"Science, Faculty of"@en . "Zoology, Department of"@en . "DSpace"@en . "UBCV"@en . "Michiel, Glen Patrick"@en . "2010-02-24T18:30:10Z"@en . "1978"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Gasterosteus aculeatus L., the threespine stickleback is in morphological terms, an extremely variable species. It is found in a wide variety of habitats throughout the northern hemisphere. Forms of threespine stickleback are found in anadromous and freshwater situations. Taxonomic examinations concerning the distribution of various forms of stickleback have produced equivocal results.\r\nTwo stickleback protein polymorphisms have been reported\r\nin the literature. A hemoglobin polymorphism has been found in south European stickleback, and a muscle myogen polymorphism (enzyme: creatine kinase) has been found in stickleback from western Canada. The distribution of alleles in both these situations caused the researchers to hypothesize that the polymorphisms were related to the anadromous existences of certain forms of stickleback.\r\nIn this study I tested these hypotheses with populations from southwestern British Columbia, across a range of macro-habitat types. One population of sticklebacks sampled is, apparently, a relict anadromous population. This population provided the opportunity to test a population during (or after) selective pressure to adapt to a freshwater existence. No evidence of the hemoglobin polymorphism was found, indeed some doubt was cast upon the European work due to technique aberrations. The muscle myogen polymorphism was present in a few populations, however, no basic environmental pattern emerged. The possibility of these alleles being selectively neutral is discussed.\r\nElectrophoretic patterns of the hemoglobin and muscle myogen patterns provoked the suggestion that the various forms of stickleback do indeed belong to a single species.\r\nThe pattern of the muscle myogen polymorphism (creatine kinase) was found to be inconsistent with the dimeric structure generally reported for the enzyme.\r\nThe hypothesis relating the polymorphisms to anadromous existences must be rejected. Several alternative projects are suggested."@en . "https://circle.library.ubc.ca/rest/handle/2429/20838?expand=metadata"@en . "AN EXAMINATION OF TWO REPORTED PROTEIN POLYMORPHISMS IN THREESPINE STICKLEBACK, GASTEROSTEUS ACULEATUS by GLEN PATRICK MICHIEL B.Sc. (Hons.), Carleton University, 1974 THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Department of Zoology we accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MASTER OF SCIENCE IN A p r i l , 1977 Glen Patrick Michiel, 1978 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f i nanc ia l gain sha l l not be allowed without my writ ten pe rm i ss i on . G. P. MICEIF.L ZOOLOGY. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date QCT. lk . 77 i A b s t r a c t Gasterosteus a c u l e a t u s L., the t h r e e s p i n e s t i c k l e b a c k i s i n m o r p h o l o g i c a l terms, an extremely v a r i a b l e s p e c i e s . I t i s found i n a wide v a r i e t y of h a b i t a t s throughout the northern hemisphere. Forms of t h r e e s p i n e s t i c k l e b a c k are found i n anadromous and freshwater s i t u a t i o n s . Taxonomic examinations c o n c e r n i n g the d i s t r i b u t i o n o f v a r i o u s forms of s t i c k l e b a c k have produced e q u i v o c a l r e s u l t s . Two s t i c k l e b a c k p r o t e i n polymorphisms have been r e -p o r t e d i n the l i t e r a t u r e . A hemoglobin polymorphism has been found i n south European s t i c k l e b a c k , and a muscle myogen polymorphism (enzyme: c r e a t i n e kinase) has been found i n s t i c k l e b a c k from western Canada. The d i s t r i b u t i o n of a l l e l e s i n both these s i t u a t i o n s caused the r e s e a r c h e r s to hypothesize t h a t the polymorphisms were r e l a t e d to the anadromous e x i s t e n c e s of c e r t a i n forms of s t i c k l e b a c k . In t h i s study I t e s t e d these hypotheses with p o p u l a t i o n s from southwestern B r i t i s h Columbia, ac r o s s a range of macro-h a b i t a t types. One p o p u l a t i o n of s t i c k l e b a c k s sampled i s , apparently., a r e l i c t anadromous p o p u l a t i o n . T h i s p o p u l a t i o n p r o v i d e d the o p p o r t u n i t y to t e s t a p o p u l a t i o n d u r i n g (or a f t e r ) s e l e c t i v e p r e s s u r e to adapt to a freshwater e x i s t e n c e . No evidence of the hemoglobin polymorphism was found, indeed some doubt was c a s t upon the European work due to technique a b e r r a t i o n s . The muscle myogen polymorphism was present i n a few p o p u l a t i o n s , however, no b a s i c environmental p a t t e r n emerged. The p o s s i b i l i t y of these a l l e l e s being s e l e c t i v e l y n e u t r a l i s d i s c u s s e d . E l e c t r o p h o r e t i c p a t t e r n s of the hemoglobin and muscle myogen p a t t e r n s provoked the sugg e s t i o n t h a t the v a r i o u s forms of s t i c k l e b a c k do indeed belong to a s i n g l e s p e c i e s . The p a t t e r n of the muscle myogen polymorphism ( c r e a t i n e kinase) was found t o be i n c o n s i s t e n t with the d i m e r i c s t r u c t u r e g e n e r a l l y r e p o r t e d f o r the enzyme. The hy p o t h e s i s r e l a t i n g the polymorphisms to anadromous e x i s t e n c e s must be r e j e c t e d . S e v e r a l a l t e r n a t i v e p r o j e c t s are suggested. i i i T able o f Contents A b s t r a c t i Table o f Contents i i i L i s t of Tables i v L i s t o f F i g u r e s v Acknowledgements v i CHAPTER I. GENERAL INTRODUCTION 1 CHAPTER I I . STICKLEBACK HEMOGLOBIN 11 1. I n t r o d u c t i o n 11 2. M a t e r i a l s and Methods 15 3. R e s u l t s 20 4. D i s c u s s i o n and Con c l u s i o n s 25 CHAPTER I I I . STICKLEBACK MUSCLE MYOGEN 2 7 1. I n t r o d u c t i o n 2 7 2. M a t e r i a l s and Methods 34 3. R e s u l t s 39 4. D i s c u s s i o n and Con c l u s i o n s 4 3 CHAPTER IV. GENERAL DISCUSSION 47 LITERATURE CITED 5 8 LIST OF TABLES i v TABLE I. Summary of hemoglobin sample l o c a t i o n s and r e s u l t s 21 TABLE I I . A summary of muscle myogen p o p u l a t i o n l o c a t -i o n s , h a b i t a t and form 33 TABLE I I I . A summary of Vancouver I s l a n d p o p u l a t i o n s sampled . 3 8 TABLE IV. Muscle myogen genotype f r e q u e n c i e s 40 TABLE V. A comparison of s i x s p e c i e s demonstrating a s i m i l a r muscle myogen p r o t e i n polymorphism 51 V L i s t o f F i g u r e s FIGURE 1. Three forms of Gasterosteus. a c u l e a t u s 3 FIGURE 2. P a t t e r n s o f the Reported Hemoglobin P o l y -morphism Found i n I t a l y 12 FIGURE 3. Geographic L o c a t i o n s o f S t i c k l e b a c k Pop-u l a t i o n s Sampled f o r Hemoglobin Polymorphism 17 FIGURE 4. The Aberrant G e l 22 FIGURE 5. The Muscle Myogen P a t t e r n 2 8 FIGURE 6. Geographic L o c a t i o n s of S t i c k l e b a c k Pop-u l a t i o n s Sampled f o r the Myogen Polymorphism 36 FIGURE 7. Age and Season 41 FIGURE 8. Geographic L o c a t i o n s of the Polymorphic P o p u l a t i o n s 4 4 FIGURE 9. H y p o t h e t i c a l P a t t e r n f o r Dimeric Quarter-nary S t r u c t u r e o f the Enzyme C r e a t i n e Kinase 51 v i Acknowledgements Help comes i n strange forms, to a graduate student, advice (hard to take), \"know-how\", patience, funds, con-versation and most importantly c r i t i c i s m (also hard to take). I t i s obvious to me that my debt, to the many who helped me, i s very great. I would p a r t i c u l a r l y l i k e to thank my advisor J.D. McPhail, for his encouragement, \"penelopean\" patience and advice during my studies. I would also p a r t i c u l a r l y l i k e to thank Dr. McPhail for taking the i n i t i a l gamble of accepting me as a student (when others had refused). He would probably c a l l i t \"succumbing to a study i n persistance\". I would also l i k e to thank the other members of my committee, Dr. B i l l N e i l l , and Dr. Judy Myers. Both kindly c r i t i c i s e d my work and thesis d r a f t s . Dr. Myers also supplied me with lab space. The following people contributed hours of help during my f i e l d , head and lab work, E. Black, D. Belford, M. Karen, C. Murray, A. Peacock, D. Peacock (no r e l a t i o n ) , P. Pressley, A. Robertson, G. Rossiter (my typist) and that unusual phenomenon J. Spence. Thank you a l l , for a most inter e s t i n g experience. v i i Utram b i b i s ? Aquam an undam? o r i g i n - unknown 1 CHAPTER I.' GENERAL INTRODUCTION Much s c i e n t i f i c attention has been directed towards morphological v a r i a t i o n i n the species complex Gasterosteus aculeatus, the threespine stickleback. Morphological features common to t h i s f i s h such as plate number, spine number, spine length, mating colour, size , and vertebral number have been demonstrated to be extremely variable over a range of locations, (Hagen, 19 67; McPhail, 1969; McPhail and Lindsey, 1970; Hagen and Gilbertson, 1972) . Fish with cert a i n combinations of these morphological features have been given names by taxonomists, beginning with Cuvier and Valenciennes, (1829), (cited i n Munzing, 1963,) for the sake of descriptive convenience. These forms of Gasterosteus aculeatus are described as l e i u r u s , semi-armatus and trachurus, (Fig. 1). Leiurus i s the unarmoured extreme, trachurus i s the well armoured extreme, and semi-armatus i s a moderate form between the two extremes. There has been some discussion with regard to the phylogenetic relationships among these forms, (Hagen, 1967; M i l l e r and Hubbs, 1969; Hagen and McPhail, 1970), however no consensus has been reached. One thing i s clear from t h i s controversy: stickleback are not a t y p i c a l species i n the t r a d i t i o n a l sense. I t should be noted that the forms do not s a t i s f y Mayr's (1963) c r i t e r i o n for sub-species, as the populations 2 i n B r i t i s h Columbia are not c l e a r l y g e o g r a p h i c a l l y i s o l a t e d . With the hope t h a t enzymatic work would c l a r i f y some of the p h y l o g e n e t i c r e l a t i o n s h i p s of the s p e c i e s , s e v e r a l workers have a p p l i e d the technique of e l e c t r o p h o r e s i s to p o p u l a t i o n s of s t i c k l e b a c k , ( A s p i n w a l l , p e r s . comm.; Hagen, 1967; C u c c h i , 1969; and Jones, u n p u b l i s h e d ) . From t h i s work i t appears t h a t s t i c k l e b a c k are i n v a r i a b l e a t the malate dehydrogenase and the alpha-glycerophosphate dehydrogenase l o c i , ( A s p i n w a l l , p e r s . comm.). S e v e r a l other l o c i were examined by A s p i n w a l l , with i n c o n c l u s i v e r e s u l t s . Two p o l y -morphisms have been r e p o r t e d i n the l i t e r a t u r e . Hagen (1967) r e p o r t e d t h a t one of the muscle myogen p r o t e i n s , l a t e r i d e n t i f i e d as c r e a t i n e k i n a s e (Gosselin-Rey e t al.,1968) i n the s t i c k l e b a c k was v a r i a b l e and f u r t h e r s t a t e d t h a t the v a r i a b i l i t y c o u l d be used to d i s t i n g u i s h between freshwater p o p u l a t i o n s ( l e i u r u s ) , and anadromous p o p u l a t i o n s (trachurus) i n l o c a t i o n s where the ranges of the two phases abutted. C a l l e g a r i n i and Cucchi (1968) r e p o r t e d a hemoglobin polymorphism which a l s o appeared to c o r r e l a t e w i t h freshwater and anadromous e x i s t e n c e s , i n southern Europe. Although a d e f i n i t i v e comparison between European p o p u l a t i o n s and North American p o p u l a t i o n s of Gasterosteus a c u l e a t u s has not been c a r r i e d out i t should be noted t h a t the European s t i c k l e b a c k s e l e c t r o p h o r e t i c a l l y s t u d i e d by 3 FIGURE I Three Forms of Gasterosteus Aculeatus These three forms of Gasterosteus have been given names by taxonomists for descriptive convenience. Form A i s called leiurus, B semi-armatus and C trachurus. Often this nomenclature i s based upon the number of later a l armour plates; however there i s a spectrum of forms between the extremes and the distinctions are often arbitrary. (Adapted from, McPhail and Lindsey, 1970, and Hagen and Gilbertson, 1972) 5 I t a l i a n r e s e a r c h e r s , resemble m o r p h o l o g i c a l l y , the North American l e i u r u s form. In Europe, the tr a c h u r u s form i s found along the no r t h e r n c o a s t (Scotland, Norway, U.S.S.R) and i n the Black Sea area. The l e i u r u s form i s g e n e r a l l y found i n southwestern Europe (Spain, France, I t a l y , A l b a n i a , etc.) (Munzing, 1963). C o n s i d e r i n g the unre s o l v e d c o n t r o v e r s y over the meaning of b i o c h e m i c a l v a r i a b i l i t y and the n e u t r a l i s t / n e o d a r w i n i s t arguments (Lewontin, 197 4), i t was i n t e r e s t i n g to note t h a t the o n l y known polymorphisms f o r t h i s s t i c k l e b a c k were both thought t o be en v i r o n m e n t a l l y a d a p t i v e , thereby s u p p o r t i n g the n e o d a r w i n i s t view. B a s i c a l l y , the n e u t r a l i s t view i s t h a t much of the enzymatic v a r i a b i l i t y r e v e a l e d by e l e c t r o -p h o r e t i c s t u d i e s i s i r r e l e v a n t to the w e l l being of the organism. The neodarwinist's (or s e l e c t i o n i s t ' s ) view i s t h a t n e a r l y a l l e l e c t r o i s o z y m e s are i n d i v i d u a l l y s e l e c t e d u n i t s of h e r e d i t y , t h a t s u r v i v e on the b a s i s of t h e i r u t i l i t y t o the i n d i v i d u a l s they are found i n . An examination of the l i t e r a t u r e p r o v i d e s s e v e r a l a l t e r n a t i v e s a ccounting f o r v a r i a b i l i t y p a t t e r n s . Some of these a l t e r n a t i v e s do not n e c e s s a r i l y r e f l e c t g e n e t i c d i f -f e r e n c e s between i n d i v i d u a l s . O b viously then, any r e s e a r c h w i t h p h y l o g e n e t i c i m p l i c a t i o n s must be performed i n such a way t h a t these confounding f a c t o r s are n e u t r a l i z e d . Some p o s s i b i l i t i e s accounting f o r p r o t e i n v a r i a b i l i t y are as f o l l o w s : 6 (1) F i s h Age S e v e r a l groups of r e s e a r c h e r s have r e p o r t e d o n t o g e n e t i c changes i n f i s h hemoglobin. W i l k i n s and l i e s (1966) f o r example, have found marked changes i n the e l e c t r o p h o r e t i c components of h e r r i n g (Clupea harengus) with age. S i m i l a r s i t u a t i o n s e x i s t i n the A t l a n t i c salmon (Salmo s a l a r ) (Koch e t a l . , 1964) , the roach ( R u t i l u s r u t i l u s ) , the rudd ( S c a r d i n i u s erythrophthalmus) (Perez and Maclean, 1974) and i n the e e l p o u t (Zoarus v i v i p a r u s ) ( H j o r t h , 1974). To compensate f o r t h i s phenomenon, one must examine cases of v a r i a b i l i t y to see t h a t the d i s t r i b u t i o n of polymorphisms i s not depen-dent on age. In f i s h , d e t e r m i n a t i o n of r e l a t i v e age does not p r e s e n t a g r e a t problem, as there i s u s u a l l y a r e l a t i o n -s h i p between l e n g t h and age ( w i t h i n p o p u l a t i o n s ) . (2) Seasonal and Temperature V a r i a b i l i t y Seasonal v a r i a b i l i t y i n c o n c e n t r a t i o n s of hemoglobin, hematocrit, and serum p r o t e i n have been noted by Denton and Yousef (1975), i n rainbow t r o u t (Salmo g a i r d n e r i ) . I n t e r e s t -i n g l y enough, i t was d i s c o v e r e d t h a t t h i s v a r i a t i o n i n c o n c e n t r a t i o n s took p l a c e independently of temperature and appeared to be i n response to other environmental f a c t o r s . D i e t , m e t a b o l i c a d a p t a t i o n s and a c t i v i t y were suggested as p o s s i b l e e x p l a n a t i o n s . Baldwin and Hochachka (1970) have shown t h a t rainbow t r o u t (Salmo 'gairdneri) a c c l i m a t e d to d i f f e r e n t temperature 7 regimes e x h i b i t d i f f e r e n t e l e c t r o p h o r e t i c p a t t e r n s of b r a i n a c e t y l c h o l i n e s t e r a s e . T h i s i s i n t e r e s t i n g s i n c e , i n v i t r o , many enzymes have narrow temperature optimums where t h e i r e f f i c i e n c y i s a t a maximum. (3) Environmental A d a p t a t i o n A most i n t e r e s t i n g example of apparent e l e c t r o p h o r e t i c allozyme a d a p t a t i o n was c i t e d by Powers, (1972). He demon-s t r a t e d t h a t two sympatric catostomid f i s h , Catostomis i n s i g n i s and Catostomis c l a r k i i e x h i b i t hemoglobin v a r i -a b i l i t y t h a t appears t o c o i n c i d e w i t h micro h a b i t a t a d a p t a t i o n . E x t e r n a l l y the f i s h are very s i m i l a r , but C. c l a r k i i i s normally found i n f a s t e r f l o w i n g s e c t i o n s o f streams. C. c l a r k i i has an e x t r a e l e c t r o p h o r e t i c hemoglobin component which when b i o c h e m i c a l l y c h a r a c t e r i z e d i s f u n c t i o n a l l y independent of pH,. C. i n s i g n i s components are a l l f u n c t -i o n a l l y dependent upon pH. T h i s dependence i s termed the Bohr e f f e c t and i s normally c o n s i d e r e d to be advantageous to the organism. So, why does C. c l a r k i i have t h i s pH i n -dependent component? Powers t h e o r i z e d t h a t the e x t r a component allowed C. c l a r k i i to remain i n the f a s t f l o w i n g water by m a i n t a i n i n g some oxygen supply to muscle t i s s u e , i n s p i t e of i n c r e a s e d t i s s u e a c i d i t y caused by the e x t r a muscle a c t i o n , thereby a v o i d i n g d i r e c t s p a t i a l c o m p e t i t i o n with C. i n s i g n i s . B i o c h e m i c a l evidence appears to support t h i s theory. .One would t h i n k t h a t l i v i n g i n f a s t e r f l o w i n g water 8 would involve more muscular a c t i v i t y , thereby producing more l a c t i c acid. Black (1958) has shown that when fishes are subjected to extreme exertion the b u i l d up of l a c t i c acid i n the blood can be great enough to overwhelm the buffer capacity of the blood and produce a pH change. Black also showed that t h i s pH change lasted for f a i r l y long periods of time (24 hours). Thus i t would appear that a pH independent component would be extremely useful to an organism under these circumstances. (4) Tissue V a r i a b i l i t y An i n t e r e s t i n g pattern of protein v a r i a t i o n involves v a r i a b i l i t y among tissues within an i n d i v i d u a l organism. Scholl and Eppenberger, (1972) discussed the tissue v a r i -a b i l i t y of creatine kinase i n a variety of f i s h species. Other researchers have i l l u s t r a t e d the same phenomenon with respect to creatine kinase i n frogs (Lyslova, 1971) and birds (Eppenberger, 1968). Examining a s p e c i f i c type of tissue within i n d i v i d u a l s of a population can overcome confusion generated by t h i s phenomenon. (5) H i s t o r i c a l Situations Although i t i s a d i f f i c u l t concept to i l l u s t r a t e , several researchers have attempted to show differences based upon h i s t o r i c a l separations (neutral d r i f t ) . Aspinwall (1974) has i l l u s t r a t e d gene frequency differences between odd and even year classes of c y c l i n g pink salmon, 9 Oncorhyrchus gorbuscha. Pink salmon were i d e a l f o r t h i s study because the year c l a s s e s r e p r e s e n t two i n d i v i d u a l demes. There i s no m i g r a t i o n between these p o p u l a t i o n s . I t i s assumed t h a t the two demes were f o r m e r l y one, and have become separated by h i s t o r i c a l a c c i d e n t . A s p i n w a l l a l s o assumed t h a t the s e l e c t i v e f o r c e s on each year c l a s s have been c o n s t a n t . Most i m p o r t a n t l y , he i l l u s t r a t e d t h a t the moderate amount of m i g r a t i o n between c o a s t a l r i v e r s was g r e a t enough to keep the between year v a r i a b i l i t y g r e a t e r than the v a r i a b i l i t y w i t h i n a year over a wide g e o g r a p h i c a l range. Now, f o r the two r e p o r t e d p r o t e i n polymorphisms i n s t i c k l e b a c k , both polymorphisms are r e p o r t e d l y a s s o c i a t e d with anadromous e x i s t e n c e s . Since both freshwater and s a l t w a t e r p o p u l a t i o n s are a v a i l a b l e i n southwestern B r i t i s h Columbia,, an i d e a l s i t u a t i o n e x i s t s to t e s t these hypotheses. In B r i t i s h Columbia the d i v i s i o n between the d i s t r i b u t i o n s of the forms of s t i c k l e b a c k are l e s s c l e a r than those p r e -v i o u s l y mentioned i n Europe. Normally the l e i u r u s form i s found i n freshwater and the t r a c h u r u s form i s found i n s a l t w a t e r , however t h i s d i s t i n c t i o n i s a gross g e n e r a l i z a t i o n . I f e l t t h a t by d e s i g n i n g the samples to cover the spectrum of m o r p h o l o g i c a l v a r i a t i o n , s a l t or freshwater e x i s t e n c e s , and gross h a b i t a t types one c o u l d get a c l e a r p i c t u r e of the d i s t r i b u t i o n of the polymorphisms w i t h i n a r e l a t i v e l y small g e o g r a p h i c a l area. 10 Because there were d i f f e r e n c e s i n the techniques and h i s t o r i e s of the two r e p o r t e d p r o t e i n polymorphisms I have decided t o r e p o r t my examination of them independently i n two i n d i v i d u a l chapters each w i t h b r i e f s p e c i f i c c o n c l u s i o n s , f o l l o w e d by a f i n a l chapter of g e n e r a l d i s c u s s i o n . 11 CHAPTER I I . STICKLEBACK HEMOGLOBIN 1. Introduction I t a l i a n researchers (C a l l e g a r i n i and Cucchi, 1968; C a l l e g a r i n i and Cucchi, 1969; Cucchi, 1969; Raunich et a l . , 1969; and Raunich et a l . , 1972) have examined a hemoglobin polymorphism i n European stickleback. This polymorphism involves three phenotypes A, AB and B (Fig.2). Phenotype A i s found i n non-migratory freshwater populations of stickleback from western I t a l y and southern Germany.. Populations from the v i c i n i t y of Verona are also monomorphic for phenotype A. Phenotypes AB and B then become more common i n populations as one approaches the A d r i a t i c Sea, p a r t i c u l a r l y i n brackish water situ a t i o n s . Ultimately coastal populations around Venice and Ravena have B A a greater frequency of the Hb a l l e l e than the Hb a l l e l e . Raunich et a l . , (1972) generated the hypothesis that the. H a l l e l e was related to the brackish and s a l t water ex-istence encountered by the migratory sticklebacks. They suggested that researchers from other coastal areas should investigate t h i s hypothesis to see i f the polymorphism was present i n other migratory populations. S e v e r a l questions arose when I examined t h i s work. F i r s t l y , was the polymorphism a genetic phenomenon? FIGURE 2 Patterns of the Reported Hemoglobin Polymorphism In I t a l y The component patte r n s found i n I t a l y are t y p i f i e d by 3 phenotypes, A,AB,and B.These phenotypes separate i n t o d i f f e r e n t component pat-terns at d i f f e r e n t pH values. A l l bands are anodal,S i s the s t a r t i n g p o i n t . B r i t i s h Columbia s t i c k l e b a c k are t y p i f i e d by the A p a t t e r n i n diagram (a),(Raunich et al.,1972). 13 + A A B B (a) p H 8 . 6 A A B B (b) p H 3 . 5 14 The I t a l i a n s suggested t h a t i t was. They conducted breeding experiments, the r e s u l t s of which suggested t h a t the Hb a l l e l e was the product a s i n g l e mutation. T h e i r f i g u r e s were so c l o s e to expected r a t i o s t h a t i n f a c t s t a t i s t i c s d i d not even have to be employed (an amazing f a c t c o n s i d e r i n g other people's l a c k of success (McPhail, p e r s . comm.) a t breeding experiment s u r v i v o r s h i p with western North American s t i c k l e b a c k ) . Secondly, they suggested t h a t the presence of the polymorphism i s r e l a t e d to the b r a c k i s h and s a l t water e x i s t e n c e s of mig r a t o r y p o p u l a t i o n s . The q u e s t i o n then a r i s e s , why weren't p o p u l a t i o n s from the v i c i n i t y of Rome and Naples polymorphic? The I t a l i a n work s t a t e s t h a t m o r p h o l o g i c a l f e a t u r e s were used to d i s t i n g u i s h between migra t o r y and s t a t i o n a r y p o p u l a t i o n s , and the Rome and Naples p o p u l a t i o n s a p p a r e n t l y f i t the s t a t i o n a r y p o p u l a t i o n c r i t e r i o n . These d i s t i n g u i s h i n g f e a t u r e s were not s p e c i f i e d . The t h i r d q u e s t i o n t h a t a r i s e s i s the q u e s t i o n the Europeans pose. Do oth e r p o p u l a t i o n s of s t i c k l e b a c k i n c o a s t a l s i t u a t i o n s e x h i b i t t h i s p r o t e i n polymorphism? S t i c k l e b a c k i n southwestern B r i t i s h Columbia occupy a s i m i l a r c o a s t a l s i t u a t i o n , t h e r e f o r e , I s e t out to examine p o p u l a t i o n s f o r the presence of the hemoglobin polymorphism. I f the polymorphism was pr e s e n t I planned to examine i t s d i s t r i b u t i o n on a micro-geographic s c a l e to t e s t the I t a l i a n h y p o t h e s i s t h a t the polymorphism was r e l a t e d to a b r a c k i s h water e x i s t e n c e . 15 2. Materials and Methods (a) F i e l d Techniques The stickleback were c o l l e c t e d with beach seines, pole seines and minnow traps. The f i s h were returned to the lab and kept a l i v e u n t i l immediately before the electrophoresis was to begin. Mortality during storage was extremely low. The f i s h were cold shocked then k i l l e d . Blood was extracted (Koehn, 196 9) by making an i n c i s i o n i n the an-t e r i o r ventral region into the bulbous arteriosus. Blood was drawn into a pasteur pipet containing a small amount of 4% (w/v) sodium c i t r a t e solution. The sodium c i t r a t e acted as an anticoagulant. The blood was then centrifuged to separate the erythrocytes. This operation was performed three times at 0\u00B0C at about 5,000 r.p.m. The supernatant was replaced a f t e r each centrifugation. The erythrocytes were then lysed by adding a small volume of d i s t i l l e d water to the plug of packed c e l l s . The c e l l p a r t i c l e s were again centrifuged out leaving the hemoglobin in the supernatant. (b) Electrophoretic Techniques Electrophoresis was c a r r i e d out u t i l i z i n g Whatman #4 paper inserts i n horizontal trays (after Selander and Yang, 1969(a)) with a 10.8% (w/v) starch g e l . Buffers used were similar to those used by Selander and Yang (1969(b)) for esterase electrophoresis. S p e c i f i c a l l y , the bridge buffer was composed of solution A and the gel 16 buffer was composed of solution B and A i n a 9:1 r a t i o . Solution A was composed of 0.03 M lithium hydroxide (mono-hydrate) and 0.19M boric acid, pH 8.1; solution B was 0.008M monohydrate c i t r i c acid and 0.05M TRIS, pH 8.4. Electrophoresis was carr i e d out for 2.5 hours at 350 volt s (and approximately 50 amps.) Gels were kept cool with cracked i c e . The gels were stained with the multi-purpose protein stain nigrosene (Brewer, 1970). (c) Geographical Locations (Fig. 3) G. aculeatus of the trachurus form were c o l l e c t e d , i n salt-water (Burrard Inlet) north of the Fraser River's plume, migrating into freshwater to breed ( L i t t l e Campbell River) south of the Fraser River, and from an unusual per-jnenant freshwater s i t u a t i o n (Lake Erroch) . The Lake Erroch f i s h are unusual because they completely resemble (phenotypically) the anadromous trachurus form of the f i s h and, yet are found i n freshwater. I t i s very l i k e l y that t h i s population i s a marine r e l i c t s i m i l a r to others found.in the area, such as the r e l i c t population of long finned smelt, Spirinchus thaleichthys, found i n nearby Harrison Lake, (Dryfoos, 1965). This population could be used to provide a check on any allozyme c h a r a c t e r i s t i c of either leiurus freshwater populations or trachurus anadromous populations, p a r t i c u l a r l y i f there was some evidence that the polymorphism was related to environmental adaptation. FIGURE 3 Geographic Locations of the Stickleback Populations Sampled for the Hemoglobin Polymorphism This map represents the south Vancouver Island, lower Fraser River region of B r i t i s h Columbia. Scale: 1 cm equals 9 km. Sample l o c a t i o n s : 1. Burrard In l e t ; 2. Marion Lake; 3. Mike Lake; 4. Lake Erroch; 5. Pemberton Ditch; 6. L i t t l e Campbell River ( l e i u r u s ) ; 7. L i t t l e Campbell River (trachurus); and 8. C r y s t a l Lake. Freshwater aculeatus of the leiurus form were c o l -lected from Marion Lake and Mike Lake (Lower Mainland north of the Fraser); Pemberton Ditch (Lower Mainland south of the Fraser); and Crystal Lake (Vancouver Island). I t should be noted that the Marion Lake f i s h were o r i g i n a l l y stocked with a Vancouver Island population from Chemainus Lake. 20 3. Results The v i s u a l r e s u l t s of electrophoresis corresponded to the r e s u l t s achieved by staining the gels with nigrosene. That i s , because hemoglobin i s c h a r a c t e r i s t i c a l l y red; one could see the banding patterns without staining. Staining simply improved the resolution of the bands. Further, the agreement of patterns attested to the purity and composition of the hemoglobin. The samples are summarized i n Table 1. No apparent v a r i a t i o n was detected i n any gels on the basis of age (length), sex, habitat or season. A l l gels except one (fig.4) i l l u s t r a t e d the four bands c h a r a c t e r i s t i c of I t a l i a n phenotype A. This aberrant gel was produced from a sample of lake Erroch f i s h . Considering that several hundred other f i s h examined (trapped at the same time) did not exhibit any v a r i a t i o n and the eight variable i n d i v i d u a l s were a l l formed on the same gel, i t seems unlikely that the aberrant gel represented r e a l genetic information. More w i l l be said of t h i s i n chapter four. Further experiments were performed varying the handling time of a single sample of blood. No v a r i a t i o n could be generated within a reasonable amount of time (one hour). If samples were l e f t for longer periods of time polymerization did occur but with no consistent pattern. Never were fewer than four components detected. Another p o s s i b i l i t y that I 21 TABLE I. Summary of Hemoglobin Sample Locations and Results POPULATION SALTWATER OR FRESHWATER FORM HABITAT SAMPLE SIZE DATE BLOOD TYPE Burrard I n l e t s a l t w a t e r trachurus c o a s t a l 32 Jan.1975 type A Marion Lake freshwater l e i u r u s lake 90 July,1975 type A Mike Lake freshwater l e i u r u s lake 13 Nov.1975 type A Lake Errock s a l t w a t e r r e l i c t i n freshwater trachurus l a k e 300 Jan.,May Oct.1975 June,1976 type A Pemberton D i t c h freshwater l e i u r u s stream 12 May,1976 type A L i t t l e Campbell R. freshwater l e i u r u s stream 30 spring1975 type A L i t t l e Campbell R. sa l t w a t e r trachurus c o a s t a l 105 springl975 type A C r y s t a l Lake freshwater l e i u r u s l a k e 37 summer1975 type A 22 FIGURE A The Aberrant Gel This f i g u r e i s a diagramatic representation of the aberrant gel taken from a population of Lake Erroch f i s h . A l l bands were anodal, S i s the s t a r t i n g point, pH 8.4. Four i n d i v i d u a l s were characterized by Hb with 2 components, four i n d i v i d u a l s had three components and seven in d i v i d u a l s had four components(the t y p i c a l type A pattern). 23 I I I I II i llll I III I I I I III I III I III I I I I III I llll I mi i in i II i mi i + cn c o n s i d e r e d was t h a t the f o u r e l e c t r o p h o r e t i c bands r e f l e c t e d the degree of oxygenation of the Hb molecule. Experiments u s i n g cyanide as a complexing agent (Sharp, 1973) showed t h i s not to be the case. .25 4. Discussion and Conclusions In one electrophoretic gel (15 fish) of Lake Erroch trachurus 8 individuals appeared to be aberrant. The banding pattern exhibited by these aberrant samples was similar to the I t a l i a n r e s u l t s (at pH 3.5). Three things are unfortunate about t h i s occurrence. F i r s t l y , the f i s h are k i l l e d to remove the blood. Secondly, my own observations indicated that samples could not be stored. Thirdly, t h i s incident occurred early i n the experimentation and did not appear (at the time) to be unusual. The sum of those three facts, means that there are no second chances at determining the banding pattern of a s p e c i f i c i n d i v i d u a l . Another researcher (Sharp, 1973) has reported transient situations occurring due to the innate i n s t a b i l i t y of f i s h blood. Sharp (1973) recommends either carrying out e l e c t r o -phoresis immediately upon extraction of the blood from the l i v e animals or generating met-hemoglobin by complexing the Hb molecule with cyanide. By carrying out both these a l t e r -natives I found that stickleback hemoglobin exhibits the same res u l t s when either of these alternatives are used. Considering that only a few samples (eight) on one single gel (15 individuals) showed v a r i a t i o n when over 200 ind i v i d u a l samples from the same population immediately before and af t e r the aberrant gel were monomorphic I con-cluded that some technical error had been made on that 25 p a r t i c u l a r day. Obviously then, i n B r i t i s h Columbia where s a l t - w a t e r t r a c h u r u s , freshwater t r a c h u r u s and freshwater l e i u r u s a l l e x h i b i t o n l y phenotype A ( F i g . 2 ), one must r e j e c t the g e n e r a l European hypothesis ( t h a t the polymorphism i s r e l a t e d to an anadromous exist e n c e ) . 27 CHAPTER I I I . STICKLEBACK MUSCLE MYOGEN 1. Introduction Hagen (1967) examined inten s i v e l y , morphological, ecological and electrophoretic differences between a res-i d e n t i a l population of leiurus and an anadromous population of trachurus i n the L i t t l e Campbell River i n Southwestern B r i t i s h Columbia. Hagen i l l u s t r a t e d that, i n the L i t t l e Campbell River the two forms of stickleback are e c o l o g i c a l l y is o l a t e d . Transfer experiments were performed and showed that each form seemed to be well adapted to i t s own habitat and poorly adapted to the habitat of the other. Hagen concluded that the two forms i n th i s p a r t i c u l a r habitat s i t u a t i o n , f i t Mayrs' (1963) c r i t e r i o n as i n d i v i d u a l species. Mayr (197 0) agreed with Hagen's analysis. One of the c h a r a c t e r i s t i c s Hagen u t i l i z e d was the muscle myogen electrophoretic pattern (Fig. 5). He d i s -covered that, as.with.body plates on fishes i n the L i t t l e Campbell River; electrophoretic patterns could be used to dist i n g u i s h each phase. In fact, Hagen f e l t that there was an absolute association between form and electrophoretic pattern. Jones (unpublished) followed t h i s work up by examining f i s h from Paxton Lake. He found that in t h i s lake two forms appear to co-exist. He f e l t however, that they were e c o l o g i c a l l y i s o l a t e d . Larson. (1976) supports t h i s view of the Paxton Lake stickleback. Larson i l l u s t r a t e s that two e c o l o g i c a l l y 28 FIGURE 5 The Muscle Myogen Pattern This figure i s a diagramatic representation of the allozymic patterns of muscle myogen proteins. The polymorphic enzyme has been i d e n t i f i e d as creatine kinase. S i s the s t a r t i n g point at pH 8.4. Type S i s the slow a l l e l e , SF i s the heterozygous a l l e l e and F i s the f a s t a l l e l e . 29 p H 8 . 4 S F F 30 d i f f e r e n t forms of stickleback inhabit the lake. One form i s benthic, one limnetic. Further, i t appears that t h i s ecological separation i s based upon inherent behavioral c h a r a c t e r i s t i c s . Jones u t i l i z e d hybrid index scores with muscle myogen type as one of the characters, but Jones found that the precise d i s t i n c t i o n that Hagen was able to make did not exis t . (Assuming one equates the limnetic and benthic forms described by Larson (.1976) with the trachurus and leiurus forms described by Hagen (1967)). There i s morphological evidence to support t h i s assumption, (Larson, 1976). In-stead Ives found that the benthic phase had a propensity for the F phenotype, whereas the limnetic type had a propensity for the S phenotype (Fig. 5) . A complication emerged when gene frequencies of the two populations were pooled. The pooled gene frequencies were found to coincide with Hardy-Weinburg frequencies. How two morphologically and behaviorally d i s t i n c t populations can have a common gene with a l l e l e s at a Hardy-Weinburg frequency has not been s a t i s f a c t o r i l y explained. Two important b i t s of information should also be con-sidered with regard to the work i n Paxton Lake. F i r s t l y , Paxton Lake was dammed i n 1956, e f f e c t i v e l y eliminating cutthroat trout, Salmo c l a r k i i , the only s i g n i f i c a n t predator of stickleback i n the lake. Secondly, Hay and McPhail (1975) reported that sticklebacks prefer to mate with l i k e forms. Accordingly, with the complications involved i n the Paxton Lake system, i t was plausible that Hagen 1s i n i t i a l description of electrophoretic information was indeed ac-curate ( i . e . trachurus exclusively one phenotype, leiurus the other) and that the Paxton Lake population was an aber-rant population where premating i s o l a t i n g mechanisms had broken down between anadromous f i s h trapped by the dam and the resident population. I. .therefore..decided-to., test Hagen 1 s i n i t i a l assumption that the c h a r a c t e r i s t i c phenotype F (Fig. 5) typefied fresh water leiurus whereas the phenotype S typefied anadromous trachurus. Further, as Jones' work implied, there i s often a difference between l i f e s t y l e s of the two extreme phases. Higher plated animals tend to be limnetic planktivores whereas lower plated animals tend to be benthic browsers. Accordingly, to incorporate t h i s aspect into the hypothesis a range of populations from low to high (mean) plated was examined (in ..other words, to test i f the polymorphism was related to l i f e s t y l e I assumed that increased plate number re f l e c t e d an increased pelagic nature). Fish were also examined from a variety of gross habitat types. (Table 2) I t should be noted that Jones i d e n t i f i e d the poly-morphic components as being the enzyme creatine kinase, as other researchers (Gosselin - Rey, et al., 1968) have 32 done with o t h e r f i s h e s . T h i s was performed u s i n g enzyme s p e c i f i c s t a i n s . C r e a t i n e k i n a s e i s one of the enzymes which a i d s i n the r e g e n e r a t i o n of ATP d u r i n g muscular a c t i v i t y (Bergmeyer, 1974) . c r e a t i n e phosphate + ADP >creatine + ATP 33 TABLE I I . A Summary of Muscle Myogen Population Locations, Forms and Habitats POPULATION FORM SALT/FRESHWATEE HABITAT POTENTIAL PREDATORS Burrard I n l e t trachurus saltwater coastal present Mike Lake l e i u r u s freshwater bog lake present Lake Erroch trachurus freshwater lake present Pemberton Ditch l e i u r u s freshwater stream present L i t t l e Campbell R. le i u r u s freshwater stream present L i t t l e Campbell R. trachurus saltwater c o a s t a l present Dougan Lake l e i u r u s freshwater small lake present Chemainus Lake l e i u r u s f r e s h u a t p r p i - n h a h l <= Mesachie Lake semiarmatus freshwater large lake present Beck Lake l e i u r u s freshwater lake present Diver Lake l e i u r u s freshwater small lake present Brannen Lake l e i u r u s freshwater lake present 2. M a t e r i a l s and Methods (a) F i e l d Techniques F i s h e s were c o l l e c t e d u t i l i z i n g the methods previously-d e s c r i b e d i n Chapter I I . The f i s h were e i t h e r k i l l e d immediately upon capture and f r o z e n on dry i c e or f r o z e n a f t e r blood e x t r a c t i o n . The samples were then s t o r e d u n t i l e l e c t r o p h o r e s i s was c a r r i e d out. (b) E l e c t r o p h o r e t i c Techniques At the a p p r o p r i a t e time the samples were thawed and a b l o c k of muscle was removed from the l e f t caudal r e g i o n of each f i s h . Care was taken t h a t o n l y muscle was removed. The muscle was mixed with an equal volume of 0.055M phosphate b u f f e r a t pH 7.5 and homogenized f o r approximately 10 seconds. The homogenate was c e n t r i f u g e d a t 5000 r.p.m. f o r 5 minutes i n a r e f r i g e r a t e d c e n t r i f u g e a t 0\u00B0C. Paper i n s e r t s soaked i n the supernatant were su b j e c t e d to e l e c t r o -phores i s i n s t a r c h g e l , as d e s c r i b e d i n chapter I I . The g e l s were sub j e c t e d to 300 v o l t s f o r two hours. Gels were s t a i n e d w i t h the a l l purpose p r o t e i n s t a i n nigrosene. (c) Geographical L o c a t i o n F i s h e s were c o l l e c t e d from s e v e r a l l o c a t i o n s i n south-western B r i t i s h Columbia. ( F i g . 6) 35 S t i c k l e b a c k used were, of course, those c o l l e c t e d f o r hemoglobin e l e c t r o p h o r e s i s . A l s o , f i s h e s from p o p u l a t i o n s r e f l e c t i n g a c l i n e of mean p l a t e numbers, were c o l l e c t e d from southeastern Vancouver I s l a n d (Table 3). The samples r e f l e c t e d a c r o s s - s e c t i o n of forms and h a b i t a t types. (d) Sample Storage Samples were s t o r e d a t -30\u00B0C. A small experiment was performed to t e s t a n e c d o t a l i n f o r m a t i o n t h a t samples c o u l d only be s t o r e d b r i e f l y . A sample of twelve f i s h was f r o z e n i n e a r l y October, 1975, thawed f o r e l e c t r o p h o r e s i s one week l a t e r , r e f r o z e n , and then re-examined e a r l y September, 1976. 36 FIGURE 6 Geographical Locations of Stickleback Populations Sampled for the Muscle Myogen Polymorphism Scale: 1 cm equals 9 km. Sample l o c a t i o n s : 1. Burrard Inl e t ; 2. Mike Lake; 3. Lake Erroch; 4.Pemberton Ditch; 5. L i t t l e Campbell River l e i u r u s ; 6. L i t t l e Campbell River trachurus; 7. Dougan Lake; 8. Chemainus Lake; 9. Mesachie Lake; 10. Beck Lake; 11. Diver Lake; 12. Brannen Lake; 13. Paxton Lake (studied by Jones and Larson). 37 TABLE I I I . A Summary of Vancouver Island Populations Sampled By assuming that increasing plate number per i n d i v i d u a l r e f l e c t s increasing pelagic nature one can t e s t the hypothesis that muscle myogen genotypes are r e l a t e d to l i f e s t y l e . Mean plate numbers per i n d i v i d u a l were previously determined by J.D. McPhail (pers.com.). LOCATION MEAN PLATE NO. PER INDIVIDUAL Chemainus Lake 6.0 Dougan Lake 7.3 Beck Lake 10.0 Brannen Lake 11.2 Diver Lake 11.8 Mesachie Lake 14.5 39 3. Results Banding patterns were similar to those observed by-Jones (unpublished) and Hagen (1967), (Fig. 5). Variation occurred at a single locus with three banding patterns possible; SS, SF and FF. Five of the twelve populations examined were poly-morphic. In each of these cases the gene frequencies did not d i f f e r s i g n i f i c a n t l y from the Hardy-Weinburg frequencies, (Table 4) . . The f i v e polymorphic populations were not char-acterized by any gross habitat type, (Table 4). Two creek populations, two lake populations, and one i n d i v i d u a l from an anadromous population were polymorphic. Variation was not apparently based upon age ( s i z e ) , or season. (Fig. 7 (a) (b) ) '.' With regard to storage, experiments were performed re-examining refrozen samples a f t e r a year of storage. Results after one year were, actually ,superior to the results, i n -i t i a l l y obtained. In other words, i n i t i a l l y ten individuals in a sample of twelve were c l e a r l y readable; one year l a t e r twelve samples were c l e a r l y readable with the l a t t e r s i t u -ation r e i t e r a t i n g the. interpretation of the former ten r e s u l t s . 40 TABLE IV. Muscle Myogen Frequencies Theoretical a l l e l e frequencies were calculated from the real data then expected Hardy-Weinberg frequencies were extrapolated. The resulting theoretical values were compared with the real frequencies using the Chi test. Stati s t i c a l l y there was no discrepancy between the real and theoretical values(figures in parentheses reflect expected values). POPULATION SAMPLE SIZE S (SLOW) S/F F (FAST) CHI2 AND PROBABILITY Burrard Inlet 32 32 - -Lake Erroch 45 45 - -L i t t l e Campbell R. (leiurus) 45 8(8.91) 24(22.23) 13(13.86) 0.28,P = .65 L i t t l e Campbell R. (trachurus)1975 34 33 1 -L i t t l e Campbell R. (trachurus) 1976 45 45 - -Mike Lake 13 13 - -Pemberton Ditch 12 7(7.44) 5(3.96) -(0.48) 0.78, P = .40 Chemainus Lake 30 30 - -Dougan Lake 28 12(12.88) 14(12.04) 2(2.80) 0.61, P = .45 Beck Lake 28 8(8.12) 14(14.00) 6(5.88) 0.004,P = .95 Diver Lake 30 30 - -Brannen Lake 30 30 - -Mesachie Lake 45 45 - -41 FIGURE 7 Age and Season Figure 7a.: Considering length to be synonymous with age within a given population(eg. Beck Lake) a s t a t i s t i c a l t e s t was performed to see i f there was any i n d i c a t i o n of a r e l a t i o n s h i p between the muscle myogen polymorphisms and ontogenetic development. No r e l a t i o n s h i p was found therefore i t was concluded that the presence of the various protein types was independent of age. Figure 7b.: Polymorphic gels were discovered at various times of year. This would seem to imply that the polymorphisms were not seasonally induced. 42 (A) ANALYSIS OF LENGTH (AGE) VERSUS GENOTYPE GENOTYPE S GENOTYPE SF GENOTYPE F (length i n mm.) 30.3 30.8 28.6 27.3 24.1 28.4 27.7 25.1 27.5 24.4 51.4 23.4 24.2 29.3 23.4 35.0 28.3 37.3 54.3 189.0 35.6 35.8 31.1 34.7 235.3 36.1 x = 31.5 37.1 38.4 x = 29.4 34.2 30.7 470.2 x = 33.6 ANOVA SOURCE SUM OF SQUARES DEGREES FREEDOM MEAN SQUARE BETWEEN MEANS 90.2 2 45.1 WITHIN GROUPS 1433.5 25 57.3 Test H o: F l = h = H^ = not the same Test s t a t i s t i c 2 F = Sm^ , c r i t i c a l r e g i o n F < F > 9 9 ( 2 } 2 5 ) = 5.6 Sp The value f o r F at Beck Lake i s 0.8 t h e r e f o r e one must accept the hypothesis that the means are equal. (B) Time of year sample taken polymorphic p o p u l a t i o n May 1976 Pemberton D i t c h J u l y 1973 Paxton Lake August 1976 L i t t l e Campbell R. l e i u r u s September 1976 Beck and Dougan Lake 43 4. Discussion and Conclusion Obviously, Hagen's o r i g i n a l assumption, that leiurus and trachurus are typefied by s p e c i f i c electrophoretic banding patterns, must be rejected for the species complex as a whole. Both the highest plated and lowest plated animals ( i . e . ocean trachurus vs. Chemainus Lake leiurus) are e l e c t r o p h o r e t i c a l l y s i m i l a r . Indeed, even samples from the leiurus zone of the L i t t l e Campbell River do not com-pl e t e l y r e f l e c t Hagen's o r i g i n a l impression. Further, using f i s h length within a population as an in d i c a t i o n of f i s h age one must conclude that the poly-morphism i s not ontogenetic i n nature. That i s , the poly-morphism i s present i n both small and large f i s h e s . When Jones car r i e d out his electrophoresis, f i s h smaller than 15 mm were completely homogenized. Researchers (Perriard et .al., 1972) have shown enzyme v a r i a b i l i t y between tissues, within a single i n d i v i d u a l . Indeed some researchers (Eppenberger et al.,. 1971; Lyslova, 1971) have found t h i s very phenomenon occurring with the enzyme creatine kinase. By taking muscle tissue from the l e f t caudal peduncle area th i s complication was avoided. Accordingly then, one can assume that the v a r i a b i l i t y observed was r e a l . If one examines (Fig. 8) the geographical d i s t r i b u t i o n of the protein polymorphism one notes there i s no i n d i c a t i o n of the polymorphism r e f l e c t i n g ranges of i n d i v i d u a l demes of FIGURE 8 Geographical Locations of the Polymorphic Populations Populations 4, 5, 7, 10, and 13 (as w e l l as one i n d i v i d u a l from the sample from population 6) were discovered to be polymorphic. The s i t e s of these populations are circled.No geographic trend i s apparent. .45 Gasterosteus a c u l e a t u s . A l s o , c o n s i d e r i n g a l t i t u d e as a rough estimate of i n v a s i o n time, o l d e r p o p u l a t i o n s do not d i f f e r from the assumed p a r e n t a l p o p u l a t i o n s . (ocean trachurus) A comparison of gross h a b i t a t c h a r a c t e r i s t i c s ( i . e . stream, l a k e , s a l t - w a t e r , freshwater, etc.) w i t h regard to the d i s t r i b u t i o n of the polymorphism p r o v i d e s no obvious c o r r e l a t i o n . 47 CHAPTER IV. GENERAL DISCUSSION My study c o n t a i n s r e s u l t s t h a t are important a t s e v e r a l d i f f e r e n t l e v e l s . T h i s can be summarized i n the f o l l o w i n g way. (a) 'Stickleback Hemoglobin Southwestern B r i t i s h Columbia Gasterosteus a c u l e a t u s have monomorphic hemoglobin. The t r a n s i e n t polymorphism served, on a s i n g l e o c c a s s i o n , c a s t s some doubt on the European work, p a r t i c u l a r l y when one c o n s i d e r s the s i m i l -a r i t i e s between the t r a n s i e n t p a t t e r n a t pH 8.4 and the European r e s u l t s (Raunich e t al\u00AB, 1972) a t pH 3.5. Obviously t h e i r work should be re-examined to double check t h e i r r e s u l t s . However, i t would appear u n l i k e l y t h a t t h e i r s i t u -a t i o n i s a technique a r t i f a c t , simply because the Europeans performed breeding experiments and found t h a t the o f f s p r i n g r a t i o s of the Hb polymorphism corresponded to a s i t u a t i o n w ith v a r i a t i o n i n a s i n g l e gene. I t should be noted t h a t only o f f s p r i n g were examined. I t i s c l e a r , t h a t i f t h i s aspect of s t i c k l e b a c k g e n e t i c s i s to be persued i t should be done i n the A d r i a t i c area, perhaps on a more p h y s i o l o g i c a l l e v e l . As I mentioned i n Chapter I, s t i c k l e b a c k do not seem to f i t t r a d i t i o n a l s p e c i e s paradigms. I t would appear, t h e r e -f o r e , t h a t comparative g e n e t i c i n f o r m a t i o n among widely d i s t r i b u t e d p o p u l a t i o n s has i n t e r e s t . 48 Some d i s c u s s i o n (Avise, 1975) has oc c u r r e d s u p p o r t i n g the use o f e l e c t r o p h o r e s i s i n s y s t e m a t i c s , p a r t i c u l a r l y when s e v e r a l d i f f e r e n t l o c i are examined. Other r e s e a r c h e r s have compared s p e c i e s a t a s i n g l e l o c u s . T s y u k i e t a l . , (1963) and Sharp (1973) f o r example have compared hemoglobins among v a r i o u s r e l a t e d s p e c i e s . The former group examined hemo-g l o b i n molecules of seven salmonids of the n o r t h e a s t e r n P a c i f i c Coast, and found t h a t the banding p a t t e r n s were very d i f f e r e n t f o r each s p e c i e s , both i n terms of component number and shape. Ferguson (1974) c o n s i d e r e d s e v e r a l e l e c t r o -p h o r e t i c p a t t e r n s (general p r o t e i n s , e s t e r a s e s , c r e a t i n e kinase and phosphoglucomutase) to suggest s p e c i e s s t a t u s f o r three forms of. a complex of coregonid f i s h e s . These coregonid are extremely v a r i a b l e m o r p h o l o g i c a l l y , with many of the v a r i a t i o n s , known to be env i r o n m e n t a l l y induced. With these precedents i n mind, i t appears t h a t one co u l d be j u s t i f i e d i n saying, the s i m i l a r i t y among hemoglobin banding p a t t e r s (anodal bands, three l a r g e , one small) of a l l freshwater (and some saltwater) European s t i c k l e b a c k s and a l l southern B r i t i s h Columbia s t i c k l e b a c k s p r o v i d e s r e i n f o r c i n g evidence t h a t the p o p u l a t i o n s i n q u e s t i o n belong to the same s p e c i e s , (b) S t i c k l e b a c k Muscle Myogen Two p o s s i b i l i t i e s remain with r e s p e c t to e x p l a i n i n g the d i s t r i b u t i o n of the c r e a t i v e k i n a s e a l l e l e s i n s t i c k l e b a c k p o p u l a t i o n s . 4 9 F i r s t l y , i t i s p o s s i b l e t h a t the polymorphism r e f l e c t s a d a p t a t i o n to a s u b t l e environmental c h a r a c t e r . T h i s h y p o t h e s i s i s extremely d i f f i c u l t to t e s t , and probably i m p o s s i b l e to d i s c o u n t . Secondly, the p o s s i b i l i t y a l s o e x i s t s t h a t the p o l y -morphism i s s e l e c t i v e l y n e u t r a l . There i s some evidence t o support t h i s p o i n t of view. A l l polymorphic p o p u l a t i o n s were i n Hardy-Weinburg e q u i l i b r i u m . A d m i t t e d l y the samples were s m a l l , however, wi t h f i v e polymorphic p o p u l a t i o n s , a l l i n Hardy-Weinberg e q u i l i b r i u m i t would seem not i m p l a u s i b l e t h a t the a l l e l e s are s e l e c t i v e l y n e u t r a l . F u r t h e r , R e d f i e l d e t a l . , (1972) attempted a s i m i l a r study examining a blood plasma polymorphism i n blue grouse (Dendragapus obscurus) p o p u l a t i o n s from a wide range of h a b i t a t s on Vancouver I s l a n d . R e s u l t s i n the grouse s i t u -a t i o n were s i m i l a r to those i n s t i c k l e b a c k except a l l p o p u l a t i o n s examined co n t a i n e d the polymorphism. T h i s r e s u l t was c i t e d as proof of s e l e c t i v e maintenance. Study over a p e r i o d of time, to see i f gene f r e q u e n c i e s , and l o c a t i o n s of polymorphic s t i c k l e b a c k p o p u l a t i o n s change or remain s t a b l e , would c l a r i f y t h i s s i t u a t i o n . Another i n t e r e s t i n g p o i n t i s t h a t s t i c k l e b a c k are not the f i r s t f i s h r e p o r t e d to have a muscle myogen polymorphism. I t has been s t a t e d i n the l i t e r a t u r e (Morgan and Ulanowicz, 1976; Uthe and Ryder, 1970); t h a t muscle myogen polymorphism i s extremely r a r e . One i s i n c l i n e d to comment t h a t muscle 50 myogen polymorphisms can be moved from the r a r e to the l e s s than uncommon category s i n c e a t l e a s t s i x s p e c i e s are known to have polymorphisms of t h i s type. A comparision of these s p e c i e s (Table 5) i l l u s t r a t e s the p h y l o g e n e t i c and environmental range encompassed by t h i s polymorphism. (The polymorphism has not.been p o s i t i v e l y i d e n t i f i e d as c r e a t i n e k i n a s e i n a l l these examples). Species e x h i b i t i n g the polymorphism i n c l u d e s a l t w a t e r f i s h e s , , freshwater f i s h e s , r i v e r f i s h e s , p e l a g i c ocean f i s h e s , p i s c i v o r e s and h e r b i v o r e s . The o n l y common f a c t o r among these s p e c i e s i s t h a t they are a l l t e l e o s t s , and t h i s c o u l d be a r e f l e c t i o n of the s p e c i e s sampled. T h e . c r e a t i n e k i n a s e polymorphism a l s o emphasizes the l a c k of b i o c h e m i c a l s t r u c t u r a l i n f o r m a t i o n across p h y l o -g e n e t i c l i n e s . C r e a t i n e k i n a s e i s a r e l a t i v e l y w e l l s t u d i e d enzyme because of i t s . a s s o c i a t i o n w i t h the Duchenne type of muscular dystrophy (Bergmeyer, 1974). I t has always been thought to have a d i m e r i c s t r u c t u r e (Kuby et a l . , 1962). T h i s i d e a i s not compatible with the s t i c k l e b a c k polymorphism. I f the enzyme was d i m e r i c the heterozygous form (S/F) would have co n t a i n e d three bands i n s t e a d of two ( F i g . 9). I t i s remotely p l a u s i b l e t h a t t h i s d i m e r i c s t r u c t u r e c o u l d be masked by l i m i t a t i o n s of the technique on one o c c a s s i o n , however t h i s m u l t i t u d e of examples p r o v i d e s many r e p e t i t i o n s a l l i m p l y i n g non-dimeric s t r u c t u r e . May (1975) a l s o p o i n t e d out t h i s monomeric nature of c r e a t i n e kinase i n the genus Onchorynchus. Many e l e c t r o p h o r e t i c s t u d i e s have p o i n t e d out TABLE V. A Comparison of the Six Species Demonstrating a Similar Muscle Myogen Protein Polymorphism SPECIES LOCATION HABITAT ECOTYPE FOOD REFERENCES Menidia menidia A t l a n t i c coast estuarine areas schooling copepods shrimp mysids Morgan and Ulanowicz, 1976. Liem and Scott,1966 Catostomus catostomus North America and Siberia freshwater bottom browser amphipods insect larvae McPhail and Lindsey, 1970. Tsuyuki et al.,1967. Anoplopoma fimbria North P a c i f i c saltwater pelagic piscivore Tsuyuki and Roberts, 1969. Hart,1973. Sebastodes elongatus North America P a c i f i c coast saltwater 50-200 fathoms - Tsuyuki et a l . , 1969. Hart,1973. Stizostedion vitreum central and east North America freshwater pelagic schooler piscivore Uthe and Ryder, 1970. McPhail and Lindsey,1970. Gasterosteus aculeatus northern hemisphere freshwater and anadromous benthic/pelagic ostracods larvae plankton McPhail and Lindsey, 1970. 52 FIGURE 9 Hypothetical Pattern for Dimeric Quarternary Structure of the Enzyme Creatine Kinase S represents the s t a r t i n g point. Homozygous S i s represented by component a, homozygous F i s represented by component c and heterozygous SF has both these components present plus component b (which would be a composite form of a and c ) . t S S F F 54 s i m i l a r m olecular i n c o n g r u i t i e s but few r e s e a r c h e r s seem to have i n v e s t i g a t e d f u r t h e r . For example comparisons of mammalian hemoglobin seems to have caused people to g e n e r a l i z e the p h y l o g e n e t i c c o n s i s t e n c y of the quartenary s t r u c t u r e of a l l p r o t e i n s of a l l animals. Consider the taxonomic arguments c i t e d i n t h i s chapter with regard to hemoglobin. The same argument can be a p p l i e d to the d i s t r i b u t i o n of c r e a t i n e k i n a s e . The extreme forms of s t i c k l e b a c k , l e i u r u s and t r a c h u r u s , were e l e c t r o p h o r e t i c a l l y s i m i l a r . That i s , s a l i n e p o p u l a t i o n s of t r a c h u r u s were e l e c t r o p h o r e t i c a l l y s i m i l a r t o the l e i u r u s p o p u l a t i o n s most remote from the ocean. T h i s would a l s o p r o v i d e r e i n f o r c i n g evidence to support the view t h a t l e i u r u s and t r a c h u r u s are members o f the same s p e c i e s , (c) Future,Research Consider the blood polymorphism i n Europe. As I s t a t e d e a r l i e r , the a u t h e n t i c i t y of the polymorphism should be checked. Assuming the outcome of t h i s check i s p o s i t i v e , I would then perform the f o l l o w i n g b i t s of r e s e a r c h . The f i r s t o p e r a t i o n I would perform would i n v o l v e b i o c h e m i c a l l y c h a r a c t e r i z i n g each e l e c t r o p h o r e t i c hemoglobin component found i n . G a s t e r o s t e u s a c u l e a t u s phenotypes A, AB and B ( F i g . 2). T h i s would e i t h e r support the European hypothesis or suggest some other a l t e r n a t i v e h y p o t h e s i s . F u r t h e r , I would i n c l u d e a d e t e r m i n a t i o n of the molecular weights of each of the components so t h a t a c l e a r e r under-standing of t h e i r s t r u c t u r a l nature might emerge. (Perhaps 55 even going as far as sequencing the protein). Then, armed with a new hypothesis or reinforced old hypothesis I would perform transfer experiments. I would transport offspring of known parentage into s a l t , brackish and freshwater environments to determine whether or not the oxygen capacities were gene t i c a l l y predetermined or the re s u l t of acclimatization, (which would also be a genetic phenomenon). Guernsey and Puluhowich (1975) i l l u s t r a t e d t h i s l a t t e r phenomenon i n blood oxygen capacities of the /American eel (Anguilla rostrata) i n s a l t , brackish and freshwater environments. A check for t h i s phenomenon i n B r i t i s h Columbia stickleback may also prove i n t e r e s t i n g . Would anadromous and freshwater sticklebacks (with the same electrophoretic components) have d i f f e r e n t oxygen capacities based upon e l e c t r o p h o r e t i c a l l y undetectable genetic d i f -ferences . Another possible project could involve a biochemical comparison between the hemoglobin of the threespine s t i c k l e -back (G. aculeatus) and the ninespine stickleback (Pungitius pungitius) . Lewis et al., (1972) observed that i n England these two species are often found together i n the same habitats. They are also known to have v i r t u a l l y i d e n t i c a l d i e t s , (Hynes), 1950). The question arose, how do these species avoid, competition? Lewis et a l . , observed that within those habitats, niche separation appeared to occur on the basis of oxygen a v a i l a b i l i t y . Pungitius pungitius 5; 6 appeared to be more t o l e r a n t of low oxygen l e v e l s because of p h y s i o l o g i c a l a d a p t a t i o n . Since i n Canada, areas e x i s t where these s p e c i e s are found together and a l s o areas e x i s t where they are found alone ( S c o t t and Crossman, 1973), i t would appear t h a t some i n t e r e s t i n g work c o u l d be c a r r i e d out with r e s p e c t to b i o c h e m i c a l a d a p t a t i o n , and p o s s i b l y (physio-l o g i c a l ) c h a r a c t e r displacement (Dobzhanski, 1970). Raunich et a l . , (1972) s t a t e d i n I t a l y P. P u n g i t i u s hemoglobin i s e l e c t r o p h o r e t i c a l l y s i m i l a r to the hemoglobin of non-migratory Gasterosteus a c u l e a t u s . Now, the q u e s t i o n a r i s e s what f u r t h e r work should be done with the c r e a t i n e kinase polymorphism? I f I were c o n t i n u i n g i n t h i s r e s e a r c h I would t r y to l e a r n more about the i.Yi v-u&io b i o c h e m i c a l c h a r a c t e r i s t i c s of the c r e a t i n e k i n a s e components. I would e s t a b l i s h pH optima, temperature optima pressure, e f f e c t s and molecular weights of the v a r i o u s components. T h i s might answer the q u e s t i o n whether or not the components are n e u t r a l , or g i v e i n s i g h t i n t o t h e i r f u n c t i o n , ( i f they have some s u b t l e f u n c t i o n ) . F u r t h e r , as S c h o l l and Eppenberger (1972) have p o i n t e d out, e l e c t r o -p h o r e t i c components of c r e a t i n e kinase vary among t i s s u e s w i t h i n i n d i v i d u a l f i s h e s . The q u e s t i o n a r i s e s , Why? Why should muscle have one form of c r e a t i n e k i n a s e and b r a i n , and t e s t e and h e a r t and stomach t i s s u e s a l l have other i n d i v i d u a l forms? Obviously, more knowledge on the 57 s t r u c t u r a l and b i o c h e m i c a l c h a r a c t e r i s t i c l e v e l s may p r o v i d e more i n s i g h t i n t o t h i s q u e s t i o n . Other q u e s t i o n s a r i s e i n t h i s r e g a r d . What i s the f u n c t i o n of c r e a t i n e kinase i n the b r a i n ? Do enzymes such as c r e a t i n e k i n a s e have m u l t i p l e f u n c t i o n s ? Much more i n f o r m a t i o n i s needed. 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"Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "An examination of two reported protein polymorphisms in threespine stickleback, Gasterosteus aculeatus"@en . "Text"@en . "http://hdl.handle.net/2429/20838"@en .