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 (D0.20, 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 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 •(-> a •S c t-H O H DO a B 6 •l-H CO 6 •>-< H •*-> c 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 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 o t-H PH co 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 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 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. The only period of assured assoc ia t ion with a l l of these cues i s very ear ly l i f e , and for t h i s reason, i t i s reasonable to expect that the period of incubation or shor t ly thereafter const i tu tes or contains a s e n s i t i v e period for the a c q u i s i t i o n of t h i s information. Thus, what might be deduced from a considerat ion of why salmonids might learn odours in ear ly l i f e , i s apparently confirmed by evidence, here presented, that indeed they have the capacity to do so. 186 LITERATURE CITED A l b e r t s , J.R. 1986. P o s t n a t a l development of o l f a c t o r y - g u i d e d behavior i n rodents, pp. 227-235, I_n: W. B r e i p o h l [ed.] Ontogeny of o l f a c t i o n i n v e r t e b r a t e s . S p r i n g e r - V e r l a g . B e r l i n . American P u b l i c H e a l t h A s s o c i a t i o n , American Water Works A s s o c i a t i o n , Water P o l l u t i o n C o n t r o l F e d e r a t i o n . 1985. 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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 U o CO 1/3 -a c o o