n both j u v e n i l e (Campana and N e i l s o n 1982) and l a r v a l (Laroche et a l . 1982) p l e u r o n e c t i d s . 50 CHAPTER 3. AGE-ENVIRONMENT INTERACTIONS IN THE PRODUCTION OF DAILY GROWTH INCREMENTS IN MIDSHIPMAN I n t r o d u c t i o n D a i l y growth increments i n the o t o l i t h s of f i s h e s have been observed i n a l a r g e number of s p e c i e s ( P a n n e l l a 1971; Brothers et a l . 1976; Taubert and Coble 1977; Wilson and L a r k i n 1980). These c o n c e n t r i c a l l y formed increments may be counted or measured to p r o v i d e a c h r o n o l o g i c a l r e c o r d of past f i s h growth. Information on h a t c h i n g date/age (Ralston 1976; Struhsaker and Uchiyama 1976), d a i l y growth r a t e s (Methot 1981) and t i m i n g of l i f e h i s t o r y t r a n s i t i o n s ( P a n n e l l a 1980; B r o t h e r s and McFarland 1981) has been d e r i v e d from the examination of o t o l i t h m i c r o s t r u c t u r e . Such data are d i f f i c u l t to o b t a i n from l a r v a l and j u v e n i l e f i s h e s by other means. D a i l y increments are produced through a d i e l p e r i o d i c i t y i n the d e p o s i t i o n of c a l c i u m carbonate on the o t o l i t h (Mugiya et a l . 1981). However, there i s some c o n t r o v e r s y as to the z e i t g e b e r behind the d a i l y c y c l e of d e p o s i t i o n , i f indeed one e x i s t s . In a s e r i e s of experiments upon l a r v a l Lepomis, Taubert and Coble (1977) determined that a 24 h l i g h t - d a r k c y c l e was necessary to e n t r a i n an endogenous rhythm of increment p r o d u c t i o n . R e v e r s a l of the l i g h t - d a r k c y c l e reversed the d a i l y sequence of increment formation i n l a r v a l T i l a p i a (Tanaka et a l . 1981). However, 36 h "days" and constant l i g h t c o n d i t i o n s had no e f f e c t on d a i l y increment p r o d u c t i o n i n j u v e n i l e s t a r r y f l o u n d e r s , P l a t i c h t h y s s t e l l a t u s (Campana and N e i l s o n 1982). 51 S i m i l a r l y , constant l i g h t or dark c o n d i t i o n s d i d not i n h i b i t the formation of d a i l y increments i n young chinook salmon, Oncorhynchus tshawytscha ( N e i l s o n and Geen 1982). The c o n t r a d i c t o r y r e s u l t s of the above s t u d i e s suggest that p h o t o period e f f e c t s on increment p r o d u c t i o n may vary with age or s p e c i e s of f i s h . Other environmental v a r i a b l e s may i n f l u e n c e the d a i l y rhythm of o t o l i t h d e p o s i t i o n . D i e l temperature f l u c t u a t i o n has been i m p l i c a t e d as a f a c t o r i n d a i l y increment p r o d u c t i o n of temperate stream f i s h e s (Brothers 1981), although t h i s suggestion has not been supported by other s t u d i e s (Campana and N e i l s o n 1982; N e i l s o n and Geen 1982). Feeding frequency may a l s o i n f l u e n c e o t o l i t h increment p r o d u c t i o n ; f i s h given m u l t i p l e d a i l y feedings have been r e p o r t e d to produce non-daily increments (Pannella 1980; N e i l s o n and Geen 1982), although a recent study suggests that f e e d i n g frequency does not a f f e c t d a i l y increment p r o d u c t i o n , but does i n f l u e n c e the formation of s u b d a i l y increments (Campana 1983b). Confidence i n the r e l i a b i l i t y of o t o l i t h m i c r o s t r u c t u r e examination r e q u i r e s knowledge of those f a c t o r s t hat may i n f l u e n c e o t o l i t h increment p r o d u c t i o n . C o n f l i c t i n g r e s u l t s i n the l i t e r a t u r e suggest that age i n f l u e n c e s the response of d a i l y increment p r o d u c t i o n to environmental v a r i a b l e s such as p h o t o p e r i o d and temperature. T h i s study was undertaken to t e s t that h y p o t h e s i s . P l a i n f i n midshipman ( P o r i c h t h y s notatus) were reared from the egg stage under v a r i o u s l i g h t and temperature regimes; constant c o n d i t i o n s and d i e l c y c l e s of each v a r i a b l e 52 were t e s t e d . The e f f e c t of the regimes on o t o l i t h m i c r o s t r u c t u r e was noted f o r both newly-hatched and j u v e n i l e f i s h . J u v e n i l e s were then s u b - d i v i d e d and t r a n s f e r r e d to d i f f e r e n t regimes, a l l o w i n g an examination of the i n t e r a c t i v e i n f l u e n c e of g r e a t e r age and novel environment on increment p r o d u c t i o n . M a t e r i a l s and Methods F e r t i l i z e d P o r i c h t h y s eggs were c o l l e c t e d i n t e r t i d a l l y from White Rock, B r i t i s h Columbia on June 9 and June 22, 1982. Yolk-sac l a r v a e remain attached to the rock upon which the eggs were o r i g i n a l l y d e p o s i t e d (Arora 1948), n e c e s s i t a t i n g the c o l l e c t i o n of both rocks and egg masses. Upon r e t u r n to the l a b o r a t o r y , e i g h t separate egg masses (50-250 ova each) were i s o l a t e d i n i n d i v i d u a l s a l t water a q u a r i a and maintained under a n a t u r a l photoperiod and a temperature of 13°C. Small amounts of methylene blue, streptomycin sulphate and p e n i c i l l i n G were used to c o n t r o l b a c t e r i a l and fungal i n f e c t i o n . Embryo development v a r i e d both among and w i t h i n egg masses, but the d i f f e r e n c e appeared to be l e s s than 2-3 d. On J u l y 1, egg masses were exposed to an experimental environment. Environmental regimes were s e l e c t e d to provide a d i e l p e r i o d i c i t y of e i t h e r photoperiod or temperature. A t h i r d regime maintained constant c o n d i t i o n s of both v a r i a b l e s . In t h i s manner, the i n f l u e n c e of both f a c t o r s on increment formation c o u l d be determined f o r newly-hatched l a r v a e . Regimes were as f o l l o w s : 14L:10D at a constant temperature of 19°C (14L:1OD/CT) 53 24L with 14 h at 21°C and 10 h at 19°C (24L/14T1:10T2) 24L at a constant temperature of 19°C (24L/CT) D u p l i c a t e a q u a r i a , each c o n t a i n i n g an egg mass (or 2 small masses, i f at s i m i l a r developmental s t a g e s ) , were kept i n l i g h t -p r o o f, t e m p e r a t u r e - c o n t r o l l e d c u b i c l e s under each of the above environments. A l l l i g h t i n g was f l u o r e s c e n t and temperature f l u c t u a t i o n s were t i m e r - c o n t r o l l e d . Mean temperatures approximated those of the egg c o l l e c t i o n s i t e ; d i e l temperature f l u c t u a t i o n s were present at the s i t e , but were not recorded. Aquarium water was changed at 7-10 d i n t e r v a l s . Hatching date v a r i e d among and w i t h i n egg masses, beginning between J u l y 7-1 1 ..• Release from the rock (near completion of yol k sac . r e s o r p t i o n ) was more v a r i a b l e , and occurred between J u l y 23-Aug 9. L i v e Artemia were f i r s t p r o v i d e d as food on J u l y 30 and were consumed by both r e l e a s e d and attached l a r v a e . T h e r e a f t e r , Artemia were maintained i n a l l a q u a r i a at a l l times, with the exception of two 3-d p e r i o d s when food was not a v a i I a b l e . By Aug 10, a l l f i s h were approximately 32 d o l d (post-hatch) and had become j u v e n i l e s (ie-had assumed the appearance of an a d u l t ) . To t e s t the e f f e c t of an a l t e r e d photoperiod or temperature c y c l e on j u v e n i l e s , one tank from each of the environmental regimes was s u b - d i v i d e d ( F i g . 8 ) . Approximately 25 f i s h were t r a n s f e r r e d from one aquarium ("cohort") to each of the remaining environments, while l e a v i n g 25 f i s h i n the o r i g i n a l environment as a c o n t r o l . S a g i t t a l o t o l i t h s were removed from up to 25 of the excess f i s h to determine the e f f e c t 8. Summary of experimental environmental regimes through time.. F i s h t r a n s f e r r e d to new environments on Aug 10 came from the same egg mass as that sampled on Aug 10. JULY 1-AUG 10 AUG 10 TRANSFER AUG 1 0 - S E P T 10 14L 10D/CT 24 L/ 14Ti:10T2 24L/CT 24L/14Ti10T 2 14L 10D CT 24L CT 56 of the o r i g i n a l environment on newly-hatched l a r v a e . In order to remove any i n t e r - c o h o r t v a r i a b i l i t y of hat c h i n g dates, only one of the two a v a i l a b l e c o h o r t s from each environment was sub-d i v i d e d and sampled. However, low numbers of 14L:10D/CT f i s h n e c e s s i t a t e d the t r a n s f e r of an e n t i r e c o h o r t . For p r o c e s s i n g , the s a g i t t a l o t o l i t h s were brushed f r e e of t i s s u e and glued s u l c u s - s i d e up with Krazy Glue on a standard microscope s l i d e . S a g i t t a e were ground and p o l i s h e d with m e t a l l u r g i c a l l a p p i n g f i l m ( g r i t s i z e 30 jum to 0.3 /am) u n t i l the growth increments i n the re g i o n of maximal growth were most v i s i b l e . I d e f i n e d a growth increment as a b i p a r t i t e s t r u c t u r e , c o n s i s t i n g of a narrow opaque band and an adjacent broad t r a n s l u c e n t r e g i o n . Major increments between the o t o l i t h p e r i p h e r y and the hatch check were counted at l e a s t twice through a compound microscope at a m a g n i f i c a t i o n of 400X. D u p l i c a t e counts of an o t o l i t h never d i f f e r e d by more than 10%. There was l i t t l e doubt concerning the nature of the hatch check; i t s r a d i u s matched that of r a d i i of o t o l i t h s removed at hatch. Growth increments i n 14L:10D/CT f i s h sampled Sept 10 were counted as above. However, a second s e r i e s of counts was made from the hatch check to the prominent Aug 10 check; the second data set served as a s u b s t i t u t e f o r the a c t u a l sampling of 14L:10D/CT f i s h on Aug 10. Increment counts were made from both the l e f t and r i g h t hand sid e s a g i t t a e . Since the two s i d e s d i d not d i f f e r s y s t e m a t i c a l l y under any of the environments ( p a i r e d t - t e s t , P>0.05), the means were used i n a l l data a n a l y s e s . 57 Increment widths were measured with a micrometer from photographs. Since i n d i v i d u a l o t o l i t h s o f t e n d i s p l a y e d e r r a t i c but d i s c e r n a b l e width trends through time, a measure of the s i m i l a r i t y of the widths of two adjacent d a i l y increments was c a l c u l a t e d : IRi = |wi ~ wi-1| (Wi + Wi-1)/2 where IRi i s the index of increment width r e g u l a r i t y f o r day i , and Wi i s the increment width f o r day i . Such an index g i v e s low value s when adjacent increments are s i m i l a r i n width, d e s p i t e any trends i n the data. Index v a l u e s were c a l c u l a t e d f o r a range of ages i n o t o l i t h s from a given environment. R e s u l t s Por i c h t h y s l a r v a e and j u v e n i l e s s u r v i v e d and grew under a l l l a b o r a t o r y environments. S u r v i v a l exceeded 95% a f t e r hatch. F i s h sampled approximately one mo a f t e r hatch (Aug 10) d i d not d i f f e r s i g n i f i c a n t l y i n standard l e n g t h (ANOVA, P>0.05). By the end of the study, only those f i s h maintained i n the 24L/14T1:10T2 environment were s i g n i f i c a n t l y smaller i n le n g t h ( S c h e f f e ' s t e s t , P<0.01); the d i f f e r e n c e was a p p a r e n t l y due to u n i n t e n t i o n a l overcrowding from the date of t r a n s f e r . Hatching was i n i t i a t e d s i m u l t a n e o u s l y i n two of the three i n i t i a l environments, but s t a r t e d 4 d l a t e r i n the 24L/CT a q u a r i a . The delay d i d not appear to be due to the a r t i f i c i a l environment, s i n c e embryo development among the 24L/CT egg 58 masses lagged behind that of the others at the time of c o l l e c t i o n . In the aquarium, approximately 95% of the viable ova hatched within 4 d of hatch i n i t i a t i o n . Intra-tank hatch date variance would be expected to affect the variance of increment counts. However, the 17 d range of l a r v a l release dates (from the rock) was not re f l e c t e d in the o t o l i t h microstructure. Unground sagittae derived from both pre- and post-hatch f i s h were extremely lobulated in structure. The o r i g i n of the numerous lobes was 5-10 "peripheral" nuclei, from which the majority of the growth increments emanated. A central nucleus also had growth increments associated with i t , although these were incorporated into the peripheral increments within 10-20 days/increments. A prominent hatch check occurred within 5-10 major increments of the central nucleus. The most prominent check of the older o t o l i t h s was that associated with the sub-d i v i s i o n / t r a n s f e r date of Aug 10. Many growth increments were v i s i b l e in the polished o t o l i t h s sampled after hatch. When plotted as a function of time, t o t a l increment counts were s i g n i f i c a n t l y greater than those expected of d a i l y production (P<0.05)(Fig. 9). Di e l l i g h t and temperature cycles both produced an increment:age slope of approximately 3.0, suggesting that numerous subdaily increments were being counted with any d a i l y increments present. Increment c l a r i t y , prominence and width varied substantially within an o t o l i t h . However, most increments could be assigned to one of two " l e v e l s " - v i s u a l l y prominent/relatively wide and v i s u a l l y f a i n t / r e l a t i v e l y narrow. To determine i f the f i r s t l e v e l 9. T o t a l increment count as a f u n c t i o n of time f o r f i s h sampled from a l l experimental environments. A s t r a i g h t l i n e has been f i t t e d to the data, although the r e l a t i o n s h i p i s probably c u r v i l i n e a r . N=5 f o r each data p o i n t . 2 0 0 1 160 1 4 L : 1 0 D / C T = 2 4 L / 1 4 T I : 1 0 T 2 —I— 10 — i — 20 i 3 0 — i — 4 0 — i — 5 0 6 0 7 0 A G E ( D A Y S ) 61 c o n s i s t e d p r i m a r i l y of d a i l y increments, the expected width of a d a i l y increment was c a l c u l a t e d . R a d i a l measurements ( c e n t r a l nucleus to r o s t r a l t i p ) of o t o l i t h s from a l l environments and a v a r i e t y of sampling dates p r o v i d e d approximate v a l u e s of the mean i n c r e a s e i n r a d i a l o t o l i t h growth per day (N=10 per d a t e ) . Date J u l y 23 J u l y 30 Aug 9 Sept 10 Mean o t o l i t h r a d i u s {/am) 270 430 620 875 D a i l y increments on the order of 12-23 and 5-8 jum wide, would be expected i n the f i r s t and second month post-hatch, r e s p e c t i v e l y . These expected increment widths are s i m i l a r to those observed i n the f i r s t " l e v e l " of growth increments. C r i t e r i a f o r d i s t i n g u i s h i n g d a i l y increments from s u b d a i l y , have been reported p r e v i o u s l y (Taubert and Coble 1977; Campana and N e i l s o n 1982; M a r s h a l l and Parker 1982). N e v e r t h e l e s s , no o b j e c t i v e c r i t e r i a have yet been d e f i n e d which can be a p p l i e d to a l l o t o l i t h s . In t h i s study, I have used v i s u a l prominence and increment width as guides f o r d i f f e r e n t i a t i n g d a i l y and s u b d a i l y increments. Increments a s s i g n e d as d a i l y were: 1) of s i m i l a r v i s u a l prominence ( c o n t r a s t ) to adjacent d a i l y increments (+30%), 2) of s i m i l a r increment width to adjacent d a i l y increments (+50%), 3) d i d not merge with adjacent d a i l y increments i n the nearest r a d i a l groove of the s a g i t t a . Some increments met only some of the above c r i t e r i a and were s u b j e c t i v e l y assigned as d a i l y or s u b d a i l y . The observed widths 62 of d a i l y increments, as c l a s s i f i e d above, were s i m i l a r to those expected on the b a s i s of o t o l i t h growth c a l c u l a t i o n s . D i e l L i g h t C y c l e O t o l i t h s of f i s h reared under a d i e l photoperiod and constant temperature (14L:1OD/CT) produced c l e a r d a i l y growth increments from the time of hatch. Regression of major increment number a g a i n s t elapsed time produced a slope not s i g n i f i c a n t l y d i f f e r e n t from 1.0 (P>0.05); A slope of 1.0 would i n d i c a t e t hat one increment was formed every day. Increment width v a r i e d with l o c a t i o n on the o t o l i t h and f i s h age ( F i g . 10). Subdaily increments were common at a l l ages, numbering up to 5 between adjacent d a i l y increments. They were most abundant i n the f i r s t month a f t e r hatch. The d i s t i n c t i o n between d a i l y and s u b d a i l y increments was g e n e r a l l y c l e a r ; however, increments produced 5-20 d a f t e r hatch were the most i r r e g u l a r on the o t o l i t h , and were sometimes d i f f i c u l t to i n t e r p r e t . S u b d a i l y increments tended to be prominent i n t h i s r e g i o n , so that d i s t i n c t i o n was a matter of degree ( F i g . 11A). F i s h t r a n s f e r r e d to a constant environment (24L/CT) as j u v e n i l e s produced p o s t - t r a n s f e r increments that were very d i f f e r e n t from those produced p r i o r to t r a n s f e r . P o s t - t r a n s f e r increments were v i s u a l l y f a i n t , and i n some cases, v i r t u a l l y uncountable ( F i g . 12A). Subdaily increments were a l s o p r e s e n t . T r a n s f e r to a constant environment was not a s s o c i a t e d with a r e c o g n i z a b l e l a g p e r i o d d u r i n g which increments g r a d u a l l y s h i f t e d t h e i r appearance. Increments produced w i t h i n 1-2 d of 10. D a i l y increment width as a f u n c t i o n of age f o r o t o l i t h samples from each of the three experimental environments. At a given age, mean widths do not d i f f e r s i g n i f i c a n t l y among environments, with the exc e p t i o n of value s at age 40 d (P<0.05). AGE (DAYS) 65 F i g . 11. Growth increments on the p o l i s h e d s a g i t t a e of l a r v a l midshipman. S u b d a i l y increments are v i s i b l e between some of the i n d i c a t e d d a i l y increments. D a i l y increments become more c l e a r with age, but are most pr o m i n e n t / c o n s i s t e n t i n width i n (A). Bar = 30 um. PN=peripheral nucleus. (A). Hatched under a d i e l l i g h t c y c l e . (B) . Hatched under a d i e l temperature c y c l e . (C). Hatched under a constant environment. z 12. S a g i t t a l growth increments produced before and a f t e r t r a n s f e r to a constant environment. F i s h hatched under 24L/CT produced c l e a r e r d a i l y increments than those t r a n s f e r r e d from a d i f f e r e n t environment. D a i l y increments are i n d i c a t e d , as i s d i r e c t i o n of s a g i t t a l growth (arrow). T=transfer check. Bar = 30 um. (A). 14L:10D/CT to 24L/CT. (B). 24L/14T1:10T2 to 24L/CT. (C). 24L/CT to 24L/CT. 7( 73 t r a n s f e r were v i r t u a l l y n o n - e x i s t e n t . N e v e r t h e l e s s , the increments produced were d a i l y i n nature, as i n d i c a t e d by increment counts s i m i l a r to those of f i s h kept under a d i e l l i g h t c y c l e (Table 3). D a i l y increments g r a d u a l l y became more prominent a f t e r approximately 15 d p o s t - t r a n s f e r , t h e i r v i s u a l c o n t r a s t improving u n t i l the end of the experiment. D i e l Temperature C y c l e F i s h hatched under a 24L/14T1:10T2 regime d e p o s i t e d growth increments that d i f f e r e d i n many r e s p e c t s from those produced under a c y c l i c photoperiod (14L:1OD/CT). Increments produced up to 8 d medial and d i s t a l of the p e r i p h e r a l n u c l e i were c h a r a c t e r i z e d by . a h i g h i n c i d e n c e of prominent s u b d a i l y increments ( F i g . 11B), moreso than was the case under a c y c l i c p h o t o p e r i o d . The d a i l y / s u b d a i l y c o n f u s i o n i s r e f l e c t e d i n the data of Aug 10 (Table 3), where the observed major increment count was s i g n i f i c a n t l y d i f f e r e n t from that expected of d a i l y increments (P<0.05). The high increment count i n d i c a t e s that some s u b d a i l y increments were prominent enough to be c l a s s i f i e d as d a i l y . Increments produced i n the 15-20 d before t r a n s f e r were g e n e r a l l y d i s t i n c t and r e g u l a r i n appearance. Increment width and the i n c i d e n c e of s u b d a i l y increments was s i m i l a r to that observed i n the corresponding region of the c y c l i c photoperiod o t o l i t h s ( F i g . 10). However, the appearance of the major increments was unusual i n that the opaque p o r t i o n of each increment was r e l a t i v e l y broad and s h a r p l y d e l i n e a t e d ( F i g . 13). Table ,3.. Growth increment counts i n r e l a t i o n to elapsed time f o r various experimental environments. F i s h were t r a n s f e r r e d to new environments (or kept i n the o r i g i n a l environment as a control) on Aug. 10. Aug 10 Samples Sept 10 Samples Envir 1 Days a f t e r hatch No. major increments SE Days Envir 2 a f t e r hatch No. major increments SE UL:10D/CT 34 34.3* 0.57 14L:10D/CT 65 66.7' 0.80 14L:10D/CT - - - 24L/CT 65 65.1 1.21 24L/14T1:10T2 34 41.1 1.29 24L/CT 65 71.2 0.70 24L/CT 30 49.1 1.33 24L/CT 61 76.9 1.04 24L/CT - - - 14L:10D/CT 61 72.7 1.10 24L/CT - - - 24L/14T1:10T2 61 69.3 0.92 * T h i s value was derived from 14L:10D/CT o t o l i t h s sampled Sept 10; counts were made- from the hatch check to the prominent s u b d i v i s i o n / t r a n s f e r check. 13. D a i l y growth increments produced on the s a g i t t a e of midshipman a f t e r 15-25 d of r e a r i n g under a d i e l temperature c y c l e . The increments are v i s u a l l y prominent and s h a r p l y d e l i n e a t e d r e l a t i v e to those produced under other environmental regimes. Bar = 20 fjm. 77 F i s h maintained i n the 24L/14T1:10T2 environment a f t e r Aug 10 were overcrowded and d i d not grow w e l l . As a r e s u l t , post-t r a n s f e r o t o l i t h growth was l i m i t e d , increments were very narrow and r e l i a b l e counts c o u l d not be made. However, increment counts of r e p r e s e n t a t i v e O t o l i t h s i n d i c a t e d that d a i l y increments were d e p o s i t e d a f t e r the t r a n s f e r date. F i s h t r a n s f e r r e d to a constant environment (24L/CT) as j u v e n i l e s produced p o s t - t r a n s f e r increments s i m i l a r to those of f i s h t r a n s f e r r e d from a c y c l i c p hotoperiod ( F i g . 12B). The d i f f e r e n c e between Aug and Sept increment counts corresponds to that expected of d a i l y increment d e p o s i t i o n (P>0.05)(Table 3). The f i r s t 5 p o s t - t r a n s f e r increments were f a i n t and v i r t u a l l y n o n - e x i s t e n t ; subsequent increments became more d i s t i n c t and r e g u l a r with time. Opaque r e g i o n s w i t h i n each increment never became as broad and d i s c r e t e as was observed p r i o r to t r a n s f e r . Constant Environment O t o l i t h s of f i s h hatched under constant c o n d i t i o n s (24L/CT) i n i t i a l l y resembled those of the other two environments (with res p e c t to the f i r s t 5-8 increments). The subsequent region resembled that of 24L/14T1:10T2 f i s h i n that s u b d a i l y increments were prominent ( F i g . 11C). Although the d i f f e r e n c e was not s i g n i f i c a n t ( S c h e f f e ' s t e s t , P=0.07), increment widths tended to be more i r r e g u l a r than those of 14L:10D/CT f i s h of s i m i l a r age ( F i g . 14). The c o n f u s i o n of d a i l y and s u b d a i l y increments i n the e a r l y l a r v a l r e g ion r e s u l t e d i n a high v a r i a n c e and a mean 78 F i g . 14. Index of d a i l y increment width r e g u l a r i t y as a f u n c t i o n of age f o r o t o l i t h samples from each of the three experimental environments. Bars represent +1 SE. INDEX OF WIDTH REGULARITY 80 increment count that was s i g n i f i c a n t l y higher than would be expected of d a i l y increments (P<0.05)(Table 3). A f t e r age 10-25 d, d a i l y increments decreased i n width ( F i g . 10) and became more r e g u l a r i n width ( F i g . 14) and appearance, although s u b d a i l y increments were s t i l l p r e s e n t . Increments with broad, d i s c r e t e opaque p o r t i o n s were not observed i n the 24L/CT f i s h , as they were i n the f l u c t u a t i n g temperature regime. For an unknown reason, o t o l i t h growth (but not f i s h growth) under a 24L/CT regime s i g n i f i c a n t l y exceeded that observed under 14L:10D/CT (P<0.05). F i s h remaining i n a constant environment a f t e r the Aug 10 t r a n s f e r date continued to produce d i s t i n c t increments, although d a i l y and s u b d a i l y increments were o c c a s i o n a l l y d i f f i c u l t to d i f f e r e n t i a t e . Increment width was s i g n i f i c a n t l y more i r r e g u l a r than i n the p o s t - t r a n s f e r r e g i o n of 14L:10D/CT f i s h ( t - t e s t , P<0.05) ( F i g . 14). Major increments i n the p o s t - t r a n s f e r region were d a i l y ; the r e g r e s s i o n of increment number a g a i n s t elapsed time r e s u l t e d i n a slope not s i g n i f i c a n t l y d i f f e r e n t from u n i t y (P>0.05). P o s t - t r a n s f e r increments of f i s h hatched and reared under constant c o n d i t i o n s were prominent, although i r r e g u l a r i n width ( F i g . 12C). In c o n t r a s t , increments of f i s h t r a n s f e r r e d to the constant environment as j u v e n i l e s were v i s u a l l y f a i n t , becoming more prominent a f t e r 2-3 wk. J u v e n i l e s t r a n s f e r r e d from a constant environment to a c y c l i c regime d e p o s i t e d s i m i l a r -appearing increments before and a f t e r t r a n s f e r . However, post-t r a n s f e r increments tended to be more r e g u l a r i n width than i n 81 constant environment f i s h ; the change g e n e r a l l y became apparent 2-4 d a f t e r t r a n s f e r . V i s u a l c o n t r a s t of d a i l y increments may have i n c r e a s e d i n the f l u c t u a t i n g temperature regime, but the change was not c o n s i s t e n t among a l l o t o l i t h s . No such change was evident among the p o s t - t r a n s f e r increments of f i s h s h i f t e d from 24L/CT to 14L:1OD/CT, although the i n c i d e n c e of s u b d a i l y increments appeared to decrease* F i s h t r a n s f e r r e d from the constant environment to e i t h e r of the c y c l i c regimes produced d a i l y increments a f t e r t r a n s f e r ; h i g h increment counts (Table 3) were d e r i v e d from the i r r e g u l a r , p r e - t r a n s f e r region of the o t o l i t h . Di s c u s s i o n D a i l y growth increments are d e p o s i t e d on the o t o l i t h s of midshipman under a v a r i e t y of environmental c o n d i t i o n s . My r e s u l t s i n d i c a t e that l i g h t , temperature, age and an endogenous c i r c a d i a n rhythm may a l l i n f l u e n c e the p r o d u c t i o n and/or appearance of d a i l y and s u b d a i l y increments. However, the v a r i a b l e s t e s t e d i n t e r a c t to a l a r g e degree, and t h e i r i n f l u e n c e on increment p r o d u c t i o n i s s u b j e c t to a l t e r a t i o n through time. A c y c l i c l i g h t regime i n f l u e n c e d increment p r o d u c t i o n i n l a r v a l f i s h more than any other v a r i a b l e t e s t e d . Under a n a t u r a l p hotoperiod, d a i l y increments were produced from the time of hatch. In c o n t r a s t , constant l i g h t c o n d i t i o n s d i s r u p t e d the p r o d u c t i o n of p o s t - h a t c h increments, r e s u l t i n g i n a high i n c i d e n c e of prominent n o n - d a i l y increments (>1 increment/24 h) and i r r e g u l a r increment widths. My o b s e r v a t i o n s are c o n s i s t e n t 82 with those of Taubert and Coble (1977), who observed numerous, non- d a i l y increments i n l a r v a l T i l a p i a hatched under constant l i g h t c o n d i t i o n s . Those authors concluded that l i g h t a c t ed as a z e i t g e b e r f o r an endogenous rhythm, and that without a c y c l i c p hotoperiod, d a i l y increment p r o d u c t i o n was not p o s s i b l e . My r e s u l t s only p a r t i a l l y support t h e i r c o n c l u s i o n . Photoperiod e n t r a i n e d d a i l y increment p r o d u c t i o n i n newly hatched midshipman. However, i n the absence of c y c l i c l i g h t or temperature s t i m u l i , an endogenous c i r c a d i a n rhythm of increment d e p o s i t i o n became apparent a f t e r an a c c l i m a t i o n p e r i o d of 2-4 wk. T h e r e f o r e , photoperiod a c t e d as a z e i t g e b e r f o r an endogenous rhythm dur i n g the e a r l y l a r v a l s t ages, but became unnecessary with i n c r e a s i n g age. The n o n - d a i l y increments produced a f t e r hatch i n t h i s study (and that of Taubert and Coble (1977)), probably comprised both d a i l y and s u b d a i l y increments. The combination r e s u l t e d i n the d e p o s i t i o n of more than one increment per 24 h. If a constant photoperiod was present at hatch, an endogenous rhythm of increment d e p o s i t i o n became apparent a f t e r an a c c l i m a t i o n p e r i o d . A c c l i m a t i o n a l s o o c c u r r e d when o l d e r f i s h were t r a n s f e r r e d from a n a t u r a l l i g h t c y c l e to constant l i g h t c o n d i t i o n s . However, the p a t t e r n of increment p r o d u c t i o n d u r i n g a c c l i m a t i o n d i f f e r e d at the two ages (Table 4 ) . The l a r v a l f i s h a c c l i m a t i o n p e r i o d may be analogous to that of newborn r a t s t r a n s f e r r e d from a d i e l p hotoperiod to constant c o n d i t i o n s . An arhythmic a c t i v i t y p a t t e r n continues f o r almost 2 wk i n r a t s before an endogenous c i r c a d i a n rhythm becomes apparent (Davis Oo T a b l e M Age e f f e c t s on growth increment production i n midshioman r e a r e d under three a r t i f i c i a l environments. Larvae J u v e n i l e s - l i g h t important as z e i t g e b e r - d a i l y & s u b d a i l y increments s i m i l a r d u r i n g a c c l i m a t i o n to 24L - l o n g a c c l i m a t i o n t o 24L - immature c i r c a d i a n rhythm - l i g h t not important as z e i t g e b e r - f a i n t d a i l y increments, but s u b d a i l y increments d i s s i m i l a r d u r i n g a c c l i m a t i o n t o 24L - short a c c l i m a t i o n to 2LL • - mature c i r c a d i a n rhythm 84 1981 ) . The l e n g t h of the a c c l i m a t i o n p e r i o d c o u l d not be determined with accuracy. A s h i f t i n increment appearance a f t e r t r a n s f e r from a constant to a c y c l i c environment g e n e r a l l y o c c u r r e d i n 2-5 d. The reverse t r a n s f e r r e s u l t e d i n almost non-e x i s t e n t increments for- a p e r i o d of 5 d, but the v i s u a l c o n t r a s t of the growth p a t t e r n s improved over the subsequent 10-15 d. T h e r e f o r e , the c r i t i c a l stage of the a d a p t a t i o n process appears to have been completed in 2-5 d. T h i s r e s u l t i s c o n s i s t e n t with that of Tanaka et a l . (1981), who observed a 6 d t r a n s i t o r y p e r i o d of increment formation when a 24 h l i g h t - d a r k c y c l e was suddenly r e v e r s e d . A g e - r e l a t e d changes i n endogenous c i r c a d i a n rhythms have not been examined i n f i s h e s . Mammalian s t u d i e s i n d i c a t e that endogenous rhythms o f t e n appear a f t e r b i r t h ; once pr e s e n t , c y c l e amplitude tends to increase with time u n t i l the rhythm i s "mature" (Davis 1981). P o r i c h t h y s l a r v a e hatched under constant l i g h t appear to f i t t h i s p a t t e r n . D a i l y and s u b d a i l y increments were not e a s i l y d i f f e r e n t i a t e d at f i r s t , suggesting that the c i r c a d i a n d e p o s i t i o n rhythm was not yet mature. Maturation a p p a r e n t l y o c c u r r e d by Days 10-20. E a r l y l a r v a l increments were t e m p o r a r i l y i n d i s t i n c t i n the 14L:10D/CT f i s h , suggesting that the c y c l i c p h o t o period e n t r a i n e d the maturing rhythm f a i r l y q u i c k l y . In a d d i t i o n , very young animals may be more responsive to a d i e l l i g h t c y c l e , due to a g e - r e l a t e d c h a r a c t e r s of the rhythm c y c l e (Sacher and Duffy 1978). For i n s t a n c e , the metabolic r a t e of newly hatched r a t s i s very s e n s i t i v e to 85 changes i n l i g h t l e v e l , while o l d e r r a t s are l e s s a f f e c t e d . In t h i s study, l a r v a l f i s h exposed to a constant environment took longer to produce d a i l y increments than d i d j u v e n i l e f i s h , s uggesting an analogy with the r a t study. S i m i l a r a g e - r e l a t e d r e s u l t s were repo r t e d by Gibson et a l . (1978) i n an ontogenetic study of f l a t f i s h a c t i v i t y c y c l e s . A constant photoperiod e l i m i n a t e d a d i e l a c t i v i t y c y c l e i n l a r v a l p l a i c e (Pleuronectes  p l a t e s s a ) , but had no such e f f e c t on j u v e n i l e s of the same spec i e s . I n c r e a s i n g age of midshipman was c o r r e l a t e d with d e c r e a s i n g increment width and fewer s u b d a i l y increments i n a l l environments. However, foremost among the a g e - a s s o c i a t e d e f f e c t s (Table 4) was the prominence of d a i l y increments i n j u v e n i l e s r e l a t i v e to l a r v a e . D i s t i n c t i o n between d a i l y and s u b d a i l y increments was seldom d i f f i c u l t i n j u v e n i l e s , u n l i k e the s i t u a t i o n i n l a r v a l o t o l i t h s . If t h i s a g e - r e l a t e d d i f f e r e n c e i n d a i l y increment formation i s u n i v e r s a l , d a i l y increment counts i n l a r v a e may be u n r e l i a b l e r e l a t i v e to s l i g h t l y o l d e r f i s h . T h i s suggestion has s e r i o u s i m p l i c a t i o n s f o r the a p p l i c a t i o n of growth increments i n aging l a r v a l f i s h . S i m i l a r l y , the absence of d e f i n i t i v e c r i t e r i a f o r d i f f e r e n t i a t i n g d a i l y and s u b d a i l y increments c o u l d cause problems i n aging f i e l d - c o l l e c t e d f i s h . S u b d a i l y increments can be numerous and c o n f u s i n g i n some sp e c i e s (Campana, unpublished d a t a ) . The demonstration of an a g e - r e l a t e d rhythm and the ex i s t e n c e of an a c c l i m a t i o n p e r i o d may r e s o l v e some of the c o n f l i c t i n g r e s u l t s i n the l i t e r a t u r e concerning the z e i t g e b e r 86 e f f e c t of l i g h t . In a pr e v i o u s study, a constant l i g h t regime d i d not i n f l u e n c e the p r o d u c t i o n of d a i l y increments i n j u v e n i l e s t a r r y f l o u n d e r s (Chapter 1). The f l o u n d e r s were approximately 8 mo o l d , suggesting that the necessary a c c l i m a t i o n p e r i o d would be s h o r t . In a d d i t i o n , the f i s h were exposed to the experimental environment f o r 2 wk p r i o r t o t e t r a c y c l i n e i n j e c t i o n (marking the s t a r t of the experiment); i t i s probable that a c c l i m a t i o n o c c u r r e d d u r i n g t h i s p e r i o d , r e s u l t i n g i n c l e a r d a i l y increment p r o d u c t i o n by the time the experiment began. An analogous e x p l a n a t i o n may e x p l a i n the r e s u l t s of another study, where chinook salmon eggs, r e a r e d i n darkness, produced d a i l y increments a f t e r hatch ( N e i l s o n and Geen 1982). The embryos were h e l d i n t o t a l darkness for, 50 d before hatch, suggesting that t h e i r endogenous c i r c a d i a n rhythm had time to a c c l i m a t e before hatch. A f l u c t u a t i n g temperature regime d i d not e n t r a i n increment p r o d u c t i o n under constant l i g h t c o n d i t i o n s . F i s h r e ared i n t h i s environment produced more increments than would be expected of d a i l y p r o d u c t i o n , s i m i l a r to those of 24L/CT f i s h . The v a r i a n c e of l a r v a l increment counts was s i m i l a r to that produced under a constant environment, both of which were s i g n i f i c a n t l y l a r g e r than the 14L:10D/CT v a r i a n c e ( B a r t l e t t ' s t e s t , P<0.01). Once a c c l i m a t i o n o c c u r r e d , d a i l y increments were produced through an ap p a r e n t l y endogenous p e r i o d i c i t y , and not through temperature entrainment of an i n t e r n a l c l o c k . However, the formation of a broad, o p t i c a l l y dense, s h a r p l y d e l i n e a t e d opaque zone i n post-a c c l i m a t i o n d a i l y increments, i n d i c a t e s that temperature 87 f l u c t u a t i o n d i d a f f e c t increment p r o d u c t i o n . The opaque p o r t i o n of a d a i l y increment c o n s i s t s of ca l c i u m carbonate and a prot e i n a c e o u s matrix, with the l a t t e r component predominating (Brothers 1981; Mugiya et a l . 1981). F a l l i n g temperatures, such as would occur at n i g h t , may i n c r e a s e the p r o p o r t i o n of p r o t e i n d e p o s i t e d i n the opaque r e g i o n , r e s u l t i n g i n an increment that has i n c r e a s e d v i s u a l c o n t r a s t . A c c e n t u a t i o n of c o n t r a s t renders increments v i s u a l l y prominent, and c o u l d e a s i l y be i n t e r p r e t e d as an e n t r a i n i n g mechanism. D i e l temperature f l u c t u a t i o n s n o t i c e a b l y accentuated increment c o n t r a s t i n young chinook salmon o t o l i t h s ( J . D. N e i l s o n , p e r s . comm.). A c o r r e l a t i o n of i n c r e a s i n g p r o t e i n d e p o s i t i o n with d e c r e a s i n g temperature suggests that the broad opaque zone formed d u r i n g the low temperature, 10 h, experimental " n i g h t " , o v e r l a i d the opaque zone formed under c i r c a d i a n c o n t r o l through a 3 h p e r i o d (Mugiya et a l . 1981). I f temperature does exert a "masking" e f f e c t ( E n r i g h t 1981), a low temperature-induced opaque zone would appear independently of any endogenous c i r c a d i a n rhythm of d e p o s i t i o n . T h e r e f o r e , m u l t i p l e d a i l y o s c i l l a t i o n s i n temperature c o u l d c o n c e i v a b l y produce a d i s t i n c t increment a f t e r each c y c l e , i n a d d i t i o n to the d a i l y increment formed under endogenous c o n t r o l . In some s i t u a t i o n s , the masking e f f e c t of temperature f l u c t u a t i o n s may be s u b s t a n t i a l , o b s c u r i n g most of the increments formed through the a c t i o n of an endogenous rhythm of d e p o s i t i o n (E. B. Br o t h e r s , p e r s . comm.). T h i s hypothesis i s c o n s i s t e n t with s t u d i e s that demonstrated that temperature c y c l e s do not e n t r a i n increment p r o d u c t i o n (Campana and N e i l s o n 88 1982; N e i l s o n and Geen 1982), but can i n f l u e n c e increment formation (Brothers 1981). My r e s u l t s suggest that a d i e l l i g h t c y c l e e n t r a i n s an endogenous c i r c a d i a n rhythm of increment d e p o s i t i o n . I n c r e a s i n g age m i t i g a t e d the z e i t g e b e r e f f e c t of photo p e r i o d , while temperature f l u c t u a t i o n i n f l u e n c e d increment appearance, r a t h e r than p e r i o d i c i t y . In a d d i t i o n , the i n c i d e n c e of s u b d a i l y increments i s c o r r e l a t e d with fe e d i n g p e r i o d i c i t y ( N e i l s o n and Geen 1982; Chapter 2 ) . The f a c t that so many v a r i a b l e s may a f f e c t increment d e p o s i t i o n suggests that the environment does not i n f l u e n c e the rhythm of o t o l i t h d e p o s i t i o n d i r e c t l y , but a c t s through some penultimate p r o c e s s . M e t a b o l i c r a t e i s s u s c e p t i b l e to environmental i n f l u e n c e , as w e l l as being s u b j e c t to an endogenous c i r c a d i a n rhythm (Matty 1978) th a t changes with age (Davis 1981). However, metabolic rate i s i n tu r n r e g u l a t e d by endocrine l e v e l s , and i t may be the environmental modulation of endocrine rhythms that u l t i m a t e l y c o n t r o l s increment p e r i o d i c i t y on the o t o l i t h (Menaker and B i n k l e y 1981). Endocrine s e c r e t i o n o f t e n f o l l o w s a c i r c a d i a n p a t t e r n (Simpson 1978), and in mammals at l e a s t , i s c l o s e l y l i n k e d to the c i r c a d i a n pacemaker i t s e l f (Menaker and B i n k l e y 1981). Hormones r e g u l a t e many asp e c t s of metabolism and growth, i n c l u d i n g s k e l e t a l c a l c i f i c a t i o n (Simpson 1978). T h e r e f o r e , i t seems reasonable to p o s t u l a t e that those f a c t o r s that e n t r a i n and/or moderate the c i r c a d i a n rhythm of endocrine s e c r e t i o n w i l l have a subsequent e f f e c t on increment d e p o s i t i o n i n the o t o l i t h . 89 CHAPTER 4. CALCIUM DEPOSITION AND OTOLITH CHECK FORMATION DURING PERIODS OF STRESS IN COHO SALMON I n t r o d u c t i o n The sequence of d a i l y growth increments i n o t o l i t h s of f i s h e s may provide a c h r o n o l o g i c a l r e c o r d of past growth. D a i l y growth increments are now being used to determine the age of l a r v a l and j u v e n i l e f i s h ( R a l s t o n , 1976; K e n d a l l and Gordon, 1981), b a c k - c a l c u l a t e d a i l y growth r a t e s (Methot, 1981) and may even pr o v i d e a dated r e c o r d of e c o l o g i c a l and p h y s i o l o g i c a l events through the l i f e t i m e of an i n d i v i d u a l f i s h ( P a n n e l l a , 1980; B r o t h e r s and McFarland, 1981). Checks ^ d i s c o n t i n u i t i e s or growth i n t e r r u p t i o n s ) i n the d a i l y increment sequence may r e c o r d p e r i o d s of p e r t u r b a t i o n or s t r e s s to the f i s h . Transmission of l i g h t through checks i s poor r e l a t i v e to other m i c r o s t r u c t u r a l f e a t u r e s , r e s u l t i n g i n s t r u c t u r e s which are immediately d i s t i n g u i s h a b l e from surrounding increments. Checks a l s o appear d i s t i n c t i n a c e t a t e p e e l r e p l i c a s and scanning e l e c t r o n microscope (SEM) photographs. S t r e s s due to c o l l e c t i o n , m i g r a t i o n and s t a r v a t i o n may a l l put a check on an o t o l i t h , with the i n t e n s i t y of the check a p p a r e n t l y p r o p o r t i o n a l to the degree of s t r e s s that the f i s h has undergone ( P a n n e l l a , 1980; Campana, un p u b l i s h e d ) . Since d a i l y increments provide a c h r o n o l o g i c a l h i s t o r y of past growth, the l o c a t i o n of a check on the o t o l i t h can be r e l a t e d to the date of the event i n q u e s t i o n . D a i l y growth increments i n • o t o l i t h s are b i p a r t i t e 90 s t r u c t u r e s , c o n s i s t i n g of a r e l a t i v e l y wide zone of c a l c i u m carbonate c r y s t a l s embedded in a p r o t e i n matrix, and an adjacent narrower band that i s dominated by the matrix (Dunkelberger et a l . , 1980; Mugiya et a l . , 1981). Recent experiments i n d i c a t e t h at the proteinaceous zone i s formed near dawn as a r e s u l t of a slowdown i n c a l c i u m d e p o s i t i o n (Mugiya et a l . , 1981; Tanaka et a l . , 1981). However, l i t t l e e l s e i s known of the d a i l y c y c l e of c a l c i u m d e p o s i t i o n on o t o l i t h s (Mugiya et a l . , 1981), and the processes r e s p o n s i b l e f o r check formation are not understood at a l l . P a n n e l l a (1980) has suggested that growth d i s c o n t i n u i t i e s may mark areas of o t o l i t h r e s o r p t i o n . O t o l i t h r e s o r p t i o n would r e s u l t i n an incomplete c h r o n o l o g i c a l sequence of d a i l y growth increments, and thus i n v a l i d a t e many a p p l i c a t i o n s of d a i l y increments. Checks are s u f f i c i e n t l y common i n w i l d f i s h to warrant understanding how and why they occur. The o b j e c t of t h i s study was to monitor c a l c i u m d e p o s i t i o n and/or r e s o r p t i o n on the o t o l i t h s of coho salmon (Oncorhynchus k i s u t c h ) before and a f t e r p e r i o d s of s t r e s s . The s t r e s s used i n t h i s study was s u f f i c i e n t to put a moderately i n t e n s e check on the s a g i t t a e of experimental f i s h . 91 M a t e r i a l s and Methods The experimental f i s h were c o l l e c t e d from T i n Can Creek, Vancouver, Canada and reared i n l a b o r a t o r y a q u a r i a f o r a minimum of 4 days before use. Standard lengths ranged from 2.9-4.1 cm, with a mean of 3.5 cm. Aquaria were maintained under a 14L:10D photoperiod at 10.5+0.5°C. A l l f i s h but those being s t r e s s e d were fed a commercial f i s h food twice d a i l y . Experiments i n which f i s h were immersed i n a 5 C a water comprised the bulk of t h i s study. They are b r i e f l y summarized i n Table 5. Ca-45 Immersion I t i s reasonable to assume t h a t a 5 C a pres e n t i n the f i s h i s not d e p o s i t e d i n s t a n t l y on bone and/or o t o l i t h s . To determine the l a g time i n v o l v e d , r e t e n t i o n experiments were performed i n which f i s h were immersed f o r v a r i o u s l e n g t h s of time i n a 5 C a water, f o l l o w e d by 0-24 hr i n freshwater. In the f i r s t r e t e n t i o n experiment, 19 coho were p l a c e d i n a 50 1 aquarium c o n t a i n i n g 15 pCi/1 of ft5CaC12 at 1000 hours. The r a d i o a c t i v e s o l u t i o n was c i r c u l a t e d f o r 12 hr p r i o r to f i s h immersion. F i s h were sampled a f t e r 24 and 48 hr of immersion. The remainder were removed from the aquarium a f t e r 24 h, r i n s e d b r i e f l y i n i s o t h e r m i c , uncontaminated water, and then p l a c e d i n an uncontaminated aquarium f o r a f u r t h e r 24 hr before sampling. F i s h were s a c r i f i c e d w i t h i n 5 min of sampling and t h e i r s a g i t t a e removed and prepared as d e s c r i b e d below. The r e t e n t i o n experiment was 92 Table 5. Experiments in which coho salmon were immersed for various lengths of time in Ca-45 water. Exper iment O b j e c t R e t e n t i o n t 1 - t 2 IMM/IMMc DAY/DAYc NIGHT/NIGHTc RES/RESc Test f o r l a g time of i n c o r p o r a t i o n of 45-Ca i n t o o t o l 1 t h T e s t e f f e c t of s t r e s s w h i l e f i s h immersed i n 45-Ca water T e s t e f f e c t of s t r e s s immediately a f t e r t r a n s f e r from 45-Ca water Same as DAY/DAYc T e s t f o r r e s o r p t i o n General C o n d i t i o n s Immersed t1 hr i n 45-Ca water f o l l o w e d by t2 hr i n fres h w a t e r Immersed 35 hr i n 45-Ca; IMM s t r e s s e d a f t e r 12 hr S t r e s s p e r i o d d u r i n g day S t r e s s p e r i o d d u r i n g n i g h t S t r e s s a f t e r removal from 45-Ca water and 45-Ca i n c o r p o r a t i o n has f i n i s h e d 94 repeated i n a m o d i f i e d v e r s i o n l a t e r , when one batch of f i s h was sampled a f t e r 24 hr immersion and the remainder were r i n s e d and t r a n s f e r r e d to f r e s h water f o r 12 hr. ( A l l experimental r e p l i c a t e s were c a r r i e d out i n , 5 C a water i n which experimental f i s h had a l r e a d y been t e s t e d . Due to the lower fl5Ca c o n c e n t r a t i o n present, r e p l i c a t e r a d i o a c t i v e l e v e l s are i n v a r i a b l y lower than t h e i r p r e c u r s o r . ) The e f f e c t of s t r e s s upon a 5 C a d e p o s i t i o n on the o t o l i t h s while f i s h were immersed i n a tt5Ca s o l u t i o n was determined by p l a c i n g 10 f i s h i n each of 2 i d e n t i c a l a q u a r i a at 2200 hours. " 5Ca c o n c e n t r a t i o n s were as above. The c o n t r o l f i s h ( I M M C ) were l e f t u n d i sturbed f o r 36 hr before sampling. The experimental f i s h ( I M M ) were l e f t u n d i s t u r b e d f o r 12 h, s t r e s s e d f o r 12 hr and then l e f t f o r a f u r t h e r 12 hr before sampling, a l l while i n the r a d i o a c t i v e c a l c i u m s o l u t i o n . T h i s experiment was repeated at a l a t e r date. Since the r e t e n t i o n experiments i n d i c a t e d that a 5 C a w i t h i n the f i s h remained a v a i l a b l e f o r d e p o s i t i o n on the o t o l i t h f o r at l e a s t 12 h, the o b j e c t . o f the next experiment was to determine the e f f e c t of s t r e s s on " 5Ca d e p o s i t i o n immediately a f t e r removal of the f i s h from a r a d i o a c t i v e environment. Twenty f i s h were pl a c e d i n a fl5Ca aquarium at 1000 hours. A f t e r 24 h, 10 c o n t r o l f i s h (DAYc) were r i n s e d and t r a n s f e r r e d to a f r e s h aquarium f o r 12 hr before sampling. The remaining experimental f i s h (DAY) were r i n s e d and t r a n s f e r r e d at the same time to a d i f f e r e n t aquarium, but were s t r e s s e d f o r the subsequent 12 hr ( i e - d u r i n g the day). Th i s experiment was repeated at a l a t e r 95 date. I t has been suggested ( P a n n e l l a , 1980; Mugiya et a l . , 1981) that the p r o t e i n matrix upon which a day's c a l c i u m i s d e p o s i t e d , i s produced near dawn. I f t r u e , " 5Ca d e p o s i t i o n c o u l d vary depending on the time of s t r e s s a p p l i c a t i o n . T h e r e f o r e , experiment DAY/DAYc ( d e s c r i b e d above) was repeated with a m o d i f i c a t i o n . Twenty f i s h were immersed at 1000 hours, but only f o r 12 h, a f t e r which the c o n t r o l (NIGHTc) and experimental (NIGHT) f i s h were l e f t u n d i sturbed and s t r e s s e d , r e s p e c t i v e l y , f o r the f o l l o w i n g 12 hr i e - o v e r n i g h t . To determine i f r e s o r p t i o n of p r e v i o u s l y d e p o s i t e d , 5 C a c o u l d occur under the experimental s t r e s s s i t u a t i o n , 20 f i s h were p l a c e d i n a fl5Ca aquarium at 1000 hours. A f t e r 24 h, 10 of the f i s h were r i n s e d and t r a n s f e r r e d to a f r e s h aquarium, where they were undisturbed f o r 2 1/2 days (RESc). The remaining f i s h were r i n s e d and t r a n s f e r r e d to a d i f f e r e n t aquarium, where they were l e f t f o r 2 days (RES). The f i s h were then s t r e s s e d f o r the f i n a l 12 h r . Ca-45 I n j e c t i o n To separate the e f f e c t s of s t r e s s on 4 5 C a d e p o s i t i o n from that of " 5Ca uptake, 20 coho were i n j e c t e d IP with a fl5Ca s o l u t i o n at a t i t r e of 0.125 juCi/g f i s h . I n j e c t i o n o c c u r r e d at 1400 hours,. whereupon 1/2 of the f i s h were p l a c e d i n an aquarium for 36 hr ( I c ) , and the other 1/2 were l e f t u ndisturbed i n a d i f f e r e n t aquarium f o r 24 h, followed by 12 hr of s t r e s s ( I ) . Sampling of a l l f i s h o c c u r r e d 36 hr a f t e r i n j e c t i o n . The 96 experiment was repeated l a t e r . S t r e s s S t r e s s was s t a n d a r d i z e d i n a l l of the experiments. F i s h remained i n a q u a r i a draped with black p l a s t i c , where they were v i s u a l l y i s o l a t e d from l a b o r a t o r y a c t i v i t i e s . At the beginning of a s t r e s s p e r i o d , the p l a s t i c was removed from the experimental tank, and the f i s h p l a c e d i n 150 ml p l a s t i c c o n t a i n e r s , meshed at each end. F i v e f i s h were p l a c e d i n each c o n t a i n e r and the c o n t a i n e r s were suspended d i r e c t l y over an a i r s t o n e . Over the 12 hr s t r e s s p e r i o d , the c o n t a i n e r s were l i f t e d out of the water at 30 min i n t e r v a l s , d r a i n e d of any remaining water, and the f i s h allowed to s t r u g g l e i n the c o n t a i n e r f o r 60 sec before being r e p l a c e d i n the water. T h i s l e v e l of s t r e s s appeared to be s i g n i f i c a n t to the coho, s i n c e experimental m o r t a l i t i e s only o c c u r r e d d u r i n g the s t r e s s p e r i o d . Many of the s u r v i v i n g , s t r e s s e d f i s h were weak and had d i f f i c u l t y swimming immediately a f t e r each s t r e s s c y c l e , although some i n d i v i d u a l s t o l e r a t e d i t w e l l . O t o l i t h p r e p a r a t i o n A f t e r s a c r i f i c e , a l l experimental f i s h were t r e a t e d s i m i l a r l y . The two s a g i t t a l o t o l i t h s were removed from each f i s h , brushed f r e e of t i s s u e , r i n s e d i n d i s t i l l e d water and then d r i e d overnight at 90°C. The s a g i t t a l p a i r from each f i s h was combined and t r e a t e d as a s i n g l e sample. A f t e r c o o l i n g , the 97 o t o l i t h s were weighed to the nearest 0.01 mg and then s o l u b i l i z e d as per Mahin and Lofberg (1966). R a d i o a c t i v i t y was determined i n a Beckman LS9000 l i q u i d s c i n t i l l a t i o n counter. The r e s u l t a n t DPM ( d i s i n t e g r a t i o n s per minute) values were compensated for decay, quench and chemiluminescence. A c t i v i t y l e v e l s (DPM) are d i r e c t l y p r o p o r t i o n a l to i s o t o p e c o n c e n t r a t i o n s and the terms are used synonomously i n t h i s paper. O t o l i t h check formation due to the s t r e s s p e r i o d was determined by r e a r i n g 5 f i s h i n the l a b f o r 19 days, followed by the 12 hr s t r e s s p e r i o d , and then a f u r t h e r 13 days of r e a r i n g . Throughout t h i s time, photoperiod and f e e d i n g r a t e remained const a n t , and the aquarium was v i s u a l l y i s o l a t e d from l a b o r a t o r y a c t i v i t i e s . Water temperature was recorded d a i l y . A f t e r removal of the s a g i t t a e , o t o l i t h s were ground and p o l i s h e d as d e s c r i b e d i n Campana and N e i l s o n (1982). Check p o s i t i o n was confirmed by c o u n t i n g back the a p p r o p r i a t e number of d a i l y increments from the p e r i p h e r y of the o t o l i t h . The p r o d u c t i o n of d a i l y increments in coho salmon was confirmed independently. R e s u l t s " 5Ca d e p o s i t i o n on the s a g i t t a e of immersed f i s h i n c r e a s e d l i n e a r l y with time f o r at l e a s t 48 h. F i s h that were t r a n s f e r r e d to f r e s h water a f t e r 24 h immersion continued to d e p o s i t r a d i o c a l c i u m on the o t o l i t h s f o r up to an a d d i t i o n a l 12 h. A f t e r that time,' no f u r t h e r " 5Ca was d e p o s i t e d ( F i g . 15). Upon t r a n s f e r , 47% of the 4 5 C a had yet to be d e p o s i t e d on the o t o l i t h s . 15. Mean a c t i v i t y of 4 5 - C a d e p o s i t e d onto o t o l i t h s through time.. V e r t i c a l bars represent 1 SE. Data p o i n t 1 was s t a n d a r d i z e d with r e s p e c t to c o n c u r r e n t l y performed Ret e n t i o n 24-0 experiment.• =Kept i n Ca-45 water.A=Transferred to freshwater a f t e r 24 hr i n Ca-45 water. 100 A moderately intense check was produced on the o t o l i t h on the day that the s t r e s s o c c u r r e d ( F i g . 16). Since there are only two b a s i c components to a f i s h o t o l i t h , c a l c i u m carbonate and the p r o t e i n o t o l i n , the d i s t i n c t appearance of the r e s u l t a n t check i s probably due to a change i n the p r o p o r t i o n of the d e p o s i t e d components. T h e r e f o r e , the s t r e s s used i n these experiments manifested a change i n the normal d e p o s i t i o n a l process of the s a g i t t a e . To determine i f a change in the r a t e of c a l c i u m d e p o s i t i o n was i n v o l v e d i n check formation, s t r e s s was a p p l i e d to experimental f i s h d u r i n g " 5Ca immersion. When s t r e s s was a p p l i e d f o r 12 h d u r i n g a 36 h immersion p e r i o d (Expt. IMM), 4 5 C a d e p o s i t i o n on the o t o l i t h s was s i g n i f i c a n t l y l e s s than that of the c o n t r o l s (Expt. IMMc)(P<.05)(Table 6). There was a good c o r r e l a t i o n between the l e n g t h of the s t r e s s p e r i o d and the r e d u c t i o n i n c a l c i u m d e p o s i t i o n . Expt. IMM f i s h were l e f t u n d i s t u r b e d f o r 67% of t h e i r immersion time. S a g i t t a e .from these f i s h c o n t a i n e d 59% of the a 5 C a of the c o n t r o l s . The r e d u c t i o n i n c a l c i u m d e p o s i t i o n c o u l d have occu r r e d because of s t r e s s i n h i b i t i o n of the c a l c i u m d e p o s i t i o n process, or i n h i b i t i o n of c a l c i u m uptake from the water. To d i s t i n g u i s h between these a l t e r n a t i v e s , Expt. DAY f i s h were s t r e s s e d f o r 12 h immediately a f t e r t r a n s f e r from the r a d i o a c t i v e water. Since a l a r g e p o r t i o n of " 5Ca d e p o s i t i o n occurs a f t e r removal from U 5 C a water (see above), i n h i b i t e d d e p o s i t i o n should be evident d u r i n g t h i s p e r i o d . Yet 4 5 C a l e v e l s were s i m i l a r i n s t r e s s e d and u n s t r e s s e d f i s h (P>.1)(Table 6). 16. Prepared s a g i t t a of experimental coho salmon. Checks due to c o l l e c t i o n (A), experimental s t r e s s (B) and water temperature change (C) are i n d i c a t e d . Bar = 10 /um. . 103 Table 6. Mean a c t i v i t y of Ca-45 d e p o s i t e d onto o t o l i t h s of f i s h kept under v a r i o u s experimental c o n d i t i o n s . Bracketed experiments were performed c o n c u r r e n t l y i n water of the same r a d i o a c t i v i t y . 1 = performed at a l a t e r date with a lower [Ca-45] i n the aquarium, due to pr e v i o u s experiments i n the same water. T h e r e f o r e , experiment p a i r s cannot be compared among themselves without s t a n d a r d i z a t i o n . o Experiment N Total Hrs. i n 45-Ca //hrs- stressed Total hrs. i n FW //hrs. stressed Mean DPM/mg o t o l i t h (X1000) SE Sign i f • Retention 24-0 5 24 0 29.5 1.61 24 7.95 1.27 Retention 24-0 6 0 1 - Retention 24-12 9 24 12 14.4 1 .78 24 24 56.3 8.23 Retention 24-24 5 Retention 48-0 9 48 0 72 .8 5.27 IMMc 10 36 0 48.6 7.32 . IMM 10 36//12 0 29. 2 5. 16 1 -IMMc 10 36 0 28.4 2.73 IMM 9 36//12 0 16.6 2.47 DAYc 10 24 12 34.2 2.89 1 - DAY 8 24 12//12 35.0 3.30 1- DAYc 11 24 12 13.3 1.72 DAY 11 24 ' 12//12 14.3 1 .56 1 -NIGHTc 10 12 12 6.59 0.92 NIGHT 10 12 12//12 6. 25 0.88 RESc - 9 24 60 57.7 8.88 — RES 10 24 60//12 51 .3 3.51 NS NS NS NS 105 If coho are e a s i l y s t r e s s e d , i t i s c o n c e i v a b l e that the capture, r i n s e and t r a n s f e r of the c o n t r o l f i s h s t r e s s e d them s u f f i c i e n t l y to i n h i b i t t h e i r c a l c i u m d e p o s i t i o n as much as that of the s t r e s s e d group. To t e s t t h i s h y p o t h e s i s , f i s h were immersed i n ' 4 5 C a f o r 24 h. One batch (Retention 24-12) was r i n s e d and t r a n s f e r r e d to f r e s h water for 12 h and the remainder were s a c r i f i c e d immediately (Retention 24-0). The former group d e p o s i t e d s i g n i f i c a n t l y more tt5Ca than d i d those f i s h sampled without t r a n s f e r (P<.05)(Table 6), i n d i c a t i n g that the r i n s e -t r a n s f e r procedure d i d not s t r e s s f i s h enough to i n h i b i t c a l c i u m d e p o s i t i o n . Since c a l c i u m d e p o s i t i o n on the o t o l i t h v a r i e s with d i e l p e r i o d i c i t y (Mugiya et a l . , 1981), i n h i b i t i o n of c a l c i u m d e p o s i t i o n through s t r e s s may only be p o s s i b l e at c e r t a i n hours of the day. F i s h i n experiments DAYc/DAY were s t r e s s e d a f t e r t r a n s f e r d u r i n g d a y l i g h t hours. Subsequently, the experiment was repeated with the 12 h s t r e s s p e r i o d o c c u r r i n g throughout the night (Expts. NIGHTc/NIGHT). The r e s u l t s i n d i c a t e d that time of day was not a f a c t o r i n the DAY/DAYc r e s u l t s , s i n c e the c o n t r o l (NIGHTc) and s t r e s s e d f i s h (NIGHT) d e p o s i t e d e s s e n t i a l l y the same q u a n t i t y of 4 5 C a (P>.1)(Table 6). The above r e s u l t s suggest that s t r e s s does not i n h i b i t the c a l c i u m d e p o s i t i o n a l process, but does i n h i b i t c a l c i u m uptake from the water. To t e s t t h i s h y p o t h e s i s , coho were i n j e c t e d with " 5Ca to bypass the c a l c i u m uptake mechanism. The mean 4 5 C a a c t i v i t y of the u n s t r e s s e d c o n t r o l f i s h was 4738 SE=216 DPM/mg o t o l i t h (x=3662 SE=201 f o r r e p l i c a t e #2), while that of the 106 s t r e s s e d group was 3847 SE=481 DPM/mg o t o l i t h (x=3307 SE=194 for r e p l i c a t e #2). These values are not s i g n i f i c a n t l y d i f f e r e n t (P>.05), thereby s u p p o r t i n g the h y p o t h e s i s . Experiment RES f i s h were s t r e s s e d 2 days a f t e r t r a n s f e r from 4 5 C a water to determine i f s t r e s s c o u l d e f f e c t r e s o r p t i o n of p r e v i o u s l y - d e p o s i t e d c a l c i u m . The r e t e n t i o n experiments i n d i c a t e d that a l l i n t e r n a l a s C a should have been d e p o s i t e d at the end of t h i s p e r i o d . Experimental (RES) and c o n t r o l f i s h (RESc) were not s i g n i f i c a n t l y d i f f e r e n t (P>.1)(Table 6), i n d i c a t i n g that r e s o r p t i o n d i d not occur. D i s c u s s i o n D a i l y growth increments are formed through a c i r c a d i a n p e r i o d i c i t y i n the r a t e of c a l c i u m d e p o s i t i o n (Mugiya et a l . , 1981; Tanaka et a l . , 1981). L e f t u n d i s t u r b e d , the d i e l c y c l e of c a l c i u m d e p o s i t i o n w i l l leave a s e r i e s of very s i m i l a r - a p p e a r i n g d a i l y increments on the o t o l i t h s of a young f i s h . However, o t o l i t h s from w i l d f i s h seldom form such a s e r i e s . At r e g u l a r or i r r e g u l a r i n t e r v a l s , the sequence of increments i s i n t e r r u p t e d by checks or d i s c o n t i n u i t i e s that can mark the occurrence of a s t r e s s f u l p e r i o d i n the l i f e of the f i s h . Since the checks are so v i s u a l l y d i s t i n c t i v e , they are almost c e r t a i n l y due to a d i s r u p t i o n i n the normal c y c l e of c a l c i u m and/or p r o t e i n d e p o s i t i o n on the o t o l i t h . Calcium d e p o s i t i o n on the o t o l i t h i s s i g n i f i c a n t l y reduced d u r i n g the formation of an o t o l i t h check. T h i s f a c t was made evident d u r i n g the experiment i n which the f i s h were s t r e s s e d 107 while i n the a 5 C a water. However, f i s h that were s t r e s s e d immediately a f t e r t r a n s f e r from fl5Ca water showed no r e d u c t i o n i n ft5Ca d e p o s i t i o n . Since the r e t e n t i o n experiments demonstrate that almost one h a l f of the " 5Ca i s d e p o s i t e d a f t e r t r a n s f e r from r a d i o a c t i v e water, s t r e s s must i n t e r f e r e with c a l c i u m uptake from the water, r a t h e r than the d e p o s i t i o n process i t s e l f . The i n j e c t i o n experiments support t h i s h y p o t h e s i s . Calcium pathways through the body of a t e l e o s t are p o o r l y understood, but i t i s w e l l documented that very l i t t l e c a l c i u m i s absorbed from food. The g i l l s and epidermis are the main areas of c a l c i u m uptake, p a r t i c u l a r l y i n freshwater f i s h ( S imkiss, 1974). And s i n c e c a l c i u m l e v e l s remain f a i r l y constant through time, c a l c i u m i n f l u x and e f f l u x must be under c o n s i d e r a b l e c o n t r o l ( N o r r i s et a l . , 1963). Mugiya et a l . (1981) determined t h a t there i s a strong c o r r e l a t i o n between a f i s h ' s plasma c a l c i u m l e v e l and c a l c i u m d e p o s i t i o n r a t e on the o t o l i t h . When plasma c a l c i u m i n c r e a s e d , c a l c i u m c o n c e n t r a t i o n i n the ambient water decreased, implying a d i r e c t or i n d i r e c t pathway from ambient water to blood plasma to o t o l i t h . T h e r e f o r e , they s p e c u l a t e d that the d i e l rhythm i n plasma c a l c i u m and c a l c i u m d e p o s i t i o n was due to a d i e l rhythm i n b r a n c h i a l uptake of c a l c i u m from the environment. The r e s u l t s of t h i s study s t r o n g l y support t h i s h y p o t h e s i s . I t appears that c a l c i u m uptake i s the process which u l t i m a t e l y i n f l u e n c e s c a l c i u m d e p o s i t i o n , and that uptake can be d i s r u p t e d by f a c t o r s such as s t r e s s . If the suggestion i s a c c u r a t e that c a l c i u m d e p o s i t i o n i s 108 l i n k e d to the p r o d u c t i o n of a p r o t e i n matrix (Degens et a l . , 1969; Dunkelberger et a l . , 1980), one may hypothesize two b a s i c processes by which o t o l i t h formation may occur. In n e i t h e r i s i t l i k e l y t h a t s t r e s s i n t e r f e r e s with the p r o d u c t i o n of the organic matrix. One p o s s i b i l i t y i s that formation of the p r o t e i n matrix and c a l c i u m d e p o s i t i o n occur c o n c u r r e n t l y at the same r a t e , with the d a i l y p roteinaceous zone due to a c e s s a t i o n or r e d u c t i o n i n c a l c i u m d e p o s i t i o n r a t e . If such were the case, s t r e s s a f t e r t r a n s f e r from the fl5Ca water should have reduced the r a t e of matrix formation, and consequently, the r a t e of c a l c i u m d e p o s i t i o n . T h i s d i d not occur. Another p o s s i b i l i t y i s that the organic matrix i s formed f i r s t each day, perhaps around dawn (Mugiya et a l . , 1981), and that c a l c i u m d e p o s i t i o n upon the matrix occurs afterwards ( P a n n e l l a , 1980). If t r u e , s t r e s s d u r i n g the- p e r i o d of matrix formation (Expts. NIGHTc/NIGHT) should have decreased subsequent c a l c i u m d e p o s i t i o n . T h i s d i d not occur. T h e r e f o r e , a r e d u c t i o n i n c a l c i u m d e p o s i t i o n , not matrix formation, appears to be c h a r a c t e r i s t i c of an o t o l i t h check. (A c a u t i o n a r y note must be a p p l i e d to t h i s i n t e r p r e t a t i o n ; c a l c i u m d e p o s i t i o n may not be d i s t u r b e d by reduced matrix p r o d u c t i o n , so long as the l a t t e r remains above a t h r e s h o l d l e v e l . T h e r e f o r e , s t r e s s may indeed reduce p r o t e i n d e p o s i t i o n . Since matrix p r o d u c t i o n c o u l d not be monitored i n t h i s study, t h i s h y p o thesis was not t e s t e d . ) O t o l i t h checks are w e l l etched by a c i d d u r i n g p r e p a r a t i o n of an a c e t a t e r e p l i c a or f o r viewing by SEM. Although Brothers et a l . (1976) d i s a g r e e , i t appears that the zone dominated by 109 organic matrix i s most h e a v i l y etched by a c i d ( P a n n e l l a 1980; Mugiya et a l . 1981). The a c i d e t c h i n g and the r e s u l t s of t h i s study i n d i c a t e that an o t o l i t h check i s composed of a r e l a t i v e l y u n c a l c i f i e d zone of organic matrix, making i t analogous in some ways to a l a r g e r v e r s i o n of the organic zone formed at dawn each day (Mugiya et a l . , 1981). T h i s suggestion i s supported by the o b s e r v a t i o n that stronger checks etc h widely; s i n c e p r o t e i n c o n t i n u e s to be l a i d down d u r i n g the s t r e s s p e r i o d causing the check, a long p e r i o d of s t r e s s w i l l r e s u l t i n a broad zone of p r o t e i n , a l l of which i s etched by a c i d . T h i s c o u l d e x p l a i n why the v i s u a l i n t e n s i t y of a check i s o f t e n p r o p o r t i o n a l to the magnitude and d u r a t i o n of the s t r e s s that caused i t ( P a n n e l l a , 1980; Campana, p e r s . o b s e r v . ) . In t h i s study, a 12 h s t r e s s p e r i o d d i d not r e s u l t i n any r e s o r p t i o n of o t o l i t h c a l c i u m d e p o s i t e d two days p r e v i o u s l y . T h i s does not r u l e out the p o s s i b i l i t y that o t o l i t h r e s o r p t i o n may occur under d i f f e r e n t circumstances, s i n c e c h r o n i c s t r e s s s i t u a t i o n s were not t e s t e d . However, in a separate study, Campana (Chapter 2) found no-evidence of r e s o r p t i o n i n o t o l i t h s of s t a r r y f l o u n d e r s ( P l a t i c h t h y s s t e l l a t u s ) or s t e e l h e a d t r o u t (Salmo g a i r d n e r i ) that had been s t a r v e d f o r p e r i o d s of up to a month. Neither were checks formed i n the o t o l i t h s of these f i s h at the time that s t a r v a t i o n commenced. Although no data were pr o v i d e d , P a n n e l l a (1980) suggests that o t o l i t h r e s o r p t i o n o c c a s i o n a l l y occurs i n w i l d f i s h , and that i t i s o f t e n marked by u n c o n f o r m i t i e s (checks that c r o s s other d a i l y increments). U n c o n f o r m i t i e s were not produced by the 1 10 s t r e s s d e s c r i b e d i n t h i s study; n e i t h e r have they been observed in other coho o t o l i t h s examined by the author, although they are common i n many f l a t f i s h s p e c i e s (Campana unpublished d a t a ) . Evidence f o r o t o l i t h r e s o r p t i o n has not been obtained by other r e s e a r c h e r s (Simkiss, 1974). Checks are u b i q u i t o u s among and w i t h i n o t o l i t h s of many sp e c i e s of f i s h e s . Hatching checks are o f t e n used as a temporal "benchmark" from which increment counts are made (Mars h a l l and Parker, 1982; N e i l s o n and Geen, 1982). Checks that occur a f t e r h a tching can be used to date events of i n t e r e s t , such as c o l l e c t i o n or r a p i d temperature s h i f t s , due to the s t r e s s that these events i n c u r . Checks formed on the o t o l i t h s of many i n d i v i d u a l s i n a p o p u l a t i o n on a given date can provide i n f o r m a t i o n on population-wide p r o c e s s e s . However, not a l l checks r e c o r d p e r i o d s of p e r t u r b a t i o n to the f i s h . Growth i n t e r r u p t i o n s o f t e n become numerous with i n c r e a s e d f i s h age and/or slow o t o l i t h growth. Some such checks occur only i n l o c a l i z e d r e g i o n s of the o t o l i t h , s uggesting l o c a l d i s r u p t i o n of the d e p o s i t i o n a l p r o c e s s . In a d d i t i o n , the checks that d e l i m i t f o r t n i g h t l y or monthly p a t t e r n s ( P a n n e l l a , 1980) do not appear to have a s t r e s s - i n d u c e d cause. In such cases, the reduced c a l c i u m d e p o s i t i o n that c h a r a c t e r i z e s the check may be due to i n h i b i t e d c a l c i u m uptake from an endogenous source. In a p o s s i b l y analogous s i t u a t i o n , b r a n c h i a l uptake of c a l c i u m v i r t u a l l y ceases near dawn each day d u r i n g the formation of the organic zone of a d a i l y increment. An endogenous c i r c a d i a n rhythm e n t r a i n e d to photoperiod may be i n d i r e c t l y r e s p o n s i b l e 111 f o r the reduced c a l c i u m uptake (Mugiya et a l . , 1981; Tanaka et a l . , 1981). T h e r e f o r e , i t i s p o s s i b l e that a b i o l o g i c a l c l o c k based on the phases of the moon i s s i m i l a r l y r e s p o n s i b l e f o r the lunar d i s c o n t i n u i t i e s . 1 12 CHAPTER 5. LUNAR CYCLES OF OTOLITH GROWTH IN JUVENILE STARRY FLOUNDERS I n t r o d u c t i o n D e p o s i t i o n on the o t o l i t h s of young f i s h e s occurs with a c i r c a d i a n p e r i o d i c i t y , r e s u l t i n g i n the formation of a continuous s e r i e s of incremental s t r u c t u r e s known as d a i l y growth increments ( P a n n e l l a , 1971; Brothers et a l . , 1976; Mugiya et a l . , 1981). Examination of the o t o l i t h m i c r o s t r u c t u r e thus p r o v i d e s a dated r e c o r d of past f i s h growth, and has been used for the d e t e r m i n a t i o n of age, growth r a t e and l i f e h i s t o r y i n f o r m a t i o n ( P a n n e l l a , 1980; Brothers & McFarland, 1981; Methot, 1981; Rosenberg & Haugen, 1982). Such data are o f t e n d i f f i c u l t to o b t a i n by other means. In a d d i t i o n to d a i l y growth p a t t e r n s , " l u n a r " c y c l e s of o t o l i t h growth have been r e p o r t e d i n some marine f i s h e s ( P a n n e l l a , 1971, 1974, 1980; Brothers et a l . , 1976; Rosenberg, 1982). P e r i o d i c p a t t e r n s of 14-15 or 28-30 d a i l y increments, o c c a s i o n a l l y d e l i m i t e d by checks ( d i s c o n t i n u i t i e s ) , were r e c o g n i z a b l e through c y c l i c a l d i f f e r e n c e s i n width and/or v i s u a l c o n t r a s t . D e s p i t e the i m p l i c a t i o n of t i d a l i n f l u e n c e , no processes have yet been o f f e r e d to e x p l a i n the lunar c y c l e s of o t o l i t h growth. Although lunar p a t t e r n s i n o t o l i t h s are p o o r l y understood, p l a u s i b l e mechanisms f o r d a i l y increment formation have been developed. A d i e l l i g h t c y c l e a p p a r e n t l y e n t r a i n s an endogenous c i r c a d i a n rhythm of o t o l i t h d e p o s i t i o n (Taubert & Coble, 1977; 1 13 Tanaka et a l . , 1981), with the need f o r entrainment reduced with i n c r e a s e d age (Campana & N e i l s o n , 1982; Campana, 1983c). Temperature i n f l u e n c e s both increment width and appearance (Br o t h e r s , 1981; N e i l s o n & Geen, 1982; Campana, 1983c), but does not act as a z e i t g e b e r (Campana & N e i l s o n , 1982; N e i l s o n & Geen, 1982; Campana, 1983c). Feeding r a t e a l s o a f f e c t s increment width (Struhsaker & Uchiyama, 1976; N e i l s o n & Geen, 1982). D a i l y increment p r o d u c t i o n has been v e r i f i e d i n j u v e n i l e s t a r r y f l o u n d e r s , P l a t i c h t h y s s t e l l a t u s , (Campana & N e i l s o n , 1982). Lunar p a t t e r n s a l s o e x i s t i n the o t o l i t h s of t h i s marine f i s h ( p e r s . o b s e r v . ) . I suggest that such p a t t e r n s are e x p l i c a b l e i n terms of v a r i a b l e s that are known to i n f l u e n c e d e p o s i t i o n on the o t o l i t h . The o b j e c t i v e s of t h i s study were t o : 1) v e r i f y the e x i s t e n c e of a lunar p a t t e r n i n the o t o l i t h s of w i l d f l o u n d e r s , and 2) develop a simple model of o t o l i t h growth c o n s i s t e n t with the observed p a t t e r n s . M a t e r i a l s and Methods J u v e n i l e s t a r r y f l o u n d e r s (standard length=4.5 cm) were sampled from Spanish Banks, Vancouver, Canada, a r e g i o n with a low g r a d i e n t i n t e r t i d a l zone and mixed t i d e s of up to 3.3 m range. C o l l e c t i o n s were made at 2-7 d i n t e r v a l s between 2 Sept 1981 and 2 Oct 1981, on or near low t i d e . A minimum of 20 f l o u n d e r s was c o l l e c t e d on each sampling date with a 10-m beach seine of 1.2-cm s t r e t c h mesh. Daytime samples were the norm, but night c o l l e c t i o n s were o c c a s i o n a l l y made on the same date f o r comparative purposes. 1 1 4 S a g i t t a l o t o l i t h s were removed from a l l f l o u n d e r s and prepared as d e s c r i b e d below. Food consumption was assessed through dry weights of r e c e n t l y - i n g e s t e d food. Stomach contents (between the p y l o r i s and the a n t e r i o r end of the i n t e s t i n e ) were emptied onto f i l t e r papers, d r i e d at 95° C f o r 24 hr, and weighed to the nearest mg. Since the food consumption index of stomach content/body weight was not a f u n c t i o n of body weight (P>0.1), the mean food consumption index was c a l c u l a t e d f o r each sampling date. O t o l i t h s were prepared f o r photography as per Campana (1983b), with the m o d i f i c a t i o n that o t o l i t h s were ground from both s i d e s to a t h i n s e c t i o n . Thin s e c t i o n s of o t o l i t h s can be c l e a r l y photographed i n a s i n g l e f o c a l plane, u n l i k e s i n g l e -ground p r e p a r a t i o n s . Increment widths were measured from photographs taken at 500X, while the date of formation of lunar checks was determined m i c r o s c o p i c a l l y at m a g n i f i c a t i o n s of 400-500X. To determine the mean width of each d a i l y increment formed d u r i n g the sampling p e r i o d , increment s e r i e s of 31 o t o l i t h s were measured and a s s i g n e d as to date of formation. To reduce the v a r i a n c e among o t o l i t h s , increment sequences were s t a n d a r d i z e d to a mean value f o r a 9-d p e r i o d : each increment w i t h i n a sequence was m u l t i p l i e d by the same s t a n d a r d i z a t i o n f a c t o r , thereby r e t a i n i n g r e l a t i v e width d i f f e r e n c e s among dates f o r a given o t o l i t h . Mean width was c a l c u l a t e d f o r each date from the combined, s t a n d a r d i z e d o t o l i t h d a ta. Water temperature and s a l i n i t y readings were recorded at 2-1 15 5 d i n t e r v a l s at the sampling s i t e on both h i g h and low t i d e s . However, s i n c e these values were h i g h l y c o r r e l a t e d with mean d a i l y a i r temperature and r a i n f a l l r e s p e c t i v e l y , the l a t t e r v a l u e s were used i n a l l analyses (Environment Canada, 1981). Other marine f i s h e s were sampled f o r comparative purposes from v a r i o u s l o c a t i o n s on the B r i t i s h Columbia c o a s t l i n e d u r i n g the summers of 1980 and 1981. R e s u l t s Lunar Checks The o t o l i t h m i c r o s t r u c t u r e of w i l d s t a r r y f l o u n d e r s was c h a r a c t e r i z e d by a sequence of d a i l y growth increments punctuated by prominent checks ( F i g . 17). P o s t r o s t r a l d i s c o n t i n u i t i e s were the most frequent, but were not o f t e n continuous around the o t o l i t h . R o s t r a l checks appeared to be more r e p r e s e n t a t i v e of the s a g i t t a as a whole, s i n c e they were g e n e r a l l y observed i n a l l quadrants. Check p e r i o d i c i t y was not random; when the p o s t - l a r v a l growth re c o r d of a random sample (N=19) of o t o l i t h s was examined, checks were most commonly separated by 7-8 or 14-15 d p e r i o d s ( F i g . 18). Other m u l t i p l e s of weekly i n t e r v a l s were a l s o e v i d e n t . By counting the number of d a i l y increments between the most rece n t l y - f o r m e d check and the o t o l i t h p e r i p h e r y ( r e p r e s e n t i n g the sampling d a t e ) , the date of check formation can be c a l c u l a t e d . My c a l c u l a t i o n s were c o n s i s t e n t with the p e r i o d i c i t y 17. O t o l i t h m i c r o s t r u c t u r e of a w i l d s t a r r y f l o u n d e r . C o n t r a s t of the d a i l y growth r e c o r d v a r i e s p e r i o d i c a l l y and i s punctuated by checks (C). Checks are more prominent than d e p i c t e d when viewed/photographed at a d i f f e r e n t f o c a l l e n g t h . H = high c o n t r a s t r e g i o n . Bar = 20 jum. 118 F i g . 18. Histogram d e p i c t i n g the time i n t e r v a l between checks i n o t o l i t h s of j u v e n i l e f l o u n d e r s (N = 19). to to CO Frequency cn -+- O —t— cn — i — to O —I 120 r e s u l t s of F i g . 18; i n a d d i t i o n , the dates of formation were c l o s e l y c o r r e l a t e d with the phases of the moon ( F i g . 19). The date corresponding to the most commonly observed check (Aug 31) was very c l o s e to the date of the new moon (Aug 29), while the second most frequent check (Sept 13) occur r e d on a f u l l moon. S i m i l a r i t i e s such as these c o u l d be c o i n c i d e n t a l ; however, checks i n the o t o l i t h s of s t a r r y f l o u n d e r s sampled from Bellingham Bay, Washington on 13 Aug 1980 (N=7) and 19 June 1981 (N=9) o c c u r r e d w i t h i n 1 d of a new moon. Th e r e f o r e , my r e s u l t s suggest that lunar checks are produced i n phase with the lunar c y c l e . Lunar P a t t e r n s A biweekly c y c l e of d a i l y increment c o n t r a s t was evident i n a l l flounder o t o l i t h s . At i n t e r v a l s of 14-15 d, v i s u a l c o n t r a s t between the opaque p o r t i o n of each increment and i t s adjacent t r a n s l u c e n t r e g i o n , peaked, d e c r e a s i n g to a minimum a week l a t e r ( F i g . 17). Maximum c o n t r a s t o c c u r r e d Sept 13-14 and Sept 26-27 dur i n g the sampling p e r i o d . These dates c o i n c i d e with the f u l l and new moon, r e s p e c t i v e l y , and a p e r i o d when d a i l y t i d a l range had reached a maximum. One prominent s u b d a i l y increment was evident w i t h i n each day of the high c o n t r a s t r e g i o n . If s u b d a i l y increments were produced i n the low c o n t r a s t r e g i o n , they were not apparent. 19. Date of check formation i n o t o l i t h s of j u v e n i l e f l o u n d e r s sampled at c a . 5 d i n t e r v a l s between 2 Sept and 2 Oct. The s e q u e n t i a l sampling regime tends to emphasize check frequency at e a r l i e r dates at the expense of the l a t e r d a tes. Phases of the moon are i n d i c a t e d . ( © = new moon) Frequency D CD to CO m to TJ co to - t — co — y - — i — cn — I — 0) -+--4 - t -00 - f -CO — I — o 123 Lunar C o r r e l a t e s of Feeding L i t t l e i s known of the feed i n g h a b i t s of j u v e n i l e s t a r r y f l o u n d e r s ( O r c u t t , 1950). Many f l a t f i s h feed i n t e r t i d a l l y on incoming and/or outgoing t i d e s ( T y l e r , 1971; Lockwood, 1980; Toole , 1980), and s t a r r y f l o u n d e r s probably do the same. To t e s t whether f e e d i n g occurs both day and n i g h t , f l o u n d e r s were c o l l e c t e d on the two low t i d e s of Sept 15 (one at noon, the second at mi d n i g h t ) , a date when the t i d a l h e i g h t s and ranges of both p a i r s of t i d e s were s i m i l a r . Flounders c o l l e c t e d at night c o n t a i n e d s i g n i f i c a n t l y l e s s food i n t h e i r stomachs than d i d day f i s h ( t - t e s t , P<0.05); l i t t l e of the food i n the night f i s h was f r e s h l y i n g e s t e d . T h e r e f o r e , the data suggest that j u v e n i l e f l o u n d e r s are daytime f e e d e r s . Mean food consumption v a r i e d s i g n i f i c a n t l y over the sampling p e r i o d . At approximately 2 wk i n t e r v a l s , the q u a n t i t y of ingested food peaked ( F i g . 20). Food intake was appa r e n t l y r e l a t e d to the t i d a l c y c l e ; s i n c e f l o u n d e r s feed at high t i d e d u r i n g the daytime, l i t t l e f e e d i n g time should be a v a i l a b l e on days when the high t i d e i s e i t h e r e a r l y , l a t e or s h o r t . A v a i l a b l e f e e d i n g time c y c l e d with the t i d e s (a 2 wk period) and was h i g h l y c o r r e l a t e d with food consumption ( F i g . 20). D a i l y Increment Width To determine i f increment widths r e f l e c t e d a lunar c y c l e , a random sample of o t o l i t h s (N=5) with long (60-120 d ) , u n i n t e r r u p t e d d a i l y increment sequences was analyzed with 1 24 F i g . 20. Food consumption as a f u n c t i o n of time f o r s t a r r y f l o u n d e r s sampled between 2 Sept and 2 Oct. Consumption was h i g h l y c o r r e l a t e d with a v a i l a b l e f e e d i n g time (= d u r a t i o n of daytime ebb t i d e ) . 126 periodogram a n a l y s i s . Periodograms among the o t o l i t h s were s i m i l a r . A l l increment width sequences were s i g n i f i c a n t l y d i f f e r e n t from white noise (P<0.0005) i e - c y c l e s of v a r y i n g f r e q u e n c i e s were p r e s e n t . No i n d i v i d u a l c y c l e s were s i g n i f i c a n t at P=0.05; however, a combination of c y c l e s with p e r i o d s of 28-30 d and 14-15 d was s i g n i f i c a n t (P<0.05)-. In a d d i t i o n , 14-15, 20-21 and 28-30 d c y c l e s were always the three most important rhythms i n the data s e r i e s , e x p l a i n i n g 30-40% of the data v a r i a n c e . Weekly p e r i o d s (7-8 d) were g e n e r a l l y not prominent i n the periodogram. The v a r i a b l e s that might be expected to i n f l u e n c e increment width i n c l u d e temperature, food consumption, s a l i n i t y (through a change i n the ambient Ca c o n c e n t r a t i o n ) and t i d a l mixing (through i t s e f f e c t on temperature and s a l i n i t y ) . These data were c o l l e c t e d f o r the sampling p e r i o d and i n c o r p o r a t e d i n t o a m u l t i p l e r e g r e s s i o n model: Y = 0.52T + 0.25F - 0.34R + 1.12M + 18.7 where Y = d a i l y increment width ( i n a r b i t r a r y u n i t s , s i n c e the data were s t a n d a r d i z e d ) , T = mean a i r temperature (°C), F = c o r r e l a t e of food consumption (from F i g . 20) lagged 3-5 d, R = r a i n f a l l (mm) lagged 1 d, and M = i n d i c a t o r of t i d a l mixing ( r a t i o of the two d a i l y t i d a l r a n g e s ) ( T a b l e 7 ) . R a i n f a l l data were lagged by 1 d s i n c e runoff takes time to reach an e s t u a r y . Food consumption data were lagged s i n c e f e e d i n g a f f e c t s o t o l i t h d e p o s i t i o n very slowly (Chapter 2). ( A l t e r n a t e l a g times d i d not improve the p r e d i c t i v e value of the model.) increment widths based on the r e g r e s s i o n model were s i m i l a r to those a c t u a l l y T a b l e 7. M u l t i p l e r e g r e s s i o n e q u a t i o n f o r d a i l y i n c r e m e n t w i d t h . . 2 F - p r o b a b x l i t y = 0.000 R = 0.78 V a r i a b l e C o e f f i c i e n t F - p r o b a b i l i t y T 0.52 0.007 F 0.25 0.121 R - 0 . 3 4 - 0.00 0 M 1.12 0.066 C o n s t a n t 1.8.7 0. 000 128 observed ( F i g . 21). Since the i n t r i n s i c growth r a t e of the f i s h may a l s o i n f l u e n c e o t o l i t h d e p o s i t i o n , the model i s only a p p l i c a b l e to j u v e n i l e f l o u n d e r s of the age under study. I t i s u n l i k e l y t h at o t o l i t h s growth d e c l i n e d s u b s t a n t i a l l y through the course of the study, c o n s i d e r i n g the r e l a t i v e l y short time p e r i o d i n v o l v e d . Temperature and r a i n f a l l were dominant parameters of the model, acc o u n t i n g f o r much of the d e c l i n e i n increment width through time. C o r r e l a t i o n and p a r t i a l c o r r e l a t i o n matrices confirmed the i n t e r a c t i v e e f f e c t of a i r temperature and r a i n f a l l (Table 8); i t i s probable that t h e i r c o r r e l a t i o n would have been higher i f e s t u a r i n e water temperature, and not a i r temperature, was included.. D i s c u s s i o n The l u n a r c y c l e was c o r r e l a t e d with three aspects of o t o l i t h d e p o s i t i o n i n j u v e n i l e f l o u n d e r s . Both increment width and c o n t r a s t c y c l e with a biweekly p e r i o d i c i t y , a p p a r e n t l y due to a t i d a l i n f l u e n c e on those environmental v a r i a b l e s that a f f e c t o t o l i t h growth. The e x i s t e n c e of o t o l i t h checks with a weekly or biweekly p e r i o d i c i t y cannot be so e x p l a i n e d , although check formation i s c l o s e l y c o r r e l a t e d to the phases of the moon. Check formation i s o f t e n a s s o c i a t e d with s t r e s s f u l i n c i d e n t s i n the l i f e h i s t o r y of young f i s h e s ( P a n n e l l a , 1980). S t r e s s reduces b r a n c h i a l uptake of calcium, r e s u l t i n g i n a calcium-poor s t r u c t u r e that i s v i s u a l l y prominent r e l a t i v e to the surrounding d a i l y increments (Chapter 4 ) . However, an 2 1 . Mean d a i l y increment width ( s o l i d l i n e ) as a f u n c t i o n of time. Expected increment width (dotted l i n e ) , as p r e d i c t e d from a m u l t i p l e r e g r e s s i o n model, i s a l s o p l o t t e d . Increment width I l l T a b l e 8. C o r r e l a t i o n and p a r t i a l c o r r e l a t i o n v a l u e s f o r t h e v a r i a b l e s i n t h e m u l t i p l e r e g r e s s i o n e q u a t i o n . I n c r e m e n t W i d t h C o r r e l a t i o n P a r t i a l C o r r e l a t i o n I n c r e m e n t w i d t h 1.00 -1.00 T 0.62 0.51 F 0.48 0.31 R -0.75 -0.68 M 0.44 0.36 1 32 analogous s t r e s s mechanism does not appear to e x i s t d u r i n g the formation of lunar checks. At i t s maximum, the two-week c y c l e of t i d a l m ixing/turbulence might s t r e s s an i n t e r t i d a l f i s h s u f f i c i e n t l y to form a check. Yet i t i s u n l i k e l y that such a c y c l e would r e s u l t i n weekly checks. Moreover, d i s c o n t i n u i t i e s seldom occur adjacent to one another, although such might be expected at the peak of a p a r t i c u l a r l y h i g h s e r i e s of t i d e s . Although my data do not suggest a weekly cue f o r check p r o d u c t i o n , biweekly p e r i o d s are l i k e l y e n t r a i n e d by the t i d a l c y c l e / l u n a r phases, with or without the e x i s t e n c e of an endogenous lunar rhythm. V i s u a l c o n t r a s t of an o t o l i t h growth record can be i n c r e a s e d by two p r o c e s s e s : a d i e l temperature s h i f t ( B r others, 1981; Chapter 3) and s t r e s s (Chapter 4). Both processes a p p a r e n t l y r e s u l t i n a s i m i l a r product - a s t r u c t u r e with an i n c r e a s e d p r o t e i n : c a l c i u m carbonate r a t i o . Since c o n t r a s t i n the f l o u n d e r o t o l i t h s v a r i e d with a 2 wk p e r i o d i c i t y , I suggest that the l u n a r p a t t e r n was a consequence of the temperature regime induced by a biweekly t i d a l c y c l e . Such a temperature regime i s known to e x i s t i n some i n t e r t i d a l zones (de Wilde & Berghuis, 1979). However, s i n c e f l o u n d e r s move i n and out with the t i d e s , the 15~d t i d a l c y c l e w i l l a l s o i n f l u e n c e f i s h p o s i t i o n r e l a t i v e to the sun-warmed inshore region (at high t i d e ) and the c o o l e r s u b t i d a l waters (at low t i d e ) . Maximum increment c o n t r a s t o c c u r r e d on dates when the t i d a l range was l a r g e , and consequently, the temperature d i f f e r e n c e between high and low t i d e s was at i t s g r e a t e s t (Vugts & Zimmerman, 1975). The 1 33 presence of a s i n g l e s u b d a i l y increment i n the hi g h c o n t r a s t r egion i s c o n s i s t e n t with t h i s h y p o t h e s i s . F a l l i n g nighttime temperatures i n c r e a s e the p r o t e i n r c a l c i u m r a t i o i n c o n c e r t with an endogenous c i r c a d i a n rhythm, r e s u l t i n g i n the formation of the opaque region of a d a i l y increment (Brothers, 1981; Mugiya et a l . , 1981; Chapter 3). A daytime temperature s h i f t , as would occur d u r i n g t i d a l m i g r a t i o n , should a l s o r e s u l t i n the formation of an opaque zone. A daytime opaque zone, or s u b d a i l y increment, i s evident i n many of the floun d e r o t o l i t h s . Much of the day-to-day v a r i a n c e i n increment width was e x p l a i n e d by a small number of environmental v a r i a b l e s . V a r i a t i o n i n mean a i r temperature (as m o d i f i e d by r a i n f a l l ) accounted f o r a l a r g e p r o p o r t i o n of the observed width v a r i a b i l i t y . I t i s l i k e l y t h a t water temperature would have p r o v i d e d an even c l o s e r correspondence, although the c o l l e c t i o n of such data would have r e q u i r e d continuous monitoring of the flo u n d e r through i t s i n s h o r e / o f f s h o r e m i g r a t i o n s . The a i r temperature data were l i m i t e d i n that they experienced more severe f l u c t u a t i o n s than d i d the water. In a d d i t i o n , any biweekly c y c l e s i n water temperature due to t i d a l mixing would not be r e f l e c t e d i n the a i r temperature data. The t i d a l mixing term i n the model attempted to compensate f o r the absence of t h i s e f f e c t . Food consumption by f l o u n d e r s f l u c t u a t e d markedly and c y c l i c a l l y through the sampling p e r i o d . But as a model parameter, i t accounted f o r l i t t l e of the width v a r i a n c e , even when the data s e r i e s was lagged. A poor correspondence between 134 food consumption and short-term o t o l i t h growth has p r e v i o u s l y been noted i n f l o u n d e r s (Chapter 2). A low metabolic r a t e , r e s u l t i n g i n r a p i d dampening of a short-term growth c y c l e , may be r e s p o n s i b l e f o r t h i s e f f e c t . S a l i n i t y (a c o r r e l a t e of r a i n f a l l ) had a s i g n i f i c a n t (P<0.05) but p u z z l i n g e f f e c t on increment width. Calcium d e p o s i t e d on the o t o l i t h i s d e r i v e d from the ambient water supply (Simkiss, 1974; Mugiya et a l . , 1981) where i t e x i s t s as a s a l t i n s o l u t i o n . Presumably, c a l c i u m c o n c e n t r a t i o n i s p r o p o r t i o n a l to s a l i n i t y . If c a l c i u m uptake i s p r o p o r t i o n a l to a v a i l a b l e c a l c i u m supply, then a decrease i n s a l i n i t y c o u l d r e s u l t i n reduced c a l c i u m d e p o s i t i o n on the o t o l i t h i e - a narrower increment. However, there i s evidence a g a i n s t such a r e l a t i o n s h i p ( I r i e , 1960). More l i k e l y , r a i n f a l l had a d i r e c t , n egative e f f e c t on water temperature. Periodograms of long-term o t o l i t h growth records demonstrated the presence of a prominent " l u n a r " rhythm of growth. Food consumption d i s p l a y e d a s i m i l a r p e r i o d i c i t y , although i t s importance i n the r e g r e s s i o n model was minimal. N e i t h e r a i r temperature' nor r a i n f a l l v a r i e d over a lunar c y c l e . However, t h e i r c o r r e l a t e s , water temperature and s a l i n i t y , are under a l a r g e t i d a l i n f l u e n c e . The t i d a l c y c l e induces a 14-15 d p e r i o d i c i t y i n the temperature of i n t e r t i d a l waters (Vugts & Zimmerman, 1975), and t h i s probably accounts f o r the observed biweekly rhythm of o t o l i t h growth (but not the 21-d rhythm, whose presence remains u n e x p l a i n e d ) . The s i g n i f i c a n c e of t h i s o b s e r v a t i o n may l i e i n the r e l a t i o n s h i p between o t o l i t h growth 135 and f i s h growth. Wilson & L a r k i n (1982) demonstrated that the o t o l i t h : f i s h growth r e l a t i o n s h i p was s i g n i f i c a n t and l i n e a r f o r j u v e n i l e sockeye salmon, Oncorhynchus nerka. S t a r r y f l o u n d e r s have a s i m i l a r r e l a t i o n s h i p (Campana, unpublished), suggesting t h a t the lunar c y c l e of o t o l i t h growth i s a r e f l e c t i o n of a s i m i l a r c y c l e i n f i s h growth. To my knowledge, tidall.y-modulated growth c y c l e s i n f i s h have not been documented to date. The few documented examples of lunar rhythms i n f i s h are g e n e r a l l y a s s o c i a t e d with spawning and/or migratory a c t i v i t i e s on the new or f u l l moon (reviews by Gibson, 1978; Neumann, 1981). Causal mechanisms are po o r l y understood, but i n at l e a s t some s p e c i e s , there appears to be an endogenous lunar rhythm (Grau et a l . , 1981; K a v a l i e r s , 1982). In t h i s study, lunar c y c l e s of o t o l i t h growth (increment width) and increment c o n t r a s t appear to be e x p l i c a b l e i n terms of t i d a l l y - m o d u l a t e d environmental v a r i a b l e s , p a r t i c u l a r l y temperature. I t was not necessary to p o s t u l a t e the e x i s t e n c e of an endogenous lunar rhythm. Supporting t h i s s u p p o s i t i o n was the absence of any observable lunar p a t t e r n i n the o t o l i t h s of 10 s p e c i e s of freshwater f i s h e s . Biweekly c o n t r a s t p a t t e r n s were observed i n j u v e n i l e s of the f o l l o w i n g marine s p e c i e s : L e p t o c o t t u s armatus, P s e t t i c h t h y s m e l a n o s t i c t u s , Cymatogaster aggregata, P h o l i s  o r n a t a , M a l l o t u s v i l l o s u s , Apodichthys f l a v i d u s , Embiotoca  l a t e r a l i s , C l i n o c o t t u s a c u t i c e p s and Parophrys v e t u l u s . Lunar checks were only observed i n the p l e u r o n e c t i d s p e c i e s examined; thus, t h e i r s i g n i f i c a n c e i s u n c l e a r . O t o l i t h m i c r o s t r u c t u r e examination can pro v i d e u s e f u l 136 i n f o r m a t i o n on short-term growth trends i n j u v e n i l e f i s h e s ; the lunar growth c y c l e of f l o u n d e r s would have been d i f f i c u l t to d e t e c t by other means. In a d d i t i o n , the c l a r i t y of the biweekly increment c o n t r a s t p a t t e r n s suggests t h e i r p o t e n t i a l f o r r a p i d age d e t e r m i n a t i o n of marine f i s h e s . Lunar checks are too v a r i a b l e f o r t h i s purpose, although j u v e n i l e Parophrys v e t u l u s from Oregon have been aged i n t h i s way (Rosenberg, 1982). Check p e r i o d i c i t y may vary g e o g r a p h i c a l l y , s i n c e young E n g l i s h s o l e i n the Vancouver region produce both weekly and biweekly checks (Campana, p e r s . o b s e r v . ) . 137 GENERAL CONCLUSIONS The o b j e c t i v e of t h i s t h e s i s has been to determine the major environmental and p h y s i o l o g i c a l f a c t o r s that i n f l u e n c e o t o l i t h growth. Although i n t e r - s p e c i e s d i f f e r e n c e s may e x i s t , my r e s u l t s suggest that a s i n g l e mechanism may account f o r the observed v a r i a t i o n s i n growth increment p r o d u c t i o n under the i n f l u e n c e of d i f f e r i n g environmental regimes. The same mechanism i s c o n s i s t e n t with observed p a t t e r n s i n the o t o l i t h m i c r o s t r u c t u r e of marine f i s h e s . In a d d i t i o n , the r e s u l t s of the Ca-45 experiments p r o v i d e a p l a u s i b l e e x p l a n a t i o n f o r the u b i q u i t y of non-lunar checks. With the exception of lunar checks, the major f e a t u r e s of o t o l i t h m i c r o s t r u c t u r e may now be i n t e r p r e t e d i n terms of c a u s a l events and past f i s h growth. The b a s i c f e a t u r e of d a i l y increment p r o d u c t i o n appears to be an endogenous c i r c a d i a n rhythm. D a i l y increment p r o d u c t i o n con t i n u e s i n the presence of constant l i g h t N e i l s o n and Geen 1982; Townsend and Shaw 1982; Chapters 1,3), constant temperature (Taubert and Coble 1977; N e i l s o n and Geen 1982; Chapters 1,3) and under s t a r v a t i o n c o n d i t i o n s ( M a r s h a l l and Parker 1982; Chapter 2 ) . A 24-h l i g h t - d a r k c y c l e e n t r a i n s the rhythm d u r i n g the e a r l y l i f e h i s t o r y of the f i s h , although the i n f l u e n c e of l i g h t i s minimized with i n c r e a s e d age (Chapter 3). It i s c o n c e i v a b l e that an u n c o n t r o l l e d v a r i a b l e with a 24-h p e r i o d e n t r a i n s increment formation. However, most animals use photoperiod as a z e i t g e b e r f o r endogenous rhythms ( J a c k l e t 1981; Takahashi and Zatz 1982) and f i s h are no exception (Schwassman 1971; Godin 1981). In a d d i t i o n , Tanaka et a l . (1981) 138 demonstrated that the time of formation of a d a i l y increment s h i f t e d i n response to a s h i f t i n the onset of d a y l i g h t . Since constant l i g h t may d i s t u r b the p e r i o d i c i t y of o t o l i t h growth i n very young f i s h , some workers have suggested t h a t a l i g h t - d a r k c y c l e i s an o b l i g a t o r y z e i t g e b e r . However, my r e s u l t s and those of Radtke and Dean (1982) have i n d i c a t e d that d a i l y increment production resumes a f t e r an a c c l i m a t i o n p e r i o d of v a r i a b l e l e n g t h . The a c c l i m a t i o n p e r i o d i s longest i n l a r v a l f i s h (Chapter 3). E a r l i e r , I suggested that the c i r c a d i a n p e r i o d i c i t y of o t o l i t h d e p o s i t i o n was a consequence of the endogenous rhythm of endocrine s e c r e t i o n . Endocrine s e c r e t i o n s are c l o s e l y a l l i e d to c i r c a d i a n pacemakers (Menaker and B i n k l e y 1981) and are u l t i m a t e l y r e s p o n s i b l e f o r metabolic a c t i v i t i e s and s k e l e t a l growth ( i e - d e p o s i t i o n on the o t o l i t h ) ( S i m p s o n 1978). As such, those v a r i a b l e s that i n f l u e n c e endocrine s e c r e t i o n may be expected to a f f e c t increment formation. Thus, the e f f e c t s of feeding p e r i o d i c i t y and temperature s h i f t s should be p r e d i c t e d as being those of masking agents, s i n c e they do not e n t r a i n the endogenous rhythm i t s e l f (Taubert and Coble 1977; Tanaka et a l . 1981; Chapters 1,2,3). C o n s i s t e n t with t h i s hypothesis are f i n d i n g s t h a t m u l t i p l e feedings and/or temperature s h i f t s may superimpose increments upon the d a i l y increments d e r i v e d from the c i r c a d i a n rhythm (Brothers 1981; N e i l s o n and Geen 1982; Chapter 2). Feeding p e r i o d i c i t y e f f e c t s probably act at the l e v e l of metabolic r a t e , where an e x t r a f e e d i n g induces a s h o r t -term i n c r e a s e i n metabolism, which i n turn induces a short-term 139 i n c r e a s e i n o t o l i t h d e p o s i t i o n r a t e ( i e - a s u b d a i l y increment). A low metabolic r a t e would probably dampen any consequence of a short-term a l t e r a t i o n i n f e e d i n g (Chapter 2). Temperature s h i f t s may act a n a l o g o u s l y . The opaque p o r t i o n of a d a i l y increment i s formed at n i g h t , when c a l c i u m d e p o s i t i o n i s reduced r e l a t i v e to p r o t e i n d e p o s i t i o n (Brothers 1980; Mugiya et a l . 1981). In midshipman su b j e c t e d to f a l l i n g n ight-time temperatures, the opaque p o r t i o n of each d a i l y increment became more prominent ( o p t i c a l l y dense) than i n constant temperature f i s h , suggesting that an i n c r e a s e d p r o p o r t i o n of p r o t e i n had been d e p o s i t e d . Presumably, a lower temperature served to i n h i b i t c a l c i u m d e p o s i t i o n r e l a t i v e to that of p r o t e i n . T h e r e f o r e , daytime temperature f l u c t u a t i o n s should be expected to produce increments independently of the "normal" d a i l y increment; the formation of "daytime" increments ( i e - s u b d a i l y ) has been observed i n s e v e r a l s p e c i e s ( Brothers 1981; Chapter 4; J . D. N e i l s o n p e r s . comm.). In some i n s t a n c e s , the masking e f f e c t of temperature may obscure any increments produced through endogenous d e p o s i t i o n a l p r o c e s s e s . V i s u a l prominence i s a c h a r a c t e r i s t i c of both temperature-s h i f t e d increments and o t o l i t h checks; a high p r o t e i n to c a l c i u m r a t i o appears u n u s u a l l y opaque, e s p e c i a l l y when adjacent to increments of lower c o n t r a s t . However, the processes that r e s u l t in v i s u a l prominence may d i f f e r between the two s i t u a t i o n s . The Ca-45 experiments demonstrated that s u f f i c i e n t l y - h i g h l e v e l s of s t r e s s may d i s r u p t increment formation through a r e d u c t i o n i n the c a l c i u m d e p o s i t i o n r a t e . Calcium d e p o s i t i o n from the blood 140 plasma was a p p a r e n t l y u n a f f e c t e d by s t r e s s ; i t was c a l c i u m uptake from the ambient water which was i n h i b i t e d . A reduced c a l c i u m content i n the plasma and o t o l i t h f o l l o w e d l a t e r (Mugiya et a l . 1981; Chapter 4 ) . Since osmotic imbalances o f t e n f o l l o w the i m p o s i t i o n of s t r e s s , a r e d u c t i o n i n c a l c i u m uptake should not be unexpected (Eddy 1981). However, my r e s u l t s do not support P a n n e l l a ' s (1980) c o n t e n t i o n that checks denote regions of c a l c i u m r e s o r p t i o n . Since the checks i n my experiments d i d not t a n g e n t i a l l y sever incremental s e r i e s (as they may do on occasion) ( P a n n e l l a 1980), I cannot d i s c a r d the p o s s i b i l i t y that c a l c i u m r e s o r p t i o n may occur i n some s i t u a t i o n s of check formation. In a d d i t i o n , checks formed through non-stress r e l a t e d causes may form as a r e s u l t of a l o c a l i z e d d i s r u p t i o n of d e p o s i t i o n . M i c r o s t r u c t u r a l examination of o t o l i t h s i s most i n f o r m a t i v e when the growth r e c o r d i s heterogeneous. Temporal changes i n increment width, increment appearance and check l o c a t i o n can provide u s e f u l data on the growth, environmental circumstances and l i f e h i s t o r y t r a n s i t i o n s of the s p e c i e s under study. In the l a s t s e c t i o n of my t h e s i s , I have i n t e r p r e t e d p a t t e r n s i n the o t o l i t h m i c r o s t r u c t u r e of f l o u n d e r s i n terms of environmental v a r i a b l e s of known e f f e c t . C y c l i c a l v a r i a t i o n s i n d a i l y increment c o n t r a s t were most e a s i l y e x p l a i n e d on the b a s i s of c y c l i c a l s h i f t s i n d i e l temperature f l u c t u a t i o n . D a i l y temperature s h i f t s r e s u l t i n d a i l y increments of i n c r e a s e d v i s u a l c o n t r a s t (Chapter 3). I f the i n t e r t i d a l temperature regime and the increment c o n t r a s t p a t t e r n are r e l a t e d , p e r i o d 141 lengths of both c y c l e s should be s i m i l a r , with increment c o n t r a s t and d a i l y temperature f l u c t u a t i o n r e a c h i n g a maximum on the same date. Both of these c o n d i t i o n s have been confirmed (Chapter 4). In a d d i t i o n , the formation of a s i n g l e s u b d a i l y increment per day i s c o n s i s t e n t with the temperature s h i f t a f l o u n d e r experiences d u r i n g i t s t i d a l l y - i n d u c e d daytime migrat i o n . The width of a d a i l y increment can be a f f e c t e d by both temperature and f o o d ^ a v a i l a b i l i t y (Struhsaker and Uchiyama 1976; N e i l s o n and Geen 1982). The low metabolic r a t e of f l o u n d e r s may p r e c l u d e the i n f l u e n c e of short-term v a r i a t i o n s i n food supply on o t o l i t h growth (Chapters 2,4). However, temperature and s a l i n i t y were s i g n i f i c a n t l y c o r r e l a t e d with increment width i n a model of flounder o t o l i t h growth. As d i s c u s s e d p r e v i o u s l y , both of these environmental v a r i a b l e s vary over a 15-d t i d a l c y c l e . If the i n f l u e n c e of temperature and s a l i n i t y on increment width i s as great as the model suggests, d a i l y growth should mimic the t i d a l c y c l e . T h i s p r e d i c t i o n was v e r i f i e d i n long-term o t o l i t h growth sequences through the use of s p e c t r a l a n a l y s i s . A s i m i l a r growth p a t t e r n may e x i s t i n the flounder i t s e l f (Wilson and L a r k i n 1982), suggesting that o t o l i t h growth p a t t e r n s c o u l d be used f o r monitoring short-term changes i n f i s h growth as w e l l as past environmental s h i f t s . Lunar checks were c o n s i s t e n t l y formed on f l o u n d e r o t o l i t h s on the new and f u l l moons. Despite the lunar phasing, no e n t r a i n i n g mechanism c o u l d be i d e n t i f i e d ; an endogenous lunar rhythm may or may not e x i s t . As a t o p i c f o r f u t u r e r e s e a r c h , 142 t h i s area i s overshadowed by the need f o r f u r t h e r increment formation s t u d i e s on l o n g - l i v e d p e l a g i c l a r v a e . For some reason, such l a r v a e (which i n c l u d e h e r r i n g and f l o u n d e r s ) do not always form d e f i n a b l e d a i l y increments (Geffen 1982; Laroche et a l . 1982; Lough et a l . 1982). Growth r a t e may (Geffen 1982) or may not (Rosenberg and Haugen 1982) have pla y e d a r o l e i n these r e s u l t s . C e s s a t i o n of increment d e p o s i t i o n i n j u v e n i l e f l o u n d e r s (Chapter 2) may have been caused by an analogous mechanism; however, t h i s phenomenon has not been observed in j u v e n i l e s of the other s p e c i e s s t u d i e d . 1 43 LITERATURE CITED Altman, P. L. and D. S. Dittmer [ E d s ] . 1974. B i o l o g y data book. V o l . I I I . F e d e r a t i o n of the American S o c i e t y of Experimental B i o l o g i s t s . 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