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Learning and memory of chemosensory stimuli by underyearling coho salmon Oncorhynchus kisutch (Walbaum) Courtenay, Simon Charles 1989

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LEARNING AND MEMORY OF CHEMOSENSORY STIMULI BY UNDERYEARLING COHO SALMON ONCORHYNCHUS KISUTCH (WALBAUM) by SIMON CHARLES COURTENAY B.Sc. U n i v e r s i t y of Western O n t a r i o , 1980 M.Sc. U n i v e r s i t y of Western O n t a r i o , 1982 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 October 1989 © Simon C h a r l e s Courtenay, .1989 In presenting this thesis in partial fulfilment of the requirements for an advanced freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. degree at the University of British Columbia, I agree that the Library shall make it The University of British Columbia Vancouver, Canada Department Date (D<r_7&. 3 / ^ r ? DE-6 (2/88) i i ABSTRACT T h i s study addressed the h y p o t h e s i s that coho salmon (Oncorhynchus k i s u t c h ) l e a r n and remember the odours of t h e i r n a t a l environment. During one or more of the embryo, a l e v i n and e a r l y f r y ( f i r s t 14 d) stages, f i s h were exposed to the a r t i f i c i a l odorant morpholine, n a t u r a l odorants generated by c o n s p e c i f i c s , or creek water. A f t e r an i n t e r v a l of at l e a s t 1 month the p r e f e r e n c e of f r y f o r an odorant was t e s t e d i n a Y-maze and compared to that of naive f r y . Increased p r e f e r e n c e was i n t e r p r e t e d as r e c o g n i t i o n . F i s h exposed d u r i n g any or a l l of the embryo, a l e v i n , or e a r l y f r y stages showed g r e a t e r p r e f e r e n c e f o r morpholine than naive f i s h when t e s t e d 54-125 d a f t e r exposure. In a d d i t i o n , p r e v i o u s l y exposed f i s h responded to morpholine with g r e a t e r heart r a t e r e d u c t i o n (another measure of r e c o g n i t i o n ) than naive f i s h i n t e s t s 477-532 d a f t e r exposure. Exposure to the odour of s i m i l a r l y - a g e d members of another f a m i l y of the same p o p u l a t i o n d u r i n g the a l e v i n stage, and to that of a second f a m i l y d u r i n g the e a r l y f r y stage r e s u l t e d i n i n c r e a s e d p r e f e r e n c e f o r the a l e v i n - s t a g e odour by one of four t e s t groups, f o r the f r y - s t a g e odour by another group, and f o r both odours by a t h i r d group, i n Y-maze t e s t s conducted at l e a s t 32 d a f t e r exposure. Subsequent experiments r e v e a l e d p r e f e r e n c e to be an i n s e n s i t i v e measure of r e c o g n i t i o n because i t i s g r e a t l y i n f l u e n c e d by odour c o n c e n t r a t i o n and p o s s i b l y other odour c h a r a c t e r i s t i c s which d i f f e r between f a m i l i e s . Members of the Quinsam R i v e r p o p u l a t i o n exposed throughout the embryo, a l e v i n , and e a r l y f r y stages to the odour of s i m i l a r l y - a g e d members e i t h e r the Big Qualicum River or Puntledge R i v e r p o p u l a t i o n s each p r e f e r r e d the f a m i l i a r odour when t e s t e d 69-79 d a f t e r exposure. In a d d i t i o n to f a m i l i a r i t y , p r e f e r e n c e among population-odours was found to be i n f l u e n c e d by q u a n t i t y of odour. Quinsam, B ig Qualicum, and Puntledge f i s h each p r e f e r r e d water c o n d i t i o n e d by faeces of members of t h e i r own p o p u l a t i o n over blank water and i n one of two experiments over water c o n d i t i o n e d by faeces of non-population members, suggesting that at l e a s t some of the odorants mediating i n t r a s p e c i f i c d i s c r i m i n a t i o n s are present i n f a e c e s . Exposure to creek water f o r a 1 month p e r i o d c e n t e r e d around swimup d i d not r e s u l t i n g r e a t e r p r e f e r e n c e than that shown by naive f i s h f o r creek water over w e l l water or e i t h e r of two r i v e r waters i n t e s t s performed at l e a s t 58 d a f t e r exposure. I t i s concluded that coho form long-term memories of at l e a s t some a r t i f i c i a l and n a t u r a l odours i n e a r l y l i f e and that l e a r n i n g i s not r e s t r i c t e d to a b r i e f c r i t i c a l p e r i o d of development. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES X LIST OF FIGURES xi ACKNOWLEDGEMENTS x v i i INTRODUCTION 1 Background Literature 4 GENERAL METHODS AND MATERIALS 14 The Fish 14 The Laboratory 16 Testing Recognition 18 The Apparatus 18 Protocol 22 The Measure of Preference . 24 Analysis 25 CHAPTER 1. LEARNING AND MEMORY OF THE ODOUR OF MORPHOLINE . 27 INTRODUCTION 27 PART 1: LEARNING DURING THE EMBRYO, ALEVIN, AND EARLY FRY STAGES 31 Treatment 31 Testing: Preference Response 32 Methods and Materials 32 Results 35 Testing: Cardiac Response 40 Methods and Materials 40 V Results 43 PART 2: INVESTIGATION OF THE PREFERENCE RESPONSE 45 Treatment 46 Experiment 1: Morpholine Switched Between Arms of the Y-Maze 48 Methods and Materials 48 Results 48 Interpretation 50 Experiment 2: Morpholine not Switched Between Arms of the Y-maze 51 Methods and Materials 51 Results 52 Interpretation 54 DISCUSSION 56 CHAPTER 2. THE ROLE OF LEARNING IN RECOGNITION OF FAMILY-SPECIFIC ODOURS 60 INTRODUCTION 60 PART 1: LEARNING DURING THE EMBRYO, ALEVIN, AND EARLY FRY STAGES 64 Treatment 64 Testing 67 Methods and Materials 67 Results 70 Assay 1: Familiar Sibs (FS) vs Unfamiliar Non-Sibs (UNS) 70 Assay 2: Non-Sibs present at the Embryo stage (ENS) vs Unfamiliar Non-Sibs (UNS) 70 Assay 3: Non-Sibs present at the Alevin stage (ANS) vs Unfamiliar Non-Sibs (UNS) 73 Assay 4: Non-Sibs present at the Fry stage (FrNS) vs Unfamiliar Non-Sibs (UNS) 75 Assay 5: Non-Sibs present at the Alevin Stage (ANS) vs Unfamiliar Non-Sibs (UNS), Repeat Tests 77 Comparison of Assays 3 and 5 78 PART 2: OTHER FACTORS AFFECTING PREFERENCE 81 Assay A: Repulsion vs Relative Attractiveness .... 81 Assay B: Relative Attractiveness to Other Conspecifics 83 Assay C: Homogeneity of Relative Attractiveness .. 86 Assay D: Quantity of Odour 86 DISCUSSION 90 CHAPTER 3. THE ROLE OF LEARNING IN RECOGNITION OF POPULATION-SPECIFIC ODOURS 93 INTRODUCTION 93 EXPERIMENT 1 97 Treatment 97 Testing 99 Methods and Materials 99 Results 100 Assay 1: Population-Recognition and the Effe c t of Common-Rearing 100 Assay 2: Recognition of Non-population Tankmates .105 Assay 3: Testing the Assumption that Fish do not v i i A c quire Odours from C o n s p e c i f i c s 107 Assay 4: The In f l u e n c e of Odour C o n c e n t r a t i o n on Preference 108 EXPERIMENT 2 112 Treatment 112 T e s t i n g 114 Methods and M a t e r i a l s 114 R e s u l t s 115 DISCUSSION 120 CHAPTER 4. THE INFLUENCE OF FAECES IN DISCRIMINATION OF POPULATION ODOURS 123 INTRODUCTION 123 METHODS AND MATERIALS 128 RESULTS AND DISCUSSION 129 Experiment 1: Response to Water Co n d i t i o n e d by Faeces 1 29 Experiment 2: Response to Two D i f f e r e n t Q u a n t i t i e s of Faeces 135 Experiment 3: P o p u l a t i o n D i s c r i m i n a t i o n 138 CHAPTER 5. LEARNING AND MEMORY OF THE ODOUR OF A NATURAL WATER 145 INTRODUCTION 145 TREATMENT 148 TESTING: PREFERENCE BETWEEN CREEK-WATER (CW) AND WELL-WATER (WW) 149 Methods and M a t e r i a l s 149 R e s u l t s 151 v i i i Creek-Water (CW) vs Well-Water (WW) 151 Mixtures of Creek-Water (CW) and Well-Water (WW) .151 TESTING: PREFERENCE BETWEEN CREEK-WATER (CW) AND A RIVER-WATER (NW) 153 Methods and Materials 153 Results 155 Creek-Water (CW) (stored in polyethylene carboy) .155 Effec t of Storage in the Carboy 157 Creek-Water (CW) (stored in glass bottles) 157 Effec t of Storage in Glass Bottles ..160 Creek-Water (CW) vs an unfamiliar River-Water (BQW) (both stored in glass bottles) 160 DISCUSSION 162 CHAPTER 6. GENERAL DISCUSSION 167 Synthesis of Results 167 Memory of Odours Presented in Early L i f e ....167 The Process of Learning 168 The Function of Odour Learning 178 Homing 178 Int r a s p e c i f i c Recognition 181 Literature Cited 186 Appendix 1. Analysis of Water Supplies at Rosewall Creek Hatchery 213 Appendix 2. Preliminary Y-Maze Tests of Morpholine Preference 218 Appendix 3. Sensory Mediation of Response to the Chemical Emanations of Conspecifics 222 Introduction 222 Methods and Materials 223 Fish 223 Olfactory Occlusion 223 Testing 224 Results and Discussion 225 Appendix 4. Morpholine and Other Substances as Experimental X LIST OF TABLES Table 1. Schedule of morpholine exposure 33 Table 2. Schedule of Y-maze t e s t s of p r e f e r e n c e f o r morpholine 36 Table 3. Schedule of exposure to non-sib odours 66 Table 4. Schedule of t e s t s of p r e f e r e n c e between f a m i l y -s p e c i f i c odours 68 Table 6. P r e f e r e n c e s of coho f r y from the Quinsam R. (Q), Big Qualicum R. (BQ), and Puntledge R. (P) f o r water c o n d i t i o n e d by p o p u l a t i o n members over non-population members 119 Table 7. Schedule and d e s c r i p t i o n of t e s t s of pr e f e r e n c e between creek water and w e l l water at Rosewall Creek Hatchery 150 Table 8. P h y s i c a l c h a r a c t e r i s t i c s , a l k a l i n i t y , and ions found i n Well and Creek waters at Rosewall Creek Hatchery - without and with 0.5 mg/L morpholine 215 Table 9. Ions not d e t e c t e d i n Well or Creek waters at Rosewall Creek Hatchery 216 LIST OF FIGURES F i g u r e 1. L o c a t i o n s of Rosewall Creek Hatchery and the P a c i f i c B i o l o g i c a l S t a t i o n (PBS) where experiments were performed, and the Quinsam, Big Qualicum, and Puntledge R i v e r s from which coho were o b t a i n e d 15 F i g u r e 2. The Y-maze apparatus used to t e s t p r e f e r e n c e between two odours 19 F i g u r e 3. Screen-swimming: with the snout pressed a g a i n s t the upstream screen, the f r y swims a c t i v e l y i n an apparent attempt to enter the arms of the Y-maze 21 F i g u r e 4. A dye t e s t showing e x t e n s i v e mixing i n the downstream common area of the Y-maze of waters from the two arms, r e s u l t i n g from a c t i v e swimming of the t e s t f i s h 21 F i g u r e 5. Observations were made through a s l i t i n a black p l a s t i c c u r t a i n suspended 1 m from the downstream end of the Y-mazes 23 F i g u r e 6. Between t r i a l s , water s u p p l i e s were switched between arms of each Y-maze 23 F i g u r e 7. B e h a v i o u r a l response of coho f r y to morpholine .. 37 F i g u r e 8. Decrease i n the response of C o n t r o l s to morpholine between t e s t s performed i n e a r l y May and l a t e May or June 39 F i g u r e 9. C a r d i a c responses to morpholine of coho smolts that had been exposed to morpholine dur i n g the Embryo, A l e v i n , or e a r l y Fry stage, Throughout a l l three stages, or had not been p r e v i o u s l y exposed ( C o n t r o l s ) . (Note: r e p l i c a t e s A and B of each treatment pooled.) 44 F i g u r e 10. Change of response to morpholine over 5 s e q u e n t i a l 5-min o b s e r v a t i o n p e r i o d s , when morpholine was a l t e r n a t e d between arms of the Y-maze from t r i a l to t r i a l 49 F i g u r e 11. Lack of change of response to morpholine over f i v e 5-min o b s e r v a t i o n p e r i o d s , when morpholine was not a l t e r n a t e d between arms of the Y-maze from t r i a l to t r i a l 53 F i g u r e 12. Preference between odours of F a m i l i a r Sibs (FS) and U n f a m i l i a r Non-Sibs (UNS) 71 F i g u r e 13. Preference between odours of two d i f f e r e n t Non-Si b groups, one of which (ENS) t e s t f i s h had been exposed to d u r i n g the embryo stage 72 F i g u r e 14. Preference between odours of two d i f f e r e n t Non-Sib groups, one of which (ANS) Experimentals (Shaded Bars) had been exposed to du r i n g the a l e v i n stage, whereas C o n t r o l s (Open Bars) had not 74 F i g u r e 15. Preference between odours of two d i f f e r e n t Non-Si b groups, one of which (FrNS) Experimentals (Shaded Bars) had been exposed to d u r i n g the f r y stage, whereas C o n t r o l s (Open Bars) had not 76 F i g u r e 16. Pre f e r e n c e between odours of two d i f f e r e n t Non-Si b groups, one of which (ANS) Experimentals (Shaded Bars) had been exposed to d u r i n g the a l e v i n stage, x i i i whereas C o n t r o l s (Open Bars) had not. (Tests performed 96-118 d a f t e r those of F i g u r e 14) 79 F i g u r e 17. Preference of f a m i l y A1b f o r odour of two U n f a m i l i a r N o n - S i b l i n g (UNS) groups over F a m i l i a r S i b l i n g s (FS) 82 Fi g u r e 18. Preference of f a m i l y A1b f o r odour of F a m i l i a r S i b l i n g s (FS) over blank water (BW) 84 F i g u r e 19. Preference of four coho f a m i l i e s between waters c o n d i t i o n e d by N o n - S i b l i n g groups G6b and A1b 85 F i g u r e 20. Preference of f a m i l y F5 f o r water c o n d i t i o n e d by G6b f i s h over four d i f f e r e n t groups of A1b f i s h 87 F i g u r e 21. Pref e r e n c e of F5 f r y between odours of G6b and A1b f r y : the e f f e c t of r e l a t i v e c o n c e n t r a t i o n s of the two odours 89 F i g u r e 22. Preference of Quinsam R. f r y between odours of population-members and members of the Big Qualicum R. p o p u l a t i o n (A) or Puntledge R. p o p u l a t i o n (B) 102 F i g u r e 23. Preference of Big Qualicum R. f r y between odours of population-members and members of the Quinsam R. p o p u l a t i o n (A) or Puntledge R. p o p u l a t i o n (B) 103 F i g u r e 24. Preference of Puntledge R. f r y between odours of population-members and members of the Quinsam R. p o p u l a t i o n (A), or Big Qualicum R. p o p u l a t i o n (B) 104 F i g u r e 25. Pref e r e n c e of Quinsam R. f r y between odours of Big Qualicum R. f r y and Puntledge R. f r y 106 F i g u r e 26. Preference of Quinsam R. f r y between odours of tankmate and non-tankmate B ig Qualicum R. f r y (A) or x i v Puntledge R. f r y (B) 109 F i g u r e 27. Preference of Puntledge R. f r y between odours of Quinsam R. f r y and Big Qualicum R. f r y : the i n f l u e n c e of r e l a t i v e c o n c e n t r a t i o n of odour 110 F i g u r e 28. Preference of Quinsam R. f r y between odours of population-members and members of the Big Qualicum R. p o p u l a t i o n (A), or the Puntledge R. p o p u l a t i o n (B) ..116 F i g u r e 29. Preference of B i g Qualicum R. f i s h between odours of population-members and members of the Quinsam R. p o p u l a t i o n (A), or the Puntledge R. p o p u l a t i o n (B) ..117 F i g u r e 30. Preference of Puntledge R. f i s h between odours of population-members and members of the Quinsam R. p o p u l a t i o n (A), or the B i g Qualicum R. p o p u l a t i o n (B) 118 F i g u r e 31. Preference of f r y of three p o p u l a t i o n s f o r water c o n d i t i o n e d by faeces of population-members, over blank water 131 F i g u r e 32. A t t r a c t i o n to water c o n d i t i o n e d by faeces of population-members over the 6 hour t e s t d u r a t i o n 132 F i g u r e 33. Preference of Puntledge R. f r y f o r blank water over water c o n d i t i o n e d by faeces of population-members .134 F i g u r e 34. Preference of Puntledge R. f r y f o r water c o n d i t i o n e d by faeces of 150 g population-members over faeces of 50 g population-members (A), though faeces of 50 g population-members were d e t e c t e d and p r e f e r r e d over blank water (B) 136 F i g u r e 35. Preference of Quinsam R. and Big Qualicum R. f i s h between waters c o n d i t i o n e d by faeces of members of those two p o p u l a t i o n s 139 F i g u r e 36. Pref e r e n c e of Quinsam R. and Puntledge R. f i s h between waters c o n d i t i o n e d by faeces of members of those p o p u l a t i o n s 140 F i g u r e 37. Pref e r e n c e of Big Qualicum R. and Puntledge R. f i s h between waters c o n d i t i o n e d by faeces of members of those p o p u l a t i o n s 141 F i g u r e 38. Pref e r e n c e f o r creek water - d i l u t e d to v a r i o u s c o n c e n t r a t i o n s with w e l l water, over w e l l water .152 F i g u r e 39. Pref e r e n c e f o r a mixture of 50% creek water/50% w e l l water, over a mixture of 20% creek water/80% w e l l water 154 F i g u r e 40. Pref e r e n c e f o r 100% Nanaimo R. water (NW) over 10% Rosewall Creek water (CW)/90% Nanaimo R. water 156 F i g u r e 41.,Preference of group C f i s h f o r 100% f r e s h Nanaimo R. water (NW) over 10% s t o r e d Nanaimo R. water/90% f r e s h Nanaimo R. water 158 F i g u r e 42. Pref e r e n c e f o r 100% Nanaimo R. water (NW) over 10% creek water (CW)/90% Nanaimo R. water 159 F i g u r e 43. Pref e r e n c e of group C f i s h f o r f r e s h Nanaimo R. water (NW) over 10% stored/90% f r e s h Nanaimo R. water 161 F i g u r e 44. Pref e r e n c e between Rosewall Creek water and Big Qualicum R. water, both d i l u t e d to 10% with Nanaimo R. water 1 63 F i g u r e 45. Response to morpholine i n w e l l water 219 xvi Figure 46. Number of seconds of screen-swimming by olfa c t o r i l y - o c c l u d e d f i s h and controls in a 5-min period 226 Figure 47. Response to conspecific chemical emanations by olfa c t o r i l y - o c c l u d e d f i s h and controls 227 x v i i ACKNOWLEDGEMENTS I am very g r a t e f u l to my s u p e r v i s o r s P.A. L a r k i n , T.P. Quinn and C. Groot f o r t h e i r many hours and p o s i t i v e t h i n k i n g . Thanks a l s o to my a d v i s o r y committee of R. L i l e y , J.D. McPhail, and T.G. Northcote. For advice and h e l p with care of f i s h , I thank G. B a l l , B. Humphreys, G. Johnsen and T. Moore. For h e l p i n o b t a i n i n g f i s h , I thank T. Perry and the s t a f f s of the Quinsam, Puntledge, and Big Qualicum H a t c h e r i e s . Many I n s t i t u t e inmates c o n t r i b u t e d a d v i c e and support and I thank e s p e c i a l l y Helen Hahn, Marc L a b e l l e , Andrew T r i t e s , C h r i s - "get out of my o f f i c e " - Foote, and Rick T a y l o r - one of those r a r e people who can h o n e s t l y look h i m s e l f i n the f a c e . I thank E. Brannon f o r making i t p o s s i b l e f o r me to work at the U n i v e r s i t y of Washington ( S e a t t l e ) and a t t e n d the Salmonid M i g r a t i o n and D i s t r i b u t i o n Symposium i n Norway. For he l p with chemistry I thank K. Humphreys, D. Seccomb, and e s p e c i a l l y L. W e i l e r . F i n a l l y , I thank my t i r e l e s s support team - my parents A l i s o n and Peter, my brother Stephen and s i s t e r - i n - l a w Mara, and my f i a n c e e and c o l l e a g u e (proof i n F i g u r e 6 ) , Helene Dupuis. T h i s work was supported by NSERC o p e r a t i n g grants and a GREAT award to P.A. L a r k i n , by the Department of F i s h e r i e s and Oceans Canada through C. Groot, and by an NSERC post-graduate s c h o l a r s h i p . 1 INTRODUCTION A f t e r f e e d i n g m i g r a t i o n s which may span thousands of k i l o m e t e r s , salmonid f i s h e s r e t u r n to reproduce i n t h e i r n a t a l a r e a s . The f i n a l stage of homing i s guided, at l e a s t i n p a r t , by odours l e a r n e d i n e a r l y l i f e i n a process known as i m p r i n t i n g (see Background L i t e r a t u r e below). In coho salmon (Oncorhynchus  k i s u t c h ) i m p r i n t i n g has been shown to occur j u s t p r i o r to l e a v i n g the home r i v e r system at the age of approximately 18 months (Hasler and Scholz 1983), and i t has been hypothesized that p e r i o d s of i m p r i n t i n g may a l s o occur e a r l i e r i n l i f e (Harden-Jones 1968). T h i s d i s s e r t a t i o n addresses two qu e s t i o n s aimed at i d e n t i f y i n g and c h a r a c t e r i z i n g o l f a c t o r y l e a r n i n g by coho salmon d u r i n g the p e r i o d of l i f e normally a s s o c i a t e d with the redd a r e a : 1. are long-term memories of ambient odours formed d u r i n g or s h o r t l y a f t e r i n c u b a t i o n , and i f so, 2. i s od o u r - l e a r n i n g r e s t r i c t e d t o the embryo, a l e v i n , or e a r l y f r y stage? The experimental approach was that used s u c c e s s f u l l y i n the study of e a r l y p e r c e p t u a l l e a r n i n g ( i n c l u d i n g examples of i m p r i n t i n g ) of odours by small mammals: exposure to an odour, f o l l o w e d by a p e r i o d of non-exposure, and then a t e s t of r e c o g n i t i o n . (see r e f e r e n c e s c i t e d i n Schmidt 1986; Panhuber 1986; A l b e r t s 1986). The most common and convenient measure of r e c o g n i t i o n used i n these s t u d i e s i s p r e f e r e n c e ; odours that are f a m i l i a r , whether n a t u r a l or s y n t h e t i c , are approached (e.g., i n a Y-maze) or a s s o c i a t e d with p r e f e r e n t i a l l y over u n f a m i l i a r 2 odours. S t u d i e s by Brannon (1972), Bodznick ( 1 978a) and Quinn and Hara (1986) i n d i c a t e that salmon f r y may a l s o p r e f e r f a m i l i a r over u n f a m i l i a r odours (reviewed below). Experiments were conducted with three d i f f e r e n t types of odours: morpholine, c o n s p e c i f i c odours, and the odour bouquet of a n a t u r a l creek water. The man-made odorant - morpholine was chosen because of Hasler and c o l l e a g u e s ' success i n i m p r i n t i n g coho and other salmonid smolts to i t (reviewed below). In Chapter 1, an experiment i s r e p o r t e d i n which coho were exposed to morpholine d u r i n g the embryo stage, the a l e v i n stage, or the e a r l y f r y stage. Use of c o n s p e c i f i c odours was suggested by the demonstration by Quinn and Busack (1985) and Quinn and Hara (1986) that coho f r y d i s t i n g u i s h e d between the chemical emanations of s i b l i n g s and n o n - s i b l i n g s . S i b l i n g s were p r e f e r r e d over u n f a m i l i a r n o n - s i b l i n g s , but not over f a m i l i a r (tankmate) n o n - s i b l i n g s , suggesting that p r e f e r e n c e depended on l e a r n i n g . In Chapter 2, an experiment i s r e p o r t e d i n which coho were exposed to the chemical emanations of three d i f f e r e n t groups of n o n - s i b l i n g s : one d u r i n g the embryo stage, a second d u r i n g the a l e v i n stage, and a t h i r d d u r i n g the e a r l y f r y stage. A number of p r e v i o u s s t u d i e s have r e p o r t e d evidence that w i t h i n s e v e r a l salmonid s p e c i e s i n c l u d i n g coho (Quinn and Tolson 1986), p o p u l a t i o n s emit c h a r a c t e r i s t i c chemicals and p r e f e r those of t h e i r own p o p u l a t i o n over those of others (reviewed i n Chapter 3). I t has been hypothesized that these odours or 3 "pheromones", emanating from j u v e n i l e s m i g r a t i n g to sea and r e s i d e n t i n the homestream (Nordeng 1971,1977; reviewed by S t a b e l l 1984) or d e p o s i t e d i n the g r a v e l by the migrants themselves before l e a v i n g the stream (reviewed by H o r r a l l 1981) are c r i t i c a l i n g u i d i n g the homing m i g r a t i o n of a d u l t salmonids (Nordeng 1971,1977). In Chapter 3 an experiment i s report e d , examining the r o l e of l e a r n i n g i n chemosensory p o p u l a t i o n r e c o g n i t i o n by coho f r y . Previous i n v e s t i g a t i o n s of the responses of coho to c o n s p e c i f i c chemical emanations have not examined the nature of the odorant, though s t u d i e s of other salmonids have i m p l i c a t e d i n t e s t i n a l c o ntents (reviewed i n Chapter 4). Observations made d u r i n g the experiments r e p o r t e d i n Chapters 2 and 3 suggested that faeces might be one important source of odour, and t h i s h y p o t h e s i s was t e s t e d i n a s e r i e s of experiments r e p o r t e d i n Chapter 4. A l a r g e body of evidence suggests that stream odours other than those of c o n s p e c i f i c s p r o v i d e important o l f a c t o r y cues to homing salmonids (reviewed i n Chapters 5 and 6). Chapter 5 r e p o r t s an experiment i n which coho f r y were t e s t e d f o r r e c o g n i t i o n of a n a t u r a l stream water to which they had been exposed d u r i n g the end of i n c u b a t i o n and beginning of the f r y stage. The r e s u l t s of the chap t e r s are i n t e g r a t e d i n Chapter 6, and r e l a t e d to the c e n t r a l q u e s t i o n s of whether and when odours are l e a r n e d i n e a r l y l i f e . 4 Background L i t e r a t u r e The idea that f i s h might home to remembered odours i s over 100 years o l d (Buckland 1880; Trevanius 1882 c i t e d by Hasler and Scholz 1983:13), and p r e l i m i n a r y experimental support dates back over 50 years ( C r a i g i e 1926), but i t was not u n t i l the 1950s that an " o l f a c t o r y h y p o t h e s i s " was s t a t e d e x p l i c i t l y . H a s l e r and Wisby (1951) hypothesized that each stream bears a unique odour - r e s u l t i n g from the s o i l s and v e g e t a t i o n of i t s drainage b a s i n , and i t i s t h i s odour that j u v e n i l e salmon l e a r n , remember u n t i l m a t u r i t y , and home t o . The a b i l i t y of f i s h to d i s t i n g u i s h the odours of d i f f e r e n t p l a n t s (Walker and Ha s l e r 1949) and s o i l s (Hasler and Wisby 1951) was demonstrated i n c o n d i t i o n i n g experiments with bluntnose minnows (Pimephales notatus ( R a f . ) ) . The r o l e of o l f a c t i o n i n homing was i n d i c a t e d by an experiment (Wisby and Ha s l e r 1954) i n which a d u l t coho were captured i n a creek and one of i t s t r i b u t a r i e s , and were d i s p l a c e d downstream below the confluence of the creek and t r i b u t a r y and allowed to repeat t h e i r upstream m i g r a t i o n . Most f i s h r e t u r n e d to t h e i r area of capture but f i s h d e p r i v e d of the sense of smell by o c c l u s i o n of the nares s t r a y e d at a higher r a t e i n t o the other stream. S i m i l a r experiments with coho and s i x other salmonid s p e c i e s by other i n v e s t i g a t o r s have produced s i m i l a r r e s u l t s (reviewed by Ha s l e r and Scholz 1983). A number of authors p o i n t e d out that these experiments d i d not prove that odours l e a r n e d i n e a r l y l i f e guided homing f o r 5 three reasons. F i r s t , r e t u r n to the s i t e of capture might have been guided by cues l e a r n e d d u r i n g the o r i g i n a l homing m i g r a t i o n r a t h e r than before or d u r i n g seaward m i g r a t i o n . Second, i n many cases i t was not demonstrated that the s i t e of capture was the n a t a l s i t e . T h i r d , the poor performance of o l f a c t o r i l y - o c c l u d e d f i s h c o u l d have r e s u l t e d from g e n e r a l trauma r a t h e r than l o s s of smell ( B r e t t and Groot 1963). More c o n c l u s i v e support f o r Hasler and Wisby's (1951) hypothesis came from experiments i n which H a s l e r and c o l l e a g u e s (reviewed by Hasler and Scholz 1983) exposed j u v e n i l e coho to man-made odorants, morpholine or phenethyl a l c o h o l , before r e l e a s e i n t o Lake Michigan. Eighteen months l a t e r when the f i s h matured, morpholine was metered i n t o one creek near the r e l e a s e s i t e , and phenethyl a l c o h o l i n t o another. F i s h "homed" to whichever chemical they had been p r e v i o u s l y exposed, while unexposed f i s h ( c o n t r o l s ) entered both creeks i n much lower numbers. These experiments demonstrated that j u v e n i l e coho l e a r n e d and remembered the a r t i f i c i a l odorants u n t i l m a t u r i t y . S i m i l a r r e s u l t s were obtained i n experiments with rainbow t r o u t (0. mykiss = Salmo g a i r d n e r i ) (Scholz et a l . 1975, 1978a; Cooper and Scholz 1976) and brown t r o u t (Salmo t r u t t a ) (Scholz et a l . 1978b), suggesting t h a t the mechanism of odour-l e a r n i n g was common to many, i f not a l l , salmonids. H a s l e r and Wisby (1951) hypothesized that the process by which salmonids l e a r n e d homestream odours was i m p r i n t i n g . I m p r i n t i n g i s c h a r a c t e r i z e d by two c r i t e r i a : the e x i s t e n c e of a 6 " s e n s i t i v e p e r i o d " and subsequent s t a b i l i t y of the r e s u l t of experience gained d u r i n g that p e r i o d (Immelmann 1975; Immelmann and Suomi 1981). While these c r i t e r i a are g e n e r a l l y accepted, there remains much c o n t r o v e r s y over t h e i r d e f i n i t i o n s (Bateson 1979,1987; Immelmann and Suomi 1981; Bateson and Hinde 1987). In g e n e r a l , the s e n s i t i v e p e r i o d i s agreed to be a p e r i o d of development d u r i n g which a c e r t a i n experience i s most e f f e c t i v e (Immelmann 1975); i n our case, "a time p e r i o d d u r i n g which the salmon i s most l i k e l y to l e a r n odor cues having to do with the unique i d e n t i t y of the home stream" (Madison et a l . 1973). S e n s i t i v e p e r i o d s are u s u a l l y but not always a s s o c i a t e d with e a r l y l i f e stages, and may be r i g i d l y c o n f i n e d to a very b r i e f p e r i o d of development (hours or days) ( i . e . , a " c r i t i c a l " p e r i od) or may extend over weeks or months and vary widely i n t i m i n g a c c o r d i n g to environment, i n d i v i d u a l , and s p e c i e s (Hess 1973; Immelmann 1975; Immelmann and Suomi 1981). S t a b i l i t y , or p e r s i s t e n c e , " r e f e r s to the f a c t t h a t a p a r t i c u l a r experience d u r i n g the s e n s i t i v e p e r i o d w i l l have, even in the face of c o n s i d e r a b l e experience with other o b j e c t s , a more permanent e f f e c t than the same experience encountered at a l a t e r (or e a r l i e r ) time" (Immelmann 1975). In the case of salmonids, the homestream odour, l e a r n e d d u r i n g the s e n s i t i v e p e r i o d , i s remembered u n t i l the i n d i v i d u a l has r e t u r n e d to the stream of o r i g i n (Madison et a l . 1973). The homing of coho salmon to man-made odorants encountered 18 months p r e v i o u s i s c o n s i s t e n t with the s t a b i l i t y c r i t e r i o n of i m p r i n t i n g . The other c r i t e r i o n - a s e n s i t i v e p e r i o d - has not 7 been demonstrated d i r e c t l y , but s e v e r a l l i n e s of i n d i r e c t evidence suggest that the p e r i o d immediately p r e c e d i n g exodus from the home r i v e r system i s such a p e r i o d . During t h i s p e r i o d , coho and most other anadromous salmonids undergo a s e r i e s of changes i n morphology, p h y s i o l o g y and behaviour, p r e p a r i n g them f o r oceanic l i f e (Hoar 1976; Scholz 1980; McKeown 1984). The f i r s t l i n e of evidence c o n s i s t s of experiments by a number of workers i n which f i s h were t r a n s p l a n t e d from t h e i r n a t a l t r i b u t a r y to a d i f f e r e n t t r i b u t a r y (reviewed by R i c k e r 1972; L i s t e r et a l . 1981b; H a s l e r and Scholz 1983). T r a n s p l a n t s made a f t e r f i s h smolted g e n e r a l l y r e s u l t e d i n homing to the n a t a l stream, whereas f i s h t r a n s p l a n t e d before or d u r i n g s m o l t i n g r e t u r n e d to the r e l e a s e stream. In s e v e r a l of these s t u d i e s , f i s h homed to the r e l e a s e s i t e a f t e r only b r i e f experience of i t (e.g. < 2 d - Jensen and Duncan 1971), sugges t i n g that the l e a r n i n g process was r a p i d . Some of these s t u d i e s were known to H a s l e r i n the 1940s, and on t h i s b a s i s , a l l experimental exposures were made du r i n g the smolt stage, immediately before r e l e a s i n g the f i s h i n t o Lake Michigan. The r e t u r n of the f i s h to a stream c o n t a i n i n g the chemical of exposure confirmed that the smolt stage was one p e r i o d of odour-l e a r n i n g (though not n e c e s s a r i l y the only such p e r i o d ) . Experiments conducted by Scholz (1980) suggested t h a t odours encountered d u r i n g s m o l t i f i c a t i o n may be more i n f l u e n t i a l than odours encountered before t h i s stage. Scholz hypothesized t h a t i m p r i n t i n g was i n i t i a t e d by the same p h y s i o l o g i c a l mechanism that r e g u l a t e s some of the morphological and 8 b e h a v i o u r a l changes of s m o l t i f i c a t i o n - an i n c r e a s e i n t h y r o i d a c t i v i t y (see Hoar 1976; Folmar and D i c k h o f f 1980; Scholz 1980; McKeown 1984; D i c k h o f f and S u l l i v a n 1987). T h i s was t e s t e d by s t i m u l a t i n g the t h y r o i d glands of presmolt coho with t h y r o i d -s t i m u l a t i n g hormone (TSH) while exposing the f i s h to e i t h e r morpholine or phenethyl a l c o h o l . Ten months l a t e r f i s h were r e l e a s e d i n t o a r i v e r , 150 m downstream from the c o n f l u e n c e of two t r i b u t a r i e s - one scented with morpholine and the other with phenethyl a l c o h o l . TSH-treated f i s h "homed" to the t r i b u t a r y b e a r i n g t h e i r i m p r i n t i n g odour, but non TSH-treated f i s h d i d not. Scholz concluded that o l f a c t o r y i m p r i n t i n g o c c u r r e d i n f i s h with a r t i f i c i a l l y e l e v a t e d t h y r o i d hormone l e v e l s , t h a t i s , i n f i s h undergoing " s m o l t i f i c a t i o n " . A d d i t i o n a l evidence that the smolt stage c o n t a i n s a s e n s i t i v e p e r i o d f o r o l f a c t o r y i m p r i n t i n g has r e c e n t l y been r e p o r t e d by Morin et a l . (1989a). A t l a n t i c salmon (Salmo s a l a r L.) exposed to the amino a c i d L - c y s t e i n e (a n a t u r a l l y - o c c u r r i n g o l f a c t a n t - Morin et a l . 1987a) 612-619 d a f t e r " b i r t h " showed c a r d i a c responses c o n s i s t e n t with r e c o g n i t i o n when t e s t e d 55 d l a t e r , whereas f i s h exposed e a r l i e r or l a t e r d i d not. T h i s p e r i o d was 21-28 d a f t e r the onset of changes i n parr c o l o r a t i o n and c o n d i t i o n f a c t o r , normally a s s o c i a t e d with s m o l t i f i c a t i o n . I n t e r e s t i n g l y , t h i s apparent s e n s i t i v e p e r i o d c o i n c i d e d with a b r i e f peak in t h y r o i d - h i s t o l o g i c a l a c t i v i t y (Morin et a l . 1989b). The a v a i l a b l e evidence supports H a s l e r and Wisby's (1951) 9 hypothesis t h a t salmonids imprint on homestream odours d u r i n g the smolt stage, and t h i s r e s e a r c h i s widely c i t e d as one of the most i n t e n s i v e l y s t u d i e d examples of i m p r i n t i n g of an environmental cue (Immelmann 1975). However, a c o n s i d e r a t i o n of the movements made by j u v e n i l e salmonids has l e d a number of i n v e s t i g a t o r s to suggest that the smolt stage may be only one of a number of s e n s i t i v e p e r i o d s f o r the l e a r n i n g of homestream odours (Harden-Jones 1968; Brannon 1972, 1982; Bodznick 1978a; H o r r a l l 1981; Wright 1985; Quinn 1985; S a g l i o 1986; Quinn et a l . , i n p r e s s ) . Salmonids which spend more than a few days or weeks i n freshwater before m i g r a t i n g to the sea ( i . e . , a l l except pink salmon and some p o p u l a t i o n s of chum salmon) o f t e n migrate e x t e n s i v e l y w i t h i n the home r i v e r system and may smolt f a r away from the n a t a l a r e a . Coho f r y may move upstream (Ruggles 1960; Skeesick 1970; Bustard and Narver 1975; T r i p p and McCart 1983; L i s t e r et a l . 1981a) or downstream (Cederholm and S c a r l e t t 1982) i n t o small t r i b u t a r y streams (Skeesick 1970; T r i p p and McCart 1983; Cederholm and S c a r l e t t 1982) or s i d e channels (Narver 1978) or s p r i n g - f e d ponds (Peterson 1982) or l a k e s (Johnstone et a l . 1987), and may overwinter i n these areas (Swales et a l . 1987; reviewed by L i s t e r et a l . 1981a; Northcote 1984; Sandercock, i n p r e s s ) . Yet when these f i s h r e t u r n from the sea to spawn, the a v a i l a b l e evidence i n d i c a t e s that they home to the area from which they emerged as f r y , not the area i n which they smolted ( L i s t e r et a l . I 9 8 l a , b ) . In one study, coho presmolts tagged d u r i n g the f a l l i n a headwater lake of the C h i l l i w a c k / V e d d e r R i ver system i n southern B r i t i s h Columbia, 10 were recaptured the f o l l o w i n g s p r i n g as smolts e m i g r a t i n g from small t r i b u t a r i e s to the lower r i v e r , and l a t e r as a d u l t s spawning i n the p r i n c i p a l i n l e t to the lake (Pearce 1981, c i t e d by L i s t e r et a l . 1981b). In another study, L i s t e r et a l . (1981a) r e p o r t e d that of coho tagged as smolts i n v a r i o u s t r i b u t a r i e s of the Cowichan R i v e r , B r i t i s h Columbia - 61% re t u r n e d to spawn i n l o c a t i o n s other than the tagg i n g s i t e . S i m i l a r l y , i n most p o p u l a t i o n s of sockeye (0. nerka), f r y emerge i n lake i n l e t streams and swim downstream to a nursery lake where they spend a year, smolt, and migrate to sea. S e v e r a l years l a t e r , they migrate back through the nursery lake to the n a t a l stream (Hartman and R a l e i g h 1964; F o e r s t e r 1968; Ri c k e r 1972). I f t h i s f i n a l stage of the m i g r a t i o n i s guided by remembered odours, those odours must have been l e a r n e d d u r i n g i n c u b a t i o n or very s h o r t l y a f t e r (Bodznick 1978b). S i m i l a r arguments can be made f o r other salmonids (e.g., chinook (0. tshawytscha) and chum (0. keta) salmon (Quinn 1985)). I t i s p o s s i b l e that o l f a c t o r y cues learned d u r i n g the smolt stage guide salmonids back to the home r i v e r system, where other guidance mechanisms take over f o r r e l o c a t i o n of the n a t a l area or s e l e c t i o n of the best spawning area. However, a more parsimonious and probable e x p l a n a t i o n was proposed by Harden-Jones (1968), who suggested t h a t , "Young salmon may undergo a s e r i e s of ( o l f a c t o r y ) i m p r i n t i n g processes c o r r e s p o n d i n g to each major change of environment made i n freshwater; g r a v e l bed, lake o u t l e t , t r i b u t a r y , main r i v e r . " Harden-Jones a l s o suggested t h a t d u r i n g homing, salmon may seek the l a s t i m p rinted odour 11 f i r s t , then the second l a s t - f a r t h e r upstream, and so on i n sequence. Such a mechanism would e x p l a i n why f i s h t r a n s p l a n t e d to non-natal t r i b u t a r i e s r e t u r n to those t r i b u t a r i e s and not to the n a t a l t r i b u t a r y ; the e a r l i e r imprinted odours cannot be r e l o c a t e d because they are not upstream. S i m i l a r l y , j u v e n i l e f i s h t r a n s p o r t e d downstream over a p o r t i o n of t h e i r normal seaward m i g r a t i o n may subsequently f a i l to home to the n a t a l area i f f a m i l i a r odours are not d e t e c t a b l e immediately upstream of the r e l e a s e s i t e (Jensen and Duncan 1971; V r e e l a n d et a l . 1975; Cramer 1981; S l a t i c k et a l . 1982; Vreeland and Wahle 1983; Quinn et a l . i n p r e s s , but see E b e l et a l . 1973; S l a t i c k et a l . 1975). L i s t e r et a l . (1981b) reviewed s t u d i e s of the homing and s t r a y i n g of P a c i f i c salmonids that had been r e l o c a t e d i n e a r l y l i f e and concluded that much of the v a r i a t i o n c o u l d be e x p l a i n e d by a s e q u e n t i a l i m p r i n t i n g mechanism. Desp i t e widespread agreement with the t e n e t s of the s e q u e n t i a l i m p r i n t i n g h y p othesis and c a l l s f o r s t u d i e s of t h i s process (Hara 1970,1975; Barnett 1977; Bodznick 1978a; H o r r a l l 1981; Northcote 1984; Wright 1985; S a g l i o 1986), experimental i n v e s t i g a t i o n s have been l i m i t e d to the p e r i o d of seaward m i g r a t i o n by smolts (Ebel 1980; Power and McCleave 1980; Brannon 1982; Quinn et a l . i n p r e s s ) . No i n v e s t i g a t i o n s have been p u b l i s h e d of the importance to homing of odours encountered i n very e a r l y l i f e . The f i r s t imprint i s p o s t u l a t e d to occur i n the p e r i o d d u r i n g which f i s h are r e l i a b l y a s s o c i a t e d with the n a t a l area -12 before and very s h o r t l y a f t e r emergence (Harden-Jones 1968; Brannon 1982). There i s some evidence that salmon l e a r n odours d u r i n g t h i s p e r i o d . Brannon (1972) showed that sockeye a l e v i n s p r e f e r r e d whichever of two waters they were reared i n (creek or lake water). I f switched between waters, a l e v i n s s t i l l p r e f e r r e d the o r i g i n a l water 2 weeks l a t e r , a l b e i t more weakly than i f not switched. T e s t s with f r y i n d i c a t e d that waters were d i s t i n g u i s h e d by o l f a c t i o n . I t was not c l e a r from Brannon's experiments whether odours were remembered beyond the a l e v i n stage because f r y a l l switched p r e f e r e n c e to lake water, presumably as p a r t of a mechanism f o r l o c a t i n g a nursery l a k e . Bodznick (1978a) performed s i m i l a r experiments with sockeye f r y and confirmed the inherent a t t r a c t i o n to lake water but a l s o found an e f f e c t of recent e xperience. Fry reared i n w e l l water d i d not show the strong p r e f e r e n c e f o r lake water shown by f i s h r e a red i n lake water, and i f the l a t t e r were t r a n s f e r r e d to w e l l water, t h e i r p r e f e r e n c e f o r lake water slowly d i m i n i s h e d over a 17 d p e r i o d . F u r t h e r evidence that young salmon l e a r n odours has been p r o v i d e d by Quinn and Hara (1986). Coho f r y were shown to p r e f e r the chemical emanations of s i b l i n g s over u n f a m i l i a r non-s i b l i n g s but not f a m i l i a r (tankmate) n o n - s i b l i n g s . Quinn and Hara (1986) d i d not examine the sensory modality mediating t h i s response but evidence that i t i s o l f a c t i o n i s presented i n Appendix 3. T h e r e f o r e , while the d u r a t i o n of memories formed at these times i s unknown, i t appears t h a t salmon a l e v i n s and young f r y l e a r n and p r e f e r ambient odours over u n f a m i l i a r odours. 13 It i s possible that learning may occur even e a r l i e r , during the embryo stage. Salmonid embryos appear to have the anatomical and physiological capacity to detect odours before hatch (Brannon 1972; Jahn 1972; Z i e l i n s k i and Hara 1988), and olfactants of a l l but very large molecular sizes should pass freel y through the external egg membrane into the p e r i v i t e l l i n e f l u i d bathing the embryo (Hayes 1949). 14 GENERAL METHODS AND MATERIALS The F i s h Coho were obtained as gametes, a l e v i n s or f r y from F i s h e r i e s and Oceans Canada h a t c h e r i e s l o c a t e d on three r i v e r s on the east coast of Vancouver I s l a n d : the Quinsam, Puntledge, and B i g Qualicum (Figure 1). Samples of the 1985 and 1986 brood years (BY) were used i n experiments with population-odours (Chapters 3 & 4). Samples of the Quinsam p o p u l a t i o n were used i n experiments with s i b l i n g odours (1984 BY) (Chapter 2), and morpholine (1985 & 1987 BY) (Chapter 1). Though c u l t u r e d i n h a t c h e r i e s s i n c e 1974, 1978 and 1971 r e s p e c t i v e l y , these p o p u l a t i o n s are n a t i v e to t h e i r r i v e r s ; each o r i g i n a t e d from n a t i v e broodstock and has been maintained e x c l u s i v e l y with f i s h homing to the hatchery (pers. comm., B. Pearce, Dept. F i s h e r i e s and Oceans Canada, Vancouver, B r i t i s h Columbia). S t r a y i n g between B.C. coho p o p u l a t i o n s appears to occur mainly between nearby p o p u l a t i o n s (Wehrhahn and Powell 1987), and while s t r a y i n g has been recorded between these p o p u l a t i o n s (Sandercock, i n p r e s s ) , the frequency i s low (<1%) (Quinn and Tolson 1986). Each p o p u l a t i o n i s of s u f f i c i e n t s i z e to permit l o c a l a d a p t a t i o n i n the face of estimated s t r a y i n g r a t e s (Wehrhahn and Powell 1987). The l i f e h i s t o r y of coho salmon i n c l u d i n g B.C. p o p u l a t i o n s i s reviewed by Sandercock ( i n p r e s s ) . T y p i c a l of p o p u l a t i o n s i n 1 5 F i g u r e 1. L o c a t i o n s of Rosewall Creek Hatchery and the P a c i f i c B i o l o g i c a l S t a t i o n (PBS) where experiments were performed, and the Quinsam, B i g Qualicum, and Puntledge R i v e r s from which coho were obtained . 16 B.C., young of these populations are spawned in the f a l l , and spend 4-6 months as embryos and alevins (larvae) in the stream bed. Most fry swim up from the gravel in the spring, and remain for up to 15 months in the homestream system before migrating to sea the following spring or summer. (Some fry migrate to sea e a r l i e r (Chapman 1962), but probably do not survive (reviewed by Sandercock, in press).) Some males spend only 4-6 months at sea ("jacks"), but most f i s h return home to spawn after 16 months, as 3 year olds. Movements of juvenile coho in the S t r a i t of Georgia are reviewed by Healey (1980) and movements offshore by Hartt (1980), Hartt and D e l l (1986), and Pearcy and Fisher (1988). Information on the Big Qualicum R. population i s available in L i s t e r and Walker (1966), L i s t e r and Genoe (1970), Fraser et a l . (1983) and Mace (1983). The Laboratory Experiments were performed at the P a c i f i c B i o l o g i c a l Station (PBS) (Department of Fisheries and Oceans Canada) in Nanaimo, B.C. and at the PBS research hatchery located on Rosewall Creek, 80 km NW of Nanaimo (Figure 1). Rosewall Creek Hatchery i s served by two water sources -well water (ground water) which i s pumped from a gravel aquifer 27 m below ground, and Rosewall Creek, pumped from upstream of the hatchery. With one exception (reported in Chapter 5), f i s h were incubated and reared in well water. Both well and creek waters were used in assays. 17 A d e s c r i p t i o n of the Rosewall Creek watershed and i t s h i s t o r y i s given by Smith (1969). Creek water i s pumped i n t o an e l e v a t e d r e s e r v o i r tank and i s g r a v i t y - f e d to the hatchery through PVC plumbing. Well water i s handled s i m i l a r l y , except that i t i s sprayed i n t o a r e s e r v o i r tank to be oxygenated. (Presumably s p r a y i n g a l s o removes any noxious gases such as hydrogen s u l p h i d e or methane.) A sample of each water source taken on August 4, 1987, was analyzed by Dr. J . Davidson of Quanta Trace Labs Inc. (Burnaby, B.C.) f o r metals, anions, a l k a l i n i t y , pH, conductance and s o l i d s . The r e s u l t s of these a n a l y s e s are r e p o r t e d i n f u l l i n Appendix 1. The two waters were very s i m i l a r i n a l l parameters except temperature (see Chapters 1 & 5) and pH (we l l water: 8.0-8.5 measured i n the f i e l d , 7.5 measured l a t e r i n the l a b o r a t o r y , creek water: 7.0 i n both measurements). A l s o analyzed were samples to which 0.5 mg/L morpholine had been added. T h i s a n a l y s i s v e r i f i e d that water c h e m i s t r i e s were not a l t e r e d by the c o n c e n t r a t i o n of morpholine used i n t e s t s r e p o r t e d i n Chapter 1 (see Appendix 1). The water supply at the P a c i f i c B i o l o g i c a l S t a t i o n i s d e c h l o r i n a t e d and f i l t e r e d Nanaimo C i t y water, which i s drawn from the Nanaimo River above salmon spawning grounds (Quinn and Hara 1986). The c h a r a c t e r i s t i c s of t h i s water were r e p o r t e d by Groot (1989) as f o l l o w s : t o t a l hardness as c a l c i u m carbonate: 19.2 mg/L; s p e c i f i c conductance: 59 umhos/cm; pH: 6.9; c a l c i u m : 6.6 mg/L; sodium: 3.2 mg/L; c h l o r i d e : 2.2 mg/L; magnesium: 0.62 mg/L; potassium: 0.3 mg/L; and t o t a l n i t r o g e n as n i t r i t e and 18 n i t r a t e : 0.04 mg/L. T e s t i n g R e c o g n i t i o n  The Apparatus R e c o g n i t i o n of odours was i n f e r r e d from p r e f e r e n c e s i n dyadic c h o i c e s . (The one exception was the use of c a r d i a c responses to assess r e c o g n i t i o n of morpholine, the apparatus and methods f o r which are d e s c r i b e d i n Chapter 1.) The t e s t apparatus was that used by Quinn and Busack (1985), Quinn and Hara (1986), and Quinn and Tolson (1986), and c o n s i s t e d of four Y-mazes, the arms of which r e c e i v e d water from d i f f e r e n t headtanks ( F i g u r e 2). . The Y-mazes were c o n s t r u c t e d of 1.9 cm plywood, p a i n t e d dark green except f o r the f l o o r which was l i g h t green ( a l k y d marine enamel g l o s s , C-I-L Bapco Co.). A screen of p e r f o r a t e d aluminum (3 mm h o l e s at 6 mm c e n t e r s ) (Chapter 2) or s t a i n l e s s s t e e l mesh (2 mm holes) (Chapters 1,3,4,5) separated the arms of each Y-maze from the downstream common area, and a second screen 2 cm from the downstream w a l l prevented f i s h from escaping through the d r a i n . Each headtank was a 75 L p o l y e t h y l e n e bucket with f i v e PVC bulkhead f i t t i n g s i n s t a l l e d around the w a l l , 5 cm above the bottom. Attached to four f i t t i n g s were v a l v e s , to supply water to the Y-mazes. Attached to the f i f t h f i t t i n g was an e x t e r n a l stand-pipe. P l a s t i c mesh covered the p o r t s of the bulkhead f i t t i n g s and headtanks were covered with p o l y e t h y l e n e or PVC l i d s to c o n t a i n f i s h d u r i n g Figure 2. The Y-maze apparatus used to test preference between two odours. The arms of each Y-maze receive water from d i f f e r e n t headtanks. 20 experiments with c o n s p e c i f i c odours (see Chapters 2 & 3). Each headtank r e c e i v e d 5 L/min water through v i n y l t u b i n g connected to the PVC plumbing of the hatchery. Four L/min d r a i n e d to supply the Y-mazes, and the remaining L/min overflowed through the e x t e r n a l standpipe, m a i n t a i n i n g a water l e v e l 10 cm below the rim of the headtanks (55 L volume). Water flowed from the headtanks to the Y-mazes through Tygon tubes (6 mL ID, #R3603, Norton P l a s t i c s , Akron Ohio) i n s e r t e d i n t o PVC elbows on the upstream w a l l s of the maze arms. The flow to each arm was 1 L/min, checked twice d a i l y . Water d r a i n e d from the downstream, common area of each Y-maze, through an e x t e r n a l standpipe which maintained a water depth i n the maze of 5 cm. Dye t e s t s i n d i c a t e d that the degree to which waters from each arm of the maze mixed i n the downstream common area, depended on the a c t i v i t y of the t e s t f i s h . When the f i s h was i n a c t i v e , waters flowed through the common area with v i r t u a l l y no mixing, but when f i s h swam a c t i v e l y as was usual ( F i g u r e 3), the waters mixed e x t e n s i v e l y ( F i g u r e 4 ) . Th e r e f o r e , a f i s h i n the downstream common area was r e l i a b l y e x p e r i e n c i n g unmixed water only when i t pressed i t s nose a g a i n s t the upstream screen. T h i s was an important c o n s i d e r a t i o n i n the c h o i c e of the measure of p r e f e r e n c e (see below). 21 F i g u r e 3 . Screen-swimming: with the snout p r e s s e d a g a i n s t the upstream screen, the f r y swims a c t i v e l y i n an apparent attempt to enter the arms of the Y-maze. Figu r e 4. A dye t e s t showing ex t e n s i v e mixing i n the downstream common area of the Y-maze of waters from the two arms, r e s u l t i n g from a c t i v e swimming of the t e s t f i s h . 22 P r o t o c o l Fry were t r a n s f e r r e d by dipnet from h o l d i n g tanks and one was p l a c e d i n the downstream common area of each Y-maze. P r e l i m i n a r y o b s e r v a t i o n s i n d i c a t e d that f r y were e a s i l y s t a r t l e d by movement of the observer around the Y-maze,, so o b s e r v a t i o n s were made through a s l i t i n a black p l a s t i c c u r t a i n suspended 1 m from the downstream end of the Y-mazes (F i g u r e 5). A f t e r a 5 min a c c l i m a t i o n p e r i o d , each f i s h was observed f o r a 5 min p e r i o d (see below). Only one f i s h c o u l d be observed at a time, so data were recorded from the four mazes s e q u e n t i a l l y and the order i n which mazes were observed was s y s t e m a t i c a l l y v a r i e d over t r i a l s . (Observations of i n d i v i d u a l s over a l l four s e q u e n t i a l o b s e r v a t i o n p e r i o d s i n d i c a t e d that p r e f e r e n c e s ( f o r morpholine at l e a s t ) d i d not change s i g n i f i c a n t l y over the 20 min p e r i o d (Chapter 1 - Part 2).) A f t e r the f o u r t h f i s h had been observed, f r y were removed; no f i s h was t e s t e d more than once. Before the next t r i a l , the Tygon tubes s u p p l y i n g water to the arms of each Y-maze were switched ( F i g u r e 6) and the mazes were d r a i n e d . ( I t i s common p r a c t i c e i n Y-maze assays to switch odours between arms of the maze, to c o n t r o l f o r Type 1 e r r o r s a c c r u i n g from a d i r e c t i o n a l b i a s of the f i s h or p r e f e r e n c e f o r one arm over the other because of some f a c t o r u n r e l a t e d to the test-odour (e.g., Quinn and Busack 1985; Quinn and Hara 1986; Quinn and Tolson 1986; Groot et a l . 1986).) As the mazes r e f i l l e d , new f i s h were in t r o d u c e d and the next t r i a l began. G e n e r a l l y 8 - 1 2 t r i a l s were conducted per day between 0900 and F i g u r e 5. Observations were made through a s l i t i n a black p l a s t i c c u r t a i n suspended 1 m from the downstream end of the Y-mazes. Fi g u r e 6. Between t r i a l s , water s u p p l i e s were switched between arms of each Y-maze. 24 1800 h. The Measure of Preference Observations of i n d i v i d u a l f r y p l a c e d i n the downstream common area of the Y-maze, i n d i c a t e d that a f t e r a few minutes of i n a c t i v i t y they u s u a l l y pressed t h e i r snouts a g a i n s t the upstream screen and swam a c t i v e l y i n an apparent attempt to enter the arms of the maze (F i g u r e 3). T h i s behaviour, termed "screen-swimming", had been used by Barnett (1986) as a measure of the p r e f e r e n c e of Midas c i c h l i d f r y (Cichlasoma c i t r i n e l l u m ) between maternal and p a t e r n a l chemical emanations. In the present study, p r e l i m i n a r y t e s t s i n d i c a t e d that the p r o p o r t i o n of time spent screen-swimming before each arm ( i . e . , i n each water s o u r c e ) , gave c l e a r e r i n d i c a t i o n s of p r e f e r e n c e between s i b l i n g odours and blank water or n o n s i b l i n g odours, than measures used i n p r e v i o u s s t u d i e s (Quinn and Busack 1985; Quinn and Hara 1986) such as the arm f i r s t entered, arm occupied a f t e r a c e r t a i n p e r i o d , and time spent i n each arm. (The d i r e c t i o n of p r e f e r e n c e i n d i c a t e d by a l l measures was the same.) Fry g e n e r a l l y spent 20-40% of the time screen-swimming, and the r e s t of the time swimming a g a i n s t the downstream screen and Y-maze w a l l s , or l y i n g i n a c t i v e . In t e s t s of response to morpholine (Chapter 1-Part 1), the mean number of seconds of screen-swimming i n a 5 min o b s e r v a t i o n p e r i o d was 96 (32%) (SE=1.7, N=1176). (As e x p l a i n e d under A n a l y s i s , data c o l l e c t e d from f i s h t h a t screen-swam f o r l e s s than 20 s i n a 5 min p e r i o d 25 were d e l e t e d from a n a l y s i s . For t e s t s with morpholine (Chapter 1-Part 1), 12% of the f i s h were d e l e t e d and the mean rose to 108 (36%) seconds of screen-swimming (SE=1.7, N=1036).) Older f i s h spend l e s s time i n screen-swimming than younger f i s h (see Appendix 3). A n a l y s i s The experimental v a r i a b l e f o r a n a l y s i s was the p r o p o r t i o n of screen-swimming time that was spent before each arm i n the Y-maze d u r i n g a 5 min o b s e r v a t i o n p e r i o d . F i s h that screen-swam l e s s than 20 seconds were d e l e t e d from a n a l y s i s . The i n t e n t of t h i s a r b i t r a r y minimum a c t i v i t y c r i t e r i o n was to e l i m i n a t e f i s h not motivated to respond normally to odours. T h i s p r e c a u t i o n was probably unnecessary; i n data r e p o r t e d f o r experimental f i s h i n Chapter 1-Part 1, no c o r r e l a t i o n was found between p r e f e r e n c e (the p r o p o r t i o n of screen-swimming time that was spent on the morpholine h a l f of the screen) and a c t i v i t y (number of seconds of screen-swimming) (r=0.024, P>0.20, N=764). That i s , f i s h that screen-swam f o r only a short time avoided morpholine to the same extent as f i s h that screen-swam f o r a long time. P r o p o r t i o n s were normalized by a r c s i n e squareroot t r a n s f o r m a t i o n (Zar 1984:239). S i g n i f i c a n c e of departure from the n u l l h y p o t hesis of no p r e f e r e n c e (45°, the transformed e q u i v a l e n t of 50%) was t e s t e d by t w o - t a i l e d t - t e s t (Zar 1984:126). (Tests were t w o - t a i l e d , because p r e f e r e n c e s among a l l types of odours used i n experiments, were found to be i n f l u e n c e d by a number of 26 unforeseen f a c t o r s and were t h e r e f o r e u n p r e d i c t a b l e . ) P a i r w i s e comparisons of responses (e.g., c o n t r o l vs experimental, r e p l i c a t e t e s t s ) were made with Welch's approximate t - t e s t (Zar 1984:131). Comparisons of three or more responses were made with one-way ANOVA (Zar 1984:162), f o l l o w e d by Tukey's m u l t i p l e range t e s t (Zar 1984:186). Sample means and 95% confidence l i m i t s were back-transformed to percentages f o r p r e s e n t a t i o n . 27 CHAPTER 1. LEARNING AND MEMORY OF THE ODOUR OF MORPHOLINE INTRODUCTION Morpholine (C aH 9NO) i s a s y n t h e t i c h e t e r o c y c l i c amine used i n a wide v a r i e t y of i n d u s t r i a l , a g r i c u l t u r a l and medical a p p l i c a t i o n s (Scholz et a l . 1975). Wisby (1952) chose t h i s substance f o r i m p r i n t i n g experiments because i t i s , 1. organic and p r e v i o u s experiments had suggested that the odour by which f i s h d i s t i n g u i s h streams i s orga n i c (Hasler and Wisby 1951), 2. h i g h l y s o l u b l e i n water, 3. c h e m i c a l l y s t a b l e i n the n a t u r a l environment, 4. not normally found i n n a t u r a l waters, and 5. n e i t h e r i n h e r e n t l y r e p e l l e n t nor a t t r a c t i v e at the low c o n c e n t r a t i o n (10" 6 mg/L) ap p a r e n t l y d e t e c t e d by coho f r y . (Wisby's (1952) experiments i n d i c a t e d t hat f r y were r e p e l l e d by > 10" 5 mg/L however.) Subsequent f i e l d experiments demonstrated that coho smolts d e t e c t e d and l e a r n e d a c o n c e n t r a t i o n of 5 X 10" 5 mg/L, remembered i t u n t i l m a t u r i t y , and were a t t r a c t e d to i t i n homing m i g r a t i o n (Hasler and Scholz 1983), while naive homing salmon appeared n e i t h e r a t t r a c t e d (Hasler and Scholz 1983) nor r e p u l s e d (Mazeaud 1981; Rehnberg et a l . 1985). C o n s i d e r a b l e debate over whether salmonids p e r c e i v e morpholine as an odour has been generated by the demonstration t h a t , u n l i k e other odorants, low c o n c e n t r a t i o n s of morpholine do not generate e l e c t r i c a l responses from the o l f a c t o r y bulb (reviewed by Hara et a l . 1984). The p o s i t i o n adopted i n the present study i s that while the process of o l f a c t o r y 28 t r a n s d u c t i o n appears to d i f f e r i n some resp e c t between morpholine and other odours (Hara and Brown 1979, 1982; Cooper 1982; S a g l i o 1986; Dodson and Bitterman 1989 - see Appendix 4), morpholine i s an odour i n the sense that i t s p e r c e p t i o n by coho salmon at a c o n c e n t r a t i o n of 2 X 10" 3 mg/L i s blocked by b l o c k i n g the nares ( H i r s c h 1977). A number of recent f i e l d s t u d i e s i n d i c a t e a c o n t i n u i n g i n t e r e s t i n the use of morpholine i m p r i n t i n g , f o r augmenting and g u i d i n g homing by coho salmon (e.g., Rehnberg et a l . 1985; Hillman and G a l l a n t 1986; H a s s l e r and Kucas 1988). A l l of these s t u d i e s have f o l l o w e d H a s l e r ' s theory t h a t i m p r i n t i n g normally occurs d u r i n g the smolt stage, and have exposed f i s h to morpholine d u r i n g t h i s p e r i o d . In only two l a b o r a t o r y s t u d i e s have f i s h been exposed to morpholine before the smolt stage. S t e f f e l ( c i t e d by Cooper and Hasler 1973) confirmed Wisby's (1952) o b s e r v a t i o n that naive coho f r y a v o i d 10" 5 mg/L morpholine, and found a r e v e r s a l i n p r e f e r e n c e r e s u l t e d from p r e v i o u s exposure of 12 or 36 h. T h i s e f f e c t was only temporary however; f i s h t e s t e d 3 d a f t e r exposure again avoided morpholine. Wright (1985) exposed sockeye to 5 X 10" 5 mg/L morpholine f o r 2 weeks of the eyed-egg or a l e v i n stage but i n subsequent b e h a v i o u r a l t e s t s , f r y f a i l e d to respond to morpholine and no naive f r y were t e s t e d . T h e r e f o r e there i s no evidence to date that salmonids imprint on the odour of morpholine i n e a r l y l i f e . There i s however a r e p o r t of morpholine i m p r i n t i n g d u r i n g the f i r s t 3 months of l i f e i n another long d i s t a n c e migrant - the green sea t u r t l e ( Chelonia 29 mydas) (Grassman and Owens 1987). T h i s chapter i s i n two p a r t s . Part one r e p o r t s an experiment i n which coho were exposed to morpholine dur i n g the embryo, a l e v i n , or e a r l y f r y stage, or throughout a l l of these stages. A f t e r at l e a s t 50 d of non-exposure, they were t e s t e d f o r r e c o g n i t i o n of the odorant, i n f e r r e d from comparisons with naive f i s h i n two d i f f e r e n t responses: p r e f e r e n c e , and c a r d i a c d e c e l e r a t i o n . The l a t t e r response r e q u i r e s some e x p l a n a t i o n . In the experiments by Hasler and c o l l e a g u e s , two p h y s i o l o g i c a l responses were found to be c o r r e l a t e d with the b e h a v i o u r a l response of morpholine r e c o g n i t i o n ( i . e . , a t t r a c t i o n d u r i n g homing): an i n c r e a s e d EEG response of the o l f a c t o r y bulb, and a decrease i n heart r a t e . A number of problems surrounded the EEG response (Dizon et a l . 1973; Cooper and Hasler 1974, 1976), i n c l u d i n g t hat i t was evoked only by high c o n c e n t r a t i o n s of morpholine (0.1-10 g/L), and c o u l d not be r e p l i c a t e d by other workers (Hara and Brown 1979, 1982). More im p o r t a n t l y i t was not c l e a r whether i t r e f l e c t e d r e c o g n i t i o n or merely heightened o l f a c t o r y s e n s i t i v i t y (Cooper 1982; Hara and Brown 1982). The heart r a t e response, on the other hand, was e l i c i t e d by the same low c o n c e n t r a t i o n of morpholine e l i c i t i n g a t t r a c t i o n , and i s b e l i e v e d to r e f l e c t c e n t r a l nervous system f u n c t i o n (Hasler and Scholz 1983:93; Morin et a l . 1989a). S p e c i f i c a l l y , i t i s suggested that nerve impulses r e l a y e d by the o l f a c t o r y nerve are i n t e g r a t e d i n the b r a i n which i n turn sends i n h i b i t o r y ( c h o l i n e r g i c ) nerve impulses through the v a g a l nerve to the 30 heart (Hasler and Scholz 1983). F a r r e l l 1984 reviews the p h y s i o l o g i c a l c o n t r o l of heart r a t e i n t e l e o s t s . H i r s c h (pers. comm. c i t e d by H a s l e r and Scholz 1983:30) observed that mature a d u l t coho which had been exposed to morpholine d u r i n g the smolt stage, u s u a l l y responded to morpholine and not another chemical (phenethyl a l c o h o l ) , while f i s h p r e v i o u s l y exposed to phenethyl a l c o h o l d i d the o p p o s i t e . S i m i l a r t e s t s by Muzi ( c i t e d i n H a s l e r and Scholz 1983:93) suggested that t h i s response was not seen u n t i l f i s h became r e p r o d u c t i v e l y mature. However, recent experiments by Morin et a l . (1989a) have shown that odour r e c o g n i t i o n can be d e t e c t e d i n the u n c o n d i t i o n e d c a r d i a c responses of immature salmon, i f r e l a t i v e l y h i g h c o n c e n t r a t i o n s of odour are used. T h e r e f o r e , the heart r a t e response appears to be a u s e f u l i n d i c a t o r of odour r e c o g n i t i o n . During the course of the b e h a v i o u r a l t e s t s i n t h i s experiment, i t appeared that p r e f e r e n c e s i n the Y-maze were being confounded by some f a c t o r . The nature of t h i s f a c t o r , and the true p r e f e r e n c e s of naive and p r e v i o u s l y - e x p o s e d f r y were i n v e s t i g a t e d i n the second part of t h i s c h a p ter. 31 PART U LEARNING DURING THE EMBRYO, ALEVIN, AND EARLY FRY STAGES Treatment On November 25 1985, gametes of four male and four female coho salmon from the Quinsam River Hatchery were t r a n s p o r t e d to Rosewall Creek Hatchery where r e a r i n g and subsequent t e s t i n g took p l a c e . The gametes were pooled and d i v i d e d by volume i n t o ten e q u a l - s i z e d groups. Each group was incubated i n a 30.5 cm l e n g t h of 15 cm diameter ABS p l a s t i c pipe with f i b e r g l a s s mesh glued over one end, suspended v e r t i c a l l y i n a darkened c i r c u l a r f i b e r g l a s s tank (35 cm diameter, 40 cm depth). These "baskets" were c e n t e r e d around and suspended from the c e n t r a l standpipe i n the tank. Four L/min w e l l water (8-9°C) flowed i n t o the tank o u t s i d e the basket, up through the screened bottom and out through the top of the standpipe. When eggs were eyed, groups were reduced to 600 each. At swim-up, f r y were r e l e a s e d i n t o the tanks and baskets were removed. Fry were fed to s a t i a t i o n on Oregon Moist P e l l e t s (Moore-Clarke Co., LaConner WA) f i v e times a day f o r the f i r s t 2 weeks and twice a day t h e r e a f t e r , and maintained on a 12:12 L/D p h o t o p e r i o d . A stock s o l u t i o n of morpholine was prepared by mixing 0.1 mL morpholine (BDH Chemical Co., Minimum Assay (GLC) 98.5%, M.W. 87.12, d e n s i t y 1.0005 g/mL) i n 50 L w e l l water, i n a p l a s t i c garbage can. A p e r i s t a l t i c pump d e l i v e r e d 1 mL/min of t h i s s o l u t i o n through s i l i c o n tubing to the experimental h o l d i n g tanks, where i t mixed with the 4 L/min water supply to produce a 32 f i n a l c o n c e n t r a t i o n of 5 X 10"" mg/L. Choice of t h i s c o n c e n t r a t i o n f o r exposure - 500 times the d e t e c t i o n t h r e s h o l d fo r f r y (Wisby 1952) - was a r b i t r a r y ; the s e n s i t i v i t y of salmonid embryos and a l e v i n s to morpholine i s unknown. The h o l d i n g tanks were arranged i n two r e p l i c a t e rows of 5 each: A and B. The f i r s t tank i n each row r e c e i v e d morpholine throughout the embryo, a l e v i n and f i r s t 2 weeks of the f r y stage (Throughout treatment). The second tank i n each row r e c e i v e d morpholine d u r i n g only the embryo stage (Embryo), the t h i r d d u r i n g only the a l e v i n stage ( A l e v i n ) , the f o u r t h d u r i n g only the f r y stage ( F r y ) , and the f i f t h never r e c e i v e d morpholine ( C o n t r o l ) . The dates and d u r a t i o n s of morpholine exposures are given i n Table 1. T e s t i n g : P r e f e r e n c e Response Methods and M a t e r i a l s The response of i n d i v i d u a l f r y was t e s t e d to a c h o i c e of morpholine f l o w i n g down one arm of a Y-maze, and blank water f l o w i n g down the other. Test p r o t o c o l and data a n a l y s i s f o l l o w those d e s c r i b e d i n General Methods and M a t e r i a l s . Ten or twelve t r i a l s were performed per day. The arms of the Y-mazes r e c e i v e d water from d i f f e r e n t headtanks. Beginning 90 min before and c o n t i n u i n g throughout a t e s t , a p e r i s t a l t i c pump d r i p p e d a stock s o l u t i o n of morpholine 33 Table 1. Schedule of morpholine exposure. Treatment Dates Number of Days Embryo Nov. 26 1985 - Jan. 14 1986 49 (1 day post f e r t i l i z a t i o n -10 days pre-hatch) A l e v i n Jan. 27 - Feb. 25 1986 29 (hatch - swim-up) Fry Feb. 27 - March 13 1986 14 (2 weeks a f t e r swim-up) Throughout Nov. 26 - March 13 1986 107 (1 day post f e r t i l i z a t i o n -2 weeks a f t e r swim-up) C o n t r o l 0 34 i n t o one headtank at a ra t e of 3 mL/min, where i t mixed with the 5 L/min water flow. Blank water was pumped i n t o the second headtank to c o n t r o l f o r odours from pump t u b i n g . Stock s o l u t i o n s had been prepared the preceding evening with water taken from the headtank supply, and h e l d i n s i m i l a r g l a s s r e s e r v o i r s . Headtanks were f l u s h e d overnight and the headtank scented with morpholine was v a r i e d s y s t e m a t i c a l l y from day to day. In p r e l i m i n a r y t e s t s , responses were e l i c i t e d by c o n c e n t r a t i o n s of morpholine > 0.5 mg/L, but not < 0.05 mg/L (see Appendix 2). Th e r e f o r e , a l l assays were conducted with 0.5 mg/L. D i f f e r e n t responses by c o n t r o l and experimental f i s h were observed i n p r e l i m i n i n a r y t e s t s performed i n Rosewall Creek water but not w e l l water, sup p o r t i n g Wright's (1985) suggestion that f i s h may d i s t i n g u i s h or respond to a f a m i l i a r odour only i n an u n f a m i l i a r water source (see Appendix 2 ) . T h e r e f o r e a l l t e s t s were performed i n Rosewall Creek water. Water temperature i n the Y-mazes rose from 8°C i n e a r l y May to 13°C i n e a r l y June. Each treatment group was t e s t e d on 2 days, with morpholine d r i p p e d i n t o the r i g h t headtank one day and the l e f t on the ot h e r . In no case d i d responses d i f f e r s i g n i f i c a n t l y between days, so data were pooled f o r f u r t h e r a n a l y s i s . The responses of the two r e p l i c a t e groups (A and B) of each treatment were compared with each other and with other treatments with a mixed e f f e c t s model f o r two-way ANOVA, fo l l o w e d by Tukey t e s t . The dates of t e s t s and the number of days between the end of ) 35 exposure t o m o r p h o l i n e and t e s t i n g a r e g i v e n i n T a b l e 2. R e s u l t s A l l e x p e r i m e n t a l t r e a t m e n t s p r e f e r r e d b l a n k water over m o r p h o l i n e (Embryo; P=0.0007; A l e v i n : P<0.0001; F r y : P<0.0001; Throughout: P<0.0001), whereas C o n t r o l s showed the o p p o s i t e p r e f e r e n c e (P<0.0001) ( F i g u r e 7 ) . S i g n i f i c a n t d i f f e r e n c e s were not d e t e c t e d between the responses of e x p e r i m e n t a l t r e a t m e n t s , and a l l except Embryo d i f f e r e d (P<0.05) from the response of C o n t r o l s . W h i l e no s i g n i f i c a n t r e p l i c a t e e f f e c t was d e t e c t e d (P=0.399), t h i s a n a l y s i s d i d i n d i c a t e an i n t e r a c t i o n between the t r e a t m e n t e f f e c t and r e p l i c a t e s which approached s i g n i f i c a n c e (P=0.065), r e f l e c t i n g g e n e r a l l y weaker responses by B than A r e p l i c a t e s ( F i g u r e 7 ) . ( T h i s may be a t t r i b u t a b l e t o g r e a t e r d i s t u r b a n c e of B r e p l i c a t e s , which were l o c a t e d on a main a i s l e a t the h a t c h e r y w h i l e A r e p l i c a t e s were not.) The s y s t e m a t i c d i f f e r e n c e between r e p l i c a t e r e sponses reduced the s e n s i t i v i t y of the a n a l y s i s , because t r e a t m e n t e f f e c t was t e s t e d a g a i n s t the i n t e r a c t i o n term. C o n s e q u e n t l y , d a t a c o l l e c t e d from A and B were a n a l y z e d s e p a r a t e l y . In b oth r e p l i c a t e s a s i g n i f i c a n t t r e a t m e n t e f f e c t was d e t e c t e d (P<0.0005): e x p e r i m e n t a l t r e a t m e n t s d i d not d i f f e r s i g n i f i c a n t l y from one a n o t h e r i n p r e f e r e n c e f o r b l a n k w a t e r , and a l l d i f f e r e d s i g n i f i c a n t l y from the p r e f e r e n c e f o r Table 2. Schedule of Y-maze t e s t s of p r e f e r e n c e f o r morpholine. T r e a t -ment Repli-cate Date (day/month) Minimum Number of Days Since Last Exposure Embryo  A l e v i n Fry. Throughout C o n t r o l ( e a r l y t e s t s ) C o n t r o l ( l a t e r t e s t s ) A B A B A B A B A B A B 19,20/05 11,13/06 21,22/05 14,15/06 23,24/05 16,18/06 6,7/05 10,11/05 9,13/05 8,14/05 26,27/05 19/06 1 25 1 48 85 109 71 95 54 58 37 80 70 60 50 40 30 20 86 79 97 99 96 87 94 92 73 83 Attraction i Avoidance Control Embryo Alevin Fry Throughout Treatment Group F i g u r e 7 . B e h a v i o u r a l r e s p o n s e o f c o h o f r y t o m o r p h o l i n e . F i s h h a d b e e n p r e v i o u s l y e x p o s e d t o m o r p h o l i n e d u r i n g t h e E m b r y o , A l e v i n , o r e a r l y F r y s t a g e s , T h r o u g h o u t a l l t h r e e s t a g e s , o r h a d n o t b e e n p r e v i o u s l y e x p o s e d ( C o n t r o l ) . Open b a r s : r e p l i c a t e A , S h a d e d b a r s : r e p l i c a t e B Cx ± 95% C L , Number o f f r y t e s t e d shown a b o v e b a r ) 38 morpholine shown by t h e i r r e s p e c t i v e c o n t r o l (P<0.05). The d i f f e r e n c e between t h i s a n a l y s i s and that combining r e p l i c a t e groups, i s that t h i s a n a l y s i s d e t e c t e d a d i f f e r e n c e s between the Embryo and C o n t r o l groups. The C o n t r o l s i n c l u d e d i n the above a n a l y s i s had been t e s t e d before a l l experimental groups except Throughout (Table 2). F o l l o w i n g the t e s t s of experimental groups i n each row, t h e i r r e s p e c t i v e C o n t r o l s were r e t e s t e d to ensure t h a t d i f f e r e n c e s between the responses of experimentals and C o n t r o l s were not due to a time e f f e c t or u n c o n t r o l l e d changes i n the assay. R e p l i c a t e A was t e s t e d twice and the r e s u l t s of the t e s t s being s i m i l a r (P>0.05), were pooled. R e p l i c a t e B was t e s t e d only once, as i n s u f f i c i e n t f i s h remained f o r a second t e s t . R e p l i c a t e A responded more weakly than p r e v i o u s l y (P=0.0040, Welch's t ) , f a i l i n g to show s i g n i f i c a n t p r e f e r e n c e ( F i g u r e 8). R e p l i c a t e B a l s o responded more weakly than p r e v i o u s l y though not s i g n i f i c a n t l y so, and f a i l e d to show s i g n i f i c a n t p r e f e r e n c e ( F i g u r e 8 ) . These data were s u b s t i t u t e d f o r the i n i t i a l C o n t r o l data i n the comparisons with experimental groups. S i g n i f i c a n t treatment e f f e c t s were s t i l l d e t e c t e d (A: P<0.005, B: P<0.025). In r e p l i c a t e A, the lack of p r e f e r e n c e of C o n t r o l s d i f f e r e d (P<0.05) from the p r e f e r e n c e f o r blank water shown by experimental groups, except from the weak pr e f e r e n c e shown by the Embryo group. In the l e s s responsive r e p l i c a t e B, s i g n i f i c a n t d i f f e r e n c e (P<0.05) was d e t e c t e d only between the 39 a O -a o I H a B B =5 e H -»-> d o o CU 86 79 99 51 80 70 60 50 40 30 20 Attraction A Avoidance Early May Late May/June Figure 8. Decrease in the response of Controls to morpholine between tests performed in early May and late May or June. (Dates in Table 2) Open bars: re p l i c a t e A, Shaded bars; replicate B (x ± 95% CL, Number of fry tested shown above bar) 40 lack of p r e f e r e n c e of the C o n t r o l group, and the p r e f e r e n c e f o r blank water shown by the Throughout group. These r e s u l t s suggest that while the responsiveness of C o n t r o l s had decreased s i n c e the o r i g i n a l t e s t s (perhaps r e f l e c t i n g s t r e s s from repeated d i s t u r b a n c e ) , t h e i r response to morpholine s t i l l d i f f e r e d from that of experimental f i s h . These data i n d i c a t e that p r e f e r e n c e f o r morpholine was i n f l u e n c e d by p r e v i o u s exposure, suggesting that coho l e a r n e d the odour d u r i n g the embryo, a l e v i n , and e a r l y f r y stages, and recognized i t 54-125 d l a t e r (Table 2). However, p r e f e r e n c e s recorded here were opposite those r e p o r t e d by Wisby (1952) and Cooper and H a s l e r (1973) (who r e p o r t e d avoidance by naive f i s h ) , and H asler and Scholz (1983) (who r e p o r t e d a t t r a c t i o n by p r e v i o u s l y exposed f i s h ) . The reason f o r t h i s d i f f e r e n c e was i n v e s t i g a t e d i n Part 2. T e s t i n g : C a r d i a c Response Methods and M a t e r i a l s Upon completion of the Y-maze t e s t s (June 20, 1986), f i s h remaining i n the r e p l i c a t e s of each treatment were pooled (N=400-800 per treatment) and r e a r i n g c ontinued i n w e l l water i n c i r c u l a r f i b e r g l a s s tanks (92 cm diameter, 49 cm depth, 12 L/min flow) at Rosewall Creek Hatchery. F i s h were i d e n t i f i e d only by treatment, not by r e p l i c a t e i n t h i s experiment. In January 1987, 120-200 medium-sized members of each group (approximately 41 10 cm f o r k - l e n g t h , 15 grams wet-weight) were a n a e s t h e t i z e d with 2-phenoxyethanol and f i n - c l i p p e d ( p e l v i c or adipose f i n s ) to allow subsequent i d e n t i f i c a t i o n . On A p r i l 9 1987, four f i s h of each of the f i v e treatment groups - were sent by a i r to P.-P. Morin's l a b at the P u b l i c Aquarium i n Quebec C i t y , Quebec, fo l l o w e d on A p r i l 14 by an a d d i t i o n a l 16 f i s h of each treatment group. In Quebec, treatment groups were h e l d together i n an 300 L aquarium (158 X 51 X 36 cm), f i l l e d with d e c h l o r i n a t e d c i t y water (12.5 + 0.5°C). F i s h were fed ad l i b i t u m once per day with a mixture of shrimps, crabmeat and s c a l l o p s . Photoperiod was maintained at 16:8 L/D. Te s t s were performed by P.-P. Morin. The t e s t apparatus and p r o t o c o l are d e s c r i b e d by Morin et a l . (1987b, 1989a). B r i e f l y , i n d i v i d u a l f i s h were weighed (wet weight), measured ( f o r k l e n g t h ) , i d e n t i f i e d by f i n c l i p , a n a e s t h e t i z e d (1% methyl p e n t y n o l ) , p a r a l y s e d ( d - t u b o c u r a r i n e c h l o r i d e , 2 X 10" 3 mg/g body weight, i n j e c t e d i n t o d o r s a l musculature), and immobilized i n a t e s t chamber which was equipped with e l e c t r o d e s f o r monitoring heart r a t e . Water, of the same source and temperature f i s h were h e l d i n , moved through the chamber at 20.5 mm/s. A separate water l i n e was p l a c e d i n the mouth of the f i s h t o perfuse the g i l l s (300 mL/min). A f t e r a 30 min a c c l i m a t i o n p e r i o d , r e s t i n g h eart r a t e (beats per min (BPM)) and i n t e r b e a t i n t e r v a l (IBI) were re c o r d e d over a 1 0 s p e r i o d . F i v e min l a t e r , morpholine p r e s e n t a t i o n t r i a l s began. A stock s o l u t i o n of morpholine was d e l i v e r e d to the upstream end of the t e s t chamber by a p e r i s t a l t i c pump, where i t mixed with the water 42 flow. The time and c o n c e n t r a t i o n of morpholine d e l i v e r e d to the f i s h were estimated as i n Morin et a l . (1987b). Heart r a t e was measured f o r 8 s before and 8 s d u r i n g morpholine p r e s e n t a t i o n , which l a s t e d approximately 10 s (Morin et a l . 1987b). Responses were recorded to ten s e q u e n t i a l p r e s e n t a t i o n s of morpholine, each separated by a short i n t e r v a l of random d u r a t i o n (45-135 s ) . F i s h of the C o n t r o l and Throughout groups (N=3 each) were t e s t e d on June 28,29 with a morpholine c o n c e n t r a t i o n of 33.1 mg/L (3.8 X 10" 4 M). F i s h of a l l treatment groups (N=5 each) were t e s t e d with a morpholine c o n c e n t r a t i o n of 331 mg/L between J u l y 1-5, and with a morpholine c o n c e n t r a t i o n of 3.31 mg/L between J u l y 9-13. In the l a s t two s e t s of t e s t s , one f i s h of each treatment group was t e s t e d per day, i n random ord e r . Cardiac responses were e l i c i t e d by only the h i g h e s t c o n c e n t r a t i o n of morpholine (331 mg/L), and i t i s these responses t h a t are r e p o r t e d below. F i s h used i n t h i s set of t e s t s were s i m i l a r a c r o s s treatment groups i n f o r k - l e n g t h (mean=12.6 cm, SD=0.7, range=11.6 - 13.7, N=25), wet-weight (mean=24.5 g, SD=4.4, range=17.3 - 31.8, N=25), and r e s t i n g heart r a t e (mean=95.8 BPM, SD=8.2, range=76.4 - 116.0, N=25) (P>0.05, one-way ANOVA). Heart r a t e r e d u c t i o n to an odour cue t y p i c a l l y occurs i n the context of a s i n g l e , l a r g e IBI (Morin et a l . 1987b, 1989a). The l a r g e s t IBI recorded i n the 8 s i n t e r v a l before morpholine p r e s e n t a t i o n was compared to the l a r g e s t IBI recorded i n the 8 s 43 i n t e r v a l d u r i n g morpholine p r e s e n t a t i o n . Percentage heart r a t e r e d u c t i o n was c a l c u l a t e d by the formula: 100 - ( l a r g e s t IBI d u r i n g X 100) / l a r g e s t IBI before Data were normalized f o r a n a l y s i s by squareroot (X+0.5) t r a n s f o r m a t i o n . The response of each f i s h , was taken as the average of i t s responses to the ten s e q u e n t i a l p r e s e n t a t i o n s of morpholine. (Analyses performed on the f i r s t response of each f i s h , and the l a r g e s t of the ten responses of each f i s h gave v i r t u a l l y the same r e s u l t . ) Responses of the f i v e treatment groups were compared by ANOVA, f o l l o w e d by Tukey's m u l t i p l e range t e s t . Means and 95% confidence l i m i t s were back transformed to percentage heart r a t e r e d u c t i o n f o r p r e s e n t a t i o n . R e s u l t s A l l experimental groups responded to 331 mg/L morpholine with c a r d i a c d e c e l e r a t i o n s whereas C o n t r o l s d i d not ( F i g u r e 9 ). S i g n i f i c a n t d i f f e r e n c e s (P<0.05) were d e t e c t e d between the c a r d i a c responses of C o n t r o l s and a l l experimental groups except the Fry group. Among the experimental groups, Throughout showed a s i g n i f i c a n t l y l a r g e r response than Embryo or Fry (P<0.05). These r e s u l t s support the c o n c l u s i o n from the Y-maze t e s t s that coho l e a r n e d the odour of morpholine d u r i n g the embryo, a l e v i n and e a r l y f r y stages. Furthermore, the c a r d i a c responses extend the d u r a t i o n of memory of t h i s odour from the 54-125 d 44 100 c o • rH o p ti o -t=> u o X a O rH D PH 80 -60 -40 -20 0 1— — T Control Embryo Alevin Fry Throughout Treatment Group Figure 9. Cardiac responses to morpholine of coho smolts that had been exposed to morpholine during the Embryo, Alevin, or early Fry stage, Throughout a l l three stages, or had not been previously exposed (Controls). (Note: re p l i c a t e s A and B of each treatment pooled.) (x ± 95% CL, Number of fry tested shown above bar) 45 i n d i c a t e d by Y-maze t e s t s to 477-532 d, suggesting that odours a s s o c i a t e d with the p e r i o d of l i f e normally spent i n the redd c o u l d be remembered at l e a s t u n t i l the smolt stage. PART 2: INVESTIGATION OF THE PREFERENCE RESPONSE Responses to morpholine recorded i n the Y-maze were op p o s i t e those r e p o r t e d by p r e v i o u s s t u d i e s - avoidance by naive f i s h (Wisby 1952; Cooper and H a s l e r 1973), a t t r a c t i o n by p r e v i o u s l y exposed f i s h (Hasler and Scholz 1983). A l s o , responses were e l i c i t e d only by a c o n c e n t r a t i o n of morpholine (0.5 mg/L) f a r above that which e l i c i t e d p r e f e r e n c e s i n pre v i o u s s t u d i e s (10" 5 mg/L). These f a c t s suggested the presence of some confounding f a c t o r i n the Y-maze assay i n Part 1. Two c h a r a c t e r i s t i c s of the Y-maze t e s t i n g procedure c o u l d have a f f e c t e d the responses of f i s h to morpholine. The s w i t c h i n g of morpholine from one arm of the maze to the other between t r i a l s c o u l d have r e s u l t e d i n morpholine r e s i d u e s i n the "blank" arm. Dye t e s t s i n d i c a t e d complete exchange of waters i n arms between t r i a l s , but r e s i d u e s c o u l d have adhered t o , and subsequently have been r e l e a s e d from, the arms of the maze or the screen a g a i n s t which f i s h swam. A second p o t e n t i a l confounding f a c t o r was the lengthy p e r i o d of exposure to morpholine (5-20 min) before response was measured. By c o n t r a s t , Wisby (1952) recorded response a f t e r j u s t 1 min of exposure. Conceivably i n the present study, naive 46 f i s h i n i t i a l l y avoided morpholine as i n Wisby's study, but then changed response, perhaps due to a d a p t a t i o n of o l f a c t i o n (Peeke and P e t r i n o v i c h 1984). As o l f a c t o r y p e r c e p t i o n decreased, f r y might have responded to other p r o p e r t i e s of morpholine, such as i t s t a s t e . These two p o t e n t i a l confounding f a c t o r s were examined by t e s t i n g both naive f i s h and f i s h p r e v i o u s l y exposed to morpholine i n two assays: with morpholine switched from arm to arm between t r i a l s as i n Part 1, and with morpholine not switched. In each assay, responses were recorded from each f i s h immediately a f t e r i n t r o d u c t i o n to the Y-maze (the f i r s t 5 min of exposure), and i n four subsequent c o n s e c u t i v e 5 min p e r i o d s . Treatment On March 15 1988, 600 coho a l e v i n s (spawn date: Nov. 10, 1987) were t r a n s p o r t e d from the Quinsam River Hatchery to Rosewall Creek Hatchery, where they were d i v i d e d i n t o two groups of 300 each, r e f e r r e d to as C o n t r o l and Experimental. Each group was incubated i n a basket suspended i n a darkened c i r c u l a r f i b e r g l a s s tank, s u p p l i e d with 4 L/min w e l l water as i n Part 1. F i s h swam up on March 28 at which time the i n c u b a t i o n baskets were removed. F r y were fed to s a t i a t i o n f i v e times a day f o r the f i r s t 2 weeks (ST40 mash, EWOS Canada F i s h D i e t s ) and twice a day t h e r e a f t e r ( p e l l e t s - White C r e s t M i l l s D i v i s i o n ) and maintained on a 12:12 L/D photoperiod. I t was noted on A p r i l 5 t h a t a l l but 50 i n d i v i d u a l s of the ( C o n t r o l ) group had escaped 47 when the tank overflowed. These were r e p l a c e d by 300 f r y of the same o r i g i n on A p r i l 6. The Experimental group was exposed to 5 X 1 0 " * mg/L morpholine c o n t i n u o u s l y from March 15 to A p r i l 11: a p e r i o d of one month ce n t e r e d around the t r a n s i t i o n from a l e v i n to f r y stage. A p e r i s t a l t i c pump dr i p p e d a stock s o l u t i o n of morpholine i n t o the water supply of the Experimental r e a r i n g tank at the r a t e of 1 mL/min. The stock s o l u t i o n was prepared from 0.06 mL morpholine (same source as i n Part 1) i n 30 L w e l l water i n a g l a s s aquarium. Test apparatus and p r o t o c o l were those used i n Part 1, with the f o l l o w i n g d i f f e r e n c e s . Instead of d r i p p i n g morpholine i n t o one headtank and blank water i n t o the other, a s i n g l e headtank s u p p l i e d water to both arms of a Y-maze and stock s o l u t i o n s of morpholine and blank water were d r i p p e d d i r e c t l y i n t o the upstream ends of Y-maze arms at the r a t e of 2 mL/min. Stock s o l u t i o n s were prepared immediately b e f o r e each t e s t from 0.5 mL morpholine (same supply used i n e a r l i e r exposure) i n 2 L Rosewall Creek water. A l l t e s t s were conducted i n Rosewall Creek water (8-11°C). Recordings of screen-swimming began immediately a f t e r a f i s h was in t r o d u c e d to the Y-maze and continued f o r f i v e c o n s e c u t i v e 5 min p e r i o d s . Each f i s h was t e s t e d only once. Data are r e p o r t e d here only from f i s h t h a t swam at l e a s t 20 s i n each of the f i v e time p e r i o d s (20 of 24 Experimental and 11 of 13 C o n t r o l f i s h i n the f i r s t experiment, 30 of 36 Experimental 48 and 29 of 36 C o n t r o l f i s h i n the second experiment). Methods s p e c i f i c to each experiment are d e s c r i b e d with the r e s u l t s of those experiments. A n a l y s i s f o l l o w e d that of Y-maze data i n Part 1, with the a d d i t i o n t h a t responses were compared a c r o s s the f i v e c o n s e c u t i v e o b s e r v a t i o n p e r i o d s by ANOVA with repeated measures (Zar 1984:222) fo l l o w e d by Tukey's m u l t i p l e range t e s t (Zar 1984:226). Experiment J_: Morpholine Switched Between Arms of the Y-Maze Methods and M a t e r i a l s A s i n g l e Y-maze was used. A f t e r d r i p p i n g morpholine f o r 25 min down one arm, and blank water down the other, the d r i p s were switched between arms, the maze was d r a i n e d and as i t r e f i l l e d , a f i s h was i n t r o d u c e d . The Experimental group was t e s t e d on June 20 (N=10) and June 24 (N=10). Responses d i d not d i f f e r s i g n i f i c a n t l y on the two days (Welch's approximate t t e s t s f o r each o b s e r v a t i o n p e r i o d ) , so were pooled f o r a n a l y s i s . C o n t r o l s were t e s t e d on June 21 (N=11). R e s u l t s Experimental f i s h showed a s i g n i f i c a n t change i n pr e f e r e n c e over the f i v e o b s e r v a t i o n p e r i o d s (P<0.0005) (F i g u r e 10). In the f i r s t p e r i o d , f i s h showed a weak a t t r a c t i o n to morpholine 49 Experimentals c • r H r — I o r C a r H o CO *o u GO G • r H 6 8 • i - H CZ5 s H c O r H PL, 70 60 50 40 30 70 60 50 40 30 Controls Attraction A Avoidance Attraction Avoidance Observation Period F i g u r e 10. Change of response to morpholine over 5 s e q u e n t i a l 5-min o b s e r v a t i o n p e r i o d s , when morpholine was a l t e r n a t e d between arms of the Y-maze from t r i a l to t r i a l . E x perimentals had been exposed to morpholine d u r i n g a 1 month p e r i o d centered around swim-up, whereas C o n t r o l s had not. (x ± 95% C.L., (N=20 E x p e r i m e n t a l s , 11 C o n t r o l s ) Compare to Fi g u r e 11. 50 (P=0.07) which d i f f e r e d s i g n i f i c a n t l y (P<0.05) from t h e i r avoidance i n p e r i o d s 3 (P=0.0016) and 5 (P=0.0031). C o n t r o l f i s h d i d not show s i g n i f i c a n t change i n response over the f i v e o b s e r v a t i o n p e r i o d s (Figure 10). S i g n i f i c a n t p r e f e r e n c e was d e t e c t e d only i n p e r i o d 1 (P=0.044), and t h i s was avoidance of morpholine. Responses of the Experimental and C o n t r o l groups d i f f e r e d i n the f i r s t o b s e r v a t i o n p e r i o d (P=0.0070), and i n the t h i r d (P=0.049). I n t e r p r e t a t i o n The responses of both naive and p r e v i o u s l y - e x p o s e d f i s h recorded d u r i n g p e r i o d s 2-5 are c o n s i s t e n t with those recorded i n Part 1, and are d i f f e r e n t from the i n i t i a l responses of the f i s h . T h e r e f o r e the " p r e f e r e n c e s " r e p o r t e d i n Part 1 do appear to have been confounded by some aspect of the t e s t procedure. I t i s not c l e a r from t h i s experiment whether the confounding f a c t o r was r e l a t e d to the s w i t c h i n g of morpholine from arm to arm between t r i a l s , or to prolonged exposure to morpholine. T h i s was i n v e s t i g a t e d i n Experiment 2. 51 Experiment 2: Morpholine not Switched Between Arms of the Y-maze Methods and M a t e r i a l s Two Y-mazes were used. Instead of s w i t c h i n g morpholine and blank water from arm to arm between t r i a l s of a t e s t , morpholine was pumped i n t o one arm and blank water i n t o the other f o r the e n t i r e t e s t . The f i r s t t r i a l of the t e s t was conducted with morpholine f l o w i n g down the r i g h t arm of the f i r s t Y-maze. The morpholine d r i p was then moved to the l e f t arm of the second Y-maze f o r the second t r i a l , then back to the r i g h t arm of the f i r s t maze f o r the t h i r d t r i a l and so on f o r a t o t a l of twelve t r i a l s . Water was l e f t running o v e r n i g h t through the Y-mazes, to f l u s h out any r e s i d u a l morpholine. The t e s t was repeated on the next day or day a f t e r , with morpholine s c e n t i n g the o p p o s i t e arms of each Y-maze. Data from the 2 days of t e s t i n g were pooled f o r a n a l y s i s . C o n t r o l f i s h were t e s t e d on June 4,6 (N=19), Experimental f i s h on June 7,8 (N=21) and both groups were r e t e s t e d i n a l t e r n a t i n g p a i r s of t r i a l s on June 16,17 (N=10 C o n t r o l s , N=9 E x p e r i m e n t a l s ) . Responses recorded i n e a r l y and mid-June were compared (Welch's approximate t f o r each o b s e r v a t i o n p e r i o d ) . Experimental responses d i f f e r e d i n no o b s e r v a t i o n p e r i o d , so were pooled f o r a n a l y s i s . C o n t r o l s d i f f e r e d at o b s e r v a t i o n p e r i o d 3 (P=0.029) - a v o i d i n g morpholine on June 16,17 (P=0.0084) but not on June 4,6. D e s p i t e t h i s d i f f e r e n c e , data were pooled f o r a n a l y s i s . A second d i f f e r e n c e between these t e s t s and those i n 52 Experiment 1 (and Part 1) was that i n these the upstream screen a g a i n s t which f i s h swam was c l e a n e d between t r i a l s to remove mucus. I t was c o n s i d e r e d i n p r e v i o u s t e s t s t h a t while f i s h might a v o i d or be a t t r a c t e d to mucus l e f t on the screen by p r e v i o u s l y - t e s t e d f i s h , t h i s should not b i a s the t e s t because mucus would be present on both h a l v e s of the sc r e e n . In t h i s experiment however, response to morpholine would r e s u l t i n f i s h swimming predominantly on and l e a v i n g d e p o s i t s on one h a l f of the screen, r e s u l t i n g i n an i n c r e a s i n g b i a s as the t e s t p rogressed. T h e r e f o r e , between t r i a l s the screen was removed from the Y-maze, brushed with 95 % e t h a n o l , r i n s e d with hot w e l l water, then Rosewall Creek water, and r e p l a c e d . P o l y e t h y l e n e or rubber gloves were worn while h a n d l i n g the screen. R e s u l t s Experimental f i s h showed no s i g n i f i c a n t response to morpholine i n any of the f i v e o b s e r v a t i o n p e r i o d s , and no s i g n i f i c a n t change i n response over time ( F i g u r e 11). C o n t r o l f i s h avoided morpholine i n the f i r s t (P=0.0039), f o u r t h (P=0.0140) and f i f t h (P=0.0070) o b s e r v a t i o n p e r i o d s , and showed no s i g n i f i c a n t change i n response over time ( F i g u r e 11). The response of Experimental f i s h d i f f e r e d s i g n i f i c a n t l y from that of C o n t r o l s i n p e r i o d s 1 (P=0.0008), 2 (P=0.0420), 4 (P=0.0320) and 5 (P=0.0240). The lack of response shown by Experimental f i s h d i f f e r e d 53 Experimentals c • I H *o r G a rH o T3 C • rH a a t/3 CD a c o rH PH 70 60 50 40 30 Attraction 70 60 50 40 30 Controls Avoidance Attraction A Avoidance Observation Period F i g u r e 11. Lack of change of response to morpholine over f i v e 5-min o b s e r v a t i o n p e r i o d s , when morpholine was not a l t e r n a t e d between arms of the Y-maze from t r i a l to t r i a l . E x perimentals had been exposed to morpholine dur i n g a 1 month p e r i o d c e n t e r e d around swim-up, whereas C o n t r o l s had not. (x ± 95% C.L., (N=30 Experimentals, 29 C o n t r o l s ) Compare to F i g u r e 10. 54 s i g n i f i c a n t l y from t h e i r avoidance of morpholine d u r i n g p e r i o d s 3 (P=0.0074) and 5 (P=0.018) of Experiment 1. No s i g n i f i c a n t d i f f e r e n c e s were d e t e c t e d between the responses of C o n t r o l s i n Experiments 1 and 2. I n t e r p r e t a t i o n These data suggest that the temporal change i n p r e f e r e n c e recorded i n Experiment 1 was not r e l a t e d to o l f a c t o r y a d a p t a t i o n or some other e f f e c t on t e s t f i s h of prolonged exposure to morpholine. Rather, the change r e s u l t e d from a change i n the odours presented to the f i s h , consequent of s w i t c h i n g morpholine from arm to arm between t r i a l s , and/or mucus r e s i d u e s on the screen. The e l i m i n a t i o n of s w i t c h i n g and mucus r e s i d u e s e l i m i n a t e d the change i n p r e f e r e n c e ; C o n t r o l s on average maintained the avoidance of morpholine recorded i n the i n i t i a l p e r i o d of Experiment 1, while Experimentals maintained a weak ( n o n - s i g n i f i c a n t ) p r e f e r e n c e . The mechanism by which odours changed d u r i n g the assay i n Experiment 1 i s not c l e a r . One p o s s i b i l i t y i s that morpholine r e s i d u e s adhered to the p a i n t e d arms of the Y-maze and/or the s t a i n l e s s s t e e l screen and were r e l e a s e d d u r i n g the next t r i a l , by the end of which none remained. That i s to say that the arms or screen of the Y-maze acted as a b a t t e r y , s t o r i n g and r e l e a s i n g morpholine, such that the stronger c o n c e n t r a t i o n d u r i n g any given t r i a l came from the "blank" arm. The reason f o r r e l e a s e o c c u r r i n g d u r i n g o b s e r v a t i o n p e r i o d s 2-5 but not 55 p e r i o d 1 i s u n c l e a r , but c o u l d be r e l a t e d to the f a c t that d u r i n g the f i r s t o b s e r v a t i o n p e r i o d , the Y-maze was s t i l l r e f i l l i n g a f t e r having been d r a i n e d . The suggestion that morpholine s t i c k s onto s u r f a c e s and i s g r a d u a l l y r e l e a s e d i s c o n s i s t e n t with the experience of other workers (pers. comm.-Dr. P.B. Johnsen, US Dept. A g r i c u l t u r e , Southern Regional Research Center, New Orleans, LA, USA). In a d d i t i o n , mucus on the screen may have i n f l u e n c e d f i s h , e i t h e r because of i t s own odour or t a s t e , or because of some i n t e r a c t i o n with morpholine. Whatever the mechanism, the experiments r e p o r t e d i n Part 2 suggest that the " p r e f e r e n c e s " recorded i n Part 1 were an a r t i f a c t of r e s i d u e s of morpholine and/or mucus. The true response of naive coho f r y to 0.5 mg/L i s avoidance, c o n s i s t e n t with t h e i r response to lower c o n c e n t r a t i o n s (Wisby 1952; Cooper and Hasler 1973). The t r u e response of coho f r y that were p r e v i o u s l y exposed to 5 X 10" 4 mg/L, i s a l a c k of avoidance or even weak a t t r a c t i o n . (Experiments 1 and 2 support the c o n c l u s i o n of P a r t 1 - that p r e v i o u s exposure a l t e r s p r e f e rence f o r morpholine. F i s h exposed f o r a 1 month p e r i o d centered around swim-up, responded d i f f e r e n t l y than C o n t r o l s i n t e s t s 2 months l a t e r (70 d f o r Experiment 1, 57 d f o r Experiment 2).) The d i s c o v e r y that the Y-maze assay i n the present study was i n f l u e n c e d by morpholine and/or f i s h r e s i d u e s , suggests an e x p l a n a t i o n f o r i t s f a i l u r e t o d e t e c t responses to low c o n c e n t r a t i o n s of morpholine (Appendix 2). Conceivably the e l i m i n a t i o n of such r e s i d u e s , as i n Experiment 2, would r e s u l t 56 i n responses to lower c o n c e n t r a t i o n s , c o n s i s t e n t with o b s e r v a t i o n s by Wisby (1952) and Cooper and Hasler (1973). DISCUSSION Taken together, experiments i n P a r t 1 and Part 2 i n d i c a t e t h at coho exposed to morpholine d u r i n g the embryo, a l e v i n or e a r l y f r y stages, subsequently showed a g r e a t e r p r e f e r e n c e and c a r d i a c response to the odorant than naive f i s h . Both responses are c o n s i s t e n t with r e c o g n i t i o n of a f a m i l i a r odour ( f o r p r e f e r e n c e - see Chapters 2 & 3, f o r c a r d i a c responses - H i r s c h 1977; H a s l e r and Scholz 1983; Morin et a l . 1989a). P r e l i m i n a r y p r e ference t e s t s conducted i n w e l l water i n d i c a t e d p r e f e r e n c e by both naive and p r e v i o u s l y - e x p o s e d f i s h (Appendix 2). Assuming these " p r e f e r e n c e s " were the reverse of the t r u e p r e f e r e n c e s of f i s h (Part 2), both groups avoided morpholine. T h e r e f o r e , p r e v i o u s l y - e x p o s e d f i s h were not a t t r a c t e d to morpholine i n f a m i l i a r water, as they were i n u n f a m i l i a r (Rosewall Creek) water. The data i n Appendix 2 are too few to be c o n c l u s i v e , but i t may be important to perform t e s t s of morpholine r e c o g n i t i o n i n an u n f a m i l i a r water source, as suggested by Wright (1985). R e c o g n i t i o n s of some other odours however, are r e f l e c t e d i n p r e f e r e n c e s i n f a m i l i a r ( w ell) water (Chapter 3 & 4). The high c o n c e n t r a t i o n s of morpholine used i n t e s t i n g ( p a r t i c u l a r l y c a r d i a c t e s t i n g ) may have s t i m u l a t e d sensory 57 m o d a l i t i e s other than o l f a c t i o n (Cooper 1982; Dodson and Bitterman 1989), but i t i s l i k e l y that the low c o n c e n t r a t i o n s used f o r experimental treatments were p e r c e i v e d as odours ( H i r s e h 1977). T h i s w i l l be d i f f i c u l t to demonstrate i n embryos (though perhaps p o s s i b l e with e l e c t r o p h y s i o l o g i c a l techniques such as those used by Z i e l i n s k i and Hara (1988), but can and should be t e s t e d i n a l e v i n s and f r y by an o l f a c t o r y o c c l u s i o n experiment of the type r e p o r t e d i n Appendix 3. While the lac k of response to low c o n c e n t r a t i o n s of morpholine i n the Y-maze may be e x p l a i n e d by c r o s s -contamination, i t i s not c l e a r why c a r d i a c responses were e l i c i t e d only by high c o n c e n t r a t i o n s . However, t h i s appears to be a c h a r a c t e r i s t i c of the c a r d i a c assay used, and i s not p e c u l i a r to morpholine. Morin (pers. comm.) f a i l e d to d e t e c t c a r d i a c responses i n A t l a n t i c salmon to L - c y s t e i n e at c o n c e n t r a t i o n s below 10"* M, although recent measurements of t e s t s have i n d i c a t e d o l f a c t o r y p e r c e p t i o n of 10" 8 M or even lower. Other s t u d i e s have r e p o r t e d c a r d i a c responses to lower c o n c e n t r a t i o n s of morpholine. H i r s c h (1977) r e p o r t e d c o n d i t i o n e d responses to 2 X 10" 3 mg/L, and H a s l e r and Scholz (1983) r e p o r t e d unconditioned responses to 5 X 10" 5 mg/L. T e c h n i c a l d i f f e r e n c e s between these s t u d i e s and the present study may e x p l a i n the d i f f e r e n t r e s u l t s (e.g., apparatus, p r o t o c o l ) , but the d i f f e r e n c e may l i e i n the f i s h themselves. I t appears that the c a r d i a c response v a r i e s with the age or p h y s i o l o g i c a l s t a t e of f i s h . In c o n d i t i o n i n g experiments, Morin 58 et a l . (1987b) found l a r g e r c a r d i a c responses to morpholine i n 15 month o l d , than 10 month o l d A t l a n t i c salmon, and (Morin et a l . i n press) l a r g e r c a r d i a c responses to L - c y s t e i n e by smolts than p o s t - s m o l t s . S i m i l a r l y , Muzi ( c i t e d i n Ha s l e r and Scholz 1983) d e t e c t e d c a r d i a c responses to 5 X 10" 5 mg/L morpholine only i n coho t r e a t e d with gonadotropin to mimic the p h y s i o l o g i c a l s t a t e of m a t u r i t y . H a s l e r and Scholz (1983:98) concluded t h a t , "...gonadotropin or sex hormones a c t on the CNS to e x e r t c e n t r i f u g a l c o n t r o l over the o l f a c t o r y system, perhaps e i t h e r by d e s e n s i t i z i n g the r e c e p t o r s to a l l but the imprinted odor, or a l t e r i n g c e n t r a l i n t e g r a t i o n p r o c e s s e s . " A l t e r n a t i v e l y , i t may be that c a r d i a c response to a f a m i l i a r odour depends not on o l f a c t o r y s e n s i t i v i t y per se, but on the gen e r a l l e v e l of a r o u s a l or s p e c i f i c m o t i v a t i o n of the f i s h . The c a r d i a c response may r e f l e c t not simple p e r c e p t i o n or even r e c o g n i t i o n of the s t i m u l u s , but ra t h e r the r e a c t i o n of the f i s h to that s t i m u l u s , which may vary with c o n t e x t . T h i s may e x p l a i n why naive coho d i d not respond to 331 mg/L morpholine i n the present study, when i n another study (Morin et a l . 1987b), s i m i l a r l y - a g e d naive A t l a n t i c salmon d i d . In the l a t t e r study, f i s h r e c e i v e d e l e c t r i c shocks d u r i n g t e s t i n g , which may have a l t e r e d t h e i r g e n e r a l l e v e l of a r o u s a l or m o t i v a t i o n . I n t e r p r e t a t i o n of the c a r d i a c response c l e a r l y r e q u i r e s f u r t h e r r e s e a r c h . The c r i t i c a l p o i n t f o r the study of o l f a c t o r y l e a r n i n g and memory i n salmonids i s that i t does not appear to be necessary to wait u n t i l f i s h mature to d e t e c t r e c o g n i t i o n ; f o r whatever reason, r e c o g n i t i o n i s d e t e c t a b l e i n the c a r d i a c 59 (and behavioural) responses of juveni le f i s h to high concentrations of odour. The. r e s u l t s of t h i s chapter suggest that the odour of morpholine was learned in ear ly l i f e and was remembered long-term (>1 year) consistent with an i m p r i n t i n g - l i k e form of l e a r n i n g . However, learning d i d not appear to be r e s t r i c t e d to one developmental stage, suggesting that there i s not one s ingle c r i t i c a l period for o l f a c t o r y imprinting in ear ly l i f e . This does not, of course, rule out the presence of one or more s e n s i t i v e p e r i o d s . 60 CHAPTER 2. THE ROLE OF LEARNING IN RECOGNITION OF FAMILY-SPECIFIC ODOURS INTRODUCTION I t has long been known that f i s h are capable of d i s c r i m i n a t i n g between c o n s p e c i f i c s and members of other s p e c i e s , and between v a r i o u s groups of c o n s p e c i f i c s (reviewed by Barnett 1977; Solomon 1977; McCann 1980; L i l e y 1982; Colgan 1983; B l a u s t e i n et a l . 1987). D i s c r i m i n a t i o n s may r e l y on v i s u a l cues, sounds, behaviours, or even e l e c t r i c a l s i g n a l s , (Myrberg 1980; Colgan 1983) but i n many cases chemical cues alone are s u f f i c i e n t . A number of c i c h l i d s p e c i e s have been shown to d i s c r i m i n a t e the chemical cues of t h e i r own young from those of other c o n s p e c i f i c or h e t e r o s p e c i f i c young (e.g., Kuhme 1963 - c i t e d by Colgan 1983; Myrberg 1975; McKaye and Barlow 1976). S e v e r a l f i s h s p e c i e s have been s u c c e s s f u l l y c o n d i t i o n e d to d i s c r i m i n a t e between chemical emanations of d i f f e r e n t i n d i v i d u a l c o n s p e c i f i c s (Goz 1941 - c i t e d by Colgan 1983; Todd et a l . 1967; Todd 1971; Richards 1974; Richards 1976, c i t e d by L i l e y 1982). Smell appears to be the dominant modality i n v o l v e d i n these d i s c r i m i n a t i o n s (e.g., McKaye and Barlow 1976). Many s t u d i e s with f i s h have examined the r o l e of l e a r n i n g i n r e c o g n i t i o n of v a r i o u s groups of c o n s p e c i f i c s (e.g., Noble and C u r t i s 1939; Baerends and Baerends-van Roon 1950; Myrberg 1964; Ferno and S j o l a n d e r 1973,1976; S j o l a n d e r and Ferno 1973; Kop and Heuts 1973; Weber and Weber 1976; Barlow and Rogers 61 1978; Hay 1978; Siepen and Crapon de Caprona 1986; Russock 1986). While many of these s t u d i e s suggested that l e a r n i n g processes s i m i l a r to i m p r i n t i n g were probably i n v o l v e d , they e i t h e r d i d not i n v e s t i g a t e which a t t r i b u t e s were l e a r n e d , or examined l e a r n i n g of v i s u a l cues o n l y . Two experimental s t u d i e s s p e c i f i c a l l y examined l e a r n i n g of c o n s p e c i f i c odours by young f i s h . The authors of both concluded that t h e i r r e s u l t s were c o n s i s t e n t with an i m p r i n t i n g mechanism of l e a r n i n g (sensu Immelman and Suomi 1981). Barnett (1986) found that on the f i r s t day of free-swimming, Midas c i c h l i d (Cichlasoma c i t r i n e l l u m ) f r y p r e f e r r e d the chemical emanations of the mother over those of the f a t h e r , unless reared with only the f a t h e r i n which case they p r e f e r r e d h i s chemical emanations. On the l a s t day of t e s t i n g (11 d a f t e r swim-up), an e f f e c t of r e a r i n g was s t i l l d e t e c t e d . T h i s study suggested that embryonic or l a r v a l f i s h l e a r n e d c o n s p e c i f i c odours, but i t d i d not i d e n t i f y a s e n s i t i v e p e r i o d f o r l e a r n i n g , or determine whether the l e a r n e d i n f o r m a t i o n was remembered beyond 11 days. Crapon de Caprona (1982) reared i n d i v i d u a l Haplochromis  b u r t o n i from the embryo stage i n groups of e i t h e r c o n s p e c i f i c s or s i m i l a r l y - a g e d c o n v i c t c i c h l i d s (Cichlasoma n i g r o f a s c i a t u m ) and when 4 months o l d , o f f e r e d i n d i v i d u a l s a ch o i c e of waters c o n d i t i o n e d by g r a v i d females of the two s p e c i e s . F i s h reared with c o n s p e c i f i c s p r e f e r r e d c o n s p e c i f i c s , whereas f i s h reared with c o n v i c t s e i t h e r p r e f e r r e d c o n v i c t s (males) or showed no pr e f e r e n c e ( f e m a l e s ) . S i m i l a r e f f e c t s of r e a r i n g experience 62 were seen on the p r e f e r e n c e s of males between male chemical emanations of c o n s p e c i f i c s and c o n v i c t s of the white c o l o u r morph. T h i s study d i d not i d e n t i f y a s e n s i t i v e p e r i o d f o r the l e a r n i n g of s p e c i e s - s p e c i f i c chemical cues, but d i d examine the d u r a t i o n and i r r e v e r s i b i l i t y of the e f f e c t s of l e a r n i n g . While p r e f e r e n c e s were mo d i f i e d by subsequent experience, the e f f e c t of r e a r i n g experience d u r i n g the f i r s t 4 months of l i f e was s t i l l apparent i n the response of males to female chemical emanations, 6 months l a t e r . The present study was i n s p i r e d by two s t u d i e s of the chemosensory a b i l i t y of coho f r y , to d i s t i n g u i s h s i b l i n g s from n o n - s i b l i n g s . Quinn and Busack (1985) r e p o r t e d that coho f r y p r e f e r r e d water bearing the chemical emanations of u n f a m i l i a r non-sibs over blank water, but p r e f e r r e d the emanations of s i b s , whether f a m i l i a r or not, over non-sibs. (The authors noted that the sensory modality i n v o l v e d c o u l d be e i t h e r o l f a c t i o n or t a s t e . An o l f a c t o r y o c c l u s i o n experiment r e p o r t e d i n Appendix 3 i n d i c a t e s t hat the response i s mediated by the nose.) Quinn and Busack (1985) suggested that s i b s were recognized by comparing the chemical cues of u n f a m i l i a r c o n s p e c i f i c s with a template, l e a r n e d d u r i n g i n c u b a t i o n or e a r l y r e a r i n g (Holmes and Sherman 1983). Support f o r t h i s h y p o thesis came from a subsequent experiment i n which f i s h were reared with non-sibs, maternal h a l f - s i b s , or f u l l s i b s (Quinn and Hara 1986). As i n the former study, f r y p r e f e r r e d the odours of tankmate s i b s over u n f a m i l i a r non-sibs, but not over those of tankmate non-sibs or maternal h a l f - s i b s . 63 Quinn and Busack (1985) and Quinn and Hara (1986) sp e c u l a t e d that s i b r e c o g n i t i o n i n coho might p l a y a r o l e i n s c h o o l i n g , t e r r i t o r i a l a g g r e s s i o n , mate s e l e c t i o n or homing, a l l of which would r e q u i r e long-term memory of the odours l e a r n e d d u r i n g or s h o r t l y a f t e r i n c u b a t i o n . In the above experiments, f i s h were never out of contact with tankmates, so i t was not c l e a r whether or not l e a r n e d odours were remembered long-term. In a d d i t i o n , i t was not c l e a r when i n e a r l y l i f e odours were being l e a r n e d . The present study was designed to address these two q u e s t i o n s . The present study i s i n two p a r t s . In the f i r s t p a r t , coho were exposed s e r i a l l y to the chemical emanations of three d i f f e r e n t non-sib groups of s i m i l a r age: one d u r i n g the embryo stage, a second d u r i n g the a l e v i n ( l a r v a l ) stage, and a t h i r d d u r i n g the e a r l y f r y (free-swimming) stage. Beginning 1 month a f t e r the l a s t exposure, f r y were t e s t e d f o r r e c o g n i t i o n of the odours. The c r i t e r i o n of r e c o g n i t i o n was p r e f e r e n c e over an u n f a m i l i a r odour ( t h a t of another f a m i l y ) to a degree g r e a t e r than that shown by naive s i b s . During the course of these assays, i t became apparent that p r e f e r e n c e among f a m i l y - s p e c i f i c odours was i n f l u e n c e d not only by r e c o g n i t i o n , but by some other f a c t o r ( s ) as w e l l . The nature of t h i s other f a c t o r ( s ) was e x p l o r e d i n Part 2. 64 PART ]_'. LEARNING DURING THE EMBRYO, ALEVIN, AND EARLY FRY STAGES  Treatment On November 19 1984, gametes of s i x female (here denoted A,B,C,D,F and G) and s i x male (1,2,3,4,5,6) coho salmon from the Quinsam R i v e r Hatchery were t r a n s p o r t e d to Rosewall Creek Hatchery where r e a r i n g and subsequent t e s t i n g took p l a c e i n w e l l water (8-9°C). A d i a l l e l e c r o s s was made with females C and D and males 3 and 4, producing f a m i l i e s C3, C4, D3 and D4. The eggs of each f a m i l y was d i v i d e d by volume i n t o two approximately e q u a l - s i z e d groups, one to be used as experimental f i s h ("a") and the other to be used as odour-producers and c o n t r o l f i s h ("b"). With the gametes from the other four females and males, 8 a d d i t i o n a l groups were c r e a t e d , A l ( a , b ) , B2(a,b), F5(a,b), and G6(a,b). These groups were a l l used as odour-producers. F i s h were incubated i n standard hatchery Heath t r a y s (Heath Tecna Corp., Kent, WA) u n t i l swim-up when they were t r a n s f e r r e d to h o l d i n g tanks (see below) and f e e d i n g began. Hatching o c c u r r e d January 12-18 (1985) and swim-up February 16-18. Fry were hand-fed Oregon Moist P e l l e t s (Moore-Clark, Inc., LaConner, WA) f i v e times a day f o r the f i r s t 2 weeks and twice a day t h e r e a f t e r . Overhead f l u o r e s c e n t l i g h t i n g was programmed f o r an 8:16 LD ph o t o p e r i o d . F i s h appeared h e a l t h y throughout the experiment and m o r t a l i t y was n e g l i g i b l e . Experimental groups (C3a, C4a, D3a, and D4a) were s e q u e n t i a l l y exposed to the chemical emanations of three non-sib 65 groups: one d u r i n g the embryo stage, a second d u r i n g the a l e v i n stage and a t h i r d d u r i n g the f i r s t 2 weeks of the f r y stage (Table 3). In each case, the odour group was of the same age as the experimental group, and of s i m i l a r number; at e g g - p i c k i n g (January 11, j u s t before h a t c h ) , l i v e embryos i n C and D groups numbered 316-468 and l a r g e r groups were c u l l e d to 350. F and G groups numbered 514 to 551 and were c u l l e d to 350. During the embryo and a l e v i n stages, the Heath t r a y c o n t a i n i n g the odour group was p l a c e d over the t r a y c o n t a i n i n g the experimental group. With t h i s i n c u b a t i o n system, water flows i n t o the top t r a y (approximately 7 - 8 L/min), bathes the embryos or a l e v i n s there and s p i l l s down to the t r a y below. During the f i r s t 2 weeks of the f r y stage, each experimental group was h e l d i n a c i r c u l a r f i b e r g l a s s tank (92 cm diam., 50 cm depth, flow: 12 L/min) i n one h a l f of a 60 X 30 X 30 cm basket of mesh n e t t i n g suspended around an aluminum frame (see Quinn and Hara 1986). In the other h a l f of the basket, separated from the experimental f i s h by mesh n e t t i n g , was the t h i r d odour group. A f t e r 2 weeks, each group of f i s h was moved to a c i r c u l a r f i b e r g l a s s tank (35 cm diameter, 40 cm depth, flow: 4 L/min) where i t remained u n t i l the end of the experiment. While "b" groups of C and D f a m i l i e s were exposed to each other d u r i n g the a l e v i n and e a r l y f r y stages, they were never exposed to the odours of F or G f a m i l i e s , making them s u i t a b l e c o n t r o l s f o r the a l e v i n and e a r l y f r y stage exposures of t h e i r r e s p e c t i v e "a" groups. N e i t h e r "a" nor "b" groups of C and D 66 Table 3. Schedule of exposure to non-sib odours. (Day/Month of winter 1984 - spring 1985) TEST GROUP CONDITIONING WATER DURING GROUP ENS ANS FrNS EMBRYO STAGE ALEVIN STAGE EARLY FRY STAGE (19/11-11/01) (11/01-18/02) (18/02-5/03) (53 days) (38 days) (15 days) D3a C4b G6a F5a C4a D3b G6b F5b C3a D4b F5a G6a D4a C3b F5b G6b 67 were ever exposed to the odours of f a m i l i e s A or B, l e a v i n g these odours " u n f a m i l i a r " to both experimentals and c o n t r o l s . Test ing Methods and M a t e r i a l s Fry of experimental groups (C3a, C4a, D3a, D4a) were t e s t e d f o r p r e f e r e n c e between the odour of a " f a m i l i a r " group ( e i t h e r s i b s (FS), or non-sibs that had been present d u r i n g the embryo (ENS), a l e v i n (ANS) or e a r l y f r y stage (FrNS)), and an " u n f a m i l i a r " group of non-sibs (UNS). ENS, ANS and FrNS groups f o r each experimental group are l i s t e d i n Table 3. The UNS groups used throughout were A1a and b i n t e s t s with D3a and C4a r e s p e c t i v e l y , and B2a and b i n t e s t s with C3a and D4a r e s p e c t i v e l y . Where c o n t r o l s (C3b, C4b, D3b, D4b) were t e s t e d , the same odour-groups were used as were used i n t e s t s with c o r r e s p o n d i n g experimentals. The dates of t e s t s and time i n t e r v a l s between exposure and t e s t i n g are given i n Table 4. Unless s p e c i f i e d otherwise, each t e s t was performed only once, i . e . , on one day. Test procedure f o l l o w e d that d e s c r i b e d i n General Methods and M a t e r i a l s . Waters were c o n d i t i o n e d by 100 g of f r y (14-107 f i s h , depending on f a m i l y and date) i n the headtanks. F i s h were p l a c e d i n headtanks at approximately 1800 hours on the day before t e s t i n g , and were not fed while i n the headtanks. To guard a g a i n s t accumulation of the odour of a p a r t i c u l a r group of 68 Table 4. Schedule of t e s t s of pr e f e r e n c e between f a m i l y -s p e c i f i c odours. F S = f a m i l i a r s i b l i n g s , UNS=unfamiliar n o n - s i b l i n g s , ENS=non-siblings present d u r i n g the embryo stage, ANS=non-siblings present d u r i n g the a l e v i n stage, FrNS=non-siblings present d u r i n g the e a r l y f r y stage, a: experimental group, b: c o n t r o l group ASSAY ODOURS GROUPS DATES EXPOSURE-TEST TESTED (day/month) INTERVAL (DAYS) FS/UNS ENS/UNS ANS/UNS a b 29/03-17/04 2/04-6/05 4/04-26/04 2/05-9/05 NA 81-115 45-67 NA FrNS/UNS a b 6/04-19/04 3/05-13/05 32-45 NA ANS/UNS a b 24/07-7/08 157-170 NA 69 f i s h , i n d i v i d u a l s p l a c e d i n the headtank were never of the same group as had occupied the tank immediately b e f o r e . At the end of each day of t e s t i n g the f i s h were removed and r e p l a c e d i n h o l d i n g tanks, and r e s i d u a l faeces were siphoned out of the headtanks. An i n d i v i d u a l f r y might be reused as an odour producer a f t e r at l e a s t 1 d i n h o l d i n g , or might then be used (once) as a t e s t f i s h . A t e s t u s u a l l y c o n s i s t e d of f o u r t e e n t r i a l s per day, beginning at 1000 and ending at 1700 h. A n a l y s i s of data f o l l o w e d that d e s c r i b e d i n General Methods and M a t e r i a l s w i t h the a d d i t i o n t h at f o r Assays 3-5, p r e f e r e n c e s of experimentals and c o n t r o l s of the four t e s t f a m i l i e s were compared with a mixed e f f e c t s model f o r two way ANOVA (Zar 1984:212). In c o n t r o l t e s t s with e i t h e r no f i s h i n the headtanks or the same odour-group i n both headtanks, t e s t - f i s h f a i l e d to show s i g n i f i c a n t p r e f e r e n c e (mean percent time swimming i n water from l e f t headtank: 55 ( A p r i l 20) and 54 ( J u l y 9) without f i s h i n headtanks; 50 ( A p r i l 22) and 50 ( J u l y 10) with f i s h i n both headtanks; N=47-54 f i s h per t e s t , P>0.10 f o r each t e s t ) , i n d i c a t i n g t h a t there was no systematic headtank b i a s . 70 R e s u l t s Assay 1_: F a m i l i a r Sibs (FS) vs U n f a m i l i a r Non-Sibs (UNS) Groups D3a, C4a and D4a p r e f e r r e d the odours of FS over those of UNS (P=0.0002, P<0.0001 and P<0.0001, r e s p e c t i v e l y ) (Figure 12). Group C3a showed no p r e f e r e n c e and t h i s response d i f f e r e d s i g n i f i c a n t l y from that of groups C4a and D4a (P<0.05 Tukey t e s t ) . These data p a r a l l e l those of Quinn and Busack (1985) and Quinn and Hara (1986); i n most but not a l l d i a d i c c h o i c e s , coho f r y p r e f e r the odours of FS over UNS. Quinn and Busack (1985) found that some f a m i l i e s p r e f e r r e d FS over one UNS group but not another, suggesting the i n f l u e n c e on p r e f e r e n c e of a f a c t o r ( s ) besides f a m i l i a r i t y . T h i s was i n v e s t i g a t e d i n Part 2 of t h i s study. An important p o i n t f o r the assays that f o l l o w i s that because p r e f e r e n c e i s not s o l e l y determined by f a m i l i a r i t y , l a c k of p r e f e r e n c e cannot be i n t e r p r e t e d as lack of r e c o g n i t i o n and p r e f e r e n c e alone cannot be i n t e r p r e t e d as r e c o g n i t i o n . R e c o g n i t i o n can only be i n f e r r e d i f c o n t r o l s (naive s i b s ) do not show the same p r e f e r e n c e . Assay 2: Non-Sibs present at the Embryo stage (ENS) vs  U n f a m i l i a r Non-Sibs (UNS) Groups D3a, C4a and D4a showed no p r e f e r e n c e between the odours of ENS and UNS ( F i g u r e 13). Groups C3a d i d show a 71 90 80 44 46 56 48 70 -60 50 40 -30 D3a C4a C3a D4a F a m i l y F i g u r e 12. Preference between odours of F a m i l i a r S i b s (FS) and U n f a m i l i a r Non-Sibs (UNS). (x ± 95% CL, Number of f r y ^ t e s t e d shown above bar) 72 52 49 CO z W »H cd o H C • < 6 6 CO e c o I-I 70 107 48 60 50 40 30 D3a C4a C3a D4a Family F i g u r e 13. P r e f e r e n c e between odours of two d i f f e r e n t Non-Sib groups, one of which (ENS) t e s t f i s h had been exposed to d u r i n g the egg stage. (x ± 95% CL, Number of f r y t e s t e d shown above bar) 73 p r e f e r e n c e , but p r e f e r r e d UNS over ENS (P=0.015). Because the i n i t i a l t e s t with C3a i n d i c a t e d t h i s unexpected response, a second t e s t was performed, the r e s u l t s of which d i d not d i f f e r from the i n i t i a l r e s u l t s so the pooled r e s u l t i s shown i n F i g u r e 13. The response of C3a d i f f e r e d from that of group D3a (P<0.05 Tukey T e s t ) . Because "b" groups of f a m i l i e s C3, D4, D3, and C4 had been exposed to each o t h e r s ' chemical emanations d u r i n g the a l e v i n and f r y stages, they were not a p p r o p r i a t e as c o n t r o l s a g a i n s t which to compare the responses of t h e i r r e s p e c t i v e "a" groups, and t h e r e f o r e were not t e s t e d . The l a c k of c o n t r o l s f o r t h i s experiment would have been un f o r t u n a t e i f experimentals had p r e f e r r e d ENS over UNS, as without c o n t r o l s i t would be impossible to a t t r i b u t e p r e f e r e n c e s to r e a r i n g h i s t o r y . However, the absence of such p r e f e r e n c e by any of the experimental groups s t r o n g l y suggests that the odours of non-s i b s that had been present d u r i n g the embryo stage were not subsequently regarded as f a m i l i a r . Assay 3: Non-Sibs present at the A l e v i n stage (ANS) vs  U n f a m i l i a r Non-Sibs (UNS) Experimentals of f a m i l i e s C3 and D4 p r e f e r r e d the odours of ANS over UNS (P<0.0001 f o r each) whereas c o n t r o l s d i d not, and f o r each f a m i l y the d i f f e r e n c e i n p r e f e r e n c e was s i g n i f i c a n t (P<0.0001 and P=0.0012 r e s p e c t i v e l y ) ( F i g u r e 14). Experimentals of the other two f a m i l i e s d i d not show g r e a t e r p r e f e r e n c e than 74 51 54 50 49 56 53 50 53 co < ce T3 CvJ ES 60 C CO CD s a o CD PH 90 80 70 60 50 -40 30 Prefer A N S Ll Prefer U N S D3 C4 C3 D4 F a m i l y F i g u r e 14. Preference between odours of two d i f f e r e n t Non-Sib groups, one of which (ANS) Experimentals (Shaded Bars) had been exposed to d u r i n g the a l e v i n stage, whereas C o n t r o l s (Open Bars) had not. (x ± 95% CL, Number of f r y t e s t e d shown above bar) 75 c o n t r o l s ; i n f a m i l y D3 n e i t h e r group showed s i g n i f i c a n t p r e f e r e n c e , and i n f a m i l y C4 both groups p r e f e r r e d ANS over UNS (experimentals: P=0.019, c o n t r o l s : P=0.022) (Fi g u r e 14). There was no s i g n i f i c a n t d i f f e r e n c e between experimentals and c o n t r o l s over a l l f a m i l i e s , but there was a s i g n i f i c a n t i n t e r a c t i o n between the treatment e f f e c t and f a m i l i e s (P=0.021), r e f l e c t i n g the d i f f e r e n c e among f a m i l i e s i n the r e l a t i o n s h i p between experimentals and c o n t r o l s . These data suggest that f i s h of f a m i l i e s C3 and D4 l e a r n e d the odour of f a m i l y F5 d u r i n g the a l e v i n stage and remembered i t when t e s t e d r e s p e c t i v e l y 67 and 64 d l a t e r . There was no i n d i c a t i o n though that f a m i l i e s D3 and C4 l e a r n e d the odour of f a m i l y G6. Assay 4: Non-Sibs present at the Fry stage (FrNS) vs U n f a m i l i a r  Non-Sibs (UNS) Experimentals of f a m i l i e s D3 and C3 p r e f e r r e d the odours of FrNS over UNS (P=0.0007 and P<0.0001 r e s p e c t i v e l y ) whereas c o n t r o l s d i d not, and f o r each f a m i l y the d i f f e r e n c e i n p r e f e r e n c e was s i g n i f i c a n t (P=0.0007 and P<0.0001 r e s p e c t i v e l y ( s i c ) ) ( F i g u r e 15). In c o n t r a s t , experimentals and c o n t r o l s of f a m i l y C4 both p r e f e r r e d FrNS over UNS (P=0.031 and P<0.0001 r e s p e c t i v e l y ) , the p r e f e r e n c e of experimentals a c t u a l l y being weaker than that of c o n t r o l s (P=0.0001). Experimentals and c o n t r o l s of f a m i l y D4 both p r e f e r r e d UNS over FrNS (P=0.0038 and P=0.017 r e s p e c t i v e l y ) ( F i g u r e 15). ANOVA f a i l e d to d e t e c t a 76 90 55 54 53 52 54 56 47 50 80 70 60 50 h 40 30 Prefer FrNS TT Prefer U N S 20 D3 C4 C3 D4 Fam i l y F i g u r e 15. Pr e f e r e n c e between odours of two d i f f e r e n t Non-Sib groups, one of which (FrNS) Experimentals (Shaded Bars) had been exposed to d u r i n g the f r y stage, whereas C o n t r o l s (Open Bars) had not. (x ± 95% CL, Number of f r y t e s t e d shown above bar) 77 s i g n i f i c a n t d i f f e r e n c e between experimentals and c o n t r o l s over the four f a m i l i e s , but d i d d e t e c t d i f f e r e n c e among f a m i l i e s i n treatment e f f e c t ( i n t e r a c t i o n term: P<0.001). These data suggest that f i s h of f a m i l i e s D3 and C3 l e a r n e d the odours of F5 and G6 r e s p e c t i v e l y d u r i n g the e a r l y f r y stage, and remembered them when t e s t e d r e s p e c t i v e l y 32 and 44 d l a t e r . There was no i n d i c a t i o n that f a m i l y D4 l e a r n e d the odour of G6 or that C4 l e a r n e d the odour of F5. The weaker p r e f e r e n c e shown by experimental than c o n t r o l members of C4 may r e f l e c t v a r i a b i l i t y i n the odour emitted by group A1b r a t h e r than an e f f e c t of r e a r i n g experience (see Assay 5 and Part 2). Assay 5: Non-Sibs present at the A l e v i n Stage (ANS) vs  U n f a m i l i a r Non-Sibs (UNS), Repeat T e s t s The s t a b i l i t y of p r e f e r e n c e s over time was examined by r e p e a t i n g Assay 3 - ANS vs UNS t e s t s - between 96 and 118 d a f t e r the o r i g i n a l t e s t s (157-170 d a f t e r exposure to ANS ended). Experimental and c o n t r o l groups were t e s t e d twice each, once with ANS i n the r i g h t headtank and once with ANS i n the l e f t headtank, and data from the two days of t e s t i n g were pooled. (In o n l y one case d i d r e s u l t s of the r e p l i c a t e t e s t s d i f f e r (P<0.0001); C4b p r e f e r r e d G6b over A1b i n one t e s t (P<0.0001) but not the other.) Responses were s i m i l a r to those recorded i n Assay 3. Experimentals of f a m i l i e s C3 and D4 p r e f e r r e d the odours of ANS 78 over UNS (P=0.0029 and P=0.0001 r e s p e c t i v e l y ) whereas c o n t r o l s d i d not, but the p r e f e r e n c e s were weaker than those recorded i n Assay 3 ( F i g u r e 16). S i g n i f i c a n t d i f f e r e n c e was s t i l l d e t e c t e d between the experimentals and c o n t r o l s of f a m i l y D4 (P=0.0013), but not f a m i l y C3. Ne i t h e r treatment group of f a m i l y D3 showed s i g n i f i c a n t p r e f e r e n c e , and both groups of f a m i l y C4 p r e f e r r e d ANS over UNS (P<0.0001 and P=0.0009 r e s p e c t i v e l y ) , but i n c o n t r a s t to Assay 3, experimentals showed a stronger p r e f e r e n c e than c o n t r o l s (P=0.0005). ANOVA again f a i l e d to de t e c t s i g n i f i c a n t d i f f e r e n c e between experimentals and c o n t r o l s over the four f a m i l i e s , and i n d i c a t e d a weaker d i f f e r e n c e than i n Assay 3 among f a m i l i e s i n treatment e f f e c t ( i n t e r a c t i o n term: P=0.074). These data suggest that f a m i l y D4 le a r n e d the odour of fa m i l y F5 d u r i n g the a l e v i n stage and remembered i t when t e s t e d 160 d l a t e r , and f a m i l y C3 showed a t r e n d in, t h i s d i r e c t i o n when t e s t e d 169 d l a t e r . There was no i n d i c a t i o n that f a m i l y D3 le a r n e d the odour of f a m i l y G6. The g r e a t e r p r e f e r e n c e by C4 experimentals than c o n t r o l s was c o n s i s t e n t with l e a r n i n g , but the d i f f e r e n c e between r e p l i c a t e t e s t s of c o n t r o l s l e f t t h i s c o n c l u s i o n open to doubt. Comparison of Assays 3 and 5 The r e s u l t s of Assays 3 and 5 were compared u s i n g a mixed e f f e c t s model f o r three-way ANOVA. Across the four t e s t f a m i l i e s , n e i t h e r Treatment e f f e c t (experimentals vs c o n t r o l s ) 79 co < u 60 a • rH 6 S • rH co CD s •rH H a o u a) 90 80 70 60 50 40 30 103 103 101 95 57 57 71 105 D3 C4 C3 D4 F a m i l y Figure 16. Preference between odours of two d i f f e r e n t Non-Sib groups, one of which (ANS) Experimentals (Shaded Bars) had been exposed to during the alevin stage, whereas Controls (Open Bars) had not. (Tests performed 96-118 d after those of Figure 14) (x ± 95% CL, Number of fry tested shown above bar) 80 nor Time (Assay 3 vs 5) was s i g n i f i c a n t , but both i n t e r a c t e d s i g n i f i c a n t l y with the Family e f f e c t (P=0.009 and P=0.016 r e s p e c t i v e l y ) . T h e r e f o r e , Treatment and Time e f f e c t s were examined f o r each f a m i l y s e p a r a t e l y with a f i x e d e f f e c t s model f o r two-way ANOVA. Experimentals d i f f e r e d from c o n t r o l s i n f a m i l i e s C3 and D4 (P<0.001 f o r each), but f o r each f a m i l y the response of experimentals was weaker i n the second t e s t , i n d i c a t e d by a s i g n i f i c a n t Time X Treatment i n t e r a c t i o n f o r C3 (P<0.001) and a s i g n i f i c a n t Time e f f e c t f o r D4 (P=0.006) (cf F i g u r e s 14 and 16). The decrease i n response of experimentals c o u l d r e f l e c t f o r g e t t i n g by some f r y , weaker m o t i v a t i o n to swim towards a f a m i l i a r odour, or some u n i d e n t i f i e d change i n the assay procedure. In c o n t r a s t , the responses of experimentals and c o n t r o l s of f a m i l y C4 d i f f e r e d (P=0.003) but d i d not change with time. The l a c k of decrease i n response over time suggests that the p r e f e r e n c e of C4 f i s h f o r G6b over A1b odours was i n f l u e n c e d by a f a c t o r ( s ) d i f f e r e n t from those governing the pr e f e r e n c e of C3 and D4 experimentals f o r F5 over B2 odours. F a m i l i a r i t y may have been one f a c t o r i n f l u e n c i n g C4 experimentals, but some other f a c t o r a l s o was a c t i n g . T h i s was e v i d e n t i n the p r e f e r e n c e shown by C4 c o n t r o l s . 81 PART 2: OTHER FACTORS AFFECTING PREFERENCE From the assays i n Part 1, i t was c l e a r that some f a c t o r ( s ) b esides r e c o g n i t i o n s t r o n g l y i n f l u e n c e d f r y p r e f e r e n c e s among c o n s p e c i f i c chemical emanations. Notably, group C4b p r e f e r r e d the odours of both G6b and F5b over A1b (F i g u r e s 14-16), though none of these odours was f a m i l i a r . I t appeared that the odours of group A1b were i n some way l e s s a t t r a c t i v e than those of other groups. C o n s i s t e n t with t h i s s uggestion, p r e l i m i n a r y t e s t s i n d i c a t e d that u n l i k e most f a m i l i e s (Assay 1), group A1b d i d not p r e f e r water c o n d i t i o n e d by f a m i l a r s i b s (FS) over u n f a m i l i a r non-sibs (UNS). In a t e s t conducted on A p r i l 25, A1b f i s h showed no p r e f e r e n c e between the odours of FS and members of group G6b, and i n a t e s t conducted on May 17, a c t u a l l y p r e f e r r e d the odour of group C3b over FS (P=0.042) (F i g u r e 17). The f o l l o w i n g assays were performed to examine the nature of Alb ' s " u n a t t r a c t i v e n e s s " . S i g n i f i c a n t headtank b i a s was not det e c t e d i n t e s t s without f i s h i n headtanks (mean percent time swimming i n water from l e f t headtank: 45 (November 9), 53 (November 12), 53 (December 12), N=37-45 f i s h per t e s t , N>0.2 for each). Assay A: Repul s i o n vs R e l a t i v e A t t r a c t i v e n e s s The purpose of t h i s assay was to determine whether the chemical emanations of A1b were r e p u l s i v e , or j u s t l e s s a t t r a c t i v e than those of other groups. Group A1b was given a 82 53 26 70 60 50 -40 -30 20 G6b C3b UNS Group Figure 17. Preference of family A1b for odour of two Unfamil iar N o n - S i b l i n g (UNS) groups over Famil iar S i b l i n g s (FS) . (x ± 95% CL, Number of f ry tested shown above bar) 83 c h o i c e of water c o n d i t i o n e d by FS and blank water on March 21 (FS i n r i g h t headtank) and March 27 (FS i n l e f t headtank). The odours of A1b f i s h were not r e p u l s i v e ; i n both t e s t s A1b f i s h were s t r o n g l y a t t r a c t e d to FS odours over blank water (P<0.0001 and P=0.011, r e s p e c t i v e l y ) (Figure 18). Assay B: Re l a t i v e A t t r a c t i v e n e s s to Other C o n s p e c i f i c s The purpose of t h i s assay was to examine the g e n e r a l i t y among coho f a m i l i e s of p r e f e r e n c e f o r another odour group over A1b. Groups D3b, C4b, C3b and D4b were given a c h o i c e of waters c o n d i t i o n e d by 100 g of groups G6b and A1b on December 12, 14, 16 and 18. A1b occupied the r i g h t headtank on the f i r s t and t h i r d days and the l e f t headtank on the second and f o u r t h days. Odour groups comprised the same 9 A1b i n d i v i d u a l s and 7 G6b i n d i v i d u a l s on a l l 4 days. To block f o r v a r i a t i o n between t e s t days, each t e s t group was t e s t e d i n three or four c o n s e c u t i v e t r i a l s on each t e s t day and the order i n which groups were t e s t e d was s y s t e m a t i c a l l y v a r i e d . F i s h of f a m i l i e s C4 and D4 p r e f e r r e d the odours of G6b over A1b (P=0.0056 and P=0.0001 r e s p e c t i v e l y ) , while f i s h of f a m i l y D3 and C3 d i d not (Figure 19). The responses of the four groups d i d not d i f f e r s i g n i f i c a n t l y (0.05<P<0.10) (pooled mean: 55.6%, 95% CL: 52.8-58.3%, N=184, P=0.0001), but these few data suggested that some f a m i l i e s of coho may be more s e n s i t i v e or more responsive than others to the d i f f e r e n c e between the odours 84 20 co fa in u cd ES 60 c CO u 6 a CU a <D PH 100 90 -80 70 -60 50 40 30 Test F i g u r e 18. P r e f e r e n c e of f a m i l y A1b f o r odour of F a m i l i a r S i b l i n g s (FS) over blank water (BW). (ic ± 95% CL, Number of f r y t e s t e d shown above bar) 85 o CA T3 M a ES G •rH e a E CU o t-l tu PH 70 47 47 50 40 60 50 40 30 D3b C4b C3b D4b Test G r o u p F i g u r e 19. Pref e r e n c e of four coho f a m i l i e s between waters c o n d i t i o n e d by N o n - S i b l i n g groups G6b and A1b. (x ± 95% CL, Number of f r y t e s t e d shown above bar) 86 of A1b and G6b f i s h . Assay C: Homogeneity of R e l a t i v e A t t r a c t i v e n e s s The purpose of t h i s assay was to determine whether the r e l a t i v e u n a t t r a c t i v e n e s s of the chemical emanations of group A1b was a t r a i t common to a l l A1b f i s h , or p e c u l i a r to a few. In t e s t s performed between November 10 and 15, f i s h of group F5 were given a c h o i c e of waters c o n d i t i o n e d by one of four d i f f e r e n t groups of A1b f i s h (100 g, N=7-11), and G6b f i s h (100 g, N=7-8). The r a t i o n a l e was that i f only some A1b smelled u n a t t r a c t i v e , F5 f i s h would p r e f e r G6b over A1b i n some, but not a l l four t e s t s . There was no d i f f e r e n c e among the responses of F5 f i s h i n the four t e s t s . In each, the odours of G6b f i s h were p r e f e r r e d over those of A1b f i s h (P=0.0018, 0.02, 0.05 and <0.0001 r e s p e c t i v e l y ) (Figure 20). T h e r e f o r e i t d i d not appear that r e l a t i v e u n a t t r a c t i v e n e s s was a t t r i b u t a b l e to one or two i n d i v i d u a l s but r a t h e r , was c h a r a c t e r i s t i c of a l l A1b f i s h . Assay D; Q u a n t i t y of Odour The purpose of t h i s assay was to t e s t the h y p o t h e s i s that the r e l a t i v e u n a t t r a c t i v e n e s s of A1b f i s h was r e l a t e d to a d e f i c i t i n q u a n t i t y of odour imparted to the water. Between November 17 and December 1., F5 f i s h were t e s t e d i n four t e s t s . F i r s t , f i s h were given a c h o i c e of blank water and water 87 o CA -a u cd a •H 6 6 CO e a O S-H PH 8 0 49 51 45 48 7 0 h 6 0 5 0 4 0 3 0 Prefer G6b Prefer A l b T e s t F i g u r e 20. Pref e r e n c e of f a m i l y F5 f o r water c o n d i t i o n e d by G6b f i s h over four d i f f e r e n t groups of A l b f i s h . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 88 c o n d i t i o n e d by 100 g of A1b f i s h (N=9). As expected, the A1b odours were p r e f e r r e d (mean=58%, N=46, P=0.02) i n d i c a t i n g t h at they were both d e t e c t a b l e and a t t r a c t i v e . Next, f i s h were given a c h o i c e of waters c o n d i t i o n e d by 100 g of A1b f i s h (N=11) and 57 g of G6b f i s h (N=4). U n l i k e the p r e f e r e n c e s recorded when 100 g of G6b f i s h had been used (Assay C), no p r e f e r e n c e was observed ( F i g u r e 21). Next, the G6b odour group was f u r t h e r reduced to o n l y 30 g (N=2). T h i s s i g n i f i c a n t l y (P=0.0001) changed the p r e f e r e n c e of the F5 f i s h , which now p r e f e r r e d A1b over G6b odours (P=0.0001) (F i g u r e 21). F i n a l l y , f i s h were given a c h o i c e of blank water and water c o n d i t i o n e d by the 30 g of G6b f i s h , to see whether these two f i s h produced a d e t e c t a b l e odour. F5 f i s h p r e f e r r e d the c o n d i t i o n e d over blank water (mean=58%, N=35, P=0.025). The r e s u l t s of these four t e s t s i n d i c a t e that q u a n t i t y of c o n s p e c i f i c odour g r e a t l y i n f l u e n c e s p r e f e r e n c e ; a f a i r l y small r e l a t i v e s u r p l u s can render one f a m i l y ' s chemical emanations more a t t r a c t i v e to c o n s p e c i f i c s than another's. The r e l a t i v e u n a t t r a c t i v e n e s s of group A1b may w e l l have been due to a q u a n t i t a t i v e d e f i c i t r e l a t i v e to other f a m i l i e s , and not to a q u a l i t a t i v e l y l e s s a t t r a c t i v e odour. 89 49 41 r O o rH c« 00 C e 6 •rH co a H CU o VH PH 70 Prefer G6b 60 50 40 30 20 Prefer A l b 57g 30g Weight of G6b Odour Group Figure 21. Preference of F5 f ry between odours of G6b and A1b f r y : the e f f e c t of r e l a t i v e concentrations of the two odours. Odours were produced by 100 g of A1b f r y , and 57 g ( l e f t bar) or 30 g ( r ight bar) of G6b f r y . (x ± 95% CL, Number of f ry tested shown above bar) 90 DISCUSSION The r e s u l t s of t h i s study support the c o n c l u s i o n of Quinn and Busack (1985) and Quinn and Hara (1986) that coho f r y d i s c r i m i n a t e and p r e f e r s i b l i n g over n o n - s i b l i n g odours, and that l e a r n i n g p l a y s a r o l e i n t h i s r e c o g n i t i o n . Furthermore, r e c o g n i t i o n does not appear dependent on recent exposure; f a m i l y - s p e c i f i c odours were recog n i z e d a f t e r a s e p a r a t i o n of at l e a s t 1 month, and i n one case a f t e r 5 months ( F i g u r e 16). I t appears that the p e r c e p t u a l l e a r n i n g documented i n Chapter 1 i s not p e c u l i a r to a r t i f i c i a l odorants such as morpholine, but occurs with n a t u r a l odours. While odours appeared to have been l e a r n e d d u r i n g the a l e v i n stage ( f a m i l i e s C3,D4) and e a r l y f r y stage ( f a m i l i e s C3,D3), there was no suggestion that c o n s p e c i f i c odours were l e a r n e d d u r i n g the embryo stage. In t h i s respect the l e a r n i n g of f a m i l y - s p e c i f i c odours would appear to d i f f e r from l e a r n i n g of morpholine. The d i f f e r e n c e may be a t t r i b u t a b l e to a p a u c i t y of odour d u r i n g the embryo stage, and not an i n a b i l i t y of embryos to d e t e c t and l e a r n c o n s p e c i f i c odours. In t h i s experiment, f i s h were exposed to the the chemical emanations of non-sibs of s i m i l a r age. S e v e r a l s t u d i e s have shown that p a r e n t a l c i c h l i d s w i l l o r i e n t to a water source bathing t h e i r w r i g g l e r s ( e q u i v a l e n t of a l e v i n s ) or f r y , but not eggs u n t i l a few hours before h a t c h i n g (see Myrberg 1975). (However, female t h r e e s p i n e s t i c k l e b a c k s (Gasterosteus a c u l e a t u s ) d i s c r i m i n a t e between t h e i r own eggs and those of o t h e r s , p o s s i b l y by smell 91 ( F i t z G e r a l d and Van Havre 1987; Smith and Whoriskey 1988).) Had embryos been exposed to the chemical emanations of c o n s p e c i f i c f r y i n s t e a d of other eggs, perhaps l e a r n i n g would have been apparent. A problem with the assay of r e c o g n i t i o n used i n t h i s study i s that f a m i l i a r i t y does not appear to be the onl y , or even the dominant determinant of p r e f e r e n c e among f a m i l y - s p e c i f i c odours. Coho f r y are a l s o s e n s i t i v e , some perhaps more than o t h e r s , to odour c o n c e n t r a t i o n . Fry may recognize a p r e v i o u s l y encountered odour, but p r e f e r an u n f a m i l i a r but more co n c e n t r a t e d odour. D i f f e r e n t groups of f i s h probably emit d i f f e r e n t c o n c e n t r a t i o n s of odour, and t h i s may have a ge n e t i c and/or a non-genetic b a s i s such as s t a t e of h e a l t h or s t e s s , i n which case odour emissions may vary from day to day. For t h i s reason and because the t e s t apparatus may i t s e l f i n t roduce v a r i a t i o n i n odour c o n c e n t r a t i o n (see Chapter 4), f u t u r e s t u d i e s of s i b l i n g r e c o g n i t i o n should c o n s i d e r r e p l i c a t i n g t e s t s , and t e s t i n g the responses of f i s h to the f a m i l i a r odour p a i r e d with s e v e r a l d i f f e r e n t u n f a m i l i a r odours. Such m u l t i p l e comparisons might circumvent the problem of c o n t r o l and experimental f i s h both p r e f e r r i n g one odour-group over another because of a c o n c e n t r a t i o n i n e q u i t y , such as appeared to e x i s t between the odours of groups G6a and A1b i n t h i s study. Quinn and Busack (1985) suggested that the odours of d i f f e r e n t f a m i l i e s might a l s o d i f f e r q u a l i t a t i v e l y i n a t t r a c t i v e n e s s to other f i s h . If so, m u l t i p l e comparisons might r e v e a l an e f f e c t of pr e v i o u s experience where a t e s t p a i r i n g the " f a m i l i a r " odour with a s i n g l e u n f a m i l i a r odour d i d not. 92 Where the interes t i s learning of natural odours in ear ly l i f e , rather than the ontogeny of s i b l i n g r e c o g n i t i o n , odours other than f a m i l y - s p e c i f i c odours might be more s u i t a b l e . The response to c o n s p e c i f i c odours, os tens ibly more d i f f e r e n t than those of f a m i l i e s of the same populat ion , might be less a f fec ted by factors such as small d i f f e r e n c e s in concentrat ion. This was invest igated in Chapter 3. 93 CHAPTER 3. THE ROLE OF LEARNING IN RECOGNITION OF POPULATION-SPECIFIC ODOURS INTRODUCTION I t has been suggested s e v e r a l times that salmon are guided home by p o p u l a t i o n - s p e c i f i c odours (reviewed by S t a b e l l 1984). Parker ( c i t e d i n C h i d e s t e r 1924) suggested, " i t i s p o s s i b l e that a c e r t a i n race of f i s h may g i v e o f f emanations that d i f f e r from those of other races; hence, the r e t u r n of i n d i v i d u a l races to t h e i r homestream co u l d be a t t r i b u t e d to t h e i r power to sense the f a m i l i a r emanation." White (1936) repo r t e d experiments i n which A t l a n t i c salmon f r y were p l a n t e d i n a stream that p r e v i o u s l y h e l d no salmon. Later that year a run of a d u l t salmon occu r r e d i n t h a t stream. White s p e c u l a t e d that these a d u l t s were of the same race as the p l a n t e d j u v e n i l e s . Solomon (1973) suggested that "the presence of salmon (parr?) i n a r i v e r renders i t s e s t u a r y a t t r a c t i v e to m i g r a t i n g a d u l t s i n the f i r s t i n s t a n c e , and that a metabolic product of a d i s c r e t e p o p u l a t i o n c o u l d be the odour to which a d u l t s home." Nordeng (1971) presented evidence that A r c t i c char ( S a l v e l i n u s a l p i n u s (L.) were a t t r a c t e d home by "an a t t r a c t a n t r e l e a s e d by t h e i r r e l a t i v e s i n t h a t system", and l a t e r (Nordeng 1977) extended t h i s "pheromone" hyp o t h e s i s to e x p l a i n the e n t i r e homeward m i g r a t i o n of anadromous salmonids as f o l l o w s : "The salmon homeward m i g r a t i o n s are i n i t i a t e d and d i r e c t e d by pheromone t r a i l s d e r i v e d from r e l a t e d smolt descending 94 almost c o n t i n u a l l y from t h e i r r e s p e c t i v e freshwater home l o c a l i t i e s i n to the sea d u r i n g s p r i n g and summer. Thus the descending smolt e s t a b l i s h p o p u l a t i o n - s p e c i f i c pheromone t r a i l s l e a d i n g from t h e i r r e s p e c t i v e freshwater home l o c a l i t i e s out to the salmon at sea. The maturing salmon respond i n n a t e l y to the smolt pheromones and are induced to separate from the immature salmon and s t a r t homewards. The m i g r a t i o n f o l l o w s along the smolt pheromone t r a i l s which are maintained by s t i l l descending smolt. During the f i n a l o r i e n t a t i o n the smolt pheromones may be supplemented with pheromones d r i f t i n g from the p o p u l a t i o n of young (parr) i n the r e s p e c t i v e freshwater home l o c a l i t i e s , and the r e s i d e n t p o p u l a t i o n s themselves may mark the end of the homeward r o u t e s . " The phermone hypothesis was o r i g i n a l l y proposed f o r p o p u l a t i o n s of A r c t i c char, brown t r o u t (Salmo t r u t t a L . ) , and A t l a n t i c salmon of the Salangen R i v e r system i n northern Norway, but Nordeng (1977) suggested i t might a l s o apply to P a c i f i c salmon s p e c i e s (except pink salmon (0. gorbuscha Walbaum) which because of a r i g i d 2 year l i f e c y c l e do not have young i n the home stream or m i g r a t i n g to sea at the time a d u l t s are r e t u r n i n g , but f o r which Nordeng suggested homing may be "caused by a s s o c i a t i o n with other m i g r a t i n g P a c i f i c salmon o r i g i n a t i n g i n the same r i v e r system...".) Sev e r a l s t u d i e s , prompted by the pheromone hy p o t h e s i s , t e s t e d the a b i l i t y of salmonids to d i s t i n g u i s h between the chemical emanations of c o n s p e c i f i c p o p u l a t i o n s . Doving et a l . (1974) showed that i n d i v i d u a l sensory c e l l s of the char o l f a c t o r y bulb responded d i f f e r e n t l y to the chemical emanations of d i f f e r e n t char p o p u l a t i o n s , suggesting t h a t p o p u l a t i o n s emitted d e t e c t a b l y d i f f e r e n t odours. S e l s e t and Doving (1980) re p o r t e d t hat a d u l t A r c t i c char of one p o p u l a t i o n p r e f e r r e d water c o n d i t i o n e d by the i n t e s t i n a l contents of a smolt of t h e i r 95 own p o p u l a t i o n over those of a smolt of another p o p u l a t i o n . Since then, f i v e b e h a v i o u r a l s t u d i e s have shown that salmonids d i s t i n g u i s h and p r e f e r the chemical emanations of members of t h e i r own p o p u l a t i o n over those of s i m i l a r l y - a g e d members of another c o n s p e c i f i c p o p u l a t i o n ( S t a b e l l 1982, 1987; Groot et a l . 1986; Olsen 1986a; Quinn and Tolson 1986). I n t e r p r e t a t i o n of these s t u d i e s i s l i m i t e d by three f a c t o r s (Quinn and Courtenay 1989). F i r s t , i n the s t u d i e s by S t a b e l l (1982, 1987) with A t l a n t i c salmon par r and Olsen (1986a) with A r c t i c char f r y , f i s h were e i t h e r s i b l i n g s or d e r i v e d from the same groups of p a r e n t s , so may have demonstrated s i b l i n g r a t h e r than p o p u l a t i o n r e c o g n i t i o n . Second, i n these s t u d i e s and t h a t of Groot et a l . (1986) with sockeye a d u l t s , odour producers and t e s t f i s h of a given p o p u l a t i o n were h e l d together f o r a p e r i o d before t e s t i n g , p e r m i t t i n g l e a r n i n g and subsequent r e c o g n i t i o n of the odours of s p e c i f i c i n d i v i d u a l s . A study by Quinn and Tolson (1986) with coho f r y and jacks avoided both of these l i m i t a t i o n s , demonstrating that "the a t t r a c t i o n was presumably based on some g e n e r a l i z e d i n t e r p o p u l a t i o n d i f f e r e n c e . " I t was assumed that odour d i f f e r e n c e s were g e n e t i c a l l y based, but odours c o u l d have been a c q u i r e d d u r i n g the embryo, a l e v i n or e a r l y f r y stage d u r i n g which p e r i o d s p o p u l a t i o n s were reared i n h a t c h e r i e s on d i f f e r e n t r i v e r s . A t h i r d and l a r g e r problem e x i s t s with the i n t e r p r e t a t i o n of the preceding f i v e s t u d i e s . Each r e p o r t e d the p r e f e r e n c e of members of one p o p u l a t i o n f o r the chemical emanations of t h e i r 96 own p o p u l a t i o n over those of a second p o p u l a t i o n . S t a b e l l (1982,1987) d i d not examine the preference of the second p o p u l a t i o n , but the other s t u d i e s d i d , and i n each case the second p o p u l a t i o n showed no pr e f e r e n c e or even p r e f e r r e d the f i r s t p o p u l a t i o n . T h i s c o u l d r e f l e c t a d i f f e r e n c e between the two p o p u l a t i o n s i n m o t i v a t i o n to swim towards p o p u l a t i o n members (Groot et a l . 1986) but there i s another p o s s i b i l i t y . F i s h of both p o p u l a t i o n s may have responded s i m i l a r l y , a l b e i t with d i f f e r e n t s t r e n g t h , to some q u a l i t a t i v e or q u a n t i t a t i v e d i f f e r e n c e between the two odours, and not to a recognized p o p u l a t i o n - s p e c i f i c odour. The demonstration of p o p u l a t i o n r e c o g n i t i o n r e q u i r e s that each p o p u l a t i o n p r e f e r i t s own odour over that of other p o p u l a t i o n s , and to date t h i s has not been shown. No s t u d i e s have yet i n v e s t i g a t e d the r o l e of l e a r n i n g i n r e c o g n i t i o n of p o p u l a t i o n - s p e c i f i c odours. The pheromone hyp o t h e s i s does not n e c e s s a r i l y r u l e out l e a r n i n g ( S e l s e t and Doving 1980), and the suggestion that pink salmon home to h e t e r o s p e c i f i c odours would seem to r e q u i r e l e a r n i n g . In h i s review of the pheromone h y p o t h e s i s , S t a b e l l (1984) concluded that p o p u l a t i o n r e c o g n i t i o n probably does not r e l y on l e a r n i n g , but i f i t does, l e a r n i n g should occur d u r i n g the embryo stage because a f t e r t h i s f i s h r i s k exposure to odours of other p o p u l a t i o n s l i v i n g upstream. T h i s chapter r e p o r t s the r e s u l t s of two experiments examining chemosensory p o p u l a t i o n r e c o g n i t i o n by coho f r y . F i s h 97 f o r both experiments were sampled from the same hatchery p o p u l a t i o n s s t u d i e d by Quinn and Tolso n (1986), p l u s a t h i r d , g e o g r a p h i c a l l y intermediate p o p u l a t i o n . S t r a y i n g between these three p o p u l a t i o n s i s known to be very r a r e (Quinn and Tolson 1986). The f i r s t experiment extended Quinn and Tolson's work by examining the responses of f i s h , r e ared i n the same hatchery to preclud e d i f f e r e n t i a l odour a c q u i s i t i o n , to a c h o i c e of odours of p o p u l a t i o n members and members of another p o p u l a t i o n . In a d d i t i o n , some f i s h were reared with members of another p o p u l a t i o n , to i n v e s t i g a t e the r o l e of f a m i l i a r i t y ( l e a r n i n g ) i n p o p u l a t i o n - r e c o g n i t i o n . During the course of t h i s experiment, i t became apparent that p r e f e r e n c e s among p o p u l a t i o n odours were i n f l u e n c e d by some f a c t o r ( s ) b e s i d e s f a m i l i a r i t y or ge n e t i c r e l a t e d n e s s . Odour c o n c e n t r a t i o n proved to be one f a c t o r , r a i s i n g the p o s s i b i l i t y that the c o n c e n t r a t i o n of odour emitted by f r y i s a p o p u l a t i o n - s p e c i f i c c h a r a c t e r i s t i c . T h i s was i n v e s t i g a t e d i n a second experiment. EXPERIMENT J_ Treatment The gametes of four male and four female coho salmon were t r a n s p o r t e d from each of three h a t c h e r i e s : Quinsam R i v e r (Q) (Nov. 25, 1985), B i g Qualicum R i v e r (BQ) (Nov. 26, 1985), and Puntledge R i v e r (P) (Nov. 26, 1985), to Rosewall Creek Hatchery where r e a r i n g and t e s t i n g took p l a c e . For each p o p u l a t i o n , the 98 pooled eggs were f e r t i l i z e d with the pooled m i l t , and then d i v i d e d i n t o four groups of equal volume. Each group was incubated and re a r e d u n t i l the end of the experiment i n one h a l f of a 60 X 30 X 30 cm basket of mesh net s t r e t c h e d over an aluminum frame, suspended i n a darkened c i r c u l a r f i b e r g l a s s tank (92 cm diameter, 76 cm depth, 8 L/min flow of w e l l water, 8-9°C ) . Baskets were d i v i d e d i n h a l f with mesh n e t t i n g , a l l o w i n g f r e e exchange of water between s i d e s . In three tanks both h a l v e s of the baskets were occupied by groups of the same p o p u l a t i o n so that f i s h were exposed to the odour of p o p u l a t i o n members only ( i . e . , Q/Q, BQ/BQ, P/P). In another three tanks, the ha l v e s of the baskets were occupied by groups of d i f f e r e n t p o p u l a t i o n s ( i . e . , Q/BQ, Q/P, BQ/P), so that f i s h were exposed to the odours of both t h e i r own and another p o p u l a t i o n . For b r e v i t y , groups w i l l be r e f e r r e d to as, f o r example - Q/Q meaning Quinsam f i s h reared with Quinsam f i s h , w hile Q/BQ are Quinsam f i s h r e a red with B i g Qualicum f i s h , and P/Q are Puntledge f i s h r e a red with Quinsam f i s h . Groups numbered 1408-2092 l i v e i n d i v i d u a l s on January 8 and were reduced on March 11 to 1200 each. Eggs hatched January 21-23, 1986. Fry swam up between February 24-27 and were exposed to an 8:16 LD photoperiod and hand fed Oregon Moist P e l l e t s 5 times a day f o r the f i r s t 2 weeks and 3 times a day t h e r e a f t e r . On March 13, 1986, 300 f r y of each of the groups Q/BQ, BQ/Q, Q/P, P/Q, BQ/P, and P/BQ were removed from baskets and 99 co n t i n u e d r e a r i n g apart from the i n f l u e n c e of the other p o p u l a t i o n i n a 35 cm diameter X 40 cm depth tank. These groups w i l l be r e f e r r e d to as, f o r example - Q (/BQ), meaning Quinsam f i s h r e ared with Big Qualicum f i s h u n t i l 2 weeks post swim-up, but not t h e r e a f t e r . T e s t i n g Methods and M a t e r i a l s T e s t i n g began 3 weeks a f t e r swim-up and c o n t i n u e d f o r 3 months (March 24 - June 19, 1986). Procedures f o l l o w e d those o u t l i n e d i n General Methods and M a t e r i a l s . I n d i v i d u a l f i s h were o f f e r e d a c h o i c e of waters c o n d i t i o n e d by members of d i f f e r e n t p o p u l a t i o n s . Waters were c o n d i t i o n e d by a number of f r y (29-136 depending of p o p u l a t i o n and date) t o t a l l i n g 100 g u n l e s s s p e c i f i e d otherwise, p l a c e d irt the headtanks at approximately 1600 h on the evening preceding a t e s t . Each experiment c o n s i s t e d of two t e s t s , g e n e r a l l y on c o n s e c u t i v e days, with odour groups occupying o p p o s i t e headtanks on the two days. ( I f headtank b i a s was obvious ( i . e . , same headtank p r e f e r r e d on both days) data were d i s c a r d e d , the apparatus was "cleaned" (headtanks and a s s o c i a t e d plumbing wiped with paper towel, r i n s e d with hot water, and f l u s h e d o v e r n i g h t ) and the experiment was repeated. See Chapter 4 f o r an examination of headtank b i a s . ) A f t e r each t e s t (day), f i s h were removed from headtanks, headtanks were d r a i n e d and wiped with paper towel, and drainage 100 p o r t s were b a c k f l u s h e d to remove any lodged f a e c e s . O d o u r - f i s h were returned t o h o l d i n g tanks, and may have been used again as odour-producers or as t e s t - f i s h , a f t e r at l e a s t one day i n h o l d i n g . I n d i v i d u a l s were d i s c a r d e d a f t e r t e s t i n g . Dates and p a r t i c u l a r s of experiments are d e s c r i b e d with t h e i r r e s u l t s . S t a t i s t i c a l a n a l y s i s of data f o l l o w e d that d e s c r i b e d i n General Methods and M a t e r i a l s . R e s u l t s Assay J_: P o p u l a t i o n - R e c o g n i t i o n and the E f f e c t of Common-Rearing The purpose of t h i s assay was to t e s t the p r e f e r e n c e of coho f r y f o r the odour of p o p u l a t i o n members over that of members of another p o p u l a t i o n , as a measure of r e c o g n i t i o n . P r e f e r e n c e s of f i s h reared with and without members of the other p o p u l a t i o n were compared to examine the r o l e of f a m i l i a r i t y ( l e a r n i n g ) i n p o p u l a t i o n r e c o g n i t i o n . F i s h reared with members of the other p o p u l a t i o n (e.g., BQ/Q) were t e s t e d with the odours of tankmates (BQ/Q and Q/BQ). F i s h reared without members of the other p o p u l a t i o n (e.g., BQ/BQ) were t e s t e d with the odours of tankmates (BQ/BQ) and members of the other p o p u l a t i o n reared with i t s own p o p u l a t i o n only (Q/Q). I t was assumed that common-r e a r i n g d i d not change the odour emitted by f i s h (e.g., Q/BQ smelled the same as Q/Q). T h i s assumption was t e s t e d and w i l l be d i s c u s s e d below (Assay 3 ) . Q/Q p r e f e r r e d Q over BQ odour (P=0.0069) as re p o r t e d by 101 Quinn and Tolso n (1986) (F i g u r e 22A). T h i s response was not s i g n i f i c a n t l y a l t e r e d by common-rearing with BQ; Q/BQ a l s o p r e f e r r e d Q over BQ odour (P=0.025). S i m i l a r l y , both Q/Q and Q/P p r e f e r r e d Q over P odour (P=0.0032 and P<0.0001 r e s p e c t i v e l y ) (Figure 22B). BQ/BQ showed no pre f e r e n c e between BQ and Q odours, as re p o r t e d by Quinn and Tolson (1986) (Figure 23A). T h i s response was s i g n i f i c a n t l y a l t e r e d (P<0.0001) by common-rearing; BQ/Q p r e f e r r e d Q over BQ (P<0.0001). In c o n t r a s t , common-rearing d i d not a f f e c t p r e f e r e n c e between BQ and P odours; both BQ/BQ and BQ/P p r e f e r r e d BQ over P odour (P=0.0052 and P=0.0003 r e s p e c t i v e l y ) ( F i g u r e 23B). P/P p r e f e r r e d Q over P odour (P=0.0003), and t h i s was not s i g n i f i c a n t l y a l t e r e d by common-rearing; P/Q showed the same pr e f e r e n c e (P=0.0003) (Figure 24A). P/P p r e f e r r e d BQ over P odour (P=0.012) and while P/BQ d i d not, an e f f e c t of common-r e a r i n g was not de t e c t e d (Figure 24B). These data i n d i c a t e that not a l l coho f r y p r e f e r the chemical emanations of t h e i r own p o p u l a t i o n over those of ot h e r s , so pr e f e r e n c e i n the Y-maze does not equate with p o p u l a t i o n r e c o g n i t i o n . Rather, some p o p u l a t i o n s seem more a t t r a c t i v e than others to a l l coho f r y (e.g., i n no t e s t was P or BQ odour p r e f e r r e d over Q odour). The nature of d i f f e r e n c e s i n inherent a t t r a c t i v e n e s s w i l l be d i s c u s s e d below i n Assay 4. However, the response of BQ to a c h o i c e of Q and BQ odours was a l t e r e d by common-rearing, i n d i c a t i n g t h a t f a m i l i a r i t y 1 02 G O • rH •»-> cd cx o PH a O CA -a rH cd W) c CO o H 70 104 109 104 102 60 50 Prefer Own Population 40 Prefer Other Population 30 B F i g u r e 22. P r e f e r e n c e of Quinsam R. f r y between odours of population-members and members of the B i g Qualicum R. p o p u l a t i o n (A) or Puntledge R. p o p u l a t i o n (B). Open bars: f i s h r e a r e d with population-members only. Shaded bars: f i s h r e a r e d with members of both p o p u l a t i o n s . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 1 0 3 G O • l - H +-> ci P Q. O a O T3 t-H c • t-H a a • t-H CO a • i - H H 7 0 89 108 91 100 Prefer Own Population 6 0 5 0 4 0 Prefer Other Population 3 0 B F i g u r e 23. Preference of Big Qualicum R. f r y between odours of population-members and members of the Quinsam R. p o p u l a t i o n (A) or Puntledge R. p o p u l a t i o n (B). Open bars: f i s h reared with population-members o n l y . Shaded bars: f i s h reared with members of both p o p u l a t i o n s . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 104 • a o +-> rt P CX o PH d O CA T3 l-H O H d s 6 CO CU a ' r - t H 70 102 102 97 103 60 50 4 0 30 Prefer Own Population Prefer Other Population B Figure 24. Preference of Puntledge R. fry between odours of population-members and members of the Quinsam R. population (A), or Big Qualicum R. population (B). Open bars: f i s h reared with population-members only. Shaded bars: f i s h reared with members of both populations. (x ± 95% CL, Number of fry tested shown above bar) 105 ( r e c o g n i t i o n ) i s one i n f l u e n c e on p r e f e r e n c e . I t may be that f a m i l i a r i t y i s a minor i n f l u e n c e , masked by the presence of more i n f l u e n t i a l d i f f e r e n c e s between odours. I f so, f a m i l i a r i t y might be manifested i n pre f e r e n c e between some odours, but not ot h e r s . With t h i s i n mind, one of the p o p u l a t i o n s that showed no evidence here of having l e a r n e d tankmate odours (Q) was given a d i f f e r e n t t e s t of tankmate r e c o g n i t i o n i n Assay 2. Assay 2: R e c o g n i t i o n of Non-population Tankmates The purpose of t h i s assay was to determine whether f i s h r eared with members of another p o p u l a t i o n had l e a r n e d and would recognize the odours of those tankmates. The c r i t e r i o n of r e c o g n i t i o n was s i m i l a r to that used i n Chapter 2: p r e f e r e n c e over an u n f a m i l i a r odour (that of another p o p u l a t i o n ) t o a degree g r e a t e r than that shown by naive p o p u l a t i o n members. Only Q f i s h were t e s t e d i n t h i s assay; Q/Q f i s h were naive to BQ and P odours, while Q/BQ was f a m i l i a r with BQ and Q/P was f a m i l i a r with P. Q/Q showed no pre f e r e n c e between BQ/BQ and P/P odours (Figure 25). Q/BQ p r e f e r r e d BQ/Q over P/P (P<0.0001), and Q/P showed no p r e f e r e n c e between P/Q and BQ/BQ (F i g u r e 25). The responses of the three Q groups d i f f e r e d (P<0.05), the d i f f e r e n c e l y i n g between Q/BQ and Q/P (P<0.05, Tukey t e s t ) . While the responses of n e i t h e r Q/P nor Q/BQ met the c r i t e r i o n of r e c o g n i t i o n of non-population tankmates ( d i f f e r e n c e from Q/Q), the d i f f e r e n c e between them suggests an e f f e c t of f a m i l i a r i t y . 106 o •rH "CO 60 • rH PQ Cfl *o rH c<J a • rH s e • rH CO a •rH H 70 102 105 105 104 109 60 -50 Prefer Big Qualicum 40 -Prefer Puntledge 30 Q / Q Q / B Q Test Group Q / P F i g u r e 25. Preference of Quinsam R. f r y between odours of Big Qualicum R. f r y and Puntledge R. f r y . Q/Q: f i s h reared with population-members o n l y . Q/BQ: f i s h r e ared with population-members and members of the Big Qualicum R. p o p u l a t i o n . Q/P: f i s h reared with population-members and members of the Puntledge R. p o p u l a t i o n . Shaded Bars: separated from non-population members 69-79 d p r i o r to t e s t i n g . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 107 The weakness of t h i s e f f e c t may r e f l e c t masking by some more i n f l u e n t i a l d i f f e r e n c e ( s ) between odours. To determine whether the e f f e c t of f a m i l i a r i t y depended on recent exposure, f i s h r eared with BQ or P f i s h and then separated from them at 2 weeks a f t e r swim-up (69-79 days p r e v i o u s ) were t e s t e d . Responses d i d not d i f f e r s i g n i f i c a n t l y from those of group members not separated; Q (/BQ) p r e f e r r e d BQ over P (P=0.023) and Q (/P) p r e f e r r e d P over BQ (P=0.044) (Fi g u r e 25). The response of Q (/P) d i f f e r e d from that of both Q (/BQ) and Q/Q (P<0.05, Tukey t e s t ) . I t appears that the e f f e c t of common-rearing recorded p r e v i o u s l y was independent of recent exposure. These data suggest that tankmate odours were le a r n e d d u r i n g or before the e a r l y f r y stage and were remembered f o r over 2 months. The f a c t t h at t h i s was expressed i n t h i s assay but not i n Assay 1 i n d i c a t e s that f a m i l i a r i t y as an i n f l u e n c e on p r e f e r e n c e between c o n s p e c i f i c odours, can be masked by other d i f f e r e n c e s between odours. Assay 3: T e s t i n g the Assumption that F i s h do not Acquire Odours  from C o n s p e c i f i c s An assumption made i n Assay 2 was that f i s h d i d not a c q u i r e odours from tankmates. T h i s assumption was important because while Q/BQ were t e s t e d with the odours of BQ/Q versus P/P, Q/P were t e s t e d with the odours of BQ/BQ versus P/Q. I f f i s h d i d 108 not a c q u i r e c o n s p e c i f i c odours then the d i f f e r e n t p r e f e r e n c e s of Q/BQ and Q/P must r e f l e c t l e a r n i n g and memory. However, i f BQ and P f i s h a c q u i r e d some Q odour d u r i n g common-rearing, Q f i s h may simply have p r e f e r r e d the group s m e l l i n g more l i k e Q. To t e s t t h i s assumption, Q/BQ f i s h were o f f e r e d a c h o i c e of the odours of BQ/Q and BQ/BQ, and Q/P f i s h were . o f f e r e d a c h o i c e of the odours of P/Q and P/P. In n e i t h e r t e s t d i d Q f i s h show any pr e f e r e n c e (Figure 26), su p p o r t i n g the assumption that BQ and P f i s h d i d not a c q u i r e odours from Q f i s h . Assay 4: The I n f l u e n c e of Odour Co n c e n t r a t i o n on Preference Assay 1 i n d i c a t e d that some f a c t o r ( s ) b e s i d e s f a m i l i a r i t y or g e n e t i c r e l a t e d n e s s i n f l u e n c e d p r e f e r e n c e among p o p u l a t i o n odours. Because r e l a t i v e c o n c e n t r a t i o n of odour had proven i n f l u e n t i a l i n p r e f e r e n c e s among f a m i l y - s p e c i f i c odours (Chapter 3), i t was hypothesized that i t might be i n f l u e n t i a l here a l s o . In t h i s assay, P/P f i s h were given a c h o i c e of waters c o n d i t i o n e d by Q/Q and BQ/BQ f i s h . When waters were c o n d i t i o n e d by 100 g of Q and BQ, P p r e f e r r e d Q (P=0.0058) (F i g u r e 27). However, when the Q odour group was reduced to 30 g, P p r e f e r r e d the 100 g of BQ (P<0.0001). T h i s was not because 30 g of Q f a i l e d to emit a d e t e c t a b l e c o n c e n t r a t i o n of odour; P s t r o n g l y p r e f e r r e d 30 g of Q over blank water (P<0.0001) (Figure 27). The responses of the P f i s h were s i g n i f i c a n t l y d i f f e r e n t i n each of these three t e s t s (P<0.05, Tukey t e s t ) . 109 0> •(-> a •S c t-H O H DO a B 6 •l-H CO 6 •>-< H •*-> c <u o u a> PH 70 97 98 60 50 40 30 Prefer Tankmates Prefer Non -Tankmates B F i g u r e 26. Pr e f e r e n c e of Quinsam R. f r y between odours of tankmate and non-tankmate Qualicum R. f r y (A) or Puntledge R. f r y (B). (x ± 95% CL, Number of f r y t e s t e d shown above bar) 110 90 80 70 60 50 40 h 30 20 105 96 87 Prefer Quinsam Prefer Big Qualicum B F i g u r e 27. Preference of Puntledge R. f r y between odours of Quinsam R. f r y and Big Qualicum R. f r y : the i n f l u e n c e of r e l a t i v e c o n c e n t r a t i o n of odour. A: odours of 100 g Quinsam R. f i s h , 100 g Big Qualicum R. f i s h . B: odours of 30 g Quinsam R. f i s h , 100 g Big Qualicum R. f i s h . C: odours of 30 g Quinsam R. f i s h , 0 g Big Qualicum R. f i s h , (x ± 95% CL, Number of f r y t e s t e d shown above bar) 111 These data i n d i c a t e that odour c o n c e n t r a t i o n i s i n f l u e n t i a l i n e s t a b l i s h i n g p r e f e r e n c e s between p o p u l a t i o n odours, as i t i s i n p r e f e r e n c e s between f a m i l y - s p e c i f i c odours. I t appeared i n Chapter 2 t h a t f a m i l i e s might d i f f e r i n odour c o n c e n t r a t i o n . S i m i l a r l y , i t may be that p o p u l a t i o n s d i f f e r i n c o n c e n t r a t i o n of emitted odour. P odours may have f a i l e d to a t t r a c t p o p u l a t i o n members because of a q u a n t i t a t i v e d e f i c i t and BQ/BQ f i s h may have f a i l e d to p r e f e r BQ over Q odours, because Q odours were s l i g h t l y more co n c e n t r a t e d . (In the l a t t e r case the g r e a t e r c o n c e n t r a t i o n of Q odour may have been balanced by the g r e a t e r f a m i l i a r i t y of BQ odour - a balance l o s t with f i s h f a m i l i a r with both odours (BQ/Q).) I t i s p o s s i b l e that the samples of f i s h used i n t h i s experiment d i d not a c c u r a t e l y represent the odour c o n c e n t r a t i o n s of the p o p u l a t i o n s . Each sample was d e r i v e d from the pooled gametes of four males and four females. U n t i l very r e c e n t l y , t h i s was standard hatchery p r a c t i s e i n B r i t i s h Columbia. I t was assumed that the m i l t of each of the males f e r t i l i z e d a roughly equal p r o p o r t i o n of the eggs, u n t i l two s t u d i e s showed t h i s to be i n c o r r e c t . W i t h l e r (1988) showed that when eggs of i n d i v i d u a l chinook salmon were f e r t i l i z e d with a mixture of equal volumes of m i l t from three males, i n d i v i d u a l males f e r t i l i z e d between 1.4% and 75.6% of the eggs. G h a r r e t t and S h i r l e y (1985) r e p o r t e d s i m i l a r sperm co m p e t i t i o n i n pink salmon. T h e r e f o r e , i t i s p o s s i b l e that the p o p u l a t i o n samples i n the present experiment were predominantly the o f f s p r i n g of a 112 s i n g l e male. T h i s i s an important d i s t i n c t i o n because the experiments i n Chapter 2 i n d i c a t e d t hat the odours of some f a m i l i e s were l e s s a t t r a c t i v e than those of o t h e r s , p o s s i b l y because of a q u a n t i t a t i v e d e f i c i t . The r e s t r i c t e d parentage of t h i s experiment may have r e s u l t e d i n q u a n t i t i e s (and/or q u a l i t i e s ) of odour, c h a r a c t e r i s t i c of p a r t i c u l a r males, but not n e c e s s a r i l y c h a r a c t e r i s t i c of the p o p u l a t i o n s . Conceivably a s i n g l e male c o n f e r r e d an odour d e f i c i t on the P sample, which was not c h a r a c t e r i s t i c of the whole P p o p u l a t i o n . A sample d e r i v e d of a wide parentage of each p o p u l a t i o n might produce a d i f f e r e n t r e s u l t i n the t e s t f o r p o p u l a t i o n - r e c o g n i t i o n (Assay 1). T h i s was i n v e s t i g a t e d i n Experiment 2. EXPERIMENT,2 Treatment On January 13 1987, approximately 2000 eyed coho eggs were t r a n s p o r t e d from each of the Big Qualicum (BQ), Quinsam (Q), and Puntledge (P) h a t c h e r i e s to Rosewall Creek Hatchery where r e a r i n g and t e s t i n g took p l a c e . Spawning dates, parentage and accumulated thermal u n i t s to January 13 are given i n Table 5. Pop u l a t i o n s were reared i n baskets suspended i n d i f f e r e n t darkened c i r c u l a r f i b e r g l a s s tanks as i n Experiment 1. Q and P eggs hatched on February 3, and f r y swum up on March 3. BQ eggs hatched on February 5, and f r y swum up on March 7. At swim-up, f i s h were exposed to an 8:16 LD photoperiod, and were hand-fed 1 1 3 Table 5. Spawning and i n c u b a t i o n data on coho eggs obtained from h a t c h e r i e s on the Quinsam R. (Q), B i g Qualicum R. (BQ), and Puntledge R. (P) f o r t e s t s of o l f a c t o r y p o p u l a t i o n r e c o g n i t i o n (Experiment 2, Chapter 3 ) . Parentage i n d i c a t e s the approximate number of females from which samples were drawn. F e r t i l i z a t i o n i n d i c a t e s the method of f e r t i l i z i n g eggs, e.g., 4/4 means the pooled eggs of 4 females were f e r t i l i z e d with the pooled m i l t of 4 males. ATU - i n d i c a t e s stage of development, and i s c a l c u l a t e d by m u l t i p l y i n g days by d a i l y temperature. P o p u l a t i o n Spawn Date Parentage F e r t i l i z a t i o n ATU to (day/mon/yr) (females) (males/females) 1 3 / 0 1 / 8 7 Q 1 3 / 1 1 / 8 6 30 1/1 2 7 6 BQ 5 / 1 2 / 8 6 110 1/3 2 6 5 P 2 6 / 1 1 / 8 6 40 4/4 3 3 4 114 Oregon Moist P e l l e t s (Moore-Clarke I n c . , LaConner WA) f i v e times a day for the f i r s t 2 weeks and three times a day thereaf ter . The mean s ize of 20 Q f i s h before t e s t i n g (March 24) was 3.9 cm f o r k - l e n g t h (SD=0.2) and 0.55 g wet weight (SD=0.09). The mean fork- lengths and wet-weights of samples of 30 f i s h from each population measured af ter the end of the experiment (May 6) d i d not d i f f e r s i g n i f i c a n t l y (P>0.05, ANOVA). The pooled mean f o r k -length was 4.9 cm (SD=0.3), and wet-weight was 1.34 g (SD=0.29). Test ing Methods and Mater ia ls I n d i v i d u a l f i s h were offered a choice of waters conditioned by tankmates ( their own population) and by members of another popula t ion . Waters were condit ioned by 100 g of f i s h ( i . e . , o d o u r - f i s h , N=68-144 depending on population and date) as in Experiment 1. Each experiment was conducted over two consecutive days, with tankmates in the l e f t headtank on one day, and the r ight headtank on the other . Despite some obvious headtank bias (preference for the same headtank on both test days) , no experiments were repeated. Following a t e s t , odour-f i s h were replaced in holding tanks, but were separated from other f i s h and were not used again as odour- or t e s t - f i s h . Experiments were performed between March 31 and A p r i l 16, 1987. Each experiment was repeated a f t e r a 10-24 day i n t e r v a l , between A p r i l 17 and May 3. Analys is followed that described 115 f o r Experiment 1. R e s u l t s Q, BQ and P coho f r y a l l p r e f e r r e d the odour of members of t h e i r own p o p u l a t i o n over the odour of e i t h e r of the other two p o p u l a t i o n s ( F i g u r e s 28-30, s i g n i f i c a n c e l e v e l s i n Table 6). Each p o p u l a t i o n was t e s t e d with each odour combination twice, and i t i s the pooled response that i s shown i n F i g u r e s 28-30. R e s u l t s of the two experiments d i f f e r e d s i g n i f i c a n t l y i n no case except f o r those i n which Q f r y were o f f e r e d a c h o i c e of Q and BQ odours. Both experiments i n d i c a t e d strong (P<0.001) pr e f e r e n c e f o r Q, but one weaker than the other (P-0.038). T h i s was probably r e l a t e d to "headtank b i a s " i n one of the two experiments; the component t e s t s of t h i s experiment gave d i f f e r e n t responses (P=0.029). The only other i n s t a n c e of s i g n i f i c a n t headtank b i a s was i n one of the two experiments i n which BQ f i s h were o f f e r e d a c h o i c e of BQ and P odours (P=0.0084) but t h i s d i d not render the experiment's r e s u l t s i g n i f i c a n t l y d i f f e r e n t from that of the other experiment. The nature of headtank b i a s i s d i s c u s s e d i n Chapter 4. These data suggest that coho f r y of a l l p o p u l a t i o n s recognize the odours of p o p u l a t i o n members and p r e f e r them over the odours of other p o p u l a t i o n s . I t does not appear to be the case that the odours of some p o p u l a t i o n s are i n h e r e n t l y more a t t r a c t i v e than those of o t h e r s , e i t h e r q u a l i t a t i v e l y or q u a n t i t a t i v e l y . T h e r e f o r e i t appears that the lack of 1 16 188 o • rt *-> a o, o C O •a rt S 6 CO o s • rt H 70 184 Prefer Own Population 60 50 40 Prefer Other Population 30 B F i g u r e 28. P r e f e r e n c e of Quinsam R. f r y between odours of population-members and members of the B i g Qualicum R. p o p u l a t i o n ( A ) , o r the P u n t l e d g e R. p o p u l a t i o n ( B ) . (x ± 95% CL, Number of f r y t e s t e d shown above b ar) 1 1 7 o • i - H CO a o C o SO t-H cd c s 6 CO 6 H 70 167 164 60 50 40 30 Prefer Own Population Prefer Other Population B F i g u r e 29. Preference of Big Qualicum R. f i s h between odours of population-members and members of the Quinsam R. p o p u l a t i o n (A), or the Puntledge R. p o p u l a t i o n (B). (x ± 95% CL, Number of f r y t e s t e d shown above bar) 18 179 179 o -t-> a a o OH a O CA -a t-H a S B • rt CO s • r t H 70 Prefer Own Population 60 50 40 Prefer Other Population 30 B Figure 30. Preference of Puntledge R. f i s h between odours of population-members and members of the Quinsam R. population (A), or the Big Qualicum R. population (B). (x ± 95% CL, Number of f r y tested shown above bar) 1 1 9 Table 6. P r e f e r e n c e s of coho f r y from the Quinsam R. (Q), Big Qualicum R. (BQ), and Puntledge R. (P) f o r water c o n d i t i o n e d by p o p u l a t i o n members over non-population members. * i n d i c a t e s which odour was p r e f e r r e d , and P i n d i c a t e s the s i g n i f i c a n c e of the p r e f e r e n c e ( t w o - t a i l e d t - t e s t ) . Mean p r e f e r e n c e s , 95% co n f i d e n c e l i m i t s , and sample s i z e s are shown i n F i g u r e s 24-26. Test F i s h Odour Choice Q * Q/BQ 0.0004 * Q/P <0.0001 BQ * BQ/Q <0.0001 * BQ/P 0.0110 P * P/Q * P/BQ <0.0001 0.0015 120 p o p u l a t i o n r e c o g n i t i o n recorded i n Experiment 1 (Assay 1) was an experimental a r t i f a c t , probably r e l a t e d to r e s t r i c t e d parentage. I t cannot be r u l e d out that f i s h i n the present experiment a c q u i r e d d i f f e r e n t r i v e r i n e odours dur i n g the embryo stage. However, the f a c t that common-rearing d i d not appear to a l t e r the chemical emanations of f i s h i n Experiment 1 (Assay 3) suggests that odours are not a c q u i r e d from the environment. DISCUSSION Taken together with the study by Quinn and Tolson (1986), the r e s u l t s of Experiment 2 are the most c o n v i n c i n g evidence to date that salmonids are capable of r e c o g n i z i n g the odour of members of t h e i r own p o p u l a t i o n . Coho appear to p r e f e r the odour of t h e i r own p o p u l a t i o n over that of other c o n s p e c i f i c p o p u l a t i o n s , but the responses recorded i n Experiment 1 i n d i c a t e that t h i s tendency i s not robust; p o p u l a t i o n membership i s not of i t s e l f enough to render the odour of one group of f i s h p r e f e r a b l e to that of another. T h i s i s not to imply t h a t p o p u l a t i o n - s p e c i f i c odours are not recognized, but rather t h a t other f a c t o r s i n f l u e n c e p r e f e r e n c e , and can be more i n f l u e n t i a l than r e l a t e d n e s s or f a m i l i a r i t y . T h i s f i n d i n g i s c o n s i s t e n t with and h e l p s e x p l a i n the r e s u l t s of p r e v i o u s s t u d i e s i n t h i s f i e l d (see I n t r o d u c t i o n ) . One f a c t o r i n f l u e n c i n g p r e f e r e n c e among p o p u l a t i o n - s p e c i f i c odours i s odour c o n c e n t r a t i o n ; higher being p r e f e r r e d over lower, at l e a s t w i t h i n the range employed i n t h i s study. T h i s 121 i s s t r i k i n g l y s i m i l a r t o the s i t u a t i o n with f a m i l y odours (Chapter 2), and suggests that p r e f e r e n c e s among c o n s p e c i f i c odours are governed by the same f a c t o r s at both l e v e l s . Consequently, d i f f i c u l t i e s with using c o n s p e c i f i c odours i n the study of l e a r n i n g and memory, are not avoided by u s i n g odours d i f f e r i n g at the p o p u l a t i o n r a t h e r than the f a m i l i a l l e v e l . Tankmate odours appeared to have been l e a r n e d d u r i n g or s h o r t l y a f t e r i n c u b a t i o n , and the memory t r a c e was s t a b l e f o r 2 months (Experiment 1, Assay 2). However, because of the p o t e n t i a l i n f l u e n c e of sperm c o m p e t i t i o n i n Experiment 1, i t i s not c l e a r whether the template l e a r n e d was p o p u l a t i o n - s p e c i f i c , or p a t e r n a l s i b - s p e c i f i c . T h e r e f o r e t h i s study may not n e c e s s a r i l y i n d i c a t e that a p o p u l a t i o n - s p e c i f i c odour template i s l e a r n e d , but does support the c o n c l u s i o n of Chapter 2 that long-term memories of c o n s p e c i f i c odours are formed d u r i n g and/or s h o r t l y a f t e r i n c u b a t i o n . I t has been shown i n s e v e r a l animals that odours are a c q u i r e d from the environment (P o r t e r et a l . 1981; Linsenmair 1987; Morel and Blum 1988). For example, c o l o n y - s p e c i f i c odours of s o c i a l i n s e c t s are i n f l u e n c e d by both g e n e t i c s and by the environment (Crosland 1989a), i n c l u d i n g c o n s p e c i f i c s (Crosland 1989b). I t has been shown that odours emitted by f i s h can be i n f l u e n c e d by d i e t (Bryant and Atema 1987), but i t has g e n e r a l l y been assumed that odours are not a c q u i r e d from c o n s p e c i f i c s (e.g., Quinn and Hara 1986, Chapter 2 of the present s t u d y ) , as Waldman (1985) showed to be the case with toad t a d p o l e s . The 122 present study supports t h i s assumption (Assay 3, Experiment 1). 123 CHAPTER 4. THE INFLUENCE OF FAECES IN DISCRIMINATION OF POPULATION ODOURS INTRODUCTION In t e s t i n g the p r e f e r e n c e s of coho f r y among p o p u l a t i o n s and f a m i l i e s , odours were pro v i d e d by f i s h p l a c e d i n the headtanks approximately 16 h before a t e s t began. The reason f o r p l a c i n g the f i s h i n the headtanks w e l l i n advance of the t e s t was to a v o i d any t r a n s i e n t d i f f e r e n c e s i n odour that might r e s u l t from h a n d l i n g s t r e s s . (Admittedly t h i s may not have e l i m i n a t e d a l l e f f e c t s of h a n d l i n g ; c e r t a i n parameters such as those a s s o c i a t e d with the immune system may take weeks to r e t u r n to p r e - t r a n s f e r l e v e l s a f t e r moving coho from one tank to another (personal communication, C.B. Schreck, Oregon Stage U n i v e r s i t y , C o r v a l l i s Oregon).) One consequence of t h i s p r a c t i c e was t h a t faeces accumulated on the bottom of the headtanks. Two i n c i d e n t a l o b s e r v a t i o n s suggested that the accumulations of faeces i n f l u e n c e d t e s t f i s h . F i r s t , f o r some t e s t s the p r e f e r e n c e s recorded from the four Y-mazes were very d i f f e r e n t . For example, f i s h t e s t e d i n maze 1 might c o n s i s t e n t l y p r e f e r the l e f t headtank, while f i s h t e s t e d i n the other three mazes c o n s i s t e n t l y p r e f e r r e d the r i g h t headtank. Yet the next day, f i s h t e s t e d i n a l l mazes might p r e f e r the r i g h t headtank. Many f a c t o r s c o u l d produce such a b i a s but i t was a l s o observed that because of water flow p a t t e r n s i n the headtanks, faeces sometimes accumulated nearer 124 some drainage p o r t s than o t h e r s . Indeed, d u r i n g some t e s t s small amounts of faeces were observed i n a Y-maze, having washed out of a headtank. Under these circumstances, any i n f l u e n c e of faeces on the o l f a c t o r y p r e f e r e n c e of f r y would be unequally d i s t r i b u t e d among Y-mazes. The second o b s e r v a t i o n was that r e p l i c a t e t e s t s of an experiment o c c a s i o n a l l y produced very d i f f e r e n t responses. For example, f i s h might s t r o n g l y p r e f e r the l e f t headtank i n both t e s t s , d e s p i t e the occupants of the l e f t and r i g h t headtanks having been re v e r s e d between t e s t s . C l e a r l y the t e s t f i s h were more i n f l u e n c e d by some c h a r a c t e r i s t i c of the headtank, than by the f i s h i n i t . T h i s "headtank b i a s " may have been the r e s u l t of r e s i d u e s d e p o s i t e d by f i s h d u r i n g p r e v i o u s t e s t s , or perhaps by odorants produced by b a c t e r i a (see S e l s e t 1980). However, the o b s e r v a t i o n that small q u a n t i t i e s of faeces sometimes washed out of a headtank suggested an a l t e r n a t i v e e x p l a n a t i o n ; the d i s t r i b u t i o n and r e t e n t i o n of faeces i n the headtanks were i n f l u e n c e d by the p a t t e r n of water flow i n the tank, and t h i s was not r i g i d l y s t a n d a r d i z e d i n these experiments. These o b s e r v a t i o n s are not the only reasons f o r s u s p e c t i n g that faeces might i n f l u e n c e p r e f e r e n c e . S e l s e t and Doving (1980) r e p o r t e d that a d u l t A r c t i c char were a t t r a c t e d to c o n s p e c i f i c i n t e s t i n a l c ontents and b i l e i n some t e s t s . One f r a c t i o n of the i n t e s t i n a l contents that proved h i g h l y a t t r a c t i v e to char c o n t a i n e d as a major component the b i l e a c i d - c h o l i c a c i d ( S e l s e t 1980). Doving et a l . (1980) r e p o r t e d that 125 the c o n c e n t r a t i o n of 3-alpha-hydroxy s t e r o i d s (of which b i l e a c i d s are one group) e x c r e t e d by a char would be s u f f i c i e n t f o r d e t e c t i o n by c o n s p e c i f i c s . I t has been suggested that b i l e a c i d s are the important o l f a c t a n t mediating both a t t r a c t i o n to c o n s p e c i f i c odours, and d i s c r i m i n a t i o n between c o n s p e c i f i c odour i n salmonids ( S e l s e t 1980). T h i s has not been demonstrated, but i s supported by the f o l l o w i n g c i r c u m s t a n t i a l evidence. B i l e s a l t d e r i v a t i v e s are among the most potent odorants known f o r f i s h , with t h r e s h o l d s of 10" 8 to 10" 9 M r e p o r t e d from e l e c t r o p h y s i o l o g i c a l s t u d i e s (Doving et a l . 1980; Hara et a l . 1984; Groot et a l . 1986; Quinn and Hara 1986; Sorenson et a l . 1987), and 1 0 " 1 5 M from a b e h a v i o u r a l study (Jones and Hara 1985). Apart from hormonal s t e r o i d s , (Sorenson et a l . 1987), only amino a c i d s have been found to be such potent o l f a c t a n t s f o r f i s h , and these do not appear to mediate a t t r a c t i o n to c o n s p e c i f i c odour i n A r c t i c char (Olsen 1986b). Groot et a l . (1986) demonstrated that the e l e c t r o - o l f a c t o g r a m response of sockeye salmon to water c o n d i t i o n e d by c o n s p e c i f i c s was n e a r l y e l i m i n a t e d when the o l f a c t o r y organ was cross-adapted to the b i l e a c i d t a u r o c h o l i c a c i d , but not the amino a c i d L-c y s t e i n e . While the composition of g a l l b l a d d e r - and f a e c a l - b i l e of salmonids has been r e p o r t e d (e.g., Denton et a l . 1974; Sacquet et a l . 1979), i t has not been demonstrated that the blend of b i l e a c i d s or t h e i r d e r i v a t i v e s d i f f e r s between c o n s p e c i f i c p o p u l a t i o n s . S t a b e l l et a l . (1982) r e p o r t e d molecular v a r i a t i o n among the i n t e s t i n a l contents of three p o p u l a t i o n s of A t l a n t i c 126 salmon, which c o u l d have been d i f f e r e n t d i s t r i b u t i o n s of b i l e a c i d s . P o p u l a t i o n r e c o g n i t i o n by i n t e s t i n a l products has a l s o not been demonstrated. F i s k n e s and Doving (1982) used samples of the i n t e s t i n a l contents c o l l e c t e d by S t a b e l l et a l . (1982) to t e s t the hypothesis that f i s h are more s e n s i t i v e to the odours of p o p u l a t i o n members than other c o n s p e c i f i c s . E l e c t r o p h y s i o l o g i c a l r e c o r d i n g s of induced waves from the o l f a c t o r y bulb i n d i c a t e d that a l l f i s h were e q u a l l y s e n s i t i v e to i n t e s t i n a l samples from a l l donors. ( S i m i l a r l y , an e l e c t r o -o l f a c t o g r a m study by Groot et a l . (1986) showed that mature sockeye were not more s e n s i t i v e to the odours of p o p u l a t i o n members than other c o n s p e c i f i c s . ) Two b e h a v i o u r a l s t u d i e s have i n v e s t i g a t e d p o p u l a t i o n r e c o g n i t i o n by i n t e s t i n a l c o n t e n t s . S e l s e t and Doving (1980) r e p o r t e d that a d u l t char p r e f e r r e d water c o n d i t i o n e d by the i n t e s t i n a l contents of a smolt of t h e i r own p o p u l a t i o n over those of a smolt from another p o p u l a t i o n , but p o i n t e d out that the l a t t e r sample was not f r e s h and might have l o s t i t s a t t r a c t i v e p r o p e r t y . S t a b e l l (1987) r e p o r t e d that one p o p u l a t i o n of A t l a n t i c salmon parr p r e f e r r e d a water e x t r a c t of i n t e s t i n a l contents or an ethanol e x t r a c t of b i l e taken from members of t h e i r own p o p u l a t i o n to s i m i l a r samples from another p o p u l a t i o n . The r e s u l t s of these s t u d i e s are c o n s i s t e n t with but not proof of p o p u l a t i o n r e c o g n i t i o n , because i n each case only one p o p u l a t i o n was t e s t e d . P r e f e r e n c e s c o u l d have 127 r e f l e c t e d q u a l i t a t i v e or q u a n t i t a t i v e d i f f e r e n c e s between odours to which a l l f i s h would have responded s i m i l a r l y . S e v e r a l authors have s p e c u l a t e d that because some b i l e a c i d s are s t a b l e , are absorbed by organic matter and minerals i n the water, and are " s t i c k y " , they might p r o v i d e f i s h with a p o p u l a t i o n - s p e c i f i c or even t e r r i t o r i a l s u b s t r a t e marker (Doving et a l . 1980; S e l s e t 1980; F o s t e r 1985; Olsen 1986b; S a g l i o 1986; S t a b e l l 1987; Quinn and Courtenay 1989). F o s t e r (1985) r e p o r t e d that lake t r o u t ( S a l v e l i n u s namaycush) p r e f e r e n t i a l l y spawned on r e e f s upon which he had p l a c e d faeces of young of the year c o n s p e c i f i c s , suggesting that c o n s p e c i f i c chemosensory cues emanating from the s u b s t r a t e may i n f l u e n c e c h o i c e of nest s i t e . S t a b e l l (1987) found that members of one p o p u l a t i o n of A t l a n t i c salmon parr p r e f e r r e d water c o n d i t i o n e d by an e x t r a c t taken from the g r a v e l s u b s t r a t e of t h e i r h o l d i n g tank, to t h a t from the s u b s t r a t e of a tank h o l d i n g members of another p o p u l a t i o n . T h i s study addressed the f o l l o w i n g three q u e s t i o n s : 1. do c o n s p e c i f i c faeces render water f l o w i n g through a headtank a t t r a c t i v e or r e p u l s i v e to coho f r y , and i f so, 2. do f r y d i s t i n g u i s h d i f f e r e n t c o n c e n t r a t i o n s of faeces, and, 3. do f r y d i s t i n q u i s h chemical emanations of faeces of members of t h e i r own p o p u l a t i o n from those of members of another p o p u l a t i o n ? 128 METHODS AND MATERIALS F i s h used i n these experiments were members of the Quinsam R. (Q), Big Qualicum R. (BQ) and Puntledge R. (P) p o p u l a t i o n s , s u r p l u s from Experiment 1, Chapter 3. S i z e s of the f i s h before the present experiments (May 6,1987) are given i n the Treatment s e c t i o n of t h a t experiment. The mean f o r k - l e n g t h s and wet-weights of 20 f i s h of each p o p u l a t i o n measured a f t e r the present experiments (August 29) were not d i f f e r e n t (ANOVA). The pooled mean f o r k - l e n g t h was 8.8 cm (SD=7.4), and wet-weight was 7.93 g (SD=2.25). The t e s t procedure was s i m i l a r to that employed i n Chapter 3, except that faeces i n s t e a d of f i s h were p l a c e d i n the headtanks to c o n d i t i o n water. At 1500-1600 hours on the day before a t e s t , f i s h were p l a c e d i n one of two g l a s s a q u a r i a of dimensions 60 X 30 X 25 cm depth. Aquaria r e c e i v e d 5 L/min w e l l water, and d r a i n e d from the s u r f a c e through a c e n t r a l standpipe. Faeces accumulated on the aquarium bottom while u r i n e or other s o l u b l e matter probably d i d not; a high s a l i n i t y dye s o l u t i o n p i p e t t e d s l o w l y onto the aquarium bottom d i s p e r s e d throughout the tank w i t h i n 10 min and was no longer v i s i b l e i n 20 min. At 0800-0900 on the morning of a t e s t , faeces were siphoned from the bottom of the aquarium with a g l a s s tube i n t o a p l a s t i c f l a s k . The faeces and 1200 mL of aquarium water were siphoned onto the bottom of one headtank. A f t e r the siphon tube and f l a s k had been r i n s e d twice with hot w e l l water, 1200 mL of water was siphoned from the bottom of the aquarium, and p l a c e d 129 i n the second headtank, p r o v i d i n g a c o n t r o l f o r the a d d i t i o n t o the f i r s t headtank of substances other than f a e c e s . In the case of Experiment 3, the second headtank r e c e i v e d faeces i n 1200 mL of water - from f i s h of another p o p u l a t i o n , h e l d o v e r n i g h t i n a second aquarium. T e s t s were conducted between 0900 and 1500 hours. A f t e r a t e s t , headtanks were clea n e d as d e s c r i b e d i n Chapter 3, and l e f t to f l u s h o v e r n i g h t . Before new occupants were introduced to the aq u a r i a ( f o r faeces accumulation), the former occupants were removed, aq u a r i a were d r a i n e d and wiped with paper towel, r i n s e d , r e f i l l e d , and l e f t f o r s e v e r a l hours to f l u s h . Unless otherwise s p e c i f i e d , a l l experiments c o n s i s t e d of data c o l l e c t e d on two, g e n e r a l l y s e q u e n t i a l , days - with the faeces of one p o p u l a t i o n i n the l e f t headtank one day (and no fa e c e s , l e s s f a e c e s , or faeces of another p o p u l a t i o n i n the r i g h t headtank), and the reverse on the other day. A n a l y s i s f o l l o w e d that d e s c r i b e d i n Chapters 2 and 3. RESULTS AND DISCUSSION Experiment _1_: Response to Water C o n d i t i o n e d by Faeces To estimate the q u a n t i t i e s of faeces used i n t h i s experiment, on May 8 two groups of 100 g of Q f i s h (N=57,59) were h e l d f o r 17 hours, a f t e r which faeces were c o l l e c t e d and 130 accumulated on a f i l t e r paper. Wet weights of faeces were 0.953 and 1.223 g, r e s p e c t i v e l y , and weights a f t e r 5.5 hours of d r y i n g at 27.6-46.5°C were 0.198 and 0.203 g, r e s p e c t i v e l y . Coho f r y of a l l three p o p u l a t i o n s s t r o n g l y p r e f e r r e d water c o n d i t i o n e d by faeces from 100 g of (tankmate) members of t h e i r p o p u l a t i o n (N=56-64), over blank water ( F i g u r e 31, P<0.0001 f o r each p o p u l a t i o n ) . These experiments were conducted between May 5 and 13. While Q and P f i s h showed a stronger p r e f e r e n c e than BQ f i s h (ANCOVA, P<0.05; Tukey T e s t , P<0.05), the three were s i m i l a r i n showing no decrease i n response over the 6 hour d u r a t i o n of the t e s t ( F i g u r e 32). T h i s suggests t h a t the odorant was abundant i n faeces and was r e l e a s e d g r a d u a l l y . F o l l o w i n g two of the above t e s t s , faeces were l e f t i n the headtank o v e r n i g h t and t e s t i n g continued on the f o l l o w i n g day. In the f i r s t t e s t (May 9), Q f i s h showed no response (mean of 20 fish=51%, P=0.78), though on the preceding day the response had been st r o n g (mean of 44 fish=66%, P<0.0001). In the second t e s t (May 14), P f i s h p r e f e r r e d the f a e c e s - c o n d i t i o n e d water (mean of 28 fish=58%, P=0.033), though l e s s s t r o n g l y than on the preceding day (mean of 42 fish=70%, P<0.0001). I t appeared t h e r e f o r e t h a t the c o n c e n t r a t i o n of f a e c a l odour was d i m i n i s h e d by a day of r i n s i n g , but not always to an i n d i s t i n g u i s h a b l e l e v e l . The response of P f r y to f a e c a l odour was r e t e s t e d on 131 C A O O <D PH C A T3 rt !* a • rt a a • rt CO o a • rt H a U «H 4> PH 80 86 89 82 70 60 50 -40 -30 Attraction Avoidance Quinsam Big Puntledge Qualicum Population Figure 31. Preference of fry of three populations for water conditioned by faeces of population-members, over blank water. (Tests performed May 5-13) (x ± 95% CL, Number of fry tested shown above bar) 1 32 15 14 14 14 15 14 CO O O O cd U. to •o ea 60 c e c o o OH 80 r 70 60 50 B Q 80 70 60 50 80 70 60 50 13 p 14 15 12 16 12 16 16 14 14 Attraction 15 14 Attraction Attraction 3 4 Hour of Test F i g u r e 32. A t t r a c t i o n to water c o n d i t i o n e d by faeces of population-members over the 6 hour t e s t d u r a t i o n . Q: Quinsam R. f r y , BQ: B i g Qualicum R. f r y , P: Puntledge R. f r y . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 133 August 25 and 26. Instead of the strong a t t r a c t i o n recorded i n May, f r y avoided water c o n d i t i o n e d by the faeces of 100 g (N=15) of tankmates ( F i g u r e 33, P<0.0001). The change i n p r e f e r e n c e f o r f a e c a l odour, between May and August, may be r e a l or only apparent. I f r e a l , i t may r e f l e c t a change i n the p r e f e r e n c e of the f i s h , or a change i n the q u a n t i t y or q u a l i t y of odour emanating from the f a e c e s . In e i t h e r case, faeces c o u l d not be the only source of odour mediating a t t r a c t i o n to c o n s p e c i f i c s because p r e v i o u s experiments had i n d i c a t e d that c o n s p e c i f i c odours were s t i l l found a t t r a c t i v e i n l a t e November (Chapter 2) and the f o l l o w i n g February (Appendix 3). Other s t u d i e s with salmonids have a l s o shown that a t t r a c t i o n to the odours of c o n s p e c i f i c s i s not l i m i t e d to young f r y (Pete 1977; Quinn et a l . 1983; Quinn and Tolson 1986; Groot et a l . 1986). Olsen (1986b) r e p o r t e d that char f r y responded to amino a c i d s and ammonia i n q u a n t i t i e s s i m i l a r to those produced d u r i n g experiments i n which f r y were a t t r a c t e d to c o n s p e c i f i c odours. Both odours were avoided however, sugge s t i n g that on i t s own, n e i t h e r mediated a t t r a c t i o n . I t may be that f i s h respond to the whole bouquet of c o n s p e c i f i c odours. A l t e r n a t e l y , i t may be that n e i t h e r the odour nor the p r e f e r e n c e of the f i s h changed, and the apparent change was an a r t i f a c t of the experimental procedure. Conceivably, an i n c r e a s e i n the c o n c e n t r a t i o n of f a e c a l odorant, and/or s l i g h t l y warmer water temperatures i n August produced a " b a t t e r y e f f e c t " 1 34 58 CA <D O <D Cd fa CA T3 cd =5 O H 00 e a a •l-H CO <D E 70 60 Attraction 50 40 c o fa Avoidance 30 F i g u r e 33. P r e f e r e n c e of Puntledge R. f r y f o r blank water over water c o n d i t i o n e d by faeces of population-members. (Test performed August 25-26) (x ± 95% CL, Number of f r y t e s t e d shown above bar) 135 ( s t i c k i n g and r e l e a s i n g of r e s i d u e s ) of the kind p o s t u l a t e d to e x p l a i n the r e s u l t s of the morpholine experiments. (Temperature i n the Y-maze was not r o u t i n e l y monitored, but was 9.8°C at 1400 h on August 27 compared to 8.4°C on A p r i l 24.) The primary odorants i n faeces are l i k e l y b i l e a c i d s which are "renowned" fo r t h e i r adherent p r o p e r t i e s (Doving et a l . 1980). R e s o l u t i o n of whether the p r e f e r e n c e of coho f r y f o r faeces does change d u r i n g t h e i r f i r s t summer w i l l r e q u i r e a s e r i e s of experiments over that time p e r i o d , examining responses to a range of c o n c e n t r a t i o n s , while p r e v e n t i n g c r o s s - c o n t a m i n a t i o n . If a change i n response i s s t i l l observed, o l d e r f i s h should be t e s t e d with the faeces of younger f i s h to determine whether the change l i e s i n p r e f e r e n c e , or i n the odorant. Experiment 2: Response to Two D i f f e r e n t Q u a n t i t i e s of Faeces The hypothesis that d i f f e r e n t i a l d i s t r i b u t i o n and r e t e n t i o n of faeces i n headtanks i n f l u e n c e s the p r e f e r e n c e of t e s t f i s h , r e q u i r e s that f i s h respond not merely to a c h o i c e of the presence versus absence of faeces, but to a d i f f e r e n c e i n f a e c a l c o n c e n t r a t i o n . T h i s was t e s t e d on June 16 and 20 with P f i s h by p l a c i n g the faeces c o l l e c t e d from 150 g of tankmates (N=45 i n one t e s t , 46 i n the other) i n one headtank and the faeces c o l l e c t e d from 50 g of tankmates (N=14,16) i n the o t h e r . The water c o n d i t i o n e d by the l a r g e r amount of faeces was p r e f e r r e d ( F i g u r e 34A, P<0.0001). 1 36 to C • i — i a a •I-H CO a o o IU o rt PH t-H o -a t-H C3 80 70 60 A 50 -40 73 Prefer Faeces of 150 g Fish Prefer Faeces of 50 g Fish 30 60 C a a •i-H CO cu a a> o t-H PH co <U O rt PH co *o t-H rt 80 70 60 50 40 30 B 77 63 Prefer Faeces of 50 g Fish Prefer Blank Water F i g u r e 34. Preference of Puntledge R. f r y f o r water c o n d i t i o n e d by faeces of 150 g population-members over faeces of 50 g population-members (A), though faeces of 50 g population-members were d e t e c t e d and p r e f e r r e d over blank water (B). In B, Open bar: June 21 /22 , Shaded bar: J u l y 6/7. (x ± 95% CL, Number of f r y t e s t e d shown above bar) ' 1 37 To ensure that the smal l e r q u a n t i t y of faeces imparted a d e t e c t a b l e odour, on June 21 and 22, f i s h were o f f e r e d a ch o i c e of blank water and water c o n d i t i o n e d by faeces c o l l e c t e d from 50 g of tankmates (N=16,16). The faeces were d e t e c t e d and p r e f e r r e d over blank water ( F i g u r e 34B - open bar, P=0.0003). The two component t e s t s of t h i s experiment d i f f e r e d (P<0.0001); f i s h showed no pre f e r e n c e f o r faeces i n the l e f t headtank, but s t r o n g l y p r e f e r r e d the r i g h t headtank when i t co n t a i n e d the faeces on the f o l l o w i n g day. T h i s i n d i c a t e s that headtank b i a s cannot be e x p l a i n e d s o l e l y by the r e l a t i v e q u a n t i t y of faeces i n the two headtanks duri n g a t e s t . I t may be that i n some cases t e s t s are i n f l u e n c e d by odour r e s i d u e s remaining on the w a l l s of the headtanks and a s s o c i a t e d plumbing from a p r e v i o u s t e s t . I f so, f i s h i n the f i r s t t e s t of t h i s experiment might have d e t e c t e d f a e c a l odour from the r i g h t headtank, d e p o s i t e d d u r i n g the p r e v i o u s day when i t h e l d the faeces of 150 g of f i s h . ( I n c i d e n t a l l y , a f a e c a l r e s i d u e would not n e c e s s a r i l y smell s i m i l a r to f r e s h faeces; Doving et a l . (1980) s t a t e d that some b i l e a c i d s are r e a d i l y degraded and would have only a short term e f f e c t on f i s h while others are more s t a b l e . ) In any case, t h i s experiment was repeated on J u l y 6 and 7 (faeces c o l l e c t e d from 11, 12 f i s h ) , and a s i m i l a r r e s u l t , without s i g n i f i c a n t d i f f e r e n c e between r e p l i c a t e t e s t s , was recorded ( F i g u r e 34B shaded bar, P<0.0001). 138 Experiment 3: P o p u l a t i o n D i s c r i m i n a t i o n Experiment 1 suggested that faeces are one source of o l f a c t o r y stimulus i n experiments with c o n s p e c i f i c odours, and Experiment 2 i n d i c a t e d that f i s h d i s c e r n and p r e f e r the g r e a t e r of two c o n c e n t r a t i o n s of faeces, j u s t as they p r e f e r water c o n d i t i o n e d by the g r e a t e r of two c o n c e n t r a t i o n s of f i s h (Chapters 2 and 3). These r e s u l t s r a i s e d the p o s s i b i l i t y t h a t faeces c o n t a i n s the s p e c i f i c odorants that allow f i s h to d i s c r i m i n a t e between waters h o l d i n g members of t h e i r own and another p o p u l a t i o n (Chapter 3, Experiment 1). F i s h were o f f e r e d a choice of waters c o n d i t i o n e d by faeces of members of t h e i r own and another p o p u l a t i o n . Faeces were c o l l e c t e d from 100 g of each p o p u l a t i o n (N=34-55). On each of the two days of an experiment, members of both p o p u l a t i o n s were t e s t e d , i n a l t e r n a t i n g p a i r s of t r i a l s . Experiments were performed with the three odour combinations: Q/BQ, Q/P, and BQ/P between May 17 and June 11. Q f i s h p r e f e r r e d Q over BQ faeces (P=0.039) and BQ f i s h d i d the reverse (P=0.0077) (F i g u r e 35A). ( S i g n i f i c a n c e of d i f f e r e n c e of responses: P=0.0007.) S i m i l a r l y , Q f i s h p r e f e r r e d Q over P faeces (P=0.055) and P d i d the reverse (P=0.0082) (Fi g u r e 36A). ( S i g n i f i c a n c e of d i f f e r e n c e of responses: P=0.0012.) However, i n a s i n g l e t e s t c o n t r a s t i n g BQ and P fae c e s , f i s h of n e i t h e r p o p u l a t i o n showed any p r e f e r e n c e ( F i g u r e 37A). T h i s t e s t was repeated with faeces c o l l e c t e d from 139 CA O o cu a Gt c/5 a • rH a C/3 rH a a e •rH eu 6 a cu o VH eu A 45 46 60 50 40 B 60 50 40 42 41 Prefer Quinsam V Prefer Big Qualicum Prefer Quinsam • Prefer Big Qualicum Quinsam Big Qualicum Population Figure 35. Preference of Quinsam R. and Big Qualicum R. f i s h between waters conditioned by faeces of members of those two populations. A: Test conducted June 10-11. B: Test conducted July 14-15. (x ± 95% CL, Number of fry tested shown above bar) 140 C O o o .6 C O c o C A C fi fi fi A 44 41 60 50 40 B 46 44 60 50 <- 40 C o o Quinsam Puntledge Population Prefer Quinsam Prefer Puntledge Prefer Quinsam 1 Prefer Puntledge F i g u r e 36. Pref e r e n c e of Quinsam R. and Puntledge R. f i s h between waters c o n d i t i o n e d by faeces of members of those p o p u l a t i o n s . A: Test conducted May 17-18. B: Test conducted June 25-26. (x ± 95% CL, Number of f r y t e s t e d shown above bar) 141 A 44 20 21 44 24 23 70 -60 -50 -Prefer Big Qualicum A 40 - Prefer Puntledge B 45 60 50 -46 1 1 1 — — 1 Prefer Big Qualicum A 40 Prefer Puntledge Big Qualicum Puntledge Population F i g u r e 37. Preference of Big Qualicum R. and Puntledge R. f i s h between waters c o n d i t i o n e d by faeces of members of those p o p u l a t i o n s . A: T e s t s conducted May 19-22. B: Tes t s conducted June 23-24. In A, f o r each p o p u l a t i o n the three bars represent from l e f t to r i g h t : t e s t s with faeces c o l l e c t e d from 30, 100, and 300 g f i s h . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 142 300 g of each p o p u l a t i o n (N=134,140), and again no p r e f e r e n c e s were observed (Figure 37A). F i n a l l y , a p a i r of t e s t s was performed with the faeces c o l l e c t e d from 30 g of each p o p u l a t i o n (N=13-15). In t h i s experiment BQ f i s h p r e f e r r e d BQ over P faeces (P=0.0029) and P d i d the reverse (P=0.060) (Figure 37A). ( S i g n i f i c a n c e of d i f f e r e n c e of responses: P=0.0006). These experiments were repeated between June 23 and J u l y 15, using the faeces c o l l e c t e d from 100 g of f i s h (N=20-36) i n a l l c ases. No p r e f e r e n c e s were shown by members of any p o p u l a t i o n , and no d i f f e r e n c e s were recorded between the responses of the two p o p u l a t i o n s t e s t e d together i n each experiment ( F i g u r e s 35B, 36B, and 37B). The l o s s of d i s t i n c t i o n between the i n i t i a l and repeat experiments may have r e s u l t e d from the same f a c t o r that a f f e c t e d a t t r a c t i o n to f a e c a l odour i n Experiment 1 - a change i n the p r e f e r e n c e of the f i s h , a change i n the q u a n t i t y or q u a l i t y of f a e c a l odorant, or c r o s s - c o n t a m i n a t i o n of odours i n the Y-maze in the l a t e r t e s t s . I suspect that c r o s s - c o n t a m i n a t i o n may have been more s i g n i f i c a n t i n the l a t e r than e a r l i e r t e s t s because of s l i g h t l y warmer water temperatures and/or because the l a r g e r faeces broke apa r t more with h a n d l i n g , and may have r e l e a s e d more odour. The suggestion that h i g h c o n c e n t r a t i o n s of f a e c a l odorant i n t e r f e r r e d with p o p u l a t i o n d i s c r i m i n a t i o n i s supported by the o b s e r v a t i o n that i n the e a r l y t e s t s , BQ and P f i s h d i s c r i m i n a t e d between small but not l a r g e amounts of faeces ( F i g u r e 37A). A b e t t e r assay of faeces d i s c r i m i n a t i o n , would be 143 that d e s c r i b e d i n Experiment 1 , one i n which the p o t e n t i a l f o r c r o s s - c o n t a m i n a t i o n was e l i m i n a t e d , and the responses of f i s h to v a r i o u s c o n c e n t r a t i o n s of faeces were recorded throughout the s p r i n g and summer. In c o n c l u s i o n , the r e s u l t s of t h i s chapter suggest that f a e c a l matter i s a potent source of c o n s p e c i f i c odour. Fry d i s c r i m i n a t e between d i f f e r e n t c o n c e n t r a t i o n s of f a e c a l odorant, n e c e s s i t a t i n g r e g u l a t i o n of the r e t e n t i o n and exposure to water of faeces i n f u t u r e experiments i n v o l v i n g f i s h odours. Lack of such r e g u l a t i o n i n p r e v i o u s experiments, i n c l u d i n g those r e p o r t e d i n Chapters 2 and 3, probably accounted f o r a c o n s i d e r a b l e p r o p o r t i o n of the v a r i a b i l i t y i n responses noted w i t h i n and between t e s t s . Odorant appears to emanate from faeces f o r at l e a s t a day a f t e r d e p o s i t i o n (presumably longer i f d e p o s i t e d i n areas of low water f l o w ) , s u p p o r t i n g suggestions that faeces c o u l d f u n c t i o n as s u b s t r a t e markers (see I n t r o d u c t i o n ) . Such s u b s t r a t e marking i s known i n other animals; Werner et a l . (1987) r e p o r t e d that green iguanas p r e f e r r e d the odour of faeces of k i n over non-kin, and suggested that faeces might be used to mark the environment to exclude u n r e l a t e d c o n s p e c i f i c s . I t appeared i n the present study that f a e c a l odorant may be s u f f i c i e n t f o r p o p u l a t i o n d i s c r i m i n a t i o n , suggesting that i n t e s t i n a l products are not only the source of a t t r a c t a n t to c o n s p e c i f i c odours, but may a l s o mediate i n t r a s p e c i f i c d i s c r i m i n a t i o n s . A d d i t i o n a l experiments w i l l be r e q u i r e d to 144 v e r i f y t h i s and to determine whether p r e f e r e n c e s f o r f a e c a l odours change over the f i r s t summer of l i f e . I t seems more l i k e l y that they do not, and the apparent change i n the present study r e s u l t e d from c r o s s - c o n t a m i n a t i o n i n the t e s t apparatus. N e v e r t h e l e s s , the p o s s i b i l i t y that p r e f e r e n c e s are a f f e c t e d by age, odour c o n c e n t r a t i o n or some other f a c t o r i s suggested by the f a c t that two s t u d i e s o f the response of j u v e n i l e salmonids to one of the b i l e a c i d s - t a u r o c h o l i c a c i d , r e p o r t e d o p p o s i t e p r e f e r e n c e s . Jones and Hara (1985) r e p o r t e d weak a t t r a c t i o n i n A r c t i c char, while Royce-Malmgren and Watson (1987) r e p o r t e d r e p u l s i o n i n A t l a n t i c salmon. The complexity surrounding odour p r e f e r e n c e s and t h e i r study i s i l l u s t r a t e d by the demonstration that p r e f e r e n c e s of A t l a n t i c salmon par r f o r c e r t a i n amino a c i d s can be reversed, simply by reducing the pH of the water (Royce-Malmgren and Watson 1987). 145 CHAPTER 5. LEARNING AND MEMORY OF THE ODOUR OF A NATURAL WATER INTRODUCTION Has l e r and Wisby (1951) hypothesized that the f l o r a , rocks and s o i l s of a watershed impart a d i s t i n c t i v e odour to each stream, which salmon l e a r n before m i g r a t i n g to sea, and home to as a d u l t s . The hypothesis was supported by the demonstration that bluntnose minnows d i s t i n g u i s h e d waters c o l l e c t e d from two Wisconsin streams by o l f a c t i o n , and that the c h a r a c t e r i s t i c odours were i d e n t i f i a b l e by the f i s h year round. Subsequent s t u d i e s have confirmed that f i s h , i n c l u d i n g salmon, can d i s t i n g u i s h between d i f f e r e n t n a t u r a l waters (e.g., I d l e r et a l . 1961; Fagurlund et a l . 1963; McBride et a l . 1964; Walker 1967; M i l e s 1968; Jensen and Duncan 1971; S u t t e r l i n and Gray 1973;. Sandoval 1980; Brannon et a l . 1984) and that the d i s t i n c t i o n i s made by o l f a c t i o n (e.g., Groves et a l . 1968; Bertmar and T o f t 1969; Delacy et a l . 1969; Thunberg 1971; Brannon 1972; Dodson and Leggett 1974; Bodznick 1978a; F r e t w e l l 1985). There i s c o n s i d e r a b l e evidence that salmon l e a r n the chemical s i g n a t u r e of the ambient water d u r i n g s m o l t i n g and home to i t as a d u l t s (e.g., Donaldson and A l l e n 1958; Harden-Jones 1968; Jensen and Duncan 1971). C i r c u m s t a n t i a l evidence suggests th a t environmental odours may a l s o be l e a r n e d and remembered e a r l i e r i n l i f e . J u v e n i l e c u t t h r o a t t r o u t (Salmo c l a r k i L . ) , A t l a n t i c salmon, and coho a l l i n h a b i t p a r t i c u l a r areas of 146 streams or lagoons f o r long p e r i o d s , to which they home when ex p e r i m e n t a l l y d i s p l a c e d ( M i l l e r 1954; Saunders and Gee 1964; Day 1966; G a r c i a de L e a n i z 1989), or a f t e r seasonal h a b i t a t s h i f t s (Naslund 1989). Downstream displacement appears to r e s u l t i n more p r e c i s e homing than upstream displacement, suggesting t h a t smells may be i n f l u e n t i a l ( M i l l e r 1954; G a r c i a de Leaniz 1989). The importance of o l f a c t i o n i n non-r e p r o d u c t i v e homing has not been demonstrated i n salmonids, but has been i n another stream r e s i d e n t , the longear s u n f i s h (Lepomis m e g a l o t i s megalotis Raf.) (Gunning 1959). Homing by salmon parr may be f a c i l i t a t e d by faeces d e p o s i t e d i n the s u b s t r a t e ( S t a b e l l 1987), and/or by other l o c a l i z e d odours which f i s h l e a r n and remember. Experimental evidence that j u v e n i l e salmonids l e a r n the odours of n a t u r a l waters i s l i m i t e d to Brannon's (1972) and Bodznick's (1978a) s t u d i e s of the r o l e of o l f a c t i o n i n the lakeward m i g r a t i o n of sockeye f r y (see I n t r o d u c t i o n ) . Brannon concluded t h a t a l e v i n s l e a r n the c h a r a c t e r i s t i c odour of i n c u b a t i o n water, and remember i t f o r at l e a s t 14 d (see a l s o Wright 1985). Near the time of emergence, an i n h e r e n t p r e f e r e n c e f o r lake water appears, presumably as p a r t of a mechanism f o r l o c a t i n g a nursery l a k e . Bodznick (1978a) confirmed the inherent p r e f e r e n c e of f r y f o r lake water but found that recent experience was a l s o i n f l u e n t i a l . S w itching lake water-reared f r y to w e l l water r e s u l t e d i n a g r a d u a l disappearance of t h e i r p r e f e r e n c e f o r lake over w e l l water. Seventeen days l a t e r the f i s h showed the same lack of p r e f e r e n c e 147 as f i s h reared e n t i r e l y i n w e l l water. While Brannon's (1972) and Bodznick's (1978a) experiments suggest that the odours of n a t u r a l waters are l e a r n e d by salmonid a l e v i n s and young f r y , they do not i n d i c a t e whether these odours are remembered f o r more than 14-17 d, because the measure of r e c o g n i t i o n -p r e f e r e n c e - becomes dominated by inherent p r e f e r e n c e f o r l a k e water and r e c e n t l y encountered water. The o b j e c t i v e of the present study was to determine whether coho salmon l e a r n the chemical s i g n a t u r e of i n c u b a t i o n water and remember i t long-term. Coho were exposed to one n a t u r a l water -Rosewall Creek Water (CW) d u r i n g the a l e v i n and e a r l y f r y stages, and a f t e r 2 months of subsequent r e a r i n g i n w e l l water (WW), were t e s t e d f o r r e c o g n i t i o n of CW. R e c o g n i t i o n was d e f i n e d as p r e f e r e n c e over WW s i g n i f i c a n t l y g r e a t e r than t h a t shown by f r y r e a r e d e n t i r e l y i n WW. During the course of these t e s t s , i t became c l e a r that CW was i n h e r e n t l y more a t t r a c t i v e to f r y than WW, masking any e f f e c t of r e c o g n i t i o n that might e x i s t . T h e r e f o r e , an a l t e r n a t i v e assay of CW r e c o g n i t i o n was d e v i s e d ; f r y were t e s t e d f o r p r e f e r e n c e between an u n f a m i l i a r water -Nanaimo River water and a mixture of CW and Nanaimo River water. I t was hypothesized that f i s h p r e v i o u s l y exposed to CW would p r e f e r the mixture i f they recognized CW, whereas c o n t r o l s would not. 148 TREATMENT On November 25 1985, the eggs of four female and m i l t of four male coho salmon were t r a n s p o r t e d from the Quinsam River Hatchery to Rosewall Creek Hatchery. Gametes were pooled and d i v i d e d by volume i n t o two equal groups, one of which was used f o r t h i s experiment. The eggs were incubated i n a 60 X 30 X 30 cm basket of mesh n e t t i n g s t r e t c h e d around an aluminum frame (see Quinn and Hara 1986), suspended i n a darkened c i r c u l a r f i b e r g l a s s tank of 92 cm diameter with 50 cm of water. The tank was f l u s h e d with 8 L/min w e l l water (WW) of temperature 7.8-8.0°C. On the day a f t e r eggs hatched, (January 23, 1986), the a l e v i n s were d i v i d e d i n t o two groups. One group of 2400 continued r e a r i n g i n WW (Group W). The second group of 800 (Group C) was moved to a basket suspended i n another tank, i n which, over a p e r i o d of s e v e r a l hours, WW was r e p l a c e d with water pumped from Rosewall Creek (8 L/min). Creek water (CW) was c o o l e r than w e l l water, (4.1°C on January 23 to 6.0°C on March 31), and slowed the r a t e of development of Group C. Group W swam up and began fee d i n g on February 27, whereas Group C d i d not swim up u n t i l March 17 and d i d not begirt f e e d i n g u n t i l March 24. The water supply to Group C was slowly r e t u r n e d to WW on March 31, 2 weeks a f t e r swim-up. Fry were hand-fed Oregon Moist P e l l e t s (Moore-Clark Inc., LaConner WA) f i v e times a day f o r the f i r s t 2 weeks and three 149 times a day t h e r e a f t e r . Overhead f l u o r e s c e n t l i g h t i n g was programmed f o r an 8:16 L/D photoperiod. TESTING: PREFERENCE BETWEEN CREEK-WATER (CW) AND WELL-WATER (WW) Methods and M a t e r i a l s The p r e f e r e n c e of Groups C and W between WW and CW was t e s t e d between May 29 and June 8, 1986 at Rosewall Creek Hatchery. Except where noted otherwise, t e s t apparatus, p r o t o c o l , and data a n a l y s i s f o l l o w e d those d e s c r i b e d i n General Methods and M a t e r i a l s . The minimum number of days between t e s t i n g Group C and the end of exposure to CW was 58. Because the t e s t apparatus was l o c a t e d i n a d i f f e r e n t b u i l d i n g from the r e a r i n g tanks, CW was t r a n s p o r t e d to the t e s t apparatus from the v a l v e f o r m e r l y used to supply Group C, through a f i r e - h o s e . F i s h to be t e s t e d were moved to c i r c u l a r f i b e r g l a s s h o l d i n g tanks (35.5 cm ID, 40 cm depth of WW) near the t e s t apparatus, 1 h before t e s t s began. The arms of the Y-mazes r e c e i v e d water from d i f f e r e n t headtanks. For each t e s t , the water f l o w i n g through each headtank and temperatures of the waters i n the Y-maze and h o l d i n g tank are given i n Table 7. Members of both groups were t e s t e d on each day, one group f o r the f i r s t seven t r i a l s , the other f o r the l a s t seven, with the order a l t e r n a t e d d a i l y beginning with Group C f i r s t . 1 50 Table 7. Schedule and d e s c r i p t i o n of t e s t s of p r e f e r e n c e between creek water and w e l l water at Rosewall Creek Hatchery. LHT: l e f t headtank, RHT: r i g h t headtank. Temperatures were measured at the end of each t e s t ( 1 7 2 0 -1800 h ) . Under percent creek water, 20 i n d i c a t e s 2 0 % of the 5 L/min flow was creek water, 8 0 % was w e l l water. Date Percent Creek Water Flowing Through Temperatures Y-Maze ( ° C ) H o l d i n g Tank LHT RHT LHT RHT 2 9 / 0 5 / 8 6 0 1 00 9. 1 10.5 9.1 3 0 / 0 5 / 8 6 20 0 9.5 9.0 9.3 3 1 / 0 5 / 8 6 20 50 9.6 10.3 9.1 7 / 0 6 / 8 6 0 5 8.3 8.4 8.5 8 / 0 6 / 8 6 1 0 8.5 8.5 8.7 151 R e s u l t s Creek-Water (CW) vs Well-Water (WW) There was no s i g n i f i c a n t d i f f e r e n c e between the responses of Groups C and W to a c h o i c e of CW and WW; both p r e f e r r e d CW (P<0.0001) (Figure 38). One p o t e n t i a l e x p l a n a t i o n f o r the g r e a t e r a t t r a c t i v e n e s s of CW was i t s temperature; CW was 1.4°C nearer the p r e f e r r e d temperature range of 12-14°C ( B r e t t 1952) than WW (Table 7). To r u l e out the p o s s i b i l i t y t h a t p r e f e r e n c e s were being made on the b a s i s of temperature, the c o n c e n t r a t i o n of CW was reduced i n subsequent t e s t s by d i l u t i o n with WW. D i l u t i o n to 20% reduced the d i f f e r e n c e to 0.5°C, 5% to 0.1°C and 1% to l e s s than 0.1°C (Table 7). In each t e s t , Groups.C and W responded s i m i l a r l y , p r e f e r r i n g CW over WW (P<0.01) (F i g u r e 38). T h e r e f o r e the p r e f e r e n c e f o r CW over WW d i d not appear r e l a t e d to temperature. Rather, CW appeared to be i n h e r e n t l y more a t t r a c t i v e than WW to f r y , and no e f f e c t of p r i o r exposure to CW was d e t e c t e d i n Group C. Mixtures of Creek-Water (CW) and Well-Water (WW) In t h i s assay, Group C and Group W f i s h were t e s t e d f o r response to two mixtures of WW and CW - one c o n t a i n i n g more CW than the other (50% vs 20%). I t was hypothesized that d i s c r i m i n a t i o n of mixtures might r e q u i r e g r e a t e r s e n s i t i v i t y to CW than d i s c r i m i n a t i o n of the presence versus absence of CW, and 152 M CD -t-> cd £ M CD CD JH u cd [2 C 6 6 CO CD a •i—i H 100 9 0 -8 0 -7 0 6 0 5 0 4 0 -3 0 26 25 21 24 23 23 25 25 1 0 0 2 0 % Creek Water in One Arm of the Y-Maze F i g u r e 38. Pre f e r e n c e f o r creek water - d i l u t e d to v a r i o u s c o n c e n t r a t i o n s with w e l l water, over w e l l water. Shaded bars: f i s h exposed to creek water f o r 1 month around swim-up (Group C), Open b a r s : f i s h r e a r e d e n t i r e l y i n w e l l water (Group W). (x ± 95% CL, Number of f r y t e s t e d shown above bar) 153 that Group C might be more s e n s i t i v e or more h i g h l y motivated to approach CW than Group W. There was no s i g n i f i c a n t d i f f e r e n c e between the responses of Groups C and W; both groups p r e f e r r e d the stronger c o n c e n t r a t i o n of CW (P<0.001) (F i g u r e 39). T h e r e f o r e , i t appeared that both groups were e q u a l l y s e n s i t i v e to the g r e a t e r c o n c e n t r a t i o n of CW and e q u a l l y motivated to swim towards i t . TESTING: PREFERENCE BETWEEN CREEK-WATER (CW) AND A RIVER-WATER (NW) Methods and M a t e r i a l s On January 8, 1987, 200 f i s h of each group were t r a n s p o r t e d from Rosewall Creek Hatchery to the P a c i f i c B i o l o g i c a l S t a t i o n (Nanaimo, B.C.) (Figure 1), where they were h e l d i n c i r c u l a r f i b e r g l a s s h o l d i n g tanks (35.5 cm ID, 40 cm water depth) at a d e n s i t y of 100 per tank. (Mean s i z e of ten f i s h of Group C: 11.8 cm fork l e n g t h (SD=1.6), 10.6 g wet weight (SD-1.3).) Each tank r e c e i v e d 4 L/min Nanaimo R i v e r water (6.5-7.0°C). (See General Methods and M a t e r i a l s f o r chemical c h a r a c t e r i s t i c s of P a c i f i c B i o l o g i c a l S t a t i o n water.) Between January 9 and 18, 1987, the p r e f e r e n c e s of Groups W and C were t e s t e d between CW d i l u t e d to 10% with NW, and pure NW. The t e s t i n g procedure f o l l o w e d that d e s c r i b e d i n General Methods and M a t e r i a l s , except that only one Y-maze was used, and 1 54 u CD •*-> cd M CD CD u u o -o cd E2 d a 6 CD a •i—i H 24 23 90 80 70 60 50 40 30 Prefer 50% Creek Water Prefer 20% Creek Water W Treatment Group Figure 39. Preference for a mixture of 50% creek water/50% well water, over a mixture of 20% creek water/80% well water. Rest of legend as in Figure 38. 155 both arms of the maze r e c e i v e d 900 mL/min NW from a s i n g l e headtank. CW was d r i p p e d i n t o the upstream end of one arm at the r a t e of 100 mL/min, with a p e r i s t a l t i c pump. A s i m i l a r q u a n t i t y of NW water from the headtank was d r i p p e d i n t o the other arm, to c o n t r o l f o r odours of the pump t u b i n g . CW had been c o l l e c t e d from Rosewall Creek Hatchery 48 h before the t e s t , and s t o r e d i n a 50 L p o l y e t h y l e n e carboy (Nalge Co., Rochester NY) i n the dark at 3°C. (The carboy had been used p r e v i o u s l y f o r water storage, and was r i n s e d f r e q u e n t l y over s e v e r a l days before use.) I n d i v i d u a l f i s h were p l a c e d i n the Y-maze, l e f t f o r 5 min, observed f o r 5 min, and d i s c a r d e d . Before the next t r i a l , p e r i s t a l t i c pump tubes were switched between arms of the maze, and the maze was d r a i n e d . When the maze was roughly h a l f r e f i l l e d , the next f i s h was in t r o d u c e d . R e s u l t s Creek-Water (CW) ( s t o r e d i n p o l y e t h y l e n e carboy) There was no s i g n i f i c a n t d i f f e r e n c e between the responses of Groups C and W to CW; both groups avoided i t (P=0.0049 and 0.0170 r e s p e c t i v e l y ) (Figure 40). The avoidance of CW was d i f f i c u l t to r e c o n c i l e with the pr e f e r e n c e shown f o r i t over WW in assays at Rosewall Creek Hatchery. I t was hypothesized that the water sample had been a l t e r e d by 48 h of storage i n the po l y e t h y l e n e carboy. T h i s h y p othesis was t e s t e d i n the f o l l o w i n g assay. 1 56 <u +-> a M 4) (D U V T3 O H c a a C O O a •I-H H 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 Prefer 10% C W / 90% N W Prefer 100% N W W Treatment Group F i g u r e 40. P r e f e r e n c e f o r 100% Nanaimo R. water (NW) over 10% Rosewall Creek water (CW)/90% Nanaimo R. water. Creek water had been s t o r e d f o r 48 h i n a p o l y e t h y l e n e carboy. Rest of legend as i n F i g u r e 38. 157 E f f e c t of Storage i n the Carboy The response of Group C was t e s t e d to NW that had been s t o r e d f o r 24 h i n the carboy. F r e s h NW was pumped i n t o the other arm of the Y-maze to c o n t r o l f o r odours of the pump t u b i n g . NW s t o r e d i n the carboy was avoided (P=0.0066) (Figure 41) suggesting that the carboy imparted odours to the water which r e p e l l e d the f i s h . Creek-Water (CW) ( s t o r e d i n g l a s s b o t t l e s ) In t h i s assay, Groups C and W were t e s t e d f o r response to CW, t r a n s p o r t e d from Rosewall Creek Hatchery i n 10 4 L used, commercially c l e a n e d wine b o t t l e s . Before use, b o t t l e s were r i n s e d r e p e a t e d l y with hot NW and l e f t to soak o v e r n i g h t . B o t t l e s were r i n s e d twice with CW before samples were taken. Samples were s t o r e d f o r 18 h i n the dark at 3°C before use. F i s h responded s i m i l a r l y to CW s t o r e d i n the g l a s s b o t t l e s as they had to CW s t o r e d i n the carboy. There was no s i g n i f i c a n t d i f f e r e n c e between the responses of Groups C and W; both groups avoided CW (P<0.0001 and 0.0055 r e s p e c t i v e l y ) ( F i g u r e 42). These r e s u l t s suggest that the avoidance of CW s t o r e d i n the p o l y e t h y l e n e carboy was not caused by odours imparted by the carboy. Instead, i t appears that storage (ageing) a l t e r e d water samples, r e n d e r i n g them l e s s a t t r a c t i v e 1 58 12 CU u O •4-> CO co T3 »-i c d 60 C •i-H a a CO a H 70 60 r-50 h 40 30 20 10 h 0 F i g u r e 41. Pre f e r e n c e of group C f i s h f o r 100% f r e s h Nanaimo R. water (NW) over 10% s t o r e d Nanaimo R. water/90% f r e s h Nanaimo R. water. Stored water had been h e l d f o r 24 h i n a p o l y e t h y l e n e carboy. Rest of legend as i n F i g u r e 38. 1 59 <D -*-> M u t-H u CO l H a 60 g e e • ^H e H 70 60 50 40 30 20 10 0 Prefer 10% C W / 90% N W Prefer 100% N W W Treatment Group F i g u r e 42. Preference f o r 100% Nanaimo R. water (NW) over 10% creek water (CW)/90% Nanaimo R. water. Creek water had been s t o r e d f o r 18 h i n g l a s s b o t t l e s . Rest of legend as i n F i g u r e 38. 160 to f i s h . E f f e c t of Storage i n G l a s s B o t t l e s The hypothesis that storage rendered water samples l e s s a t t r a c t i v e to f r y was t e s t e d by s t o r i n g NW f o r 18 h i n the g l a s s b o t t l e s , and then g i v i n g f r y a c h o i c e of the s t o r e d NW pumped i n t o one arm of the Y-maze, and f r e s h NW pumped i n t o the other arm. F i s h avoided the s t o r e d NW (P=0.022) ( F i g u r e 43), sup p o r t i n g the hypothesis that water samples change d u r i n g storage, and become l e s s a t t r a c t i v e to f i s h . Creek-Water (CW) vs an u n f a m i l i a r River-Water (BQW) (both s t o r e d  i n g l a s s b o t t l e s ) The r e s u l t s of the above assays suggested t h a t storage rendered water samples l e s s a t t r a c t i v e to f i s h . I t was reasoned that t h i s e f f e c t of storage c o u l d not be e l i m i n a t e d , but c o u l d be compensated by pumping CW i n t o one arm of the Y-maze, and a s i m i l a r l y s t o r e d but d i f f e r e n t water i n t o the other arm. The d i f f e r e n t water was Big Qualicum R i v e r Water (BQW) c o l l e c t e d from the B i g Qualicum R i v e r Hatchery, 15 km southeast of Rosewall Creek Hatchery. (The chemical c h a r a c t e r i s t i c s of BQW are r e p o r t e d by M i l l e r et a l . 1987 as f o l l o w s : pH: 7.7; c o n d u c t i v i t y : 84.2 umbos/cm; f i l t e r a b l e r e s i d u e : 67 mg/L; a l k a l i n i t y : 37.2 mg/L as c a l c i u m carbonate; major c a t i o n s 1 6 1 10 *o CD I-I o ••-> CO co *a cd a •i—l s s ? CO s • I—I r-i 70 60 50 40 30 20 10 0 Prefer 10% Stored/ 90% Fresh N W Prefer 100% Fresh N W F i g u r e 43. Preference of group C f i s h f o r f r e s h Nanaimo R. water (NW) over 10% stored/90% f r e s h Nanaimo R. water. Stored water had been h e l d f o r 18 h i n g l a s s b o t t l e s . Rest of legend as i n F i g u r e 38. 162 (mg/L): c a l c i u m (12.2), magnesium (1.9), pottasium (0.2), s i l i c o n (3.4) sodium (1.6); major anions (mg/L): c h l o r i d e (1.3), n i t r a t e (0.03), s u l f a t e (2.1). These f i g u r e s apply to a sample taken i n August (1979), but samples c o l l e c t e d year round are s i m i l a r . ) CW and BQW were c o l l e c t e d i n the g l a s s b o t t l e s , w i t h i n 1 h of each o t h e r . Groups C and W were t e s t e d once with waters c o l l e c t e d 16 h before the t e s t and s t o r e d i n the dark at 3°C, and once with waters c o l l e c t e d 2 h before the t e s t . The responses of Groups C and W were not s i g n i f i c a n t l y d i f f e r e n t : both groups weakly (not s i g n i f i c a n t l y ) p r e f e r r e d CW over BQW ( F i g u r e 44). There was no s i g n i f i c a n t d i f f e r e n c e between responses to waters c o l l e c t e d 16 h and 2 h before the t e s t , f o r e i t h e r group. Data pooled a c r o s s the two t e s t s i n d i c a t e s a s i g n i f i c a n t p r e f e r e n c e by both groups f o r CW over BQW (P=0.035 (C), P=0.033 (W)). There i s no suggestion i n these data that Group C responded d i f f e r e n t l y than Group W to CW, and t h e r e f o r e no evidence that Group C remembered the odour of CW, from the a l e v i n and e a r l y f r y stages. DISCUSSION The response of coho f r y to a ch o i c e of n a t u r a l waters appears s i m i l a r to that r e p o r t e d by Brannon (1972) and Bodznick (1978a) f o r sockeye f r y : c l e a r p r e f e r e n c e s based on some c h a r a c t e r i s t i c ( s ) of the waters other than t h e i r f a m i l i a r i t y to the f i s h . As Bodznick (1978a) noted, t h i s does not imply t h a t the odour of n a t a l water i s not remembered, but does suggest 1 6 3 u CD •4-> cd M CD CD rH u (A cd 60 c 6 a CD s •rH H 90 80 70 60 50 40 30 8 10 10 10 18 20 Prefer Rosewall Creek Water Prefer Qualicum R. Water B A + B Test F i g u r e 44. Preference between Rosewall Creek water and Big Qualicum R. water, both d i l u t e d to 10% with Nanaimo R. water. Creek water and Big Qualicum R. water had been s t o r e d i n g l a s s b o t t l e s f o r .16 h (A) or 2 h (B). Rest of legend as i n F i g u r e 38. 164 that l e a r n i n g of these p a r t i c u l a r odours cannot be s t u d i e d through the p r e f e r e n c e s of f r y . If f a m i l i a r i t y does not determine p r e f e r e n c e between n a t u r a l waters, what does? CW and WW d i f f e r e d s l i g h t l y i n pH and probably i n f r e e carbon d i o x i d e c o n c e n t r a t i o n (Appendix 1), but these d i f f e r e n c e s probably d i d not determine p r e f e r e n c e . Pure WW and 99% WW/1% CW would have d i f f e r e d by l e s s than 0.01 pH u n i t s and 0.1 mg/L f r e e carbon d i o x i d e , yet were c l e a r l y d i s t i n g u i s h e d ( F i g u r e 38). I t seems more l i k e l y that CW c o n t a i n e d a potent a t t r a c t a n t not present, or present i n very low c o n c e n t r a t i o n i n WW. Calcium, i d e n t i f i e d by Bodznick (1978c) as h i g h l y a t t r a c t i v e t o sockeye f r y , was present i n s i m i l a r c o n c e n t r a t i o n s i n CW and WW (Appendix 1), so c o u l d not e x p l a i n the g r e a t e r a t t r a c t i v n e s s of CW. In f a c t , WW proved very s i m i l a r to CW i n i t s whole i n o r g a n i c composition (Appendix 1), r a i s i n g the p o s s i b i l i t y t h a t WW i s d e r i v e d of CW seepage through the creek bed. While seepage through g r a v e l and sand beds might not a l t e r the i n o r g a n i c composition of the water, i t c o u l d reduce the c o n c e n t r a t i o n of o r g a n i c s through b a c t e r i a l d e gradation and a d s o r p t i o n (personal communications, Dr. J . Atwater, Dept. C i v i l and Mechanical E n g i n e e r i n g , U n i v e r s i t y of B r i t i s h Columbia, Vancouver; Dr. K. H a l l , Westwater Research Center, U n i v e r s i t y of B r i t i s h Columbia, Vancouver). No measurements were made of the organic compositions of CW and WW, but c o n c e i v a b l y coho f r y responded to a g r e a t e r c o n c e n t r a t i o n i n CW of c o n s t i t u e n t s such as amino a c i d s and b i l e a c i d s emanating from Rosewall Creek's f i s h 165 community, which i n c l u d e s coho salmon. R e s u l t s of pre v i o u s s t u d i e s suggest that storage does not e l i m i n a t e the odours by which f i s h d i s t i n g u i s h n a t u r a l waters (e.g., H a s l e r and Wisby 1951; I d l e r et a l . 1961; Fagurlund et a l . 1963; McBride et a l . 1964; Walker 1967), but that at l e a s t one important odour component i s v o l a t i l e ( I d l e r et a l . 1961; Fagurlund et a l . 1963). Loss of a h i g h l y v o l a t i l e a t t r a c t a n t might e x p l a i n the reduced a t t r a c t i v e n e s s of s t o r e d water i n the present study. A l t e r n a t i v e l y , i t may be th a t some other r a p i d p h y s i c a l or chemical change r e s u l t e d from s t o r a g e . M i l e s (1968) r e p o r t e d that the a t t r a c t i v e n e s s of stream water to e l v e r s of the American e e l ( A n g u i l l a r o s t r a t a ) was hal v e d by storage i n a stoppered g l a s s b o t t l e f o r 1 d, and reduced to one t h i r d a f t e r 2 d. Water s t e r i l i s e d i n an aut o c l a v e d i d not l o s e a t t r a c t i v e n e s s , suggesting that the decrease i n a t t r a c t i v e n e s s was r e l a t e d t o b a c t e r i a l degradation of the a t t r a c t i v e component of the water. The a t t r a c t i v e component appeared to be p a r t i c u l a t e , as f i l t e r i n g the water through a 45 micron membrane reduced a t t r a c t i v e n e s s . However, a e r a t i o n of f i l t e r e d (and u n f i l t e r e d ) water i n c r e a s e d a t t r a c t i v e n e s s , from which M i l e s (1968) concluded that the p a r t i c u l a t e a t t r a c t a n t c o u l d be formed from d i s s o l v e d matter. Perhaps the most i n t e r e s t i n g r e s u l t i n t h i s chapter i s that coho f r y d i s c r i m i n a t e d between not only the presence and absence of a n a t u r a l water (Figure 38), but a l s o between two d i f f e r e n t d i l u t i o n s of i t (Figure 39). F r e t w e l l (1985) re p o r t e d that 166 a d u l t sockeye salmon a l s o show t h i s a b i l i t y ; f i s h d i s t i n g u i s h e d and p r e f e r r e d homestream water d i l u t e d 20% over homestream water d i l u t e d 50%. The response was shown to depend on o l f a c t i o n , and to be a p r e f e r e n c e f o r the g r e a t e r c o n c e n t r a t i o n of homestream water r a t h e r than an avoidance of the d i l u t a n t water. F r e t w e l l (1985) p o i n t e d out that t h i s o b s e r v a t i o n i m p l i e s a more s o p h i s t i c a t e d mechanism f o r r i v e r i n e homing than merely p o s i t i v e r h e o t a x i s i n the presence of homestream odour (as suggested by Johnsen and H a s l e r 1980). Johnsen and Hasler (1980) noted that i n f o l l o w i n g an odour t r a i l upstream coho salmon moved i n t o and out of the odour plume, and suggested that t h i s z i g - z a g swimming allowed the f i s h to a v o i d a d a p t a t i o n of the o l f a c t o r y sense. I t i s now known that some neurons i n the o l f a c t o r y system of f i s h do not adapt to odours, suggesting that z i g - z a g swimming may be unnecessary f o r m a i n t a i n i n g s e n s a t i o n of at l e a s t some odours (Doving 1989). F i g u r e 39 of the present study supports t h i s suggestion; f r y r e t a i n e d the a b i l i t y to d i s t i n g u i s h two c o n c e n t r a t i o n s of CW a f t e r a 5 to 20 minute p r e - t e s t p e r i o d . Doving (1989) suggested t h a t the true purpose of z i g - z a g swimming may be to gain sensory i n f o r m a t i o n other than o l f a c t o r y i n f o r m a t i o n . I t may a l s o be t h a t z i g - z a g swimming serves the f u n c t i o n of keeping the f i s h i n the g r e a t e s t c o n c e n t r a t i o n of odorant. 167 CHAPTER 6. GENERAL DISCUSSION Sy n t h e s i s of R e s u l t s Memory of Odours Presented i n E a r l y L i f e The r e s u l t s of Chapter 1 suggested that coho l e a r n e d the odour of morpholine d u r i n g each of the embryo, a l e v i n and e a r l y f r y stages, and showed a d i f f e r e n t b e h a v i o u r a l response than naive f i s h 54 t o 125 d a f t e r exposure. The s t a b i l i t y of the e f f e c t of morpholine exposure ( l e a r n i n g ) was r e f l e c t e d i n c a r d i a c responses 477 to 532 d a f t e r exposure. The r e p r o d u c a b i l i t y of the e f f e c t was confirmed when f i s h exposed f o r a 1 month p e r i o d at swim-up, showed b e h a v i o u r a l responses d i f f e r e n t than naive f i s h i n t e s t s 57 to 70 d l a t e r . Chapter 2 supports the i n t e r p r e t a t i o n t h a t the e f f e c t of morpholine exposure on subsequent p r e f e r e n c e was o d o u r - l e a r n i n g , by demonstrating a s i m i l a r e f f e c t of exposure to a n a t u r a l odour. F a m i l y - s p e c i f i c chemical emanations ( i . e . , odours -Appendix 3) presented d u r i n g the a l e v i n stage were a p p a r e n t l y r e c o g n i s e d 64 d, 67 d, and 160 d l a t e r , and odours presented d u r i n g the e a r l y f r y stage were a p p a r e n t l y r e c o g n i s e d 32 d and 44 d l a t e r . I t was not c l e a r from t h i s experiment whether l e a r n i n g of c o n s p e c i f i c odours occurs before h a t c h i n g . C h a r a c t e r i s t i c odours, or at l e a s t important c o n s t i t u e n t s of them, appear to be i n t e s t i n a l i n o r i g i n (Chapter 4) and may be produced by embryos i n q u a n t i t i e s too small to i n f l u e n c e other 168 embryos. T h i s suggests that i n nature, l e a r n i n g of s i b l i n g odours may not occur u n t i l a f t e r hatch, but does not r u l e out l e a r n i n g d u r i n g the embryo stage of odours emanating from y e a r l i n g p o p u l a t i o n members as suggested by S t a b e l l (1984). Chapter 3 presented f u r t h e r evidence that long-term memories of c o n s p e c i f i c odours - i n t h i s case p o p u l a t i o n -s p e c i f i c odours - were formed d u r i n g or s h o r t l y (< 14 d) a f t e r i n c u b a t i o n and were r e f l e c t e d i n p r e f e r e n c e s 69-79 d l a t e r (Assay 2). Chapter 5 f a i l e d to demonstrate l e a r n i n g of the "odour bouquet" of a stream d u r i n g the a l e v i n and e a r l y f r y stages. Pr e f e r e n c e among n a t u r a l waters appeared to be determined by f a c t o r s other than t h e i r f a m i l i a r i t y . In t h i s r e s p e c t , coho f r y appear to respond s i m i l a r l y to sockeye f r y (Brannon 1972; Bodznick 1978a). The Process of L e a r n i n g With r e s p e c t to the q u e s t i o n of temporal or developmental r e s t r i c t i o n of l e a r n i n g , the l e a r n i n g of f a m i l y - s p e c i f i c odours (Chapter 2) c o u l d a l l have o c c u r r e d around the time of swim-up (as c o u l d l e a r n i n g of p o p u l a t i o n - s p e c i f i c odours - Chapter 3). The data suggested that one f a m i l y l e a r n e d the odour presented d u r i n g the a l e v i n stage, while another l e a r n e d the odour presented d u r i n g the e a r l y f r y stage, and a t h i r d f a m i l y l e a r n e d odours presented i n both stages. Conceivably, on the day that 169 odours were changed, the three f a m i l i e s were at s l i g h t l y d i f f e r e n t stages of development; the f i r s t having moved beyond the c r i t i c a l p e r i o d while the second had not yet e n t e r e d i t and the t h i r d was intermediate and imprinted on both odours. T h i s i m p l i e s that two odours encountered s e q u e n t i a l l y d u r i n g a s i n g l e s e n s i t i v e or c r i t i c a l p e r i o d are both i m p r i n t e d . In b i r d s , the act of i m p r i n t i n g may b r i n g the s e n s i t i v e p e r i o d to a c l o s e , making subsequent i m p r i n t i n g d i f f i c u l t (Boakes and Panter 1985). However, experiments with spiny mice (Acomys c a h i r i n u s ) suggest that subsequent i m p r i n t i n g may occur. In spiny mice there i s a s e n s i t i v e p e r i o d i n the f i r s t 3 d a f t e r b i r t h f o r l e a r n i n g of odours, and odours encountered d u r i n g t h i s p e r i o d are subsequently p r e f e r r e d over u n f a m i l i a r odours ( P o r t e r and E t s c o r n 1974, 1976). I f two odours are presented s e q u e n t i a l l y d u r i n g t h i s p e r i o d - the f i r s t i s p r e f e r r e d over the second un l e s s the second i s presented f o r a longer p e r i o d than the f i r s t , i n which case no p r e f e r e n c e i s seen between the two ( P o r t e r and E t s c o r n 1975). T h i s r e s u l t suggests a "primacy" e f f e c t - the f i r s t odour i s more e a s i l y imprinted, but may a l s o suggest that the second odour was l e a r n e d . While the r e s u l t s of Chapters 2 and 3 were not n e c e s s a r i l y i n c o n s i s t e n t with r e s t r i c t i o n of l e a r n i n g to the time of swim-up, the r e s u l t s of Chapter 1 were. The odour of morpholine was a p p a r e n t l y l e a r n e d d u r i n g the e a r l y f r y , a l e v i n , and even d u r i n g the embryo stage. A t e c h n i c a l p o i n t about the morpholine experiment r e p o r t e d 170 i n Chapter 1 - Part 1 deserves comment. I t was suggested i n Chapter 1 that morpholine forms r e s i d u e s on s u r f a c e s which are l a t e r r e l e a s e d . T h i s p r o p e r t y r a i s e s the p o s s i b i l i t y t h a t r e s i d u e s p e r s i s t e d i n r e a r i n g tanks beyond r e p o r t e d exposure p e r i o d s , and c o u l d have been l e a r n e d l a t e r than suggested. However, given the low c o n c e n t r a t i o n of morpholine used (5 X 10" 5 mg/L) and high r a t e of water flow (4 L/min) through the small (40 L) tanks, r e s i d u e s are u n l i k e l y to have p e r s i s t e d f o r long. During embryo stage exposure, i n t r o d u c t i o n of morpholine was stopped 10 d before eggs hatched (Table 1), r e n d e r i n g i t u n l i k e l y that r e s i d u e s were s t i l l present to be d e t e c t e d by a l e v i n s . The data presented i n Chapter 1 are to my knowledge the f i r s t evidence that f i s h form long-term memories of odours d u r i n g the embryonic and l a r v a l stages, but s i m i l a r a b i l i t i e s have been shown i n other animals. While in u t e r o , r a t f e t u s e s l e a r n chemical cues, probably s m e l l s , of the mother and even of her d i e t , and a f t e r b i r t h , these cues are p r e f e r r e d by the pups over u n f a m i l i a r chemical cues (Hepper 1987, 1988; Smotherman and Robinson 1988). Assuming that morpholine and n a t u r a l odours are l e a r n e d s i m i l a r l y (Hasler and Scholz 1983), s e v e r a l t h i n g s can be s a i d about the l e a r n i n g p r o c e s s . F i r s t , i t does not appear to be r e s t r i c t e d to a s i n g l e , b r i e f , d e v e l o p m e n t a l l y - f i x e d " c r i t i c a l p e r i o d " such as swim-up (although swim-up and other times c o u l d be s e n s i t i v e p e r i o d s ( H o r r a l l 1981)). Second, l e a r n i n g of one 171 odour does not appear to prevent subsequent l e a r n i n g of other odours (Chapter 2). Brannon (1972) reached the same c o n c l u s i o n in h i s experiments with sockeye a l e v i n s , and Bodznick's (1978a) experiments with sockeye f r y a l s o appear to show t h i s ( i . e . , p r e f e r e n c e among water sources changed with e x p e r i e n c e ) . T h i r d , u n l i k e the very r a p i d o d o u r - l e a r n i n g of the smolt stage (e.g., M i g h e l l (1975, c i t e d by H a s l e r and Scholz 1983) found that 4 h was a s u f f i c i e n t p e r i o d f o r h o l d i n g coho smolts i n a new stream to ensure homing to i t ) , o d o u r - l e a r n i n g i n e a r l y l i f e may r e q u i r e longer exposure. The t h i r d p o i n t i s s p e c u l a t i v e , and a r i s e s from comparison of the morpholine experiment with a p r e l i m i n a r y study by S t e f f e l ( r e p o r t e d i n Cooper and Hasler 1973). U n l i k e the present study in which coho were exposed to morpholine f o r at l e a s t 14 d (Table 1), S t e f f e l exposed coho f r y to 5 X 10" 5 mg/L morpholine fo r only 12 or 36 h. Fry t e s t e d i n a Y-maze immediately a f t e r or 24 h a f t e r exposure d i d not a v o i d morpholine as naive f i s h d i d - i n d i c a t i n g a short term e f f e c t of exposure, but t h i s e f f e c t disappeared i f t e s t i n g was postponed f o r 72 h. Sample s i z e s i n S t e f f e l ' s study were s m a l l , and, on i t s own, the d i f f e r e n c e i n r e s u l t from the present study i s not c o n v i n c i n g . In t h e i r study of o l f a c t o r y i m p r i n t i n g by green sea t u r t l e s , Grassman and Owens (1987) a l s o concluded that long-term exposure was necessary f o r l e a r n i n g . T u r t l e s exposed to morpholine or phenethyl a l c o h o l throughout the f i r s t 3 months of l i f e (the embryo and h a t c h l i n g stages) subsequently showed 172 p r e f e r e n c e f o r the i m p r i n t i n g odour, while t u r t l e s exposed du r i n g only the embryo or h a t c h l i n g stages d i d not. Grassman and Owens concluded t h a t , " E i t h e r we are d e a l i n g with an i m p r i n t i n g process with a prolonged c r i t i c a l p e r i o d or we may be d e a l i n g with some other developmental p s y c h o b i o l o g i c a l mechanism that d i f f e r s s l i g h t l y from i m p r i n t i n g . " In e i t h e r case, the e f f e c t does not appear to be p e c u l i a r to the odour morpholine* The experiments of Brannon (1972) and Bodznick (1978a) a l s o support the suggestion that odour l e a r n i n g by young salmon i s a slow p r o c e s s . Brannon (1972) r e p o r t e d that while a l e v i n s that had been switched from one water source to another 14 d p r e v i o u s l y s t i l l p r e f e r r e d t h e i r o r i g i n a l i n c u b a t i o n water, they d i d so more weakly than a l e v i n s not switched. T h i s r e s u l t admits to many i n t e r p r e t a t i o n s , but Brannon's was that while a l e v i n s s t i l l regarded the f i r s t water as the more f a m i l i a r of the two, the second was being l e a r n e d , becoming f a m i l i a r , and hence i n c r e a s i n g i n a t t r a c t i v e n e s s . S i m i l a r l y , Bodznick (1978a) showed that f r y switched from lake to w e l l water took 17 d to l o s e t h e i r p r e f e r e n c e f o r lake over w e l l water, and show the l a c k of p r e f e r e n c e c h a r a c t e r i s t i c of f i s h r e a r e d e n t i r e l y i n w e l l water. Bodznick suggested that p r e f e r e n c e s were the r e s u l t of both an inherent p r e f e r e n c e f o r lake water ( f r y reared i n w e l l water a c q u i r e d i t w i t h i n 1 d ) , and of a l e a r n e d p r e f e r e n c e f o r r e c e n t l y encountered water. Taken together, the experiments of Brannon (1972) and Bodznick (1978a) suggest that the f u l l e f f e c t s of l e a r n i n g may r e q u i r e more than 2 weeks of exposure at t h i s e a r l y stage of l i f e . 173 The c o n t r i b u t i o n s of the experiments i n t h i s d i s s e r t a t i o n are t w o f o l d : f i r s t , the demonstration that coho f r y and smolts remember o l f a c t o r y c h a r a c t i s t i c s of t h e i r n a t a l environment, and second, the demonstration that t h i s l e a r n i n g i s observable i n t h e i r c a r d i a c and p r e f e r e n c e responses. Using these responses, i t should be p o s s i b l e to determine whether the process of l e a r n i n g i s i m p r i n t i n g . I f the process through which odours are l e a r n e d i n e a r l y l i f e i s i m p r i n t i n g , a t e m p o r a l l y - or d e v e l o p m e n t a l l y - r e s t r i c t e d p e r i o d e x i s t s d u r i n g which exposure to an odour r e s u l t s i n a more s t a b l e e f f e c t than the same d u r a t i o n of exposure e i t h e r e a r l i e r or l a t e r (Immelmann and Suomi 1981). The demonstration of s t a b i l i t y of e f f e c t r e q u i r e s that f i s h exposed at d i f f e r e n t times be t e s t e d both at the same age (and t h e r e f o r e d i f f e r e n t i n t e r v a l s from exposure) and same i n t e r v a l from exposure (hence d i f f e r e n t ages) (Bateson and Hinde 1987). I t was on the b a s i s of these c r i t e r i a that Morin et a l . (1989a) concluded that a s e n s i t i v e p e r i o d f o r o l f a c t o r y i m p r i n t i n g (SPOI) of L - c y s t e i n e o c c u r r e d d u r i n g the smolt stage i n A t l a n t i c salmon. I t i s important to p o i n t out that the s e n s i t i v e p e r i o d i d e n t i f i e d by Morin et a l . (1989a) r e l a t e d to the response of c a r d i a c d e c e l e r a t i o n by immature f i s h , and might or might not a l s o r e l a t e to homing behaviour. S i m i l a r l y , a s e n s i t i v e p e r i o d f o r the l e a r n i n g that i n f l u e n c e s p r e f e r e n c e and c a r d i a c responses of coho f r y may or may not be a s e n s i t i v e p e r i o d f o r the a c q u i s i t i o n of i n f o r m a t i o n used i n homing. 174 Two hypotheses worthy of i n v e s t i g a t i o n a r e : 1. there i s a f a i r l y b r i e f (< 2 weeks) SPOI a s s o c i a t e d with a l e v i n / p a r r t r a n s i t i o n and 2. there i s a long SPOI, beginning i n the l a t e embryo stage, and ending d u r i n g the f i r s t summer. The f i r s t h y p o t h esis has been suggested by a number of i n v e s t i g a t o r s i n r e l a t i o n to the homing response (e.g., Harden-Jones 1968; H o r r a l l 1981). T h i s hypothesis i s c o n s i s t e n t with S c h o l z ' s (1980) hypothesis of t h y r o i d involvement i n i m p r i n t i n g ; swim-up i n coho as w e l l as chum, pink, chinook and A t l a n t i c salmon i s a s s o c i a t e d with a peak i n plasma t h y r o x i n e (T4), though not 3 , 5 , 3 ' - t r i i o d o - L - t h y r o n i n e (T3) ( S u l l i v a n et a l . 1983; D i c k h o f f and S u l l i v a n 1987; S u l l i v a n et a l . 1987). B r i e f d u r a t i o n of s e n s i t i v i t y would a l s o be c o n s i s t e n t with the b r i e f (< 8 d) SPOI i d e n t i f i e d i n A t l a n t i c salmon smolts by Morin et a l . (1989a). The second hypothesis r e q u i r e s some e x p l a n a t i o n . Long s e n s i t i v e p e r i o d s f o r the a c q u i s i t i o n of c e r t a i n kinds of i n f o r m a t i o n are known to occur i n other animals. For example, the s e n s i t i v e p e r i o d f o r sexual i m p r i n t i n g i n g r e y l a g geese (Anser anser) p e r s i s t s through at l e a s t the f i r s t 150 d of l i f e (Schutz 1969). Coho in the present study l e a r n e d the odour of morpholine d u r i n g the embryo, a l e v i n and e a r l y f r y stages, r a i s i n g the p o s s i b i l i t y of a long s e n s i t i v e p e r i o d beginning i n the embryo stage. T h i s h y p o t h e s i s i s c o n s i s t e n t with Scholz's (1980) hypothesis of t h y r o i d involvement i n i m p r i n t i n g , as t h y r o i d hormones have been shown to be present i n the y o l k of coho eggs, and i n the blood of a l e v i n s ( S u l l i v a n et a l . 1987). 175 During the f i r s t s p r i n g of l i f e , coho f r y experience a peak i n t h y r o i d a c t i v i t y of s i m i l a r t i m i n g and d u r a t i o n t o the peak experienced d u r i n g the second s p r i n g (the smolt s t a g e ) , though of s m a l l e r s i z e ( D i c k h o f f et a l . 1982). Termination of the s e n s i t i v e p e r i o d a f t e r the s p r i n g i s suggested by a decrease i n plasma T4 c o n c e n t r a t i o n i n l a t e May, and T3 c o n c e n t r a t i o n i n l a t e J u l y . D i c k h o f f et a l . (1982) r e p o r t e d that d u r i n g the v e r n a l p e r i o d of t h y r o i d a c t i v i t y , coho p l a c e d i n a novel water source responded with a b r i e f (1 d) e l e v a t i o n i n plasma T4 c o n c e n t r a t i o n . C o n t r o l experiments i n d i c a t e d that t h i s • e f f e c t was not due to e i t h e r h a n d l i n g s t r e s s or s o c i a l f a c t o r s . The authors s p e c u l a t e d that t h i s response might s i g n a l "a mechanism fo r f a c i l i t a t i n g i m p r i n t i n g on chemical cues i n the water d u r i n g downstream m i g r a t i o n " . P r e l i m i n a r y experiments suggested that t h i s response to novel water does not occur o u t s i d e of the v e r n a l p e r i o d , and f u r t h e r experiments to c o n f i r m t h i s are planned ( p e r s o n a l communication, Dr. W.W. D i c k h o f f , U.S. N a t i o n a l Marine F i s h e r i e s S e r v i c e , S e a t t l e , WA). A number of q u e s t i o n s about the mechanism of o l f a c t o r y i m p r i n t i n g are r a i s e d by the f a c t t h a t salmonids do not always home. Quinn and Fre s h (1984) noted that "The phenomenon of s t r a y i n g i n salmon i s g e n e r a l l y ignored, and r e l i a b l e estimates of the a c t u a l p r o p o r t i o n s of salmon that home are v i r t u a l l y n o n e x i s t e n t . " However, that s t r a y i n g occurs i s obvious from the f a c t t h a t salmonids have r e c o l o n i z e d areas that were g l a c i a t e d 176 u n t i l 10,000 years ago. Most of the p o p u l a t i o n s of coho salmon along the g l a c i a t e d P a c i f i c coast of North America, south of the Alaska P e n i n s u l a are b e l i e v e d to be the r e s u l t of p o s t g l a c i a l d i s p e r a l from a refuge i n the Columbia River system (McPhail and Lindsey 1970:172). The r a p i d i t y with which t h i s process can occur i s i l l u s t r a t e d by the. c o l o n i z a t i o n of the Great Lakes by pink salmon. A s i n g l e i n t r o d u c t i o n of 21,000 f i n g e r l i n g s to a t r i b u t a r y of Lake Superior i n 1956 gave r i s e to spawning p o p u l a t i o n s i n a l l of the Great Lakes a f t e r only 12 g e n e r a t i o n s (Kwain 1987; Emery 1981). The cause of s t r a y i n g i s unknown (Quinn 1984), but two hypotheses have been proposed which generate d i f f e r e n t p r e d i c t i o n s about the mechanism of o l f a c t o r y i m p r i n t i n g . Quinn (1984) hypothesized that homing and s t r a y i n g are a l t e r n a t i v e l i f e - h i s t o r y s t r a t e g i e s which e x i s t i n dynamic balance; the homing genome being favoured i n s t a b l e freshwater environments and the s t r a y i n g genome i n unstable freshwater environments. Quinn d i d not s p e c u l a t e about what d i f f e r s between s t r a y e r s and homers, but i t has been suggested that the d i f f e r e n c e may l i e i n the i m p r i n t i n g mechanism. Immelmann (1975) noted that i n d i v i d u a l v a r i a t i o n , presumed to have a g e n e t i c b a s i s , has been d e t e c t e d i n the " i m p r i n t a b i l i t y " of some animals to c e r t a i n o b j e c t s , and suggested that s i m i l a r v a r i a b i l i t y might e x i s t i n i m p r i n t i n g of h a b i t a t c h a r a c t e r i s t i c s . I n d i v i d u a l s i m p r i n t i n g s t r o n g l y would adhere s t r i c t l y to that environment, while i n d i v i d u a l s not i m p r i n t i n g s t r o n g l y "represent some kind of a " p o o l " , the members of which are able to c o l o n i z e new n i c h e s , 177 e.g. i n a changing environment or i n marginal zones of the d i s t r i b u t i o n a l area ..." (Immelmann 1975). T h i s hypothesis p r e d i c t s that c o n s i d e r a b l e v a r i a b i l i t y e x i s t s i n the a b i l i t y of salmonids to l e a r n or imprint odours, and s t r a y s are f i s h t h a t f a i l to i m p r i n t . (Dodson (1988) a l s o s p e c u l a t e d that s t r a y i n g r e f l e c t s a l a c k of i m p r i n t i n g , but suggested that " s t r a y i n g may not be an a l t e r n a t i v e s t r a t e g y but simply a m a n i f e s t a t i o n of l e s s p r e c i s e homing.") A very d i f f e r e n t e x p l a n a t i o n f o r s t r a y i n g by salmonids was o f f e r e d by Baker (1982). Baker suggested that the e x t e n s i v e movements made by j u v e n i l e s around the home r i v e r system before seaward m i g r a t i o n , are e x p l o r a t i o n s d u r i n g which the s u i t a b i l i t y of v a r i o u s areas as spawning s i t e s or r e a r i n g s i t e s f o r progeny are assessed, and remembered. Con c e i v a b l y , salmon f r y assess environmental q u a l i t y through t h e i r r a t e of growth (Thorpe 1987) or through some p h y s i o l o g i c a l c o r r e l a t e such as t h y r o i d a c t i v i t y ( D i c k h o f f and S u l l i v a n 1987). A d d i t i o n a l e x p l o r a t i o n , of more d i s t a n t areas, may occur d u r i n g homing m i g r a t i o n , e x p l a i n i n g the w e l l known " t e s t i n g " of non-natal streams. S t r a y s , Baker suggests, are not f a i l e d i m p r i n t e r s , but f i s h t h a t have "judged" the n a t a l area to be an i n f e r i o r spawning s i t e to a s i t e d i s c o v e r e d d u r i n g e x p l o r a t i o n s . T h i s e x p l a n a t i o n r e q u i r e s somewhat more a n a l y t i c a l c a p a b i l i t y than most might be prepared to a s s i g n to f i s h , but i t has a c e r t a i n charm as a p r o p o s i t i o n to d i s p r o v e . Three p r e d i c t i o n s are generated by Baker's h y p o t h e s i s . 178 F i r s t , i n d i v i d u a l v a r i a t i o n i n l e a r n i n g a b i l i t y s h o u l d be low; a l l i n d i v i d u a l s s h o u l d be a b l e t o r e l o c a t e the n a t a l a r e a . Second, odours (and o t h e r h a b i t a t c h a r a c t e r i s t i c s ) s h o u l d be l e a r n e d whenever e n c o u n t e r e d . As coho f r y of some p o p u l a t i o n s make e x t e n s i v e movements i n a l l se a s o n s , t h i s presumably r u l e s out a d e v e l o p m e n t a l l y or t e m p o r a l l y r e s t r i c t e d i m p r i n t i n g mechanism. T h i r d , w h i l e odours s h o u l d be remembered t o f a c i l i t a t e homing, o t h e r h a b i t a t c h a r a c t e r i s t i c s s h o u l d a l s o be remembered, and the a s s o c i a t i o n of the odour and o t h e r c h a r a c t e r i s t i c s s h o u l d be remembered. The i d e a t h a t odours may not be the o n l y n a t a l a r e a c h a r a c t e r i s t i c l e a r n e d by sal m o n i d s has been e x p r e s s e d by o t h e r a u t h o r s ( e . g . , H o r r a l l 1981). The c o n t r a s t of the p r e d i c t i o n s of Baker's (1982) and Immelmann's (1975) hypotheses i l l u s t r a t e j u s t how much remains t o be l e a r n e d about the mechanisms of o l f a c t o r y i m p r i n t i n g . The F u n c t i o n of Odour L e a r n i n g Homing The d e m o n s t r a t i o n t h a t odours a r e l e a r n e d i n e a r l y l i f e i s not of i t s e l f e v i d e n c e t h a t t h i s i n f o r m a t i o n i s used i n r e p r o d u c t i v e homing. However, e v i d e n c e t h a t one s a l m o n i d - the l a k e t r o u t - homes t o odours l e a r n e d i n v e r y e a r l y l i f e has r e c e n t l y been produced by Dr. R. H o r r a l l ( p e r s o n a l communication, U n i v e r s i t y of W i s c o n s i n , Madison WN). H o r r a l l (1981) 179 " . . . a r t i f i c i a l l y imprinted two groups of lake t r o u t to phenethyl a l c o h o l : one group d u r i n g the p e r i o d from the eyed-egg stage u n t i l the e a r l y f i n g e r l i n g stage; the other group at the y e a r l i n g stage 1 mo before s t o c k i n g . These f i s h , along with c o n t r o l s , were stocked i n t o Lake Michigan f o r a f i e l d t e s t u s ing s y n t h e t i c decoy odors. Each group c o n t a i n e d about 64000 f i s h and a small number of each group was r e t a i n e d i n the l a b o r a t o r y f o r b e h a v i o r a l experiments. Two of the o b j e c t i v e s of these s t u d i e s are to determine c r i t i c a l p e r i o d s f o r o l f a c t o r y i m p r i n t i n g and to assess the r e l a t i v e importance of n a t u r a l and s y n t h e t i c odors i n the decoy experiments." H o r r a l l i s p r e s e n t l y p r e p a r i n g the r e s u l t s of t h i s study f o r p u b l i c a t i o n , and has k i n d l y given me permission to c i t e them. For 4 years (1984-1987), dur i n g the f a l l spawning season when experimental f i s h were expected to "home", phenethyl a l c o h o l was d r i p p e d from buoys p l a c e d 8-11 km from the s t o c k i n g s i t e . M o n i t o r i n g of f i s h a t t r a c t e d to the buoys i n d i c a t e d " r e t u r n s " of approximately twice as many of the e a r l y exposed group as c o n t r o l s . The y e a r l i n g exposed group ( f i s h exposed at age 15.5-17 months) was caught, i n numbers not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s . H o r r a l l concluded t h a t lake t r o u t l e a r n the o l f a c t o r y c h a r a c t e r i s t i c s of the n a t a l area at some time before l e a v i n g i t as f r y which g e n e r a l l y occurs around 6 weeks a f t e r swim-up. Lake t r o u t d i f f e r from coho and other anadromous salmonids i n a number of ways which might a f f e c t the t i m i n g of o l f a c t o r y l e a r n i n g , n o t a b l y i n not undergoing the process of s m o l t i f i c a t i o n . An i n v e s t i g a t i o n of the importance of n a t a l odours i n homing by coho, i n v o l v i n g both f i e l d and l a b o r a t o r y s t u d i e s i s p r e s e n t l y underway at the U n i v e r s i t y of Washington (personal communication, Mr. A. Dittman and Dr. T.P. Quinn, 180 School of F i s h e r i e s , U n i v e r s i t y of Washington, S e a t t l e WA). Rec o g n i t i o n of p r e v i o u s l y encountered odours may a l s o f u n c t i o n i n non-reproductive homing. I t was noted i n Chapter 5 that salmonid f r y that are e x p e r i m e n t a l l y d i s p l a c e d w i t h i n the homestream o f t e n r e t u r n to the area of capture, and i t was suggested that remembered odours may f a c i l i t a t e t h i s "homing". I n v e s t i g a t i o n s of the r o l e of o l f a c t o r y l e a r n i n g i n salmonid homing may be r e l e v a n t i n a l a r g e r taxonomic c o n t e x t . Reproductive and non-reproductive homing has been demonstrated or i n f e r r e d i n a wide range of other f i s h s p e c i e s (see Gerking 1959; Thunberg 1971; Smith 1985; Me l v i n et a l . 1986; H o r r a l l 1981), and o l f a c t i o n appears to be important i n many cases (reviewed by Smith 1985:88). In a d d i t i o n , the r o l e of f a m i l i a r odours may not be l i m i t e d to streams and r i v e r s . For example, McCleave et a l . (1987) suggested that e e l s ( A n g u i l l a spp.) might as l a r v a e imprint on a d i s t i n c t i v e odour i n the southern water mass of the Sargasso Sea, and as homing a d u l t s , cease m i g r a t i o n upon encountering t h i s f a m i l i a r home area odour. S i m i l a r l y , Harden-Jones (1980) suggested that p l a i c e ( Pleuronectes p l a t e s s a ) and c e r t a i n other marine s p e c i e s that appear to home to c e r t a i n c o a s t a l marine areas f o r spawning might have imprinted very e a r l y i n l i f e on the odour of groundwater seepage, and subsequently use t h i s odour as a l o c a l landmark. H a r t t and D e l l (1986) suggested that i n j u v e n i l e salmonids, the process of s e q u e n t i a l i m p r i n t i n g might continue d u r i n g m i g r a t i o n through c o a s t a l waters. Odours emanating from 181 the v a r i o u s r i v e r s passed by j u v e n i l e s might be l e a r n e d , and might subsequently f a c i l i t a t e homing. Remembered r i v e r odours may even p r o v i d e d i r e c t i o n a l i n f o r m a t i o n at sea. Using u l t r a s o n i c t r a n s m i t t e r s , Westerberg (1982a,b, 1984) t r a c k e d homing A t l a n t i c salmon through c o a s t a l waters, and noted that the f i s h made v e r t i c a l m i g r a t i o n s between the sea s u r f a c e and thermocline One f i s h rendered anosmic made more of these m i g r a t i o n s than other f i s h . Westerberg noted that d i s t i n c t t h i n l a y e r s of waters may extend f o r k i l o m e t e r s i n the sea, and suggested that homing salmon search through the l a y e r s f o r homestream water. Once l o c a t e d , the f i s h might o r i e n t a g a i n s t the d i r e c t i o n of l o c a l shear to r e l o c a t e the home e s t u a r y . A subsequent study v e r i f i e d t h a t anosmic f i s h make more v e r t i c a l e x c u r s i o n s , and provided evidence that the f i s h c o u l d d i s c r i m i n a t e the odours of the d i f f e r e n t water l a y e r s (Doving et a l . 1985). Homing sockeye salmon i n c o a s t a l waters a l s o make v e r t i c a l m i g r a t i o n s and i n some cases appear to p r e f e r depths at which water l a y e r s occur (Quinn and t e r H a r t 1987; Quinn et a l . 1989). I n t r a s p e c i f i c R e c o g n i t i o n The r e s u l t s of experiments i n Chapters 2 and 3 i n d i c a t e d t h at one c l a s s of odours l e a r n e d i n e a r l y l i f e i s that of c o n s p e c i f i c s , c o n f i r m i n g the r e p o r t of Quinn and Hara (1986) and demonstrating that t h i s l e a r n i n g r e s u l t s i n long-term memory. The impetus f o r most of the p r e v i o u s s t u d i e s of i n t r a s p e c i f i c 182 r e c o g n i t i o n by salmonids has been the pheromone homing hypothesis (Nordeng 1971,1977), with i t s c e n t r a l tenet t h a t p o p u l a t i o n - s p e c i f i c odours f a c i l i t a t e homing. I r o n i c a l l y , as the evidence mounts that p o p u l a t i o n - s p e c i f i c odours e x i s t and permit p o p u l a t i o n r e c o g n i t i o n , an i n c r e a s i n g body of evidence suggests t h a t homing does not depend on these odours. S u c c e s s f u l i m p r i n t i n g and decoying of homing salmon with a r t i f i c i a l odorants ( S u t t e r l i n et a l . 1982; H a s l e r and Scholz 1983), and n a t u r a l waters (Donaldson and A l l e n 1958; Jensen and Duncan 1971; H a s l e r and Scholz 1983; Brannon and Quinn - i n press) i n the absence of c o n s p e c i f i c s has l e d to g e n e r a l acceptance t h a t c o n s p e c i f i c odours are not e s s e n t i a l . Other evidence suggests that c o n s p e c i f i c odours are not even of primary importance i n homing. Groot et a l . (1986) produced evidence t h a t the oceanic phase of homing by sockeye salmon i s n e i t h e r i n i t i a t e d nor guided by the odours of j u v e n i l e population-members, and l a b o r a t o r y s t u d i e s with coho salmon have suggested t h a t the freshwater phase i s a l s o guided by other odours (Quinn et a l . 1983; Brannon et a l . 1984). F i e l d i n v e s t i g a t i o n s of the importance of f i s h odours to homing are l o g i s t i c a l l y d i f f i c u l t and consequently few. In one study Black and Dempson (1986) r e p o r t e d that c a p t u r i n g 49 homing A r c t i c char and h o l d i n g them i n a t r i b u t a r y p r e v i o u s l y u n i n h a b i t a t e d by anadromous f i s h , f a i l e d to a t t r a c t any other char i n t o t h a t t r i b u t a r y . Brannon and Quinn ( i n press) r e p o r t e d that coho reared i n Lake Washington returned to t h e i r r e a r i n g s i t e , bypassing a hatchery at the U n i v e r s i t y of Washington i n which 183 s i b l i n g s and other a d u l t s of t h e i r p o p u l a t i o n were being h e l d . The migrants had no choice but to swim w i t h i n about 100 m of the hatchery, so there can be l i t t l e doubt that i t s odours were d e t e c t a b l e . L e a r n i n g of c o n s p e c i f i c odours i n e a r l y l i f e c o u l d be an i n c i d e n t a l consequence of n a t a l area i m p r i n t i n g f o r homing, or of an a b i l i t y to l e a r n and a v o i d the odours of h e t e r o s p e c i f i c competitors (Hoglund et a l . 1975) or p r e d a t o r s (Rehnberg and Schreck 1987). A l t e r n a t i v e l y , l e a r n i n g and subsequent r e c o g n i t i o n of s i b and/or population-odours may have been s e l e c t e d f o r . Quinn and Courtenay (1989) suggested that o l f a c t o r y r e c o g n i t i o n might serve a f u n c t i o n i n mate s e l e c t i o n , f a c i l i t a t i n g avoidance of both s i b s and s t r a y s from other p o p u l a t i o n s . S i b l i n g r e c o g n i t i o n c o u l d a l s o serve j u v e n i l e s i n s c h o o l i n g or t e r r i t o r i a l i n t e r a c t i o n s (Quinn and Busack 1985). Olsen (1989) suggested that s i b l i n g r e c o g n i t i o n by j u v e n i l e A r c t i c char might f a c i l i t a t e s h o a l i n g (aggregating without the equal spacing and p o l a r i z a t i o n c h a r a c t e r i s t i c of s c h o o l i n g ( P i t c h e r 1983, 1986), and K e l l e y (1988) suggested the same f o r s t r i p e d bass (Morone s a x a t i l i s ) . I t i s w e l l documented that some anuran tadpoles form s i b l i n g a g g r e g a t i o n s , (Waldman 1982; O'Hara and B l a u s t e i n 1985), and Van Havre and F i t z G e r a l d (1988) r e p o r t e d that 18-20 d o l d f r y of the t h r e e s p i n e s t i c k l e b a c k (Gasterosteus  a c u l e a t u s L.) p r e f e r e n t i a l l y shoaled with k i n over non-kin. To the extent t h a t s c h o o l i n g reduces r i s k of p r e d a t i o n ( P i t c h e r 184 1980) and p r e d a t o r s p r e f e r e n t i a l l y s e l e c t odd i n d i v i d u a l s from s c h o o l s (Shaw 1978), an i n d i v i d u a l may be s a f e s t i n a school of s i b s . In a d d i t i o n , i n c r e a s i n g the school s i z e may confer g r e a t e r s a f e t y on other members, i n c r e a s i n g the i n d i v i d u a l ' s i n c l u s i v e f i t n e s s (Hamilton 1964) i f the members are s i b s . Other b e n e f i t s may a l s o r e s u l t from s c h o o l i n g or s h o a l i n g , i n c l u d i n g improved performance i n swimming (Weihs 1973), f o r a g i n g ( P i t c h e r et a l . 1982), and reducing e c t o p a r a s i t i s m ( P o u l i n and F i t z G e r a l d 1989). I t has been shown that i n some p o p u l a t i o n s of coho, many f r y are unable to e s t a b l i s h f e e d i n g t e r r i t o r i e s and are c a r r i e d out to sea, where i t i s presumed they p e r i s h (Chapman 1962). The i n c l u s i v e f i t n e s s of a t e r r i t o r y h older might under these c o n d i t i o n s be i n c r e a s e d by a l l o w i n g a s i b to e s t a b l i s h an a d j o i n i n g t e r r i t o r y , while e x c l u d i n g non-sibs. In c o n c l u s i o n , the c h a r a c t e r i s t i c of salmonid b i o l o g y that would appear to p l a c e a premium on the a b i l i t y to l e a r n odours i n e a r l y l i f e - both n a t a l area odours and c o n s p e c i f i c odours i s that salmon are great c o l o n i z e r s . Mayr (1974) and Immelmann (1975) argued that the most important- advantages c o n f e r r e d by l e a r n i n g over g e n e t i c a l l y coded i n s t r u c t i o n s , are a g r e a t e r i n f o r m a t i o n c a p a c i t y , and g r e a t e r a b i l i t y to adapt to novel c i r c u m s t a n c e s . Both p o t e n t i a l s are important to the o f f s p r i n g of a s t r a y ( c o l o n i z e r ) which f i n d s i t s e l f i n an e x c e l l e n t r e a r i n g and spawning h a b i t a t which i s not the a n c e s t r a l stream. The a b i l i t y t o l e a r n l o c a l h a b i t a t c h a r a c t e r i s t i c s a l l o w s r e t u r n 185 to t h i s area, and learning c o n s p e c i f i c c h a r a c t e r i s t i c s allows s e l e c t i o n of a mate bearing l o c a l adaptations. 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C was sampled from a f i r e h o s e connected to the v a l v e from which water was taken f o r experiments rep o r t e d i n Chapters 1 and 5. W was sampled from one of the Tygon tubes d r a i n i n g one headtank. A 1 L sample of each was taken f o r a n a l y s i s of anions, a l k a l i n i t y pH, and s o l i d s . A 250 mL sample of each was taken f o r a n a l y s i s of c a t i o n s , and these samples were a c i d i f i e d with 0.5% n i t r i c a c i d to prevent p r e c i p i t a t i o n on the w a l l s of the c o n t a i n e r . C o n t a i n e r s were new p o l y e t h y l e n e b o t t l e s (Nalge Co., Rochester NY), r i n s e d three times with sample water before f i l l i n g . A second set of samples of each water was taken, and to these samples 0.5 mg/L morpholine was added. The purpose of these samples was to determine whether the c o n c e n t r a t i o n of morpholine used i n t e s t s r e p o r t e d i n Chapter 1 a l t e r e d water c h e m i s t r i e s . Samples were t r a n s p o r t e d i n a c o o l e r , and d e l i v e r e d 4 h l a t e r to Dr. J . Davidson of Quanta Trace L a b o r a t o r i e s Inc., Burnaby B.C. At the time of sampling, the temperature and pH of each water was measured. C was 14.1°C, W was 8.9°C. The pH of C was between 8.0 and 8.5, the pH of W was 7.0 (Hach Broad Range (1-14) pH Test K i t ) . (The c o l o r i m e t r i c k i t may have given a s l i g h t l y h i g h reading f o r C; measurement on August 13 with a pH D10 - 2PK meter (Western S c i e n t i f i c S e r v i c e s Ltd.) gave readings of 7.6 f o r C and 7.2 f o r W.) 214 C and W were similar in the concentrations of cations and anions present (Table 8), and in ions absent (Table 9). C contained s l i g h t l y more iron and less n i t r a t e than W. The concentrations of several cations and anions were higher in morpholine-treated than blank samples and iron was lower, but these differences were small and may r e f l e c t sampling or measurement error. Both waters contained neg l i g i b l e concentrations of particulate matter (suspended s o l i d s ) , and similar concentrations of dissolved s o l i d s . The a l k a l i n i t y of both waters were similar but the pH of W was 0.5 units less basic than C, suggesting a higher l e v e l of free carbon dioxide. (Nomographic estimation (American Public Health Association et a l . 1985) of free carbon dioxide based on data in Table 8: 6 mg/L (W), 2 mg/L (C). Estimation from f i e l d temperatures and pH s y i e l d s l e v e l s of 8 vs <1 mg/L i f Hach k i t pH s are used, 5 vs 2 mg/L i f pH meter recordings are used.) In summary, the similar chemistries of C and W suggest a common o r i g i n . Oxygen leve l s were recorded on August 15 and 17, 1987, with a Cole-Parmer oxygen/temperature d i g i t a l meter. W sampled from one Tygon tube from one of the headtanks of the Y-maze apparatus was 8.9 - 9.1°C, and held 10.0-10.5 mg/L oxygen (90-92% saturated). W water in the fiberglass holding-tanks in which coho fry were being reared was 8.4 - 8.8°C, and held 9.2-10.5 mg/L oxygen (79-91% saturated). (The low temperature and high oxygen concentration indicate good water turnover in holding tanks.) C sampled from a firehose attached to the valve from 215 Table 8. P h y s i c a l c h a r a c t e r i s t i c s , a l k a l i n i t y , and ions found i n Well and Creek waters at Rosewall Creek Hatchery - without and with 0.5 mg/L morpholine . The c o n c e n t r a t i o n s of c a t i o n s were measured by I n d u c t i v e l y Coupled Argon Plasma - O p t i c a l Emission Spectroscopy (ICAP-OAS). Anions were measured by Ion Exchange Chromatography (IEC), and ammonia by S e l e c t i v e Ion E l e c t r o d e ( S I E ) . Measure Well Water Creek Water Without With Without With Morph. Morph. Morph. Morph. P h y s i c a l Tests pH 7.0 7.0 7.5 7.5 Conduc. (uS/cm) 71.5 72.0 72.0 7 1.0 T o t a l S o l i d s (ppm) 54. 58. 58. 60. D i s s . S o l i d s (ppm) 54. 58. 58. 60. Susp. S o l i d s (ppm) <2. <2. <2. <2. A l k a l i n i t y (CaC03) A l k a l i n i t y 31.0 33.0 32.0 33.0 OH NIL NIL NIL NIL C03 NIL NIL NIL NIL HC03 31.0 33.0 32.0 33.0 C a t i o n s (mg/L) Aluminum (Al) 0.016 0.017 0.017 0.023 Bar i urn (Ba) 0. 0003 0.0005 0.0009 0.0009 Boron (B) <0 . 001 0.058 0.014 0.009 Calc i urn (Ca) 9.07 9.63 10.1 10.4 Copper (Cu) 0.0022 0.0019 0.0018 0.0013 I ron (Fe) 0.0004 <0.0003 0.0032 0.001 0 Magnesium (Mg) 1 .76 1 .86 1 .65 1 .70 Molybdenum (Mo) 0.001 0.002 0.002 0.001 Phosphorous (P) 0.006 0.006 0.008 0.007 Potassium (K) 0. 09 0.10 0.09 0.10 S i l i c o n ( S i ) 3.50 5. 37 3.06 3.18 Sodi urn (Na) 1 .27 1 .37 1 .35 1 .39 St r o n t ium (Sr) 0.0112 0.0120 0.0135 0.0139 Zinc (Zn) <0.0002 0.0008 0.0006 0.0005 Anions (mg/L) C h l o r i d e (Cl) 1.41 1 .24 1 .46 1 . 52 N i t r a t e (N03-N) 0.17 0.19 0.03 0.03 S u l f a t e (S04) 0.85 1 .00 0.83 0.88 216 Table 9. Ions not det e c t e d i n Well or Creek waters at Rosewall Creek Hatchery. (Methods as i n Table 8) D e t e c t i o n T h r e s h o l d Ion (mg/L) C a t i o n s 0.0001 B e r y l l i u m (Be) Manganese (Mn) Titanium ( T i ) 0.0002 Cadmium (Cd) Chromium (Cr) S i l v e r (Ag) Vanadium (V) 0.0005 Cobalt (Co) N i c k e l (Ni) Zirconium (Zr) 0.005 Antimony (Sb) L i t h i u m ( L i ) Mercury (Hg) Selenium (Se) Thorium (Th) 0.002 Lead (Pb) 0.02 A r s e n i c (As) Uranium (U) Anions 0.02 Ammonia (NH3-N) 0.03 F l u o r i d e (F) 0.05 Bromide (Br) Phosphate (P04-P) 0.1 N i t r i t e (N02-N) 217 which water was drawn f o r experiments i n Chapters 1 and 5, was 13.8°C, and h e l d 9.0 mg/L oxygen (86% s a t u r a t e d ) . 218 APPENDIX 2. PRELIMINARY Y-MAZE TESTS OF MORPHOLINE PREFERENCE Tes t s i n Well Water T e s t s were performed with f i s h that had been exposed to 5 X 10"" mg/L morpholine throughout the embryo, a l e v i n and f i r s t 14 d of the f r y stage, and with f i s h that had not been p r e v i o u s l y exposed to morpholine. A f u l l d e s c r i p t i o n of these groups, designated Throughout and C o n t r o l , r e s p e c t i v e l y i s given i n Chapter 1. R e p l i c a t e A groups were used i n a l l t e s t s except f o r two with r e p l i c a t e B Throughout f i s h (5 X 10" 3 mg/L and one t e s t with 5 X 10"* mg/L). The purpose of these t e s t s was to e s t a b l i s h the minimum c o n c e n t r a t i o n of morpholine to which f r y would respond. Methods and m a t e r i a l s f o l l o w e d those d e s c r i b e d i n Chapter 1, Part 1. T e s t s were performed i n w e l l water (the water f i s h were reared in) between A p r i l 14 and June 10, 1986. The minimum number of days between the l a s t exposure of the Throughout groups to morpholine and t e s t i n g was 32 d. Temperatures i n the Y-mazes were s i m i l a r to those i n h o l d i n g tanks (8-9°C). Fry were n e i t h e r a t t r a c t e d nor r e p e l l e d by 5 X 10"" mg/L, 5 X 10" 3 mg/L, or 5 X 10" 2 mg/L morpholine (Figure 45). Throughout f i s h were a t t r a c t e d (P=0.0006) to 0.5 mg/L i n one t e s t , but showed no response i n three other t e s t s . C o n t r o l s showed no response to 0.5 mg/L i n any of four t e s t s . Both Throughout and C o n t r o l f i s h were a t t r a c t e d to 5 mg/L (P=0.0067, P=0.0005 r e s p e c t i v e l y ) . Throughout f i s h were weakly a t t r a c t e d 219 30 34 43 35 37 33 47 52 30 35 C 'y—l a o I n S a CD o V-i CD PH 80 70 60 50 40 80 70 60 50 40 B n 1 " 32 38 33 38 52 52 33 Attraction Avoidance Attraction Avoidance 0.0005 0.005 0.05 0.5 50 Morpholine Concentration (mg/L) F i g u r e 45. Response to morpholine i n w e l l water. A: f i s h exposed to morpholine, Throughout the egg, a l e v i n and e a r l y f r y stages. B: C o n t r o l s . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 220 (P=0.0710) to 50 mg/L. These r e s u l t s suggested that h i g h c o n c e n t r a t i o n s of morpholine acted as a general a t t r a c t a n t f o r both p r e v i o u s l y -exposed and naive f i s h , while lower c o n c e n t r a t i o n s e l i c i t e d no response. (Presumably the confounding f a c t o r a f f e c t i n g t e s t s i n creek water a f f e c t e d these t e s t s a l s o , such that the true response of f i s h was avoidance of morpholine r a t h e r than the apparent a t t r a c t i o n - see Chapter 1 - Part 2.) T e s t s i n Rosewall Creek Water The idea of t e s t i n g f i s h i n Rosewall Creek water came from the experimental study of m o r p h o l i n e - i m p r i n t i n g by sockeye embryos and a l e v i n s by Wright (1985). Wright f a i l e d to det e c t a t t r a c t i o n or avoidance of morpholine by p r e v i o u s l y - e x p o s e d f i s h t e s t e d i n f a m i l i a r water ( d e c h l o r i n a t e d S e a t t l e C i t y water). He hypothesized that the d i f f e r e n c e between f a m i l i a r c i t y water with and without morpholine was e i t h e r too s u b t l e f o r f r y to d i s t i n g u i s h , or too s l i g h t to warrant d i f f e r e n t i a t i o n . In an u n f a m i l i a r water t h i s might not be the case; morpholine might stand out as the only f a m i l i a r odour. T h e r e f o r e , Wright t e s t e d f i s h i n u n f a m i l i a r Lake Washington water. No o v e r a l l p r e f e r e n c e or avoidance of morpholine was d e t e c t e d , but there was a s i g n i f i c a n t i n t e r t r i a l h e t e r o g e n e i t y which Wright i n t e r p r e t e d as i n d i c a t i n g response to morpholine. 221 While i n s u f f i c i e n t to conclude that f r y r e c o g n i z e d the odour of morpholine, Wright's data suggested that f i s h may have been more responsive to morpholine i n u n f a m i l i a r water. T h e r e f o r e , t e s t s were conducted i n the other water source a v a i l a b l e at Rosewall Creek Hatchery - Creek water. Creek water i s s i m i l a r to w e l l water i n i n o r g a n i c composition, but the two are c l e a r l y d i s t i n g u i s h e d by coho f r y (Chapter 5 ) . I t was hypothesized that i n Creek water, Throughout f i s h would respond to morpholine at c o n c e n t r a t i o n s below that at which i t became a g e n e r a l a t t r a c t a n t f o r C o n t r o l s . On May 5, Throughout f i s h ( r e p l i c a t e A) were t e s t e d f o r response to 5 X 10" 3 mg/L morpholine. No response was d e t e c t e d (mean response of 39 fish=51%, P=0.76). On the next two days, response to 0.5 mg/L morpholine was t e s t e d . Response was d e t e c t e d , and, s u r p r i s i n g l y , f i s h avoided the morpholine (data r e p o r t e d i n Chapter 1). On the next two days, the response of C o n t r o l s to 0.5 mg/L morpholine was e v a l u a t e d and found to be o p p o s i t e that of Throughout f i s h ; C o n t r o l s were a t t r a c t e d . On the b a s i s of these data, the other r e p l i c a t e s of Throughout and C o n t r o l treatments were t e s t e d , f o l l o w e d by the other experimental groups. These t e s t s confirmed the d i f f e r e n c e i n response of p r e v i o u s l y - e x p o s e d and naive f i s h , s u p p o r t i n g Wright's hypothesis that r e c o g n i t i o n of morpholine i s expressed on l y i n u n f a m i l i a r water. 222 APPENDIX 3. SENSORY MEDIATION OF RESPONSE TO THE CHEMICAL EMANATIONS OF CONSPECIFICS I n t r o d u c t i o n A number of s t u d i e s have r e p o r t e d that salmonids are a t t r a c t e d to the chemical emanations of c o n s p e c i f i c s ( S e l s e t and Doving 1980; Quinn et a l . 1983; Olsen 1985, 1986a; Olsen and Hoglund 1985; Groot et a l . 1986). One study of j u v e n i l e A r c t i c char ( S a l v e l i n u s a l p i n u s ) (Hoglund and Astrand 1973) r e p o r t e d that t h i s response disappeared when the o l f a c t o r y e p i t h e l i u m was burned, i n d i c a t i n g that the chemical emanations of t h i s s p e c i e s were d e t e c t e d by c o n s p e c i f i c s as odours - sensu H i r s c h (1977). ("Odor i s used as a p r o p e r t y of the chemical substance p e r c e i v e d by the nose, whether mediated by the o l f a c t o r y or t r i g e m i n a l nerves." Free nerve endings of the t r i g e m i n a l nerve are l e s s s e n s i t i v e than o l f a c t o r y r e c e p t o r s to most but not a l l chemicals (Koster 1971:129).) Three s t u d i e s have shown that j u v e n i l e coho are a t t r a c t e d to and d i f f e r e n t i a t e between d i f f e r e n t c o n s p e c i f i c emanations (Quinn and Busack 1985; Quinn and Hara 1986; Quinn and Tolson 1986), but none of these i n v e s t i g a t e d the sensory modality i n v o l v e d . Quinn and Busack (1985) noted that because of recent evidence by Hara et a l . (1984) that salmonid t a s t e r e c e p t o r s are extremely s e n s i t i v e to c e r t a i n b i l e a c i d s , i t c o u l d not be assumed that c o n s p e c i f i c emanations were d e t e c t e d by o l f a c t i o n . 223 The purpose of t h i s experiment was to determine whether coho f r y depend on o l f a c t i o n f o r p e r c e p t i o n of the chemical emanations of c o n s p e c i f i c s . T h i s was t e s t e d by comparing the responses to water c o n d i t i o n e d by c o n s p e c i f i c s , of f r y with and without occluded nares. Methods and M a t e r i a l s F i s h F i s h used were from the Fry experimental group of the morpholine experiment (Chapter 1-Part 1). These f i s h were d e r i v e d of the pooled gametes of four male and four female coho from the Quinsam hatchery. O l f a c t o r y O c c l u s i o n F i s h to be o l f a c t o r i l y occluded (Experimentals) were a n a e s t h e t i z e d with 2-phenoxyethanol. ( T h i s a n a e s t h e t i c does not impair chemosensory responses of salmonids (Quinn et a l . 1988).) Petroleum j e l l y ( V a s e l i n e ) c o l o u r e d blue with food-dye to i n c r e a s e i t s v i s i b i l i t y was i n j e c t e d i n t o each p o s t e r i o r nare with a s y r i n g e f i t t e d with a b l u n t e d l a r g e - b o r e needle. When petroleum j e l l y i s s u e d from the a n t e r i o r nare, the o l f a c t o r y p i t was c o n s i d e r e d f i l l e d . ( T h i s i s a common method of o l f a c t o r y o c c l u s i o n with f i s h (e.g., Wisby and Ha s l e r 1954; Brannon 1972; McCleave and LaBar 1972; Royce-Malmgren and 224 Watson 1987).) A number of other f r y ( C o n t r o l s ) were s i m i l a r l y handled and a n a e s t h e t i z e d , without having anything i n j e c t e d i n t o t h e i r o l f a c t o r y p i t s . Experimentals and C o n t r o l s were h e l d together i n a c i r c u l a r f i b e r g l a s s tank (35 cm diameter, 40 cm water depth, 4 L/min flow of w e l l water) f o r 48 h before t e s t i n g . No m o r t a l i t y or i l l e f f e c t s from t h i s procedure were observed. T h i r t y experimental and t h i r t y c o n t r o l f r y were prepared on each of February 16, 17, 19 and 20, 1987. T e s t i n g F i s h were t e s t e d f o r p r e f e r e n c e between w e l l water c o n d i t i o n e d by 100 g (N=9-10) of t h e i r former tankmates, and blank w e l l water. The experimental apparatus, p r o t o c o l and data a n a l y s i s are a l l as e x p l a i n e d i n General Methods and M a t e r i a l s , and i n Chapter 2 and 3. Odour-producers were p l a c e d i n one headtank on the evening p r e c e d i n g the t e s t : the r i g h t headtank f o r t e s t s conducted on February 18 and 21, the l e f t headtank f o r t e s t s conducted on February 19 and 22. Experimental and C o n t r o l f i s h were t e s t e d together and i d e n t i f i e d a f t e r each t r i a l by i n j e c t i n g water i n t o the nares, d i s l o d g i n g the v a s e l i n e plugs from Experimentals. Within treatment groups, responses were s i m i l a r across the four days of t e s t i n g (P>0.05, ANOVA), so data were pooled f o r comparison of Experimentals and C o n t r o l s . 225 R e s u l t s and D i s c u s s i o n O c c l u s i o n of the o l f a c t o r y p i t s d i d not e l i m i n a t e s c r e e n -swimming behaviour, or even reduce i t s d u r a t i o n (Figure 46) (comparision of C o n t r o l s and Experimentals: P=0.75). However, o c c l u s i o n d i d e l i m i n a t e p r e f e r e n c e f o r c o n s p e c i f i c emanations. C o n t r o l s showed s i g n i f i c a n t p r e f e r e n c e (P<0.0001), whereas Experimentals d i d not, and the d i f f e r e n c e between the two responses was s i g n i f i c a n t (P<0.0001) (F i g u r e 47). These r e s u l t s suggest that the response of coho f r y to c o n s p e c i f i c emanations depends on o l f a c t i o n , and that c o n s p e c i f i c emanations can be r e f e r r e d to as odours. 226 89 77 60 c a S CO I a <D 4> U o CO 1/3 -a c o o <u CO 80 60 40 20 0 Occluded Nares Control Treatment Group gure 46. Number of seconds of occluded f i s h and c o n t r o l s screen-swimming by in a 5-min p e r i o d . o l f a c t o r i l y -(x ± 95% CL Number of f r y t e s t e d shown above bar) 89 77 Attraction Avoidance Occluded Nares Control Treatment Group e 47. Response to c o n s p e c i f i c chemical emanations by o l f a c t o r i l y - o c c l u d e d f i s h and c o n t r o l s . (x ± 95% CL, Number of f r y t e s t e d shown above bar) 228 APPENDIX 4. MORPHOLINE AND OTHER SUBSTANCES AS EXPERIMENTAL ODOURS The r e s u l t s of the present study suggested that morpholine i s b e t t e r s u i t e d than c o n s p e c i f i c odours or stream waters, to the study of o l f a c t o r y l e a r n i n g by salmonids. However, morpholine has some u n d e s i r a b l e p r o p e r t i e s of which i n v e s t i g a t o r s should be aware. F i r s t , the widespread use of morpholine i n manufacturing and agrochemicals s i n c e 1945 has probably r e s u l t e d i n contamination of many n a t u r a l waters world wide (personal communication, Dr. G. Barthalmus, Dept. Zoology, North C a r o l i n a S t a t e U n i v e r s i t y , R a l e i g h , N . C ) . F i s h may be exposed to morpholine before experimental exposure, and i n f i e l d experiments may respond to l o c a l accumulations. ( I n t e r e s t i n g l y , l a r g e q u a n t i t i e s of morpholine were being used to c l e a n the steam condensors of a power p l a n t i n the immediate area of Hasler and c o l l e a g u e s ' study s i t e i n Lake Michigan, but a p p a r e n t l y d i d not i n t e r f e r e with t h e i r experiments (personal communications, Dr. P.B. Johnsen, U.S. Dept. A g r i c u l t u r e , A g r i c u l t u r e Research S e r v i c e , New Orleans, LA; Dr. R.M. H o r r a l l , U n i v e r s i t y of Wisconsin, Madison WN).) Second, as o u t l i n e d i n the I n t r o d u c t i o n to Chapter 1, there i s some q u e s t i o n whether morpholine i n t e r a c t s with the o l f a c t o r y system of f i s h i n the same manner as other known o l f a c t a n t s (Hara et a l . 1984). S p e c i f i c a l l y , e l e c t r o p h y s i o l o g i c a l s t u d i e s 229 of the o l f a c t o r y bulb have f a i l e d to i n d i c a t e any response to c o n c e n t r a t i o n s of morpholine to which f i s h homed i n the experiments by H a s l e r and c o l l e a g u e s , or even to the higher c o n c e n t r a t i o n s used i n the present study. Dodson and Bitterman (1989) have r e c e n t l y suggested an i n t r i g u i n g r e s o l u t i o n of the b e h a v i o u r a l and e l e c t r o p h y s i o l o g i c a l data. They suggest that a low c o n c e n t r a t i o n of morpholine may not be d e t e c t e d of i t s e l f , but combines with other odours to form a unique compound odour which i s d e t e c t e d . T h i s suggestion was supported by the demonstration that g o l d f i s h ( C a r a s s i u s auratus) d i d not d i s c r i m i n a t e 5 X 10"* M (44 mg/L) morpholine from blank water, but d i d d i s c r i m i n a t e a mixture of t h i s c o n c e n t r a t i o n of morpholine and 5 X 10" 3 M L - s e r i n e from the L - s e r i n e component alone. The authors note that the high c o n c e n t r a t i o n of morpholine was not d e t e c t e d only by o l f a c t i o n , and i t s pH of 8.5 may have i o n i z e d L - s e r i n e . N e v e r t h e l e s s , t h i s h y p o t h e s i s deserves f u r t h e r a t t e n t i o n . B e h a v i o u r a l responses to morpholine at the c o n c e n t r a t i o n a p p a r e n t l y l e a r n e d (5 X 10"* mg/L) were not d e t e c t e d i n the present study. T h i s was probably due to c r o s s - c o n t a m i n a t i o n i n the Y-maze, r a t h e r than f a i l u r e of the f i s h to d e t e c t and respond to t h i s c o n c e n t r a t i o n , because both Wisby (1952) and Cooper and H a s l e r (1973) r e p o r t e d p r e f e r e n c e responses to a c o n c e n t r a t i o n ten times l e s s than t h i s . T h e r e f o r e , i t should be p o s s i b l e to e l i m i n a t e c r o s s - c o n t a m i n a t i o n i n the Y-maze assay, o b t a i n a response to low c o n c e n t r a t i o n s of morpholine, and t e s t whether morpholine i s d e t e c t e d as an odour. 230 Involvement of the nares can be t e s t e d by o c c l u s i o n as d e s c r i b e d in Appendix 3, and c o n f i r m a t i o n of the r o l e of the o l f a c t o r y system can be t e s t e d by s e c t i o n of the o l f a c t o r y t r a c t (e.g., Dodson and Bitterman 1989) or c a u t e r i z a t i o n of the o l f a c t o r y e p i t h e l i u m (e.g., Olsen 1986b). The t h i r d and most s e r i o u s problem with morpholine i s that i t may pose a h e a l t h hazard to both f i s h and i n v e s t i g a t o r s . The molecule i s very r e a c t i v e and i s e a s i l y converted to c a r c i n o g e n i c n i t r o s o m o r p h o l i n e s by b a c t e r i a i n the gut of animals (personal communication, Dr. G. Barthalmus). In a d d i t i o n , a h i g h c o n c e n t r a t i o n (300 mg/L) has been shown to k i l l pigmented c e l l s and a l t e r the chemistry of nerve c e l l s that t r a n s m i t dopamine (Noga et a l . 1986; p e r s o n a l communication, Dr. G. Barthalmus). There was some concern i n the present study that extended exposure to morpholine i n the e a r l y l i f e stages of coho might a f f e c t the development of the o l f a c t o r y system. The Merck index (Windholz 1976) d e s c r i b e s morpholine as an i r r i t a n t to the eyes, s k i n and mucous membranes i n humans, and Hara and Brown (1979) suggested that i n f i s h , i t "may penetrate i n t o the o l f a c t o r y e p i t h e l i u m and cause a n o n s p e c i f i c i r r i t a t i o n a l e f f e c t " , even c a u s i n g " d e s t r u c t i o n of s p e c i f i c membrane s t r u c t u r e s , or t o t a l c e l l damage" at high c o n c e n t r a t i o n . Two l i n e s of evidence suggest that the o l f a c t o r y systems of f i s h i n the present study were not damaged by prolonged exposure to 5 X 10"* mg/L morpholine. F i r s t , the o l f a c t o r y membranes of 231 f i v e f i s h of each treatment group sampled on August 24, 1987, were examined by scanning e l e c t r o n microscopy. F i s h exposed d u r i n g the embryo stage, a l e v i n stage, e a r l y f r y stage, or throughout a l l three stages were found to have a normal number and appearance of receptor c e l l s , i n d i s t i n g u i s h a b l e from those of c o n t r o l f i s h . Second, f i s h that had been exposed d u r i n g the e a r l y f r y stage were used i n the o l f a c t o r y o c c l u s i o n experiment r e p o r t e d i n Appendix 3, i n which they demonstrated an o l f a c t o r y response to c o n s p e c i f i c odours. Two other chemicals have been used or proposed f o r use i n salmonid i m p r i n t i n g experiments, but these have problems of t h e i r own. Phenethyl a l c o h o l ( C 8 H 1 0 0 ) occurs n a t u r a l l y i n a number of o i l s and i s widely used as a rose essence and f l a v o r (Windholz 1976). Hasler and c o l l e a g u e s chose i t f o r use i n i m p r i n t i n g experiments on the b a s i s of a r e p o r t by Teichmann (1962) that i t was d e t e c t e d by rainbow t r o u t at the low c o n c e n t r a t i o n of 4.1 X 10" 8 M. The i m p r i n t i n g experiments demonstrated that coho smolts d e t e c t e d and l e a r n e d t h i s c o n c e n t r a t i o n of odour, and as a d u l t s , homed to i t (Hasler and Scholz 1983). On the b a s i s of these r e s u l t s , phenethyl a l c o h o l has been used i n subsequent i m p r i n t i n g experiments (e.g., p e r s o n a l communication - A. Dittman, School of F i s h e r i e s , U n i v e r s i t y of Washington, S e a t t l e WA). H o r r a l l ( p ersonal communication, U n i v e r s i t y of Wisconsin, Madison WN) used phenethyl a l c o h o l rather than morpholine i n a recent i m p r i n t i n g experiment i n v o l v i n g hatchery reared lake t r o u t , because morpholine was not approved f o r use i n Wisconsin h a t c h e r i e s . ) 232 U n d e s i r a b l e p r o p e r t i e s of phenethyl a l c o h o l i n c l u d e that i t i s r e l a t i v e l y i n s o l u b l e i n water, has a s t r o n g l o c a l a n a e s t h e t i c e f f e c t on humans, and i n experiments with mice has caused severe c e n t r a l nervous system damage (Scholz et a l . 1975). In a d d i t i o n , i t shares with morpholine the c h a r a c t e r i s t i c of f a i l i n g to produce an e l e c t r o p h y s i o l o g i c a l response from the o l f a c t o r y bulb, at the c o n c e n t r a t i o n used f o r i m p r i n t i n g (Hara 1973). Sandoval (1980) suggested phenol (C 6H 60) as an i m p r i n t a n t , because i t i s h i g h l y s o l u b l e i n water, should be s t a b l e under stream c o n d i t i o n s , and i s d e t e c t e d by j u v e n i l e coho at the low c o n c e n t r a t i o n of 10" 5 mg/L but i s n e i t h e r a t t r a c t i v e nor r e p u l s i v e at c o n c e n t r a t i o n s l e s s than 10" 2 mg/L (at which c o n c e n t r a t i o n i t i s r e p u l s i v e ) . However, phenol and p h e n o l i c compounds are widely used as g e n e r a l d i s i n f e c t a n t s , t o p i c a l a n a e s t h e t i c s , and a n t i s e p t i c s , and phenol i t s e l f i s both c a u s t i c and extremely t o x i c to humans i f i n g e s t e d or absorbed through the s k i n (Windholz 1976). A n a t u r a l a l t e r n a t i v e to morpholine f o r l a b s t u d i e s appears to be the amino a c i d L - c y s t e i n e . Based on a suggestion from Hara, Morin used t h i s odour i n h i s experiments on o l f a c t o r y i m p r i n t i n g by A t l a n t i c salmon (Morin et a l . 1987a, 1989a). L-c y s t e i n e has been shown to be h i g h l y e f f e c t i v e i n evoking o l f a c t o r y (and perhaps not g u s t a t o r y ) r e c e p t o r responses i n salmonids, (see r e f e r e n c e s i n Morin et a l . 1987a). Another 233 important c h a r a c t e r i s t i c of L - c y s t e i n e i s presumably that u n l i k e some of the other odorous amino a c i d s , i t i s not e x c r e t e d or s e c r e t e d by the f i s h themselves (Hara et a l . 1984; Olsen 1986b). Ther e f o r e , L - c y s t e i n e would seem to be w e l l s u i t e d to l a b o r a t o r y s t u d i e s of o l f a c t o r y l e a r n i n g , but perhaps not to f i e l d s t u d i e s where the odour may occur n a t u r a l l y . S i m i l a r l y , one or more b i l e a c i d s or t h e i r d e r i v a t i v e s , s i m i l a r to but d i f f e r e n t from those e x c r e t e d by salmonids might make an e x c e l l e n t experimental odorant f o r the l a b o r a t o r y (see Chapter 4). PUBLICATIONS Quinn, T.P. and S.C. Courtenay. 1989. I n t r a s p e c i f i c chemosensory d i s c r i m i n a t i o n i n salmonid f i s h e s : a l t e r n a t i v e e x p l a n a t i o n s , pp. 35-41, I_n: E.Brannon and B.Jonsson, (eds.) Proceedings of the salmonid m i g r a t i o n and d i s t r i b u t i o n symposium. U n i v e r s i t y of Washington Press. S e a t t l e . Courtenay, S.C. 1985. Simultaneous m u l t i n e s t i n g by the fo u r s p i n e s t i c k l e b a c k , Apeltes quadracus. Canadian F i e l d N a t u r a l i s t 99(3): 360-363. K a v a l i e r s , M., S.C. Courtenay, and M. H i r s t . 1984. Opiates i n f l u e n c e b e h a v i o r a l thermoregulation i n the c u r l y - t a i l e d l i z a r d , L e iocephalus c a r i n a t u s . Physiology and Behavior 32(2): 221-224. Courtenay, S.C. and M.H.A. Keenleyside. 1983. Nest s i t e s e l e c t i o n by the f o u r s p i n e s t i c k l e b a c k , Apeltes quadracus ( M i t c h i l l ) . Canadian J o u r n a l of Zoology 61: 1443-1447. Courtenay, S.C. and M.H.A. Keenle y s i d e . 1983. Wriggler-hanging: a response to hypoxia by brood-rearing H e r o t i l a p i a m u l t i -spinosa ( T e l e o s t e i , C i c h l i d a e ) . Behaviour 85(3/4): 183-197. 

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