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UBC Theses and Dissertations

Effects of crowding and water borne stressors on five fishes with different social and ecological habits Furnell, Donald J. 1982

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EFFECTS OF CROWDING AND WATER BORNE STRESSORS ON FIVE FISHES WITH DIFFERENT SOCIAL AND ECOLOGICAL HABITS B.Sc. Hon.,University Of V i c t o r i a , V i c t o r i a , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF 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 1 October 1982 by DONALD J . FURNELL THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in Donald J . F u r n e l l , 1 982 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of c^oo/.o&^ The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i ABSTRACT The r e s p o n s e s o f f i v e e t h o l o g i c a l l y d i s t i n c t f i s h e s t o c r o w d i n g a n d w a t e r b o r n s t r e s s o r s were e x a m i n e d . I n i t i a l e x p e r i m e n t s u s i n g one s t o c k i n g d e n s i t y i n d i c a t e d a c t i v a t e d c a r b o n removed a w a t e r b o r n s t r e s s o r i n h i b i t i n g g r o w t h i n Corynopoma r i i s e i b u t n o t R i v u l u s h a r t i . L a t e r e x p e r i m e n t s u s i n g B r a c h y d a n i o r e r i o , A s t r o n o t u s o c e l l a t u s , a n d B e t t a  s p l e n d e n s c o n f i r m e d t h e r e m o v a l o f a w a t e r b o r n s t r e s s o r by a c t i v a t e d c a r b o n f o r a l l b u t B e t t a s p l e n d e n s . The s t r e s s o r ' s e f f e c t a p p e a r e d t o d e p e n d on a s p e c i e s ' n a t u r a l h a b i t a t . When c u l t u r e d , f i s h e s a d a p t e d t o s m a l l t r a n s i e n t w a t e r b o d i e s t o l e r a t e d o r d i d n o t s e c r e t e s t r e s s o r s ; t h o s e f r o m p e r m a n e n t , r a p i d l y d i l u t i n g e n v i r o n m e n t s were i n h i b i t e d i n g r o w t h o r s u r v i v a l . R e s p o n s e t o c r o w d i n g o r s o c i a l i n t e r a c t i o n was e x a m i n e d i n B r a c h y d a n i o r e r i o , A s t r o n o t u s o c e l l a t u s a n d B e t t a s p l e n d e n s . R e d u c t i o n o f g r o w t h o r s u r v i v a l was d i r e c t l y r e l a t e d t o s t o c k i n g d e n s i t y i n B r a c h y d a n i o r e r i o a n d A s t r o n o t u s o c e l l a t u s , b u t t h e e f f e c t was l e s s t h a n h a l f t h a t o f w a t e r b o r n s t r e s s o r s . I n A s t r o n o t u s o c e l l a t u s c r o w d i n g s t r e s s r e a c h e d a t h r e s h o l d b e y o n d w h i c h i n c r e a s e d d e n s i t y h a d no e f f e c t . S o c i a l i n t e r a c t i o n i n B e t t a s p l e n d e n s d i d n o t i n h i b i t g r o w t h , b u t c a u s e d an i n c r e a s e i n e n e r g y c o n s u m p t i o n . No r e l a t i o n s h i p b e t w e e n a s p e c i e s ' s o c i a l h a b i t s a n d r e s p o n s e t o c r o w d i n g c o u l d be d i s c e r n e d . I n B r a c h y d a n i o r e r i o and A s t r o n o t u s o c e l l a t u s , t h e c o m b i n e d e f f e c t s were c o m p e n s a t o r y o r a d d i t i v e , r e s p e c t i v e l y , when c r o w d i n g a nd w a t e r b o r n s t r e s s o r s were s i m u l t a n e o u s l y e x a m i n e d . i i i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i I n t r o d u c t i o n 1 Space competition '. . 2 Water q u a l i t y 4 Research o b j e c t i v e s 9 Methods 11 F i l t e r m a t e r i a l i d e n t i f i c a t i o n 12 Density experiments i n f i l t e r e d and u n f i l t e r e d water ... 14 Measurement of responses to treatments 18 R e s u l t s 20 F i l t e r m a t e r i a l i d e n t i f i c a t i o n 20 Density experiments i n f i l t e r e d and u n f i l t e r e d water ... 23 Brachydanio r e r i o 23 Astronotus o c e l l a t u s 34 Betta splendens 42 D i s c u s s i o n 50 Corynopoma r i i se i i 50 R i v u l u s h a r t i 51 Brachydan i o r e r i o 52 Astronotus o c e l l a t u s ' 53 Betta splendens 55 Summary 56 L i t e r a t u r e c i t e d 60 Appendix I 67 V LIST OF TABLES Table 1. S p e c i e s - s p e c i f i c maintenance d e t a i l s 18 Table 2. V a r i a b l e a u t o c o r r e l a t i o n check, Brachydanio r e r i o 26 Table 3. Growth a n a l y s i s f o r Brachydanio r e r i o 34 Table 4. V a r i a b l e a u t o c o r r e l a t i o n check, Astronotus o c e l l a t u s 38 Table 5. Growth a n a l y s i s f o r Astronotus o c e l l a t u s 41 Table 6. V a r i a b l e a u t o c o r r e l a t i o n check, Be t t a splendens .. 4'2 Table 7. P r o p o r t i o n of Betta splendens with bubblenests ... 45 Table 8. F i n a l mean weights of Bett a splendens 48 Table 9. Growth a n a l y s i s f o r Betta splendens 49 Table 10. Summary of responses and h a b i t s 57 v i L I S T OF FIGURES F i g u r e 1. P h a s e 1 e x p e r i m e n t a l t a n k 13 F i g u r e 2. P h a s e 2 e x p e r i m e n t a l t a n k 15 F i g u r e 3. F i n a l mean l e n g t h s o f Corynopoma r i i s e i 21 F i g u r e 4. F i n a l mean l e n g t h s o f R i v u l u s h a r t i 21 F i g u r e 5. B r a c h y d a n i o r e r i o , r e p l i c a t e 1, low s t o c k i n g d e n s i t y 24 F i g u r e 6. B r a c h y d a n i o r e r i o , r e p l i c a t e 1 , mid' s t o c k i n g ' d e n s i t y 24 F i g u r e 7. B r a c h y d a n i o r e r i o , r e p l i c a t e 1, h i g h s t o c k i n g d e n s i t y 24 F i g u r e 8. B r a c h y d a n i o r e r i o , p o o l e d r e p l i c a t e s 2 a n d 3, low d e n s i t y 27 F i g u r e 9. B r a c h y d a n i o r e r i o , p o o l e d r e p l i c a t e s 2 a n d 3, m i d d e n s i t y 27 F i g u r e 10. B r a c h y d a n i o r e r i o , p o o l e d r e p l i c a t e s 2 a n d 3, h i g h d e n s i t y 27 F i g u r e 11. B r a c h y d a n i o r e r i o , r e p l i c a t e 1, a l l d e n s i t i e s , w i t h o u t AC f i l t r a t i o n 29 F i g u r e 12. B r a c h y d a n i o r e r i o , r e p l i c a t e 1, a l l d e n s i t i e s , w i t h AC f i l t r a t i o n 29 F i g u r e 13. B r a c h y d a n i o r e r i o , p o o l e d r e p l i c a t e s 2 a n d 3, a l l d e n s i t i e s , w i t h o u t AC f i l t r a t i o n 31 F i g u r e 14. B r a c h y d a n i o r e r i o , p o o l e d r e p l i c a t e s 2 and 3, a l l d e n s i t i e s , w i t h AC f i l t r a t i o n 31 F i g u r e 15. P r o p o r t i o n of Astronotus o c e l l a t u s s u r v i v i n g at low d e n s i t y 36 F i g u r e 16. P r o p o r t i o n of Astronotus o c e l l a t u s s u r v i v i n g at mid d e n s i t y 36 F i g u r e 17. P r o p o r t i o n of Astronotus o c e l l a t u s s u r v i v i n g at h i g h d e n s i t y 36 F i g u r e 18. P r o p o r t i o n of Astronotus o c e l l a t u s s u r v i v i n g , a l l d e n s i t i e s , with AC f i l t r a t i o n 39 F i g u r e 19. P r o p o r t i o n of Astronotus o c e l l a t u s s u r v i v i n g , a l l d e n s i t i e s , without AC f i l t r a t i o n 39 F i g u r e 20. B e t t a splendens with v i s u a l c o n t a c t 43-F i g u r e 21. B e t t a splendens without v i s u a l c o n t a c t 43 F i g u r e 22. B e t t a splendens with AC f i l t r a t i o n 46 F i g u r e 23. B e t t a splendens without AC f i l t r a t i o n 46 v i i i ACKNOWLEDGEMENTS: Glen Z e l l e r and K. T u r b e r f i e l d o f the American K i l l f i s h A s s o c i a t i o n were i n v a l u a b l e i n su p p l y i n g r e a r i n g a d v i c e and stock. Dr. N . L i l e y p r o v i d e d r a r e a d u l t s f o r breeding u n c u l t u r e d experimental stock and in f o r m a t i o n on t h e i r c o l l e c t i o n and h a b i t s . Maureen Baker l e n t a w i l l i n g hand when the d a i l y f e e d i n g schedule c o u l d not be met. Instrumental water a n a l y s i s was provided by Dr. P. H a r r i s o n ' s l a b o r a t o r y . The r e s e a r c h was funded by a N a t u r a l Sciences and En g i n e e r i n g Research C o u n c i l grant to Dr. N.J. Wilimovsky who added welcome ad v i c e , encouragement and l a b o r a t o r y space without which t h i s work co u l d not have been completed. $SIGNOFF 1 INTRODUCTION The h e a l t h , growth, and r e p r o d u c t i o n of f i s h e s are synchronously i n f l u e n c e d by many agents. In nature, they may be broadly c a t e g o r i z e d as co m p e t i t i o n , p r e d a t i o n and the p h y s i c a l - c h e m i c a l a q u a t i c environment. Depending on the system, they vary i n i n t e n s i t y and d i f f e r e n t s p e c i e s have evolved d i f f e r e n t a d a p t a t i o n s to d e a l with them. When f i s h e s are c u l t u r e d , an attempt i s made to i d e n t i f y and optimize these agents. In aq u a r i a and many l a r g e f i s h c u l t u r e o p e r a t i o n s , p r e d a t i o n can be' e l i m i n a t e d or r e s t r i c t e d to f a r below n a t u r a l l e v e l s . Removing p r e d a t o r s i n c r e a s e s c o m p e t i t i o n and degradation of the a q u a t i c environment. Tr o p h i c competition can be negated by fe e d i n g to s a t i a t i o n and removing excess food (Magnuson 1962). Competition f o r space i s not as r e a d i l y c o n t r o l l e d . I t v a r i e s with c o n t a i n e r s i z e and shape, may have t h r e s h o l d s of e f f e c t i v e n e s s (Brown 1946a,b,c, Chen and Prowse 1964, Minchin 1972) or may have no e f f e c t . A b i o t i c f a c t o r s in the aqua t i c environment are l a r g e l y under c o n t r o l of c u l t u r i s t s , although optimal water q u a l i t y i s d i f f i c u l t to r e a l i z e . Research i n t o space competition and water q u a l i t y has produced c o n f l i c t i n g r e s u l t s which may be r e l a t e d to s p e c i f i c a d a p t a t i o n s of the d i f f e r e n t f i s h e s i n v o l v e d . T h i s r e s e a r c h seeks to q u a n t i f y and compare the e f f e c t s and i n t e r a c t i o n s of these f a c t o r s among sp e c i e s of d i f f e r e n t h a b i t s and environments. 2 SPACE COMPETITION E a r l y crowding r e s e a r c h oh a q u a t i c animals studied" t a d p o l e s and s n a i l s as w e l l as f i s h . K a w a j i r i (1928) found rainbow t r o u t s t o c k i n g d e n s i t y d i r e c t l y r e l a t e d to s u r v i v a l , but i n v e r s e l y r e l a t e d to growth; f i n a l biomass at a l l d e n s i t i e s reached an e q u i l i b r i u m . Breder and Coates (1932) concluded that a c o n t a i n e r c o u l d only support a f i x e d number of guppies even i f the o r i g i n a l stock d e n s i t i e s were much d i f f e r e n t . Rugh (1934) s p e c u l a t e d that i n c r e a s e d c o l l i s i o n s by t a d p o l e s , stocked at higher d e n s i t y , caused gr e a t e r a c t i v i t y and reduced growth. Wright (1960) drew t h i s c o n c l u s i o n f o r s n a i l s . B i l l i a r d et a l . (1981) do not s p e c i f y c o l l i s i o n s or c o n t a c t s , but c o n s i d e r v i s u a l i n t e r a c t i o n i n f i s h e s a cause of crowding s t r e s s . Crowding s t r e s s may be unequally d i s t r i b u t e d w i t h i n a s o c i a l h i e r a r c h y and depend on h i e r a r c h y type. Using Salmo  t r u t t a , Brown (1946a,b,c) e s t a b l i s h e d that crowding s t r e s s was mediated through s o c i a l dominance and the inverse r e l a t i o n s h i p between growth and s t o c k i n g d e n s i t y had an asymptote. Studying medaka, Magnuson (1962) saw v a r y i n g d e n s i t i e s only a f f e c t e d growth when t r o p h i c competition o c c u r r e d . In reviewing crowding s t r e s s i n mammals, Andrews (1979) d i s c u s s e s the crowding r e s p o n s e - s o c i a l h i e r a r c h y r e l a t i o n s h i p i n view of b e h a v i o u r a l type. M a i n t a i n i n g f i s h at equal d e n s i t y and v a r y i n g c o n t a i n e r s i z e may negate crowding e f f e c t s on growth (Chen and Prowse 1964) or c o n v e r s e l y , enhance i t (Minchin 1972). Greene (1964) 3 r e c o g n i z e d t h e p o t e n t i a l f o r an i n t e r a c t i o n o f f a c t o r s a n d s u g g e s t e d c r o w d i n g a n d s e c r e t o r y p r o d u c t s may a c t s y n e r g i s t i c a l l y . R e c e n t work i n w h i c h n i t r o g e n o u s e x c r e t o r y p r o d u c t s were c o n t r o l l e d showed c r o w d i n g r e d u c e d g r o w t h ; t h e e f f e c t was c a u s e d by r e d u c e d a p p e t i t e o r e f f i c i e n c y o f f o o d u t i l i z a t i o n ( J a c k s o n a n d Payne 1 9 8 1 ) . D e v e l o p m e n t o f a n a l y t i c a l t e c h n i q u e s h a s made p o s s i b l e e x a m i n a t i o n o f p r i m a r y a n d s e c o n d a r y r e s p o n s e s t o s t r e s s (Murphy 1967, S t r a n g e e t a l . 1 9 7 8 ) . S c h r e c k (1981)' f o u n d c r o w d i n g d e c r e a s e d g r o w t h i n c o h o s a l m o n , b u t s e c o n d a r y s t r e s s i n d i c a t o r s w e r e l a c k i n g . Wedemeyer (1976) f o u n d s e c o n d a r y i n d i c a t o r s o n l y l a s t e d a week a f t e r s t o c k i n g . T h i s s u g g e s t s e i t h e r f i s h h a b i t u a t e t o c r o w d i n g s t r e s s o r s e c o n d a r y i n d i c a t o r s q u a n t i f y h a n d l i n g r a t h e r t h a n c r o w d i n g s t r e s s a n d t h e d e n s i t i e s u s e d c a u s e d no s t r e s s . To s t u d y e f f e c t s o f s p a c e c o m p e t i t i o n , e i t h e r t h e s p a c e o r t h e number o f f i s h i n i t c a n be v a r i e d . B e c a u s e i t i s p o s s i b l e t o i n f l u e n c e f i s h by c o n t a i n e r s h a p e , t h e o p t i o n o f a l t e r i n g v o l u m e w i t h o u t d i s t u r b i n g s h a p e i s i m p o s s i b l e . E f f e c t s o f s p a c e c o m p e t i t i o n may b e s t be e x a m i n e d by l e a v i n g c o n t a i n e r s i z e c o n s t a n t a n d v a r y i n g f i s h d e n s i t y i n t h a t f i x e d s p a c e . R e s p o n s e s t o c r o w d i n g a r e w e l l s t u d i e d , a l t h o u g h e x p e r i m e n t a l v a r i a t i o n o f o t h e r f a c t o r s makes c o m p a r i s o n d i f f i c u l t . I n f u r t h e r d i s c u s s i o n , c r o w d i n g , s t o c k i n g d e n s i t y , a n d s p a c e c o m p e t i t i o n w i l l r e f e r s y n o n y m o u s l y t o v a r i a t i o n s o f number p e r u n i t v o l u m e i n 4 i d e n t i c a l c o n t a i n e r s . WATER QUALITY R e s e a r c h i n t o w a t e r b o r n e f a c t o r s h a s h i s t o r i c a l l y c e n t e r e d a r o u n d c e r t a i n g r o u p s o f w o r k e r s . E a r l y i n t e n s i v e i n v e s t i g a t i o n was done by W.C. A l l e e a nd c o l l e a g u e s i n C h i c a g o d u r i n g t h e 1 9 3 0 s . They were c o n c e r n e d w i t h e x a m i n i n g g r o w t h enhancement i n homo and h e t e r o t y p i c a l l y c o n d i t i o n e d w a t e r ( i e . w a t e r i n w h i c h c o n s p e c i f i c s a nd o t h e r s p e c i e s , r e s p e c t i v e l y , h a d been h e l d ) . I n f o u r p a p e r s ( A l l e e e t a l . 1934, 1936, 1940, L i v e n g o o d 1937) t h e y p r e s e n t e d o b s e r v a t i o n s on enhancement o f g r o w t h by h o m o t y p i c w a t e r i n g o l d f i s h , p l a t i e s , a n d b l a c k b u l l h e a d c a t f i s h . W a t e r a c c u m u l a t e d ammonia, o r g a n i c n i t r o g e n , t o t a l a n d f i x e d o r g a n i c r e s i d u e s , a nd s u l f a t e , c h l o r i d e , s o d i u m , c a l c i u m , p o t a s s i u m , a n d magnesium i o n s i n r e s p o n s e t o i n t r o d u c t i o n o f f i s h e s . They a t t r i b u t e d g r o w t h enhancement t o an o r g a n i c compound a l t h o u g h a u t o c l a v i n g d i d n o t i n a c t i v a t e i t . E v a n s (1 9 3 6 ) e s t a b l i s h e d t h a t v i t a m i n s were n o t i n v o l v e d a s h a d been s p e c u l a t e d . A s u b s t a n c e w i t h a s i m i l a r e f f e c t a t m a s s i v e d i l u t i o n c o u l d be e x t r a c t e d f r o m g o l d f i s h s k i n ( A l l e e e t a l . 1 9 3 6 ) . W a t e r c o n d i t i o n e d by m u s s e l s a l s o e n h a n c e d g r o w t h , b u t n o t f i n r e g e n e r a t i o n , i n p l a t i e s (Shaw 1 9 3 2 ) . These s t u d i e s were t h e l a s t t o a t t r i b u t e g r o w t h enhancement t o a w a t e r b o r n e f a c t o r ; s u b s e q u e n t r e s e a r c h s u g g e s t e d an i n v e r s e r e l a t i o n s h i p . I t i s n o t e w o r t h y t h a t c o n d i t i o n i n g i n t h e s e e x p e r i m e n t s e n t a i l e d h o l d i n g f i s h i n 5 water f o r only 22 hours and agents had l i t t l e time f o r accumulation to c o n c e n t r a t i o n s s t u d i e d in l a t e r work. The next i n v e s t i g a t o r s to take up t h i s problem worked in the l a b o r a t o r y of Meryl S. Rose. Lynn and Edelman (1936) r e c o g n i z e d water q u a l i t y a f f e c t e d metamorphosis in Rana l a r v a e and Rose (1959a,b, 1960) continued t h i s work in f i s h e s and t a d p o l e s . Richards (1958) found t a d p o l e s ' growth i n h i b i t e d by agents removable with ageing water 26 days, h e a t i n g , f r e e z i n g , c e n t r i f u g a t i o n , f i l t r a t i o n , or s o n i c a t i o n . She a s s i g n e d the' f a c t o r to a l g a l - l i k e c e l l s which were l a t e r (Richards 1962) c l a s s i f i e d as p a r a s i t e s . Passed i n f e c e s , they c o u l d be c u l t u r e d . Using c u l t u r e technique, Akin (1966) found the c e l l s l o s t i n h i b i t o r y e f f e c t i n t h r e e g e n e r a t i o n s and tadpoles i n h i b i t e d each o t h e r ' s growth with or without access to d i e t a r y f e c a l c e l l s . She proposed the " c e l l s were c a r r i e r s which l a t e r r e l e a s e d i n t o the water a t a d p o l e - s e c r e t e d i n h i b i t o r y f a c t o r . The u n i d e n t f i e d c e l l s were o b l i g a t e to growth i n h i b i t i o n . Rose (1959a,b) expanded i n v e s t i g a t i o n s to f i s h e s and found guppies and whiteclouds i n h i b i t e d growth of c o n s p e c i f i c s , but were not i n t e r s p e c i f i c a l l y e f f e c t i v e . In c o n f i r m i n g the Rana work, Rose (1960) i n d i c a t e d growth i n h i b i t i o n was stronger i n smaller p o p u l a t i o n members and n e g l i g i b l e in the l a r g e s t . T h i s suggests water borne agents d i s t r i b u t e t h e i r e f f e c t unequally over a s o c i a l h i e r a r c h y as does s p a c i a l crowding. Swingle (l956a,b) r e p o r t e d a water borne r e p r o d u c t i v e 6 i n h i b i t o r y substance i n ponds s e p a r a t e l y h o l d i n g g o l d f i s h , c arp and b u f f a l o f i s h . I t prevented r e p r o d u c t i o n u n t i l f i s h e s were p l a c e d i n t o f r e s h water. Whiteside and Richan (1969) made a f u r t h e r c o n f i r m a t i o n of Swingle's o b s e r v a t i o n s . In a s s o c i a t i o n with Swingle, Greene (1964) found a r e p r o d u c t i v e s e l f i n h i b i t o r s e c r e t e d i n t o water by Brachydanio r e r i o . S i m i l a r responses were e l i c i t e d by other fami l y members, but not by u n r e l a t e d c e n t r a r c h i d s . He a t t r i b u t e d much of the e f f e c t to ammonia, e r r o n e o u s l y b e l i e v i n g a c t i v a t e d carbon removed' ammonia (Hassler 1974). He does, however, p o i n t out p o s s i b l e i n t e r a c t i o n s between space c o m p e t i t i o n and s e c r e t o r y p r o d u c t s . The d i f f i c u l t y i n pond s t u d i e s i s the i n a b i l i t y to c o n t r o l f a c t o r s such as ammonia or oxygen d e p l e t i o n . More r e c e n t l y , water borne compounds have been p a r t i a l l y i s o l a t e d which, i n v a r y i n g degree, have negative e f f e c t s on a q u a t i c animals. Wright (i960) used a c t i v a t e d carbon to remove a water borne f a c t o r from s n a i l water. He a l s o i d e n t i f i e d a need to examine water borne agents at d i f f e r e n t d e n s i t i e s and observe f a c t o r i n t e r a c t i o n s . In a study which accounted f o r ammonia in ponds, Tang (1963) found i n h i b i t i o n i n Swingle's experiments was probably caused by ammonia. Yu and Perlmutter (1970) f i l t e r e d water h o l d i n g Brachydan io r e r i o over methyl c h l o r o f o r m and removed a growth i n h i b i t o r . Water from blue gouramis ( T r i c h o g a s t e r  t r i c h o p t e r u s ) produced no e f f e c t on Brachydanio r e r i o . Using a b i o c h e m i c a l assay, Pfuderer et a l . (1974) i s o l a t e d a 7 h e a r t - r a t e d e p r e s s e n t i n mono and d i g l y c e r i d e a n d s t e r o l f r a c t i o n s o f c o n s p e c i f i c ' s w a t e r e x t r a c t i o n s . F r a n c i s e t a l . ( 1 9 7 4 ) e x t r a c t e d a f a c t o r f r o m g o l d f i s h a n d c a r p w a t e r , u s i n g a c t i v a t e d c a r b o n , t r i c h l o r o e t h a n e , and c a r b o n t e t r a c h l o r i d e . I t i n h i b i t e d g r o w t h i n e i t h e r s p e c i e s , b u t n o t i n d i s t a n t l y r e l a t e d s u n f i s h and g u p p i e s . Chen and M a r t i n i c h ( 1 9 7 5) r e p o r t e d a c o m b i n a t i o n o f two w a t e r b o r n e f a c t o r s was r e q u i r e d t o i n d u c e o v u l a t i o n i n B r a c h y d a n i o  r e r i o . The f i r s t was r e m o v a l o f an i n h i b i t o r by u s i n g f r e s h w a t e r , t h e o t h e r was' p r e s e n c e o f a m a l e pheromone. T h i s * s u g g e s t s a s y n e r g i s m o f f a c t o r s . S m i t h ( 1 9 7 7 ) t e s t e d t h e h y p o t h e s i s t h a t mucus s e c r e t i o n s c a u s e d i n c o m p a t a b l i t y o f d i f f e r e n t f i s h e s h e l d t o g e t h e r . He u s e d a b l o o d c l o t t i n g a s s a y t o show m u l l e t mucus c a u s e d a n t i g e n i c r e a c t i o n s i n T i l a p i a b l o o d c e l l s . T h e r e was, h o w e v e r , no i n t r a s p e c i f i c r e s p o n s e . I n f u r t h e r i n t e r s p e c i f i c t e s t s , m i l k f i s h c e l l s c l o t t e d i n r e s p o n s e t o T i l a p i a a n d l u t j a n i d mucus, b u t a g a i n had no i n t r a s p e c i f i c r e a c t i o n s . T h i s i n t e r s p e c i f i c e f f e c t i s d i s s i m i l a r t o p r e v i o u s s p e c i e s - s p e c i f i c i n h i b i t o r s a n d t h e r e i s no e m p i r i c a l e v i d e n c e o f g r o w t h a l t e r a t i o n due t o i t . H e n d e r s o n - A r z a p a l o a n d S t i c k n e y ( 1 9 8 0 ) i s o l a t e d a h y p e r s e n s i t i v e a n a p h a l a c t i c component i n s l i m e and w a t e r o f T. m o z a m b i c a . I t was s p e c i f i c w i t h i n t h e g e n u s , b u t n o t t o an u n r e l a t e d c a t f i s h . S o l v e n t e x t r a c t i o n a n d e l e c t r o p h o r e s i s d e m o n s t r a t e d i t m i g r a t e d a s a B - g l o b u l i n . I t c a u s e d i n c r e a s e d m o r t a l i t y and c o u l d n o t be e x t r a c t e d f r o m c l o s e l y r e l a t e d T. a u r e a . A t l o w d e n s i t y i t s e f f e c t a p p e a r e d 8 reduced. T h i s study i s however, confused by the f a c t that the authors d e s c r i b e t h e i r system as a flow through (40 minutes per turnover), although the f a c t o r took 4-11 weeks to accumulate. To g e n e r a l i z e , both space competition and s e c r e t o r y products can be c o n s i d e r e d s t r e s s o r s . They have been shown to produce v a r i o u s r e a c t i o n s i n f i s h e s depending on s p e c i e s , probably a c t i n g through the primary, secondary, and t e r t i a r y pathways of s t r e s s response (Love 1974, Mazeaud et a l . 1977). E f f e c t s are e x p l a i n e d by the gen e r a l a d a p t a t i o n syndrome (Selye 1956) encompassing phases of alarm, r e s i s t a n c e and f i n a l l y e x haustion. A l l stages are of concern to an a q u a c u l t u r i s t because they channel energy f o r growth i n t o other metabolic a c t i v i t i e s . S t r e s s o r s may i n t e r a c t or act i n d e p e n d e n t l y . p * l o b v i o u s l y there i s no general agreement between authors on the nature of i n h i b i t o r y substances or space c o m p e t i t i o n . How then do a q u a c u l t u r i s t s respond to optimize growth in t h e i r stock? Oxygen demand i s of primary importance (and consequently carbon d i o x i d e ) . These can l a r g e l y be c o n t r o l l e d by a e r a t i o n (Brockway 1950, Willoughby 1968). I t i s g e n e r a l l y recognized that ammonia i s t o x i c (Brockway 1950, Westers 1977, H i l l a b y and Rand a l l 1979) and i t can be t r e a t e d i n r e c i r c u l a t i n g systems by ion exchange or b a c t e r i a l f i l t r a t i o n (Spotte 1970, 1979, Speece 1973, L i a o and Mayo 1974). The l a t t e r method has the advantage of d i g e s t i n g organic m a t e r i a l s . T h i s lowers b i o l o g i c a l oxygen demand (BOD), p r i m a r i l y a s s o c i a t e d with 9 suspended s o l i d s such as waste food (Speece 1973), but does not e l i m i n a t e i t . Some of the remaining f r a c t i o n can be removed with micromechanical f i l t r a t i o n ( i e . diatomaceous e a r t h ) , but r e f r a c t o r y products remain (Spotte 1979). These are of i n c r e a s i n g importance as water re-use becomes an economic n e c e s s i t y i n heated systems (Mayer and Kramer 1973). T h e i r e f f e c t s on f i s h need e l u c i d a t i o n . To quote Henderson-Arzapalo and S t i c k n e y (1980), "Further r e s e a r c h i n t o t h i s [water borne s t r e s s o r s ] i s needed to determine i t s e f f e c t w i t h i n f i s h s p e c i e s and i t s i m p l i c a t i o n s i n natural" and f i s h c u l t u r e ecosystems." A c u l t u r e ecosystem must a l s o c o n s i d e r s t o c k i n g d e n s i t y and i n t e r a c t i o n of both f a c t o r s . RESEARCH OBJECTIVES The f o r e g o i n g i n f o r m a t i o n opens at l e a s t four q u e s t i o n s which bear i n v e s t i g a t i o n . They concern e f f e c t s of water borne agents other than those normally accounted f o r by c u l t u r a l proceedures, e f f e c t s of space c o m p e t i t i o n , i n t e r a c t i o n s between these f a c t o r s and f i n a l l y d i s t r i b u t i o n of e f f e c t s i n r e l a t i o n to d i f f e r e n t s p e c i e s ' a d a p t a t i o n s . T h i s r e s e a r c h w i l l t h e r e f o r e address the f o l l o w i n g four q u e s t i o n s : 1. Using standard water treatment techniques (to c o n t r o l ammonia, BOD, and oxygen c o n c e n t r a t i o n s ) , i s there an accumulation of water borne agents which i n f l u e n c e f i s h e s h e l d in c l o s e d systems? 10 2. Are f i s h e s a f f e c t e d by s t o c k i n g d e n s i t y when h e l d i n i d e n t i c a l water and c o n t a i n e r s and fed to excess? 3. What i s the r e l a t i v e e f f e c t i v e n e s s of these f a c t o r s and together are they compensatory or depensatory? 4. Do d i f f e r e n t s p e c i e s respond to water borne agents and space competition d i f f e r e n t l y ? 11 METHODS The h a b i t s , zoogeography, aquarium h i s t o r y , and stock sources of the f o l l o w i n g experimental s p e c i e s are summarized i n appendix I: Corynopoma r i i s e i ( G i l l ) , R i v u l u s hart i (Boulenger), Brachydanio re r i o (Hamilton-Buchanan), Astronotus o c e l l a t u s ( A g a s s i z ) , and Betta splendens (Regan). Two phases of experiments were conducted. The f i r s t i d e n t i f i e d f i l t e r s l i k e l y to remove water borne s t r e s s o r s . Two s p e c i e s with d i f f e r e n t h a b i t s (Corynopoma r i i s e i and R i v u l u s h a r t i ) were r a i s e d s e p a r a t e l y at an equal low d e n s i t y each with one of three f i l t e r mediums p l u s an u n f i l t e r e d c o n t r o l . Growth i n e x p e r i m e n t a l l y f i l t e r e d tanks was then compared with u n f i l t e r e d c o n t r o l f i s h . Phase 2 experiments examined e f f e c t s of s t o c k i n g d e n s i t y i n the presence and absence of water borne s t r e s s o r s . These experiments used one f i l t e r m a t e r i a l , a f f e c t i n g growth i n phase 1 experiments, to remove the water borne s t r e s s o r s from water h o l d i n g three e t h o l o g i c a l l y d i v e r g e n t f i s h s p e c i e s at three s t o c k i n g d e n s i t i e s . The e f f e c t of water borne s t r e s s o r - c r o w d i n g s t r e s s i n t e r a c t i o n was t e s t e d by comparison to simultaneously run u n f i l t e r e d c o n t r o l s at three d e n s i t i e s . Growth, normal s u r v i v a l and/or c h a l l e n g e s u r v i v a l were used to index p h y s i o l o g i c a l e f f e c t s of s t o c k i n g d e n s i t y and removal of water borne s t r e s s o r s . 1 2 FILTER MATERIAL IDENTIFICATION The three t e s t f i l t e r m a t e r i a l s , a c t i v a t e d carbon (AC), diatomaceous e a r t h (DE) and s t r o n g l y a c i d i c c a t i o n exchange r e s i n s (IR) were s e l e c t e d based on the range of compounds each removed and t h e i r s u i t a b i l i t y to f a s t flow aquarium use. In s e l e c t i n g AC, DE, and IR i t was hoped a s u f f i c i e n t range of substances would be removed to i n c l u d e any a f f e c t i n g f i s h e s . These were h e l d i n c o n v e n t i o n a l motor d r i v e n e x t e r n a l aquarium f i l t e r s operated 2-3 hours twice d a i l y a f t e r f e e d i n g . In a d d i t i o n to experimental f i l t e r s , each tank was equipped with b i o l o g i c a l sponge f i l t e r s f o r n i t r i f i c a t i o n of ammonia, a i r stones, g r a v e l , s y n t h e t i c p l a n t s ( f o r R i v u l u s h a r t i ) , and two Corydoras aneus to consume excess food. Rearing tanks (Figure 1) were c r e a t e d by d i v i d i n g a l a r g e r 200 L aquarium i n t o four s e a l e d chambers with a l a r g e heated r e s e r v o i r to maintain constant temperature. In two separate runs, 20 4-5 week o l d f r y of Corynopoma r i i s e i and l a t e r R i v u l u s h a r t i were p l a c e d i n t o e x p e r i m e n t a l l y f i l t e r e d tanks and the u n f i l t e r e d c o n t r o l a l l f i l l e d with c l e a n , aged tap water p l u s 180 ml of seawater (Hattingh et a l . 1975). Fry were randomly assigned to treatments from a s i n g l e l a r g e tank h o l d i n g more than twice the 80 f i s h r e q u i r e d . Fry were fed twice d a i l y , to excess, with Artemia n a u p l i i , t u b i f e x worms, commercial dry food, Enchytraeus a l b i d u s , or beefheart p r e p a r a t i o n . They were maintained at 25-27° C with a 12-16 hour photoperiod. Weekly, 30% of the water was 13 Figure 1. Configuration of the phase 1 experimental tank. A-D are sealed 18 L treatment tanks. E is one of 3 external power filters. F is the heated water resevoir. replaced with fresh tap water at the same temperature. After approximately six weeks (Rivulus harti-41 days, Corynopoma  r i i s e i - 3 9 days) a l l fishes were anesthetized with quinaldine and a f i n a l standard length taken, with vernier c a l i p e r s , to the nearest mm. Daily checks during the experiment showed pH ranged from 6.5-7.0, dissolved oxygen stayed near saturation and ammonia level s were below 0.25 ppm. Oxygen was analyzed using a modified Winkler technique; nutrients and pH were determined c o l o r i m e t r i c a l l y . 14 DENSITY EXPERIMENTS IN FILTERED AND UNFILTERED WATER Phase I experiments demonstrated AC removed a growth i n h i b i t o r y substance from water h o l d i n g Corynopoma r i i s e i , but not R i v u l u s h a r t i . T h i s made AC a promising m a t e r i a l to employ i n more ex t e n s i v e t e s t i n g and i n d i c a t e d R i v u l u s h a r t i e i t h e r t o l e r a t e d or d i d not s e c r e t e a growth i n h i b i t o r y substance a f f e c t i v e at t h i s s t o c k i n g d e n s i t y or AC, DE, and IR were inca p a b l e of removing i t . R e s u l t s suggested examining a range of s p e c i e s and s t o c k i n g d e n s i t i e s , o b t a i n i n g r e p l i c a t e d i n f o r m a t i o n . P r a c t i c a l c o n s t r a i n t s l i m i t e d t e s t i n g of f i l t e r m a t e r i a l s to AC, although c a t i o n exchange r e s i n s and b i o l o g i c a l f i l t e r s were f i t t e d to a l l systems f o r ammonia c o n t r o l and r e d u c t i o n of BOD. Phase 2 experiments, using Astronotus o c e l l a t u s and Brachydanio r e r i o , employed a s e r i e s of 30 L a q u a r i a d i v i d e d by screens i n t o three i n d i v i d u a l compartments in a l i n e a r arrangement (Figure 2). Each tank was f i t t e d with a 660 L/hr power f i l t e r m o d i f i e d to accept AC and c a t i o n r e s i n f i l t e r c a r t r i d g e s . P o l y e s t e r f i l t e r bags, s u p p l i e d with the power f i l t e r s , were allowed to develop a b a c t e r i a l coat t o a i d i n n i t r i f i c a t i o n , reduce BOD, and p r o v i d e mechanical f i l t r a t i o n . AC c a r t r i d g e s were recharged with f r e s h wood c h a r c o a l every three days. IR c a r t r i d g e s were a l s o r e p l a c e d every three days. Spent r e s i n s were scrubbed in s c a l d i n g water and r e a c t i v a t e d by soaking overnight i n double the bed volume (500ml) of 10% sodium c h l o r i d e s o l u t i o n . The frequency of IR f i l t e r changes was based on p r e l i m i n a r y runs Figure 2. Configuration of a single phase 2 experimental tank with an activated carbon cartridge installed. Direction of water flow indicated by a thick arrow. A, is a turbulence reduction compartment. B-D, are rearing compartments from low to high density respectively. E, is an activated carbon cartridge. F, is a cation exchange resin cartridge. G. is a 660L/Hr filter pump. H Ln 16 with the l a r g e s t s p e c i e s (Astronotus o c e l l a t u s ) . A f t e r s i x days ammonia l e v e l s i n c r e a s e d , which demonstrated that r e s i n s were s a t u r a t e d and no longer e f f e c t i v e . Screens were washed weekly i n s c a l d i n g water and excess food thoroughly siphoned from a q u a r i a a f t e r each f e e d i n g , the siphon water passed through a f i n e net and returned to the tank. Water i n each compartment was a g i t a t e d by a i r s t o n e s and d i s s o l v e d oxygen c o n c e n t r a t i o n s h e l d near s a t u r a t i o n . Constant equal temperature was achieved by immersing tanks in* a common water bath with' a heater balanced a g a i n s t a slow f r e s h i n f l o w (< 1L/hr). A 12-14 hour photoperiod was maintained by timed f l o u r e s c e n t l i g h t s . Three r e p l i c a t e s were conducted with Brachydanio r e r i o (three AC f i l t e r e d and three u n f i l t e r e d c o n t r o l s ) i n two separate runs. Larger f i s h were used and a longer ( s i x week) experiment done with a s i n g l e p a i r of tanks. L a t e r , two simultaneous r e p l i c a t e s were done with s l i g h t l y s maller Brachydanio rer i o . T h i s p e r m i t t e d t e s t i n g the system before committing a l a r g e r batch of j u v e n i l e s to experiments. Due to d i f f e r e n c e s i n i n i t i a l s i z e , r e p l i c a t e s have been analyzed as two separate groups in f o l l o w i n g s e c t i o n s . R e p l i c a t e runs with a l l other s p e c i e s were synchronous. Only Brachydanio r e r i o and Astronotus o c e l l a t u s were reared i n aquarium systems i l l u s t r a t e d i n F i g u r e 2. These employed high flow r a t e pumps u n s u i t a b l e to the f i n a l experimental s p e c i e s , Betta splendens, normally an i n h a b i t a n t of stagnant waters. Furthermore, i t was 17 i m p o s s i b l e t o k e e p more t h a n one m a l e B e t t a s p l e n d e n s i n a s i n g l e e n c l o s u r e due t o t h e i r s e v e r e f i g h t i n g t e n d e n c i e s . C o n s e q u e n t l y t h i s s p e c i e s was r e a r e d i n t h e t r a d i t i o n a l manner u s i n g one L j a r s e a c h h o l d i n g a s i n g l e f i s h . To e x a m i n e t h e e f f e c t o f s o c i a l i n t e r a c t i o n , some f i s h were r e a r e d i n j a r s p l a c e d s i d e by s i d e v i e w i n g t h e n e i g h b o u r i n g f i s h w h i l e o t h e r s were r a i s e d i n j a r s c o v e r e d by d a r k g r e e n p l a s t i c p r o h i b i t i n g v i s u a l c o n t a c t . S i x t y B e t t a s p l e n d e n s were r a i s e d i n t h i s manner. To e x a m i n e e f f e c t s o f w a t e r b o r n e s t r e s s o r s , t h e s i x t y j a r s ' were d i v i d e d ' i n t o two g r o u p s . One was f i l l e d w i t h w a t e r s t o r e d i n a l a r g e h o l d i n g t a n k f i l t e r e d w i t h AC, b i o l o g i c a l , a nd m e c h a n i c a l mediums, t h e o t h e r g r o u p f i l l e d f r o m a s e c o n d t a n k w i t h o u t an AC f i l t e r . The 30 j a r s i n e a c h w a t e r g r o u p were e q u a l l y s p l i t i n t o v i s u a l c o n t a c t a n d non c o n t a c t g r o u p s . W a t e r i n t h e j a r s was r e p l a c e d o n c e o r t w i c e d a i l y w i t h h o l d i n g t a n k w a t e r , a f t e r f e e d i n g . The d i s c a r d e d w a t e r and e x c e s s f o o d was r e t u r n e d t o t h e h o l d i n g t a n k f o r n i t r i f i c a t i o n o f ammonia a n d m e c h a n i c a l f i l t r a t i o n . T h i s a l l o w e d s t r e s s o r s t o a c c u m u l a t e o n l y f o r a s h o r t t i m e b e t w e e n c l e a n i n g s i n AC f i l t e r e d j a r s , n o t t h e e n t i r e f i v e week e x p e r i m e n t a s i n t h e c a s e o f non AC f i l t e r e d g r o u p s . No a e r a t i o n was s u p p l i e d b e c a u s e B e t t a s p l e n d e n s a r e o b l i g a t e a i r b r e a t h e r s w i t h w e l l d e v e l o p e d l a b y r i n t h o r g a n s . S p e c i f i c m a i n t e n a n c e d e t a i l s f o r e a c h s p e c i e s a r e g i v e n i n t a b l e 1. 18 Table 1. Maintenance d e t a i l s f o r each s p e c i e s i n phase-2 experiments. Temperature Food Feedings/day pH Ammonia Maximum (ppm) D i s s o l v e d Oxygen Photoperiod Densit i e s ( f i sh per e n c l o s u r e ) : Low Mid High R e p l i c a t e s : C o n t r o l AC F i l t e r e d Brachydanio  r e r i o 26 C Artemia n a u p l i i , Tetra-min, E. a l b i d u s , Microworms 2 7.0 0.10 Saturated 12 hr 5 10 20 3 3 Astronotus  o c e l l a t u s 26 C Tubi fex, Tetra-min, Beef heart p r e p a r a t i o n 1-2 7.0 0.25 Saturated 12 hr 4 8 1 6 2 2 Betta'  splendens 30 C Artemia n a u p l i i , Tetra-min, E. a l b i d u s , Microworms 1-2 6.5-7.0 0.10 Not t e s t e d 14 hr N/A N/A N/A 30 30 MEASUREMENT OF RESPONSES TO TREATMENTS Three parameters were monitored to assess e f f e c t s of treatments: s u r v i v a l , growth, and/or a b i l i t y to withstand thermal c h a l l e n g e . These are t e r t i a r y responses to s t r e s s (Wedemeyer and McLeay 1981) and although only r e f l e c t i v e of i n t e r n a l primary and secondary changes, they are of the most immediate concern to a q u a c u l t u r i s t s . S u r v i v a l was monitored d a i l y by simply removing dead f i s h and r e c o r d i n g the date and f i s h e s ' standard l e n g t h . Thermal c h a l l e n g e was a p p l i e d at the end of experiments to i n c r e a s e s t r e s s on s u r v i v o r s and consequently m o r t a l i t y . Thermal c h a l l e n g e f o r Brachydanio r e r i o i n v o l v e d r a i s i n g temperatures 3° C every day u n t i l 35° C was reached. Because 19 Astronotus o c e l l a t u s i s t o l e r a n t of high temperatures they were lowered to produce s t r e s s . Temperatures were allowed to f a l l 3° C per day u n t i l 17° C was reached. Feeding was d i s c o n t i n u e d d u r i n g thermal c h a l l e n g e t r i a l s . Thermal c h a l l e n g e y i e l d e d l i t t l e i n f o r m a t i o n f o r Brachydanio r e r i o or Astronotus o c e l l a t u s due to r a p i d , random m o r t a l i t y . T h e r e f o r e , weight l o s s d u r i n g s t a r v a t i o n was examined i n Betta splendens to index energy consumption i n response to s t r e s s . Growth was monitored weekly by p h o t o g r a p h i c a l l y ( A l l e e et a l . 1934) r e c o r d i n g a l l f i s h e s ' standard' l e n g t h s . F i s h were p l a c e d i n a shallow (2cm) c o n t a i n e r , l i g h t e d from below through a 1mm g r i d and photographed from above (Kodak T r i - X . Pan, ASA 400 black and white f i l m shot at 1/250 sec with a V i v i t a r S e r i e s 1 Au t o f o c u s i n g Macrozoom l e n s at a f i l m to s u b j e c t d i s t a n c e of 44 cm). Negatives were p l a c e d in. s l i d e h o l d e r s and p r o j e c t e d onto a screen. The p r o j e c t o r was at a d i s t a n c e which gave a convenient s c a l e on a wheeled map measurer; m a g n i f i c a t i o n u s u a l l y approximated 15 diameters. The map measurer was run down the mid-dorsal l i n e from snout to the confluence of body and caudal f i n . T h i s permitted measurement of f i s h photographed i n a curved p o s i t i o n . Comparison between r e p l i c a t e d measurements of p r e v i o u s l y photographed dead f i s h to measurements on a micrometer microscope stage gave equal means a t 0.1mm accuracy (t=0.13 with 8 DF) with p r e c i s i o n of 0.12mm2 (variance of t r i p l i c a t e measurements). 20 RESULTS FILTER MATERIAL IDENTIFICATION F i g u r e 3 i l l u s t r a t e s mean f i n a l lengths of Corynopoma  r i i s e i i n four d i f f e r e n t l y f i l t e r e d tanks. Those reared i n the AC f i l t e r e d tank showed a h i g h l y s i g n i f i c a n t g r e a t e r f i n a l mean l e n g t h than other groups. A n a l y s i s of v a r i a n c e , i n c l u d i n g a l l 4 groups, gave a h i g h l y s i g n i f i c a n t F(21.773 with 3,63 DF) f o r the t e s t of equal means. A s i m i l a r a n a l y s i s , e x c l u d i n g the AC group gave an i n s i g n i f i c a n t F(2.308 with 2,46 DF) f o r the same t e s t . D e l e t i n g any other group always gave a h i g h l y s i g n i f i c a n t F among the remaining t h r e e . L i t t l e d i f f e r e n c e i n s u r v i v a l from the o r i g i n a l 20 f i s h was observed between treatments, although g r e a t e s t s u r v i v a l was recorded i n the AC f i l t e r e d group (N i n F i g u r e 3). No gross d i f f e r e n c e s were noted between any group's behaviour, a c t i v i t y l e v e l or f e e d i n g ; a l l appeared healthy and normal. F i g u r e 4 i l l u s t r a t e s mean f i n a l lengths of R i v u l u s  h a r t i i n the four experimental r e a r i n g tanks. No treatment had an e f f e c t on growth. A n a l y s i s of v a r i a n c e i n c l u d i n g a l l four groups showed no s i g n i f i c a n t d i f f e r e n c e between means (F=1.956 with 3,67 DF). S u r v i v a l i n the AC group was higher than in other treatments, but not s i g n i f i c a n t l y so. 21 F i g u r e 3 . F i n a l mean l e n g t h s o f Corynopoma r i s e i i a f t e r 6 weeks g r o w t h w i t h d i f f e r e n t f i l t e r m a t e r i a l s . F i g u r e 4 . F i n a l mean l e n g t h s o f R i v u l u s h a r t i a f t e r 6 weeks g r o w t h w i t h d i f f e r e n t f i l t e r m a t e r i a l s . 28 26 24 22 _ 2 0 I « o Lu U 112 LU < 5 8 LL 6 4 2 VERTICAL LINES = 1 STANDARD DEVIATION n=18 n=17 to n=16 n=16 FILTER MATERIAL 34 32 30 28 26 24 ^22 JE20 to S 18 _i z 16 < ^14 _i < 12 z LL. 10 8 6 4 2 VERTICAL LINES = 1 STANDARD DEVIATION n=18 to n=19 n=16 n=18 FILTER MATERIAL 23 DENSITY EXPERIMENTS IN FILTERED AND UNFILTERED WATER Brachydanio r e r i o Growth of Brachydanio r e r i o was examined using m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s . The standard p a r t i a l r e g r e s s i o n c o e f f i c i e n t s express the r e l a t i v e c o n t r i b u t i o n to the dependent v a r i a b l e ( i e . length) each independent v a r i a b l e makes when a l l others are h e l d constant (Snedecor and Cochran 1967, A f i f i and Azen 1972, Zar 1974, Anon. 1976, Edwards 1976, M o s t e l l e r and Tukey 1977). The method depends, in p a r t , upon independent v a r i a b l e s being o r t h o g o n a l . If a c o r r e l a t i o n between t h e i r v a l u e s e x i s t s , i t i s impossible to speak of changing one v a r i a b l e without a f f e c t i n g o t h e r s . In t h i s a n a l y s i s , time, s t o c k i n g d e n s i t y , and f i l t e r treatment were independent v a r i a b l e s . M u l t i p l e r e g r e s s i o n , a l t e r n a t e l y using each of time, d e n s i t y , and treatment (coded as 1 f o r a c o n t r o l and 2 f o r AC f i l t e r e d ) as the dependent v a r i a b l e and others as independent v a r i a b l e s showed no r e l a t i o n s h i p s e x i s t e d . Table 2 g i v e s the N, R 2, F s t a t i s t i c , DF and the p r o b a b i l i t y of F f o r each a n a l y s i s . Although i t i s d i f f i c u l t to i l l u s t r a t e m u l t i d i m e n s i o n a l r e l a t i o n s h i p s , F i g u r e s 5-7 show the e f f e c t of f i l t e r treatment on l e n g t h s e p a r a t e l y f o r each d e n s i t y i n the i n i t i a l experimental r e p l i c a t e . I t should be noted that v a r i a n c e of the means was used i n f i g u r e s r a t h e r than the c o n v e n t i o n a l standard d e v i a t i o n which was too small to d i s p l a y . By p r e s e n t i n g data f o r each d e n s i t y s e p a r a t e l y , the 24 F i g u r e 5. Mean l e n g t h s o f B r a c h y d a n i o r e r i o i n r e p l i c a t e 1 s t o c k e d a t a d e n s i t y o f 5 f i s h p e r e n c l o s u r e . . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. F i g u r e 6. Mean l e n g t h s o f B r a c h y d a n i o r e r i o i n r e p l i c a t e 1 s t o c k e d a t a d e n s i t y o f 10 f i s h p e r e n c l o s u r e . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. F i g u r e 7. Mean l e n g t h s o f B r a c h y d a n i o r e r i o i n r e p l i c a t e 1 s t o c k e d a t a d e n s i t y o f 20 f i s h p e r e n c l o s u r e . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. 35 i 5 FPT 5 §30 o z 25 U J z < £ 2 0 15 • AC FILTERED • CONTROLS : i 1 n = 5 5 , ,1 1 » ' 1 2 3 4 5 6 WEEK 35 §30 o L525 < £ 2 0 15 10 FPT • AC FILTERED • CONTROLS 10 10 4 10 10 9 n=10 10 g • •  1 2 3 4 5 WEEK 35 | 3 0 x t— • o z 2 5 i L U _ 1 Z < UJ20 15 20 FPT • AC FILTERED • CONTROLS 20 2 0 2 0 n=20 20 20 16 i , ( i '* 0 1 2 3 4 5 6 WEEK 26 T a b l e 2. R e g r e s s i o n s t a t i s t i c s f o r e x a m i n i n g t h e a u t o c o r r e l a t i o n o f i n d e p e n d e n t v a r i a b l e s t i m e , t r e a t m e n t a nd d e n s i t y f o r B r a c h y d a n i o  r e r i o e x p e r i m e n t s . R e g r e s s i o n N R 2 F DF P ( F ) V a r i a b l e ( D e p e n d e n t ; I n d e p e n d e n t ) R e p l i c a t e j _ T i m e ; D e n s i t y , T r e a t m e n t 458 0.00011 0.025 2,455 0.975 D e n s i t y ; T r e a t m e n t , T i m e 458 0.00004 0.009 2,455 0.990 T r e a t m e n t , - T i m e , D e n s i t y 458 0.00007 0.015 2,455 0.985 R e p l i c a t e s 2&3 T i m e ; D e n s i t y , T r e a t m e n t 549 0.00209 0.572 2,546 0.565 D e n s i t y ; T r e a t m e n t , T i m e 549 0.00210 0.575 2,546 0.563 T r e a t m e n t , - T i m e , D e n s i t y 549 0.00019 0.051 2,546 0.950 i l l u s t r a t i o n s e f f e c t i v e l y h o l d d e n s i t y c o n s t a n t . F i g u r e s 8-10 i l l u s t r a t e t h i s f o r p o o l e d r e s u l t s f r o m r e p l i c a t e s 2 and 3. T h e s e f i g u r e s c l e a r l y show t h e m a r k e d e f f e c t o f AC f i l t r a t i o n on g r o w t h o f B r a c h y d a n i o r e r i o i n a l l r e p l i c a t e s . The e f f e c t i s l e s s p r o n o u n c e d i n t h e h i g h e s t d e n s i t y t r e a t m e n t ( F i g u r e s 7 a n d 1 0 ) , e s p e c i a l l y i n t h e f i r s t e x p e r i m e n t a l r e p l i c a t e . G r e a t e r g r o w t h i n AC f i l t e r e d t a n k s o c c u r r e d d e s p i t e s i m i l a r low ammonia c o n c e n t r a t i o n s , e x c e s s f o o d , a n d i d e n t i c a l pH and d i s s o l v e d o x y g e n c o n c e n t r a t i o n s . T h i s d e m o n s t r a t e d AC removed a w a t e r b o r n e s t r e s s o r i n h i b i t i n g g r o w t h . F i g u r e s 11-12 show t h e e f f e c t o f s t o c k i n g d e n s i t y on g r o w t h f o r t h e f i r s t e x p e r i m e n t a l r e p l i c a t e . F i g u r e s 13-14 i l l u s t r a t e p o o l e d r e s u l t s f o r t h e l a s t two r e p l i c a t e s i n a s i m i l a r f a s h i o n . E f f e c t s o f d e n s i t y a r e n o t n e a r l y a s p r o n o u n c e d a s t h o s e o f f i l t e r m a t e r i a l . They a r e a b s e n t o r i n c o n s i s t e n t o v e r t i m e i n u n f i l t e r e d c o n t r o l t a n k s ( F i g u r e 27 F i g u r e 8. Mean l e n g t h s o f B r a c h y d a n i o r e r i o i n r e p l i c a t e s 2 an d 3 s t o c k e d a t a d e n s i t y o f 5 f i s h p e r e n c l o s u r e . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. F i g u r e 9. Mean l e n g t h s o f B r a c h y d a n i o r e r i o i n r e p l i c a t e s 2 an d 3 s t o c k e d a t a d e n s i t y o f 10 f i s h p e r e n c l o s u r e . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. F i g u r e 10. Mean l e n g t h s o f B r a c h y d a n i o r e r i o i n r e p l i c a t e s 2 and 3 s t o c k e d a t a d e n s i t y o f 20 f i s h p e r e n c l o s u r e . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. 16 o 2 LU 12 2 < £ 10 81 10 t 1 10 ,0 T * \ 1 n=10 1 0 II 10 •AC FILTERED i CONTROLS 1 2 WEEK 16 E E U I I-o UJ 12 < £ 10 20 j 20 20 19 n-20 2 0 u •AC FILTERED •CONTROLS 0 1 2 3 WEEK 16 £ u E o z 12 < UJ 10 39 39 T 38 37 • AC FILTERED • CONTROLS • i 1 2 3 WEEK 29 F i g u r e 11. Mean l e n g t h s o f B r a c h y d a n i o r e r i o a t a l l d e n s i t i e s i n r e p l i c a t e 1 r e a r e d w i t h o u t AC f i l t r a t i o n . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. F i g u r e 12 . Mean l e n g t h s o f B r a c h y d a n i o r e r i o a t a l l d e n s i t i e s i n r e p l i c a t e 1 r e a r e d w i t h AC f i l t r a t i o n . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. 30 35 E3CH E o s2 5 < LU 20 4 15 CONTROL 1 DENSITY: A 5 FISH A 10 FISH • 20 FISH 17 n = 5 1 1 10 2 0 1 9 T * 4 T 4 16 18- I J I 4 17 17 4 17 M 2 3 WEEK 3 5 1 CHARCOAL 1 £30 E x o z LU 25 Lu20 J 15 DENSITY: A 5 FISH AIOFISH • 20 FISH 10 i ! n = 5 10 2 0 5 10 20 20 f o 20 T 10 20 10 4 4 20 10 20 1 2 3 4 5 WEEK 31 F i g u r e 13. Mean l e n g t h s o f B r a c h y d a n i o r e r i o a t a l l d e n s i t i e s i n p o o l e d r e p l i c a t e s 2 a n d 3 r e a r e d w i t h o u t f i l t r a t i o n . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. F i g u r e 14. Mean l e n g t h s o f B r a c h y d a n i o r e r i o a t a l l d e n s i t i e s i n p o o l e d r e p l i c a t e s 2 a n d 3 r e a r e d w i t h AC f i l t r a t i o n . V e r t i c a l l i n e s a r e t h e v a r i a n c e o f t h e means. POOLED CONTROLS 2&3 10 1? 20 10 n=10 20 40 t 39 1 10 2 0 H iu <f 37 H j 38 DENSITY: *5 FISH A 10 FISH • 20 FISH 0 1 2 WEEK POOLED CHARCOAL 2&3 10 20 10 39 20 n=10 39 20 10 20 t j 38 39 4 DENSITY: A5 FISH A 10 FISH • 20 FISH 1 2 WEEK 33 11 a n d 1 3 ) , b u t more o b v i o u s i n AC f i l t e r e d t a n k s ( F i g u r e 12 a n d 1 4 ) . T a b l e 3 g i v e s i n f o r m a t i o n f r o m t h e r e g r e s s i o n a n a l y s i s . A b s o l u t e v a l u e s o f s t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t s i n d e x t h e r e l a t i v e i m p o r t a n c e o f t i m e , d e n s i t y and t r e a t m e n t on g r o w t h a n d c o n f i r m r e s u l t s s e e n i n t h e f i g u r e s . A l l make a h i g h l y s i g n i f i c a n t c o n t r i b u t i o n t o e x p l a i n i n g t h e v a r i a n c e o f l e n g t h m e a s u r e m e n t s ( t - s t a t i s t i c s i n T a b l e 3) a n d t o g e t h e r a c c o u n t f o r 9 1 % and 77% ( M u l t i p l e R 2 ) o f i t i n r e p l i c a t e 1 and the* p o o l e d r e s u l t s f o r r e p l i c a t e s 2 a n d 3 r e s p e c t i v e l y . Time a c c o u n t s f o r t h e l a r g e s t p r o p o r t i o n o f l e n g t h v a r i a b i l i t y . T r e a t m e n t a c c o u n t s f o r a p p r o x i m a t e l y 57-67% t h e amount t i m e d o e s w h i l e s t o c k i n g d e n s i t y o n l y a c c o u n t s f o r 16-17% o f t h e v a r i a n c e t i m e d o e s . The o r t h o g o n a l n a t u r e o f i n d e p e n d e n t v a r i a b l e s i s f u r t h e r c o n f i r m e d by t h e v a l u e o f t h e m u l t i p l e R 2 b e i n g e q u a l t o t h e sum o f i n d i v i d u a l R 2 ' s . I n summary, AC f i l t r a t i o n had a m a r k e d e f f e c t on g r o w t h , w h e r e a s s t o c k i n g d e n s i t y had a much l e s s n o t i c a b l e e f f e c t . T h i s was o f g r e a t e r i m p o r t a n c e i n AC f i l t e r e d t a n k s t h a n i n c o n t r o l s ( F i g u r e s 1 1 - 1 4 ) . I t s u g g e s t s a p o s s i b l e m a s k i n g a c t i o n t o w a t e r b o r n e s t r e s s o r s w h i c h o v e r w h e l m s t h e e f f e c t o f s t o c k i n g d e n s i t y on g r o w t h ( i e . t h e f a c t o r s a r e c o m p e n s a t o r y ) . 34 T a b l e 3. L e a s t s q u a r e s m u l t i p l e r e g r e s s i o n a n a l y s i s f o r B r a c h y d a n i o r e r i o u s i n g l e n g t h a s t h e d e p e n d e n t v a r i a b l e a n d t i m e , s t o c k i n g d e n s i t y , a n d f i l t e r t r e a t m e n t a s i n d e p e n d e n t v a r i a b l e s ( R 2 ( 1 ) , R 2 ( 2 ) , a nd R 2 ( 3 ) r e s p e c t i v e l y ) . S o u r c e DF R e g r e s s i o n 3 E r r o r 454 T o t a l 457 M u l t i p l e R 2=0.905 R 2 ( 1 ) + R 2 ( 2 ) + R 2 ( 3 ) = 0 R e p l i c a t e ]_ SS 10237 1071 1 1308 S.E.=1. ,836+0.002+0, MS 3412.4 2.3579 5355 070 = 0 F 1447, P ( F ) <0.0001 908 S t a n d a r d V a r i a b l e P a r t i a l R e g r e s s i o n t - S t a t i s t i c C o e f f i c i e n t Time 0.95' 63.21 T r e a t m e n t 0.64 17.82 D e n s i t y -0.17 -3.57 P ( t ) < o \ o o o r <0.0001 0.0004 S o u r c e DF R e g r e s s i o n 3 E r r o r 545 T o t a l 548 M u l t i p l e R 2=0.768 R e p l i c a t e s 2&3 SS MS 1489 496.4 450 0.8251 1 939 S.E.=0.908 F P ( F ) 601.56 <0.0001 R 2 ( 1 ) + R 2 ( 2 ) + R 2 ( 3 ) = 0 . 6 9 0 + 0 . 0 1 1 + 0 . 0 7 7 = 0 . 7 7 8 S t a n d a r d V a r i a b l e P a r t i a l R e g r e s s i o n t - S t a t i s t i c P ( t ) Coef f i c i e n t Time 0.86 39.96 <0.0001 T r e a t m e n t 0.49 13.12 <0.0001 D e n s i t y -0.14 -3.37 0.0008 A s t r o n o t u s o c e l l a t u s U n l i k e B r a c h y d a n i o r e r i o , t h e i n d e x o f s t r e s s u s e d i n a n a l y s i s o f A s t r o n o t u s o c e l l a t u s was s u r v i v a l . I n i t i a l l e n g t h s were h i g h l y v a r i a b l e due t o i m p o r t a t i o n i n two s e p a r a t e b a t c h e s . F u r t h e r , many s u f f e r e d an i c h t h y o p h t h i r i u s i n f e c t i o n when r e c e i v e d . T h e s e were h e l d i n q u a r a n t i n e a n d a l l t r e a t e d w i t h m a l a c h i t e g r e e n , t e t r a c y c l i n e a n d f o r m a l d e h y d e . F i s h were n o t i n t r o d u c e d t o e x p e r i m e n t a l t a n k s 35 u n t i l a l l symptoms h a d been a b s e n t f o r one week. The d i s e a s e c a u s e d an i n c r e a s e i n s i z e v a r i a n c e a n d r e d u c e d i t s u s e f u l n e s s a s a s t r e s s i n d i c a t o r . L i t t l e g r o w t h was o b s e r v e d i n u n f i l t e r e d c o n t r o l f i s h a n d when e x a m i n e d , by r e g r e s s i o n , i n AC f i l t e r e d t a n k s r u n an e x t r a two w e e k s , o n l y 22% ( R 2 = 0 . 2 2 ) o f l e n g t h v a r i a n c e was e x p l a i n e d by t h e i n d e p e n d e n t v a r i a b l e s t i m e , t r e a t m e n t , a n d d e n s i t y . A t t h e en d o f f i v e w e eks, t h e r m a l c h a l l e n g e was a p p l i e d t o t h e r e m a i n i n g AC f i l t e r e d f i s h o n l y . R e s u l t i n g m o r t a l i t y showed no r e l a t i o n s h i p ' t o s t o c k i n g d e n s i t y , o r f i s h s i z e , a n d was v e r y r a p i d . T h u s , s u r v i v a l d u r i n g t h e e x p e r i m e n t was u s e d t o e x a m i n e t h e p a t t e r n o f s t r e s s . D a i l y s u r v i v a l was e x p r e s s e d a s a f r a c t i o n o f o r i g i n a l s t o c k i n g d e n s i t y a n d s e r v e d a s t h e d e p e n d e n t v a r i a b l e . M u l t i p l e r e g r e s s i o n a n a l y s i s was a g a i n u s e d a f t e r c h e c k i n g t h e o r t h o g o n a l n a t u r e o f i n d e p e n d e n t v a r i a b l e s t i m e , f i l t e r t r e a t m e n t , a n d s t o c k i n g d e n s i t y . I n d e p e n d e n t v a r i a b l e s showed no a u t o c o r r e l a t i o n a s i n d i c a t e d by c o r r e l a t i o n c o e f f i c i e n t s i n T a b l e 4. S u r v i v a l s t a t i s t i c s h a v e been p l o t t e d s e p a r a t e l y f o r e a c h s t o c k i n g d e n s i t y ( F i g u r e 15-17) a n d t r e a t m e n t ( F i g u r e 1 8 - 1 9 ) . T h i s r e p r e s e n t s t h e a n a l y t i c a l a p p r o a c h o f e x a m i n i n g s t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t s where a s i n g l e i n d e p e n d e n t v a r i a b l e i s a l t e r e d t o a s s e s s i t s e f f e c t on t h e d e p e n d e n t v a r i a b l e , s u r v i v a l . The e f f e c t o f f i l t e r t r e a t m e n t on s u r v i v a l i s p r o n o u n c e d ( F i g u r e 15-17) and a t a l l d e n s i t i e s , p o s i t i v e . 36 F i g u r e 15. P r o p o r t i o n o f A s t r o n o t u s o c e l l a t u s s u r v i v i n g when s t o c k e d a t a d e n s i t y o f 4 f i s h p e r e n c l o s u r e . F i g u r e 16. P r o p o r t i o n o f A s t r o n o t u s o c e l l a t u s s u r v i v i n g when s t o c k e d a t a d e n s i t y o f 8 f i s h p e r e n c l o s u r e . F i g u r e 17. P r o p o r t i o n o f A s t r o n o t u s o c e l l a t u s ' s u r v i v i n g - when s t o c k e d a t a d e n s i t y o f 16 f i s h p e r e n c l o s u r e . 0.80 K 0.60' ZD o P 0.40 cr o CL o £ 0.201 • CONTROLS • AC FILTERED 4 FISH / ENCLOSURE 8 12 16 20 TIME (DAYS) 8 FISH / ENCLOSURE A 8 12 16 20 TIME (DAYS) 3 8 T a b l e 4. R e g r e s s i o n s t a t i s t i c s f o r e x a m i n i n g t h e a u t o c o r -r e l a t i o n o f i n d e p e n d e n t v a r i a b l e s t i m e , t r e a t -m e n t , a n d d e n s i t y f o r e x p e r i m e n t a l r e p l i c a t e s 1 an d 2 f o r A s t r o n o t u s o c e l l a t u s . R e g r e s s i o n V a r i a b l e ( D e p e n d e n t i n d e p e n d e n t ) N R' DF REPLICATES 1 AND 2 P ( F ) T i m e ; D e n s i t y , T r e a t m e n t D e n s i t y ; T r e a t m e n t , T i m e T r e a t m e n t ; T i m e , D e n s i t y 240 0.00000 0.007 2,237 0.999 240 0.00000 0.000 2,237 1.000 240 0.00000 0.006 2,237 0.999 A s t r o n o t u s o c e l l a t u s , a t t h e s i z e a n d age u s e d , a r e a g g r e s s i v e , a l t h o u g h n o t s t r o n g l y t e r r i t o r i a l ; t o t a l s t o c k i n g d e n s i t y e x c e e d e d t h o s e recommended by a p p r o x i m a t e l y f i v e t i m e s ( I n n e s 1966, F r e y 1 9 7 0 ) . I t was p o s s i b l e t o m a i n t a i n t h i s d e n s i t y i n AC f i l t e r e d e n c l o s u r e s , b u t n o t i n c o n t r o l s d e s p i t e e q u a l o x y g e n c o n c e n t r a t i o n s a n d pH, an e q u a l r e l a t i v e l y l ow ammonia c o n c e n t r a t i o n a nd e x c e s s f o o d . I t s h o u l d be n o t e d t h a t t h e s e f i s h were a t l e a s t t h r e e t i m e s t h e w e i g h t o f o t h e r s p e c i e s . S t o c k i n g d e n s i t y h ad a l o w e r , b u t s i g n i f i c a n t i n f l u e n c e on s u r v i v a l . F i g u r e s 18-19 show s u r v i v a l was b e t t e r i n t h e l o w e s t s t o c k i n g d e n s i t y o f b o t h f i l t e r t r e a t m e n t s . D e s p i t e t h e a g g r e s s i v e n a t u r e o f A s t r o n o t u s o c e l l a t u s , s i z e o f d e a d f i s h d i d n o t c o r r e l a t e w i t h s e q u e n c e o f t h e i r d e a t h s . The p r o n o u n c e d e f f e c t o f f i l t e r t r e a t m e n t i s r e f l e c t e d i n t h e a b s o l u t e v a l u e o f i t s s t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t ( T a b l e 5 ) . The c o m b i n e d i n d e p e n d e n t v a r i a b l e s e x p l a i n 5 3 % o f v a r i a t i o n i n s u r v i v a l . P r o p o r t i o n a l l y , f i l t e r t r e a t m e n t h a d t h e most p r o n o u n c e d e f f e c t . As e x p e c t e d , t i m e 39 F i g u r e 18. P r o p o r t i o n o f A s t r o n o t u s o c e l l a t u s s u r v i v i n g a t a l l s t o c k i n g d e n s i t i e s w i t h AC f i l t r a t i o n . F i g u r e 19. P r o p o r t i o n o f A s t r o n o t u s o c e l l a t u s s u r v i v i n g a t a l l s t o c k i n g d e n s i t i e s w i t h o u t AC f i l t r a t i o n . 40 1.00 | 0.80 rr CO Q0.60 r-cr: o CL § 0 . 4 0 A A A A A A A A A A • • • A • A' A DENSITY: A U FISH A 8 FISH • 16 FISH 0.20 AC FILTERED CD > > CC ID t o Z O r-rr o CL o rr o_ 1.00 0.80 0.60 0.40 4 8 12 TIME (DAYS) 16 20 A A • • A DENSITY: A U FISH A 8 FISH • 16FIShl A • • • A A 0 A A A A A A 0.20 CONTROLS • • "l6 " 20 8 12 TIME (DAYS) 41 T a b l e 5. L e a s t s q u a r e s m u l t i p l e r e g r e s s i o n a n a l y s i s f o r A s t r o n o t u s o c e l l a t u s u s i n g t h e p r o p o r t i o n s u r v i v i n g a s t h e d e p e n d e n t v a r i a b l e a n d t i m e , s t o c k i n g d e n s i t y , a nd f i l t e r t r e a t m e n t a s i n d e p e n d e n t v a r i a b l e s ( R 2 ( 1 ) , R 2 ( 2 ) , R 2 ( 3 ) r e s p e c t i v e l y ) . R e p l i c a t e J_ a n d 2 S o u r c e DF SS MS F P ( F ) R e g r e s s i o n 3 10.659 3.553 89.14 <0.0001 E r r o r 236 9.407 0.040 T o t a l 239 20.066 M u l t i p l e R 2=0.531 S.E.=0.200 R 2 ( 1 ) + R 2 ( 2 ) + R 2 ( 3 ) = 0 . 2 1 6 + 0 . 0 3 3 + 0 . 2 8 2 = 0 . 5 3 1 S t a n d a r d V a r i a b l e P a r t i a l R e g r e s s i o n t - S t a t i s t i c P ( t ) C o e f f i c i e n t Time -0.562 -10.43 <0.0001 T r e a t m e n t 0.613 11.93 <0.0001 D e n s i t y -0.256 -4.07 0.0001 e x p l a i n e d an a l m o s t e q u a l l y i m p o r t a n t f r a c t i o n o f s u r v i v a l v a r i a t i o n . The c o n t r i b u t i o n o f s t o c k i n g d e n s i t y was l e s s t h a n h a l f t h a t o f t i m e o r t r e a t m e n t , a l t h o u g h i t made a s i g n i f i c a n t c o n t r i b u t i o n . A g a i n , t h e sum o f i n d i v i d u a l R 2 ' s e q u a l e d t h e m u l t i p l e R 2 i n d i c a t i n g o r t h o g o n a l i n d e p e n d e n t v a r i a b l e s . I n summary, AC f i l t r a t i o n a p p e a r e d t o remove a w a t e r b o r n e s t r e s s o r , o t h e r t h a n n i t r o g e n o u s w a s t e s , w h i c h h ad a m a r k e d e f f e c t on f i s h h e a l t h . S t o c k i n g d e n s i t y may h a v e s u p p l e m e n t e d t h e n e g a t i v e s t r e s s o f t h e w a t e r b o r n e f a c t o r a s l o w e s t d e n s i t i e s h a d g r e a t e s t s u r v i v a l . H owever, a t h i g h e r d e n s i t i e s no r e l a t i o n s h i p was a p p a r e n t . 6 42 B e t t a s p l e n d e n s B e t t a s p l e n d e n s ' g r o w t h was a l s o a n a l y z e d by m u l t i p l e l i n e a r r e g r e s s i o n m e t h o d s . T a b l e 6 t e s t s a u t o c o r r e l a t i o n o f i n d e p e n d e n t v a r i a b l e s t i m e , v i s u a l c o n t a c t t r e a t m e n t , a n d f i l t e r t r e a t m e n t . V i s u a l c o n t a c t was c o d e d 1 f o r c o n t a c t a n d 9 f o r no c o n t a c t . F i l t e r t r e a t m e n t was c o d e d 1 f o r u n f i l t e r e d c o n t r o l s a n d 2 f o r AC f i l t r a t i o n . R e g r e s s i o n T a b l e 6. R e g r e s s i o n s t a t i s t i c s f o r e x a m i n i n g t h e a u t o c o r -r e l a t i o n o f i n d e p e n d e n t v a r i a b l e s t i m e , c o n t a c t t r e a t m e n t a n d f i l t e r t r e a t m e n t f o r B e t t a s p l e n d e n s , - R e g r e s s i o n N R 2 F DF P ( F ) V a r i a b l e ( D e p e n d e n t ; I n d e p e n d e n t ) T i m e ; C o n t a c t , F i l t e r 353 0.00006 0.010 2,350 0.9901 C o n t a c t ; F i l t e r , Time 353 0.00020 0.036 2,350 0.9648 F i l t e r ; T i m e , C o n t a c t 353 0.00026 0.046 2,350 0.9555 a n a l y s i s shows no a u t o c o r r e l a t i o n b e t w e e n i n d e p e n d e n t v a r i a b l e s . F i g u r e s 20 a n d 21 i l l u s t r a t e e f f e c t o f f i l t e r t r e a t m e n t on g r o w t h o f B e t t a s p l e n d e n s . T h e r e i s no o b s e r v a b l e d i f f e r e n c e i n g r o w t h a t t r i b u t a b l e t o r e m o v a l , by AC, o f a w a t e r b o r n e compound. T h i s means e i t h e r B e t t a s p l e n d e n s d o e s n o t s e c r e t e a w a t e r b o r n e s t r e s s o r a t t h e s t o c k i n g d e n s i t i e s u s e d , t h e y a r e r e s i s t e n t t o i t s e f f e c t s , o r AC c o u l d n o t remove i t . F i g u r e s 22 a n d 23 show t h e s i m i l a r l a c k o f e f f e c t 43 F i g u r e 20. Mean l e n g t h o f B e t t a s p l e n d e n s r a i s e d i n i n d i v i d u a l j a r s , b u t a l l o w e d v i s u a l c o n t a c t w i t h a d j a c e n t f i s h e s . V e r t i c a l l i n e s a r e t h e v a r i a n c e s o f t h e means. F i g u r e 21. Mean l e n g t h o f B e t t a s p l e n d e n s r a i s e d i n i n d i v i d u a l j a r s n o t a l l o w e d v i s u a l c o n t a c t w i t h a d j a c e n t f i s h . V e r t i c a l l i n e s a r e t h e v a r i a n c e s o f t h e means. 44 40 _ 32 1 E E VISUAL CONTACT o LU 24 tt 15 15 15 15 < 16 LU 8 • AC FILTERED • CONTROLS tt 15 14 15 15 2 3 4 TIME (WEEKS) 40 -£ 32 x | 24 H LU 2 16 NON CONTACT *t H 1 u 1^ i t tt U 15 15 n = 15 15 8 4 • AC FILTERED • CONTROLS tt H 14 15 . H 15 I I • 1 • I 1 2 3 4 5 6 TIME (WEEKS) -45 v i s u a l c o n t a c t h a d on g r o w t h . T h i s r e s u l t i s s u r p r i s i n g b e c a u s e v i s u a l c o n t a c t f i s h were n o t i c a b l y more a c t i v e t h a n non c o n t a c t f i s h . The f o r m e r d i s p l a y e d g i l l f l a r i n g , t a i l w a g g i n g , a n d n e s t b u i l d i n g b e h a v i o u r s t h r o u g h o u t t h e d a y w h i l e t h e l a t t e r g e n e r a l l y hung m o t i o n l e s s . To i n d e x t h i s d i f f e r e n c e , t h e p r o p o r t i o n o f b u b b l e n e s t s f o u n d i n j a r s o f m a l e s i n e a c h g r o u p were r e c o r d e d f o r t h e f i f t h week. Mean p r o p o r t i o n s a r e r e p o r t e d i n T a b l e 7. T a b l e 7. P r o p o r t i o n o f j a r s c o n t a i n i n g m a l e s w h i c h h a d bub-b l e n e s t s (number o f m a l e s w i t h n e s t s / n u m b e r m a l e s ) O b s e r v a t i o n s made d a i l y f o r 7 d a y s (week 5 ) . G r o u p AC F i l t e r e d , C o n t a c t C o n t r o l , C o n t a c t AC F i l t e r e d , N o n c o n t a c t C o n t r o l , N o n c o n t a c t Mean P r o p o r t i o n W i t h N e s t s 0.98 0.95 0.53 0.58 N 7 7 7 7 0.0013 0.0024 0.0058 0.0049 T h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t w e e n means o f c o n t a c t g r o u p s ( t = 1 . 0 9 w i t h 12 DF) o r n o n c o n t a c t g r o u p s ( t = 1 . 2 9 w i t h 12 D F ) . When a l l f o u r means were e x a m i n e d i n a n a l y s i s o f v a r i a n c e a s i g n i f i c a n t d i f f e r e n c e e x i s t e d (F=94.30 w i t h 3,24 D F ) . T h i s s u g g e s t e d c o n t a c t g r o u p s were more a c t i v e t h a n n o n c o n t a c t t r e a t m e n t s . The o b v i o u s d e v i a t i o n s i n a c t i v i t y w i t h o u t g r o w t h d i f f e r e n c e s s u g g e s t e d i n e q u a l i t y i n e n e r g y use by c o n t a c t and n o n c o n t a c t g r o u p s . I t was n o t p o s s i b l e t o t e s t t h i s d i r e c t l y by m e a s u r i n g r a t i o n s i z e a s a l l were f e d t o e x c e s s . To q u a n t i f y 46 F i g u r e 22. Mean l e n g t h o f v i s u a l c o n t a c t a n d n o n c o n t a c t B e t t a s p l e n d e n s r e a r e d i n w a t e r f i l t e r e d t h r o u g h AC. V e r t i c a l l i n e s a r e t h e v a r i a n c e s o f t h e means. F i g u r e 2 3 . Mean l e n g t h o f v i s u a l c o n t a c t a n d n o n c o n t a c t B e t t a s p l e n d e n s r e a r e d i n w a t e r w i t h o u t AC f i l t r a t i o n . V e r t i c a l l i n e s a r e t h e v a r i a n c e s o f t h e means. 4i7; 40 32 E E x 24 o AC FILTERED U 15 111 < LU 16 8 n=15 15 VISUAL CONTACT * NON CONTACT * tt 15 U 15 tt tt 3 4 TIME (WEEKS) 40 32 i "E x 24 h-o z LU < LU 2 CONTROLS fj 15,5 n=15 ' 15 15 8 15 VISUAL CONTACT * NON CONTACT A n H »* "4 15 I 15 15 I 15 15U 2 3 4 TIME (WEEKS) 48 d i f f e r e n c e s i n e n e r g y c o n s u m p t i o n , a l l f i s h were s t a r v e d f o r one week a f t e r g r o w t h e x p e r i m e n t s e n d e d t h e n l o s s o f w e i g h t was c o m p a r e d b e t w e e n g r o u p s . T a b l e 8 g i v e s a c o m p a r i s o n o f f i n a l mean w e i g h t s . T a b l e 8. F i n a l mean w e i g h t s o f B e t t a s p l e n d e n s a f t e r 7 d a y s o f s t a r v a t i o n . G r o u p AC F i l t e r e d , C o n t a c t C o n t r o l , C o n t a c t AC F i l t e r e d , N o n c o n t a c t C o n t r o l , N o n c o n t a c t Mean W e i g h t (g) N S 2 0.61 15 0.014 0.56 15 0.014 0.7B 14 0.006 0.80 15 0.008 I t i s c l e a r f r o m t h e s e r e s u l t s t h a t B e t t a s p l e n d e n s p e r m i t t e d v i s u a l c o n t a c t consumed more e n e r g y . I n a c a t y l i t i c s t a t e , t h e i r w e i g h t d r o p p e d t h e most r a p i d l y . T h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two c o n t a c t g r o u p s ( t = 1 . 0 6 w i t h 28 DF) o r n o n c o n t a c t g r o u p s ( t = - 0 . 8 6 w i t h 27 D F ) . A n a l y s i s o f v a r i a n c e u s i n g a l l f o u r i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e b e t w e e n means (F=20.89 w i t h 3,55 DF) s u g g e s t i n g n o n c o n t a c t f i s h l o s t s i g n i f i c a n t l y l e s s w e i g h t t h a n v i s u a l c o n t a c t f i s h . E q u a l g r o w t h d u r i n g e x p e r i m e n t s c a n t h e r e f o r e be a t t r i b u t e d t o g r e a t e r f o o d c o n s u m p t i o n o r i t s b e t t e r u t i l i z a t i o n . F i n a l l y , T a b l e 9 g i v e s t h e r e s u l t s o f m u l t i p l e r e g r e s s i o n a n a l y s i s . S t a n d a r d p a r t i a l r e g r e s s i o n c o e f f i c i e n t s q u a n t i f y r e l a t i v e c o n t r i b u t i o n s o f e a c h i n d e p e n d e n t v a r i a b l e t o t h e d e p e n d e n t v a r i a b l e , l e n g t h . A n a l y s i s d e m o n s t r a t e s t h e o b v i o u s i m p o r t a n c e o f t i m e i n 49 T a b l e 9. L e a s t s q u a r e s m u l t i p l e r e g r e s s i o n a n a l y s i s f o r B e t t a s p l e n d e n s u s i n g l e n g t h a s t h e d e p e n d e n t v a r i a b l e a n d t i m e , f i l t e r t r e a t m e n t , a n d v i s u a l c o n t a c t a s i n d e p e n d e n t v a r i a b l e s ( R i 2 ' ( l ) , R'2("2)', and R 2 ( 3 ) r e s p e c t i v e l y ) . S o u r c e DF R e g r e s s i o n 3 E r r o r 349 T o t a l 352 SS 5553.7 1485.3 7039.1 MS 1851 .2 4.2560 F 434.97 M u l t i p l e R 2=0.789 S.E.=2.0630 R 2 ( 1 ) + R 2 ( 2 ) + R 2 ( 3 ) = 0 . 7 8 9 + 0 . 0 0 0 + 0 . 0 0 0 = 0 . 7 8 9 V a r i a b l e Time F i l t e r C o n t a c t S t a n d a r d P a r t i a l R e g r e s s i o n C o e f f i c i e n t 0.888 -0.002 0.007 t - S t a t i s t i c 36. 1 2 -0.04 0.13 P ( F ) <0.0001 P ( t ) <0.0001 0.9684 0.8995 e x p l a i n i n g l e n g t h v a r i a t i o n a n d a g r e e s w i t h F i g u r e s 2 0 - 2 3 , p o i n t i n g o u t t h e u n i m p o r t a n c e o f f i l t e r t r e a t m e n t a n d v i s u a l c o n t a c t . C o m b i n e d v a r i a b l e s e x p l a i n 7 9 % o f t h i s v a r i a t i o n , a l t h o u g h c o e f f i c i e n t s o f f i l t e r a n d v i s u a l c o n t a c t t r e a t m e n t s do n o t make any s i g n i f i c a n t c o n t r i b u t i o n . T o g e t h e r t h e y a c c o u n t f o r l e s s t h a n 0.1% t h e v a r i a t i o n t i m e d o e s . I n summary, n e i t h e r f i l t e r t r e a t m e n t n o r v i s u a l c o n t a c t a f f e c t g r o w t h o f B e t t a s p l e n d e n s . T h i s i n d i c a t e s r e s i s t a n c e t o o r l a c k o f a w a t e r b o r n e s t r e s s o r o r AC's i n a b i l i t y t o remove i t . V i s u a l c o n t a c t c a u s e d i n c r e a s e d a c t i v i t y a n d e n e r g y c o n s u m p t i o n b u t , b e c a u s e no d i f f e r e n c e i n g r o w t h was o b s e r v e d , f i s h e i t h e r a t e more o r u s e d f o o d more e f f i c i e n t l y . 50 DISCUSSION R e s u l t s show o b v i o u s i n t e r s p e c i f i c d i f f e r e n c e s i n r e s p o n s e t o c r o w d i n g a n d w a t e r b o r n e s t r e s s o r s . The d i s c u s s i o n a p p r o a c h e s s e p e r a t e l y , by s p e c i e s , t h e f i r s t t h r e e q u e s t i o n s o f t h e i n t r o d u c t i o n a n d i n summary t h e f i n a l q u e s t i o n i s a d d r e s s e d . Corynopoma r i i s e i i C o rynopoma r i i s e i , an o p e n w a t e r s c h o o l i n g f i s h , r e q u i r e s c l e a n , w e l l a e r a t e d s y s t e m s t o grow a n d r e p r o d u c e . S p a w n i n g demands h e a v y f e e d i n g w i t h a v a r i e t y o f l i v e f o o d s a n d i s t r i g g e r e d by a 50% w a t e r c h a n g e a n d e l e v a t e d t e m p e r a t u r e ( I n n e s 1 9 6 6 ) . The s t o c k u s e d was n o t e x p o s e d t o c u l t u r e f o r s u c c e s s i v e g e n e r a t i o n s o r c o n s e q u e n t l y s e l e c t e d t o t o l e r a t e a s y n t h e t i c e n v i r o n m e n t . W a t e r b o r n e a g e n t s h a v e a m a r k e d e f f e c t on g r o w t h o f Corynopoma r i i s e i ( F i g u r e 3 ) , b u t c a n be removed by AC f i l t r a t i o n . T h i s r e s p o n s e i s s i m i l a r t o o b s e r v a t o n s by Yu a n d P e r l m u t t e r (1970) i n B r a c h y d a n i o r e r i o a n d by G r e e n e ( 1 9 6 4 ) f o r g o l d f i s h a s w e l l a s B r a c h y d a n i o r e r i o , a l t h o u g h n e i t h e r s t u d y e m p l o y e d b i o l o g i c a l f i l t r a t i o n t o r e d u c e o r g a n i c compounds ( L a i o a n d Mayo 1974, S p o t t e 1 9 7 9 ) . Compounds i n h i b i t i n g Corynopoma r i i s e i a r e l i k e l y m i d - w e i g h t o r g a n i c c a r b o n s n o t d e g r a d a b l e by h e t e r o t r o p h i c b a c t e r i a . They may be d i r e c t l y s e c r e t e d o r o c c u r a s b r e a k d o w n p r o d u c t s o f b a c t e r i a l f i l t r a t i o n . A l t h o u g h t h i s s p e c i e s i s n o t s u b j e c t t o i n t e n s i v e c u l t u r e , a s i m i l a r e f f e c t i n 51 d o m e s t i c a t e d s p e c i e s w o u l d be o f i m p o r t a n c e . R i v u l u s h a r t i T h e s e l a r g e c y p r i n o d o n t s a r e h a r d y f i s h , e a s i l y spawned i n s m a l l a q u a r i a , a n d a d a p t e d t o l i f e i n s m a l l p u d d l e s a n d s t r e a m s . They b e n e f i t f r o m " a g e d w a t e r " ( F r e y 1970, S t e r b a 1973) w i t h a l o w pH, b u t a r e a b l e t o w i t h s t a n d w a t e r c o n d i t i o n s d e l e t e r i o u s t o o t h e r s p e c i e s . The s t o c k u s e d was r e c e n t l y i n t r o d u c e d t o d o m e s t i c a t i o n and n o t s u b j e c t t o c u l t u r a l s e l e c t i o n . E i t h e r R i v u l u s h a r t i was u n e f f e c t e d by w a t e r b o r n e a g e n t s a t t h e s e s t o c k i n g d e n s i t i e s o r AC d i d n o t remove them. T h e s e f i s h were c o n s i d e r a b l y l a r g e r t h a n Corynopoma r i i s e i ( F i g u r e s 3 and 4) so d i f f e r e n c e s i n r e s p o n s e c a n n o t be a t t r i b u t e d t o a l o w e r b i o m a s s . Yu a n d P e r l m u t t e r ( 1 9 7 0 ) f o u n d w a t e r h o l d i n g B r a c h y d a n i o r e r i o h a d no e f f e c t on R i v u l u s h a r t i , h o w e v e r , t h e y d i d n o t e x a m i n e i n t r a s p e c i f i c e f f e c t s o f R i v u l u s h a r t i c o n d i t i o n e d w a t e r . T h i s may i n d i c a t e a t o l e r a n c e t o w a t e r b o r n e a g e n t s . R i v u l u s  h a r t i ' s a b i l i t y t o t o l e r a t e p o o r w a t e r q u a l i t y w o u l d f u r t h e r a r g u e t o l e r a n c e t o w a t e r b o r n e s t r e s s o r s . I t i s a l s o p o s s i b l e t h i s s p e c i e s d i d n o t s e c r e t e w a t e r b o r n e s t r e s s o r s , n o t s u r p r i s i n g i n f i s h a d a p t e d t o s m a l l e a s i l y p o l l u t e d h a b i t a t s . N o t e d a l s o was t h e n e c e s s i t y f o r a r t i f i c i a l c o v e r ( i e . g r e e n n y l o n w o o l ) t o e l i m i n a t e n e r v o u s b e h a v i o u r . I n an i n i t i a l t r i a l w i t h o u t c o v e r , f i s h f a i l e d t o e a t o r grow. T h i s i s a somewhat q u a l i t a t i v e s u g g e s t i o n o f c r o w d i n g 52 s t r e s s . B r a c h y d a n i o r e r i o R e s u l t s f r o m AC f i l t r a t i o n i n a l l t h r e e r e p l i c a t e s t e n d t o c o n f i r m t h e r e p o r t s o f G r e e n e 1964, Yu a n d P e r l m u t t e r ( 1 9 7 0 ) , a n d Chen and M a r t i n i c h ( 1 9 7 5 ) . T h e s e a u t h o r s f o u n d B r a c h y d a n i o r e r i o s e n s i t i v e t o w a t e r b o r n e s t r e s s o r s a l t h o u g h a g a i n none u s e d b i o l o g i c a l f i l t r a t i o n t o r e d u c e o r g a n i c l o a d i n g o f t h e w a t e r . B r a c h y d a n i o r e r i o i s an open w a t e r s c h o o l i n g f i s h l i v i n g i n an e n v i r o n m e n t w h i c h d i l u t e s w a s t e s r a p i d l y . T h i s may h e l p e x p l a i n t h e s p e c i e s ' i n t o l e r a n c e t o w a t e r b o r n e a g e n t s . However, t h e s t o c k u s e d h a d been s u b j e c t t o i n t e n s i v e c u l t u r e and t h e s e l e c t i v - e p r e s s u r e o f t h i s t r e a t m e n t i s unknown. The n e g a t i v e i n f l u e n c e o f s t o c k i n g d e n s i t y i s n o t a s o b v i o u s a s i s f i l t e r t r e a t m e n t s ' . I n no r e p l i c a t e s was i t a p p a r e n t i n c o n t r o l g r o u p s ( F i g u r e s 11 and 13) where g r o w t h was s u p p r e s s e d by w a t e r b o r n e f a c t o r s . T h e r e i s h o w e v e r , a d e f i n i t e i n v e r s e r e l a t i o n s h i p b e t w e e n g r o w t h a n d d e n s i t y i n AC f i l t e r e d t a n k s ( F i g u r e s 12 and 1 4 ) . I f t h e s e f a c t o r s a c t e d s y n e r g i s t i c a l l y , t h e d e n s i t y r e l a t i o n s h i p w o u l d have been a c c e n t u a t e d i n t h e d o u b l y s t r e s s e d c o n t r o l g r o u p s . I f t h e two f a c t o r s were a d d i t i v e an e q u a l s t o c k i n g d e n s i t y s t r e s s w o u l d have been s e e n d e s p i t e f i l t e r t r e a t m e n t . The r e s u l t s d e m o n s t r a t e a c o m p e n s a t o r y r e l a t i o n s h i p b e t w e e n f a c t o r s i n w h i c h w a t e r b o r n e a g e n t s h a v e a d o m i n a n t o r m a s k i n g r o l e . 53 I t i s d i f f i c u l t t o i n t e r p r e t d i s t r i b u t i o n o f s t r e s s t h r o u g h a h i e r a r c h y . It's o c c u r r e n c e would* c a u s e an i n c r e a s e i n s i z e v a r i a t i o n o v e r t i m e (Magnuson 1 9 6 2 ) ; t h e l a r g e s t f i s h g r o w i n g much f a s t e r t h a n t h e s m a l l e s t . No p a t t e r n o f c h a n g e i n s i z e v a r i a n c e i s c o n s i s t e n t b e t ween r e p l i c a t e s a n d s i z e a t d e a t h b o r e no r e l a t i o n s h i p t o t h e s e q u e n c e o f m o r t a l i t y . A n d r e w s ( 1 9 7 9 ) a n d N o b l e (1939) n o t e c o m p l e x h i e r a r c h i c a l p a t t e r n s i n a n i m a l s w h i c h a g g r e g a t e a s o p p o s e d t o s t r a i g h t l i n e r e l a t i o n s h i p s i n more s o l i t a r y s p e c i e s . A s t r o n o t u s o c e l l a t u s H i g h m o r t a l i t y among A s t r o n o t u s o c e l l a t u s w i t h o u t AC f i l t r a t i o n c l e a r l y d e m o n s t r a t e s t h e e f f e c t o f a w a t e r b o r n e s t r e s s o r . Were e f f e c t s l e s s p r o n o u n c e d , i t i s l i k e l y t h a t g r o w t h i n h i b i t i o n w o u l d h a v e been o b s e r v e d a s f i s h c h a n n e l e d e n e r g y , w h i c h w o u l d have been u s e d f o r g r o w t h , i n t o a d a p t i n g t o t h e f a c t o r . The m a n i f e s t a t i o n o f w a t e r b o r n e s t r e s s o r s a s m o r t a l i t y a g e n t s c o u l d be r e l a t e d t o t h e s p e c i e s ' l a r g e s i z e . A s t r o n o t u s o c e l l a t u s r a n g e d b e tween 2 and 3 g, B r a c h y d a n i o r e r i o n e v e r e x c e e d e d 0.4 g. T h i s i s a s e v e n f o l d h i g h e r b i o m a s s i n e a c h e x p e r i m e n t a l r e p l i c a t e o f A s t r o n o t u s  o c e l l a t u s . The i n t e n s i v e f i l t r a t i o n m a i n t a i n e d ammonia a t low c o n c e n t r a t i o n s a n d l a r g e l y r e m o v e d s u s p e n d e d s o l i d s ( a n d c o n s e q u e n t l y BOD). The s t r e s s o r s were e i t h e r s e c r e t e d d i r e c t l y by f i s h o r o c c u r r e d a s b r e a k d o w n p r o d u c t s o f b a c t e r i a l f i l t r a t i o n . The s o u r c e m i g h t be d i s t n g i s h e d by 54 r e a r i n g t h e f i s h i n a s t e r i l e e n v i r o n m e n t w i t h o u t b a c t e r i a l f i l t r a t i o n . S t o c k i n g d e n s i t y e f f e c t s a l s o o c c u r r e d , b u t a t h a l f t h e s t r e n g t h o f f i l t e r t r e a t m e n t ( T a b l e 5 ) . T h e r e may be a t h r e s h o l d t o c r o w d i n g s t r e s s . S u r v i v a l i n t h e l o w e s t d e n s i t y g r o u p was b e t t e r i n b o t h t r e a t m e n t s . H o w e v e r , t h e r e i s no c o n s i s t e n t d i f f e r e n c e i n s u r v i v a l b e t w e e n t h e two h i g h e s t d e n s i t i e s ( F i g u r e s 1 8 - 1 9 ) . T h i s a g r e e s w i t h M i n c h i n ' s ( 1 9 7 2) f i n d i n g s u s i n g a n o t h e r c i c h l i d . No d a t a s u g g e s t an i n t e r a c t i o n b e t w e e n s o c i a l d o m i n a n c e a n d c r o w d i n g . The s e q u e n c e o f m o r t a l i t y was random o v e r f ' i s f f s i z e . S i z e r a n g e was g r e a t e r t h a n i n o t h e r s p e c i e s and w i t h t h e b e l l i c o s e n a t u r e o f A s t r o n o t u s o c e l l a t u s t h e s e r e p l i c a t e s p r o v i d e d t h e g r e a t e s t p o t e n t i a l f o r h i e r a r c h y f o r m a t i o n . I n t e r a c t i o n s b e t w e e n w a t e r b o r n e f a c t o r s a n d c r o w d i n g c a n be c o n s i d e r e d a d d i t i v e . E a c h h a d an e f f e c t on s u r v i v a l a n d b o t h e x e r c i s e s i m u l t a n e o u s i n f l u e n c e . F i g u r e s 18-19 show t h e l o w e s t d e n s i t y had g r e a t e r s u r v i v a l i n b o t h f i l t e r t r e a t m e n t s ; h i g h e r d e n s i t i e s had p o o r e r s u r v i v a l . The d e n s i t y d e p e n d e n t p a t t e r n was s u p e r i m p o s e d on b o t h f i l t e r t r e a t m e n t s and n e a r l y e q u a l . I n ,a s y n e r g i s t i c i n t e r a c t i o n , g r e a t e r d i f f e r e n c e s b e t w e e n d e n s i t i e s w o u l d be e x p e c t e d i n n o n f i l t e r e d t a n k s . T h i s d i d n o t o c c u r . The e q u a l d e n s i t y e f f e c t i n b o t h t r e a t m e n t s w o u l d s u g g e s t t h e i n t e r a c t i o n was s i m p l y a d d i t i v e . 55 B e t t a s p l e n d e n s No w a t e r b o r n e agent' c o u l d " be d e t e c t e d ' i n B e t t a  s p l e n d e n s ' t a n k s . Water c o n d i t i o n s were a p p r o x i m a t e l y s i m i l a r t o t h e p r e v i o u s s p e c i e s ; ammonia l e v e l s were l o w , pH n e u t r a l , o x y g e n c o n c e n t r a t i o n o f no i m p o r t a n c e , a n d s u s p e n d e d s o l i d s (BOD) r e d u c e d by m e c h a n i c a l a n d b i o l o g i c a l f i l t r a t i o n . The B e t t a s p l e n d e n s were i n t e r m e d i a t e i n s i z e b e t w e e n B r a c h y d a n i o r e r i o a n d A s t r o n o t u s o c e l l a t u s so t h e l a c k o f e f f e c t c a n n o t be a t t r i b u t e d t o b i o m a s s d i f f e r e n c e s . The s t o c k was o b v i o u s l y one s u b j e c t t o l o n g d o m e s t i c a t i o n , d e m o n s t r a t e d by t h e e x t r a o r d i n a r i l y e l o n g a t e d f i n s a n d b r i l l i a n t c o l o r a t i o n o f t h e b r o o d s t o c k m a l e . P e r h a p s s u c c e s s i v e g e n e r a t i o n s i n c l o s e c o n f i n e m e n t e x e r t e d s e l e c t i v e p r e s s u r e t o t o l e r a t e w a t e r b o r n e a g e n t s . H owever, t h e w i l d f o r m e v o l v e d a l a b y r i n t h o r g a n a n d i s a l s o s u i t e d t o t o l e r a t i n g p o o r q u a l i t y w a t e r . The e v i d e n c e o f Yu a n d P e r l m u t t e r ( 1 9 7 0) t h a t w a t e r f r o m a n o t h e r a n a b a n t i d h a d no e f f e c t on B r a c h y d a n i o r e r i o may s u g g e s t t h e y s e c r e t e no a g e n t s . C o n v e r s e l y , t h e s p e c i e s - s p e c i f i c n a t u r e o f many w a t e r b o r n e f a c t o r s (Rose 1960, G r e e n e 1964, F r a n c i s e t a l . 1974, P f u d e r e r e t a l . 1974) a n d t h e t o l e r a n c e o f a n a b a n t i d s t o p o o r w a t e r q u a l i t y make e v i d e n c e s u p p o r t i n g a l a c k o f f a c t o r s q u e s t i o n a b l e . S o c i a l i n t e r a c t i o n a l s o d i d n o t i n f l u e n c e g r o w t h o f t h e B e t t a s p l e n d e n s . The s i t u a t i o n i s n o t d i r e c t l y c o m p a r a b l e t o t h e p r e c e e d i n g two s p e c i e s a s p h y s i c a l c o n t a c t c o u l d n o t be p e r m i t t e d . T h i s i s h o w e v e r , t h e same v i s u a l i n t e r a c t i o n 56 d i s c u s s e d by B i l l i a r d e t a l . ( 1 9 8 1 ) a s a s o u r c e o f c r o w d i n g s t r e s s . N o b l e (1939) d e s c r i b e s m a l e B e t t a s p l e n d e n s . a s n o t h a v i n g a s o c i a l h i e r a r c h y due t o t h e i r a g g r e s s i v e n e s s a n d 55 o f 59 s u r v i v i n g f i s h were m a l e ( t h i s i s n o t u n u s u a l i n B e t t a  s p l e n d e n s ( L u c a s 1 9 8 2 ) ) . The e x p e r i m e n t a l r e s u l t s d i s p r o v e a c o n t e n t i o n by S t e r b a (1973) t h a t v i s u a l c o n t a c t i s a g r o w t h s t i m u l a n t i n B e t t a s p l e n d e n s . V i s u a l c o n t a c t o b v i o u s l y c a u s e d f a r g r e a t e r a c t i v i t y . M a l e B e t t a s p l e n d e n s u n d e r g o e x t r e m e b e h a v i o u r a nd a c t i v i t y c h a n g e s when m e e t i n g o t h e r c o n s p e c i f i c m a l e s . T h e i r a c t i v i t y d i v e r t s ' e n e r g y i n t o m e t a b o l i s m o t h e r t h a n g r o w t h a s s t a r v a t i o n e x p e r i m e n t s d e m o n s t r a t e d . B e c a u s e t h e y grew a t t h e same r a t e a s i n a c t i v e f i s h , t h e y must have had a c c e s s t o more e n e r g y . T h i s c o u l d be s u p p l i e d by i n c r e a s e d f o o d c o n s u m p t i o n o r more e f f i c i e n t u s e o f t h a t e a t e n . I n e i t h e r c a s e , i t w o u l d be o f c o n c e r n t o a c u l t u r i s t w i t h f o o d c o s t s t o c o n s i d e r . SUMMARY The r e s u l t s i n d i c a t e a r e l a t i o n s h i p e x i s t s b e t w e e n a s p e c i e s ' h a b i t s a n d i t s r e s p o n s e t o c r o w d i n g o r w a t e r b o r n e s t r e s s o r s . T a b l e 10 s u m m a r i z e s e a c h s p e c i e s ' a q u a r i u m h i s t o r y , n a t u r a l h a b i t s , a n d r e s p o n s e s t o s t r e s s o r s . The t h r e e s p e c i e s t h a t d i s p l a y e d a n e g a t i v e r e s p o n s e t o w a t e r b o r n e a g e n t s ( i e . Corynopoma r i i s e i , B r a c h y d a n i o  r e r i o , a n d A s t r o n o t u s o c e l l a t u s ) a r e f o u n d i n l a r g e , p e r m a n e n t , c h e m i c a l l y s t a b l e b o d i e s o f w a t e r w h i c h r a p i d l y d i l u t e s e c r e t i o n s . I t i s p o s s i b l e t h a t d o m e s t i c a t i o n T a b l e 1 0 . Summary o f t h e r e s p o n s e s , h a b i t s , and a q u a r i u m h i s t o r y o f r e s e a r c h s p e c i e s . S p e c i e s C . r i i s e i R. h a r t i B . r e r i o A . o c e l l a t u s B . s p l e n d e n s A q u a r i u m H i s t o r y 1 g e n e r a t i o n 0 g e n e r a t i o n s Many g e n e r a t i o n s Many g e n e r a t i o n s Many g e n e r a t i o n s N a t u r a l H a b i t s S o c i a l Water Q u a l i t y S c h o o l i n g S o l i t a r y Good G o o d -P o o r S c h o o l i n g - Good T e r r i t o r i a l E x t r e m e l y P o o r T e r r i t o r i a l H a b i t a t Permanence Pe rmanent T r a n s i e n t S c h o o l i n g Good Permanent Pe rmanent T r a n s i e n t R e l a t i o n s h i p B e t w e e n R e s p o n s e and F a c t o r D e n s i t y W a t e r I n t e r a c t i o n Unknown N e g a t i v e Unknown Unknown None Unknown N e g a t i v e N e g a t i v e C o m p e n s a t o r y N e g a t i v e N e g a t i v e A d d i t i v e None None None 58 i n f l u e n c e s t h i s r e s p o n s e by s e l e c t i n g t o l e r a n t o r n o n s e c r e t o r y i n d i v i d u a l s , however B r a c h y d a n i o r e r i o a nd A s t r o n o t u s o c e l l a t u s b o t h had l o n g a q u a r i u m h i s t o r i e s a n d were s t i l l a f f e c t e d by w a t e r b o r n e a g e n t s . C u l t u r a l s e l e c t i o n may h a v e i n c r e a s e d t o l e r a n c e , b u t t h e e f f e c t r e m a i n s s t r o n g . Two s p e c i e s n o t a f f e c t e d by t h e f i l t e r t r e a t m e n t s , R i v u l u s h a r t i a n d B e t t a s p l e n d e n s , b o t h i n h a b i t t r a n s i e n t w a t e r s more s u s c e p t i b l e t o p o l l u t i o n by s e c r e t o r y and b a c t e r i a l d e g r a d a t i o n p r o d u c t s . R i v u l u s ' h a r t i a l s o " o c c u r i n f l o w i n g w a t e r ( N . R . L i l e y p e r s . comm. 1982) w h i c h s h o u l d o b v i a t e t h e n e e d f o r t o l e r a n c e . H o w e v e r , t h i s s p e c i e s moves f r o m one w a t e r body t o o t h e r s and must t o l e r a t e a r a n g e o f h a b i t a t s . B e t t a s p l e n d e n s may have been s e l e c t e d t o r e s i s t w a t e r b o r n e a g e n t s d u r i n g i t s l o n g d o m e s t i c a t i o n , b u t t h e R i v u l u s h a r t i u s e d were b r e d f r o m w i l d s t o c k a n d showed an i d e n t i c a l r e s p o n s e . The a p p a r e n t l a c k o f r e s p o n s e may be due t o f a i l u r e o f t h e f i l t e r s t o remove s t r e s s o r s , a b s e n c e o f s e c r e t i o n s by f i s h , o r t o l e r a n c e t o t h e i r e f f e c t s . The f i r s t i s u n l i k e l y . The m e d i a u s e d i n p h a s e 1 f i l t e r s c o n t r o l a l l b u t i n o r g a n i c a n i o n s , none o f w h i c h a r e known t o a f f e c t f i s h i n c u l t u r e . T o l e r a n c e t o o r l a c k o f s e c r e t i o n s r e m a i n a s e x p l a n a t i o n s f o r R i v u l u s h a r t i a n d B e t t a s p l e n d e n s , b u t r e q u i r e f u r t h e r r e s e a r c h b e f o r e d i s c u s s i o n . No r e l a t i o n s h i p i s o b v i o u s b e t w e e n a s p e c i e s ' s o c i a l h a b i t s a n d c r o w d i n g , a l t h o u g h o n l y t h r e e s p e c i e s were e x a m i n e d . I n b o t h c a s e s where an e f f e c t was o b s e r v e d i t was 59 o n l y a f r a c t i o n t h a t o f w a t e r b o r n e f a c t o r s . T h i s may i n d i c a t e a more p l a s t i c r e s p o n s e . T h i s a r g u m e n t i s s u p p o r t e d by f i n d i n g a d e n s i t y t h r e s h o l d w h i c h d e m o n s t r a t e s t h a t a l e v e l o f h a b i t u a t i o n c a n be r e a c h e d . B e t t a s p l e n d e n s a r e a b l e t o a d j u s t t h e i r e n e r g y p a r t i t i o n i n g t o c o m p e n s a t e f o r i n c r e a s e d s o c i a l a c t i v i t y . T h i s w o u l d seem s u i t a b l e f o r s u c h an a g g r e s s i v e s p e c i e s i f i t had a b u n d a n t f o o d . I f n o t , i n c r e a s e d d i s p l a y a c t i v i t y w o u l d l e a d t o r e d u c e d f i t n e s s a s i n d i c a t e d by s t a r v a t i o n e x p e r i m e n t s . No d a t a e x i s t w h i c h a r e d i r e c t l y c o m p a r a b l e f o r B r a c h y d a n i o r e r i o a n d A s t r o n o t u s ' o c e l l a t u s a s t h e s e s p e c i e s w e re a l l o w e d p h y s i c a l c o n t a c t a n d c o n t i n u o u s d i s p l a y o p p o r t u n i t y . I t i s i n t e r e s t i n g t o n o t e d i f f e r e n c e s b e t w e e n f a c t o r i n t e r a c t i o n s i n B r a c h y d a n i o r e r i o a n d A s t r o n o t u s o c e l l a t u s . No i n t e r p r e t a t i o n i s o f f e r e d , b u t s u c h d i f f e r e n t c o m b i n e d r e s p o n s e s w o u l d be o f some i m p o r t a n c e t o c u l t u r i s t s . I f work i s done t o c o r r e c t one c o m p e n s a t o r y f a c t o r , t h e e f f o r t w i l l be w a s t e d u n l e s s t h e o t h e r i s a l s o c o n t r o l l e d . I n summary, some f i s h e s a r e i n f l u e n c e d by w a t e r b o r n e a g e n t s o t h e r t h a n t h o s e c o m p e n s a t e d by many c u l t u r a l o p e r a t i o n s . Some c a n be removed by AC. F i s h e s r e s p o n d n e g a t i v e l y t o w a t e r b o r n e a g e n t s a n d c r o w d i n g . I n two s p e c i e s t h e e f f e c t o f c r o w d i n g was a f r a c t i o n o f t h e r e s p o n s e t o w a t e r b o r n e a g e n t s ; i n a n o t h e r s p e c i e s no e f f e c t was o b s e r v e d . The i n t e r a c t i o n o f s t r e s s o r s c a n a t l e a s t be c o m p e n s a t o r y o r a d d i t i v e a n d d e p e n d s on t h e s p e c i e s e x a m i n e d . 60 LITERATURE CITED A f i f i , A.A. and S.P. A z e n . 1972. 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The t e s t i c u l a r d e v e l o p m e n t o f t h e s i l v e r c a r p , H y p o p h t h a l m i c h t h y s m o l i t r i x ( C . & V . ) , i n c a p t i v i t y i n r e l a t i o n t o t h e r e p r e s s i v e e f f e c t o f w a s t e s f r o m f i s h e s . J a p . J . I c h t h y . . 10:24-27. Wedemeyer, G.A. and D.J. M c L e a y . 1981. M e t h o d s f o r d e t e r m i n i n g t h e t o l e r a n c e o f f i s h e s t o e n v i r o n m e n t a l s t r e s s o r s . I n : S t r e s s a n d F i s h (A.D. P i c k e r i n g e d . ) . A c a d e m i c P r e s s . T o r o n t o . p p 2 4 7 - 2 7 5 . Wedemeyer, G.A. 1976. P h y s i o l o g i c a l r e s p o n s e o f j u v e n i l e c o h o s a l m o n ( O n c o r h y n c h u s k i s u t c h ) a nd r a i n b o w t r o u t ( S a l m o g a i r d n e r i ) t o h a n d l i n g a n d c r o w d i n g s t r e s s i n i n t e n s i v e c u l t u r e . J . F i s h . R e s . Bd. Can. 33:2699-2702. 66 W e s t e r s , H a r r y . 1977. R a t i o n a l d e s i g n o f h a t c h e r i e s f o r i n t e n s i v e s a l m o n i d c u l t u r e , b a s e d on m e t a b o l i c c h a r a c t e r i s t i c s . P r o g . F i s h - C u l t . 3 9 : 1 5 7 - 1 6 5 . W h i t e s i d e , B.G. a n d F r e d J . R i c h a n . 1969. R e p r e s s i v e f a c t o r s c o n t r o l l i n g r e p r o d u c t i o n i n g o l d f i s h . P r o g . F i s h - C u l t . 3 1 : 1 6 5 . W i l l o u g h b y , H a r v e y . 1968. A method f o r c a l c u l a t i n g c a r r y i n g c a p a c i t i e s o f h a t c h e r y t r o u g h s a n d p o n d s . P r o g . F i s h -C u l t . 3 0 : 1 7 3 - 1 7 4 . W r i g h t , C A . 1960. The c r o w d i n g phenomenon i n l a b o r a t o r y c o l o n i e s o f f r e s h w a t e r s n a i l s . Ann. T r o p . Med. P a r i s i t . 5 4 : 2 2 4 - 2 3 2 . Y u , Man-Lim a n d A l f r e d P e r l m u t t e r . 1970. G r o w t h i n h i b i t i n g f a c t o r s i n t h e z e b r a f i s h , B r a c h y d a n i o r e r i o , a n d t h e b l u e g o u r a m i , T r i c h o g a s t e r t r i c h o p t e r u s . G r o w t h . 34: 153-175. Z a r , J e r r o l d H. 1974. B i o s t a t i s t i c a l a n a l y s i s . P r e n t i c e -H a l l . N . J . 620p. 67 APPENDIX I Corynopoma r i i s e i ( G i l l ) Common Name: Sword T a i l C h a r a c i n Corynopoma r i i s e i i s a r i v e r i n e a n d l a c u s t r i n e s c h o o l i n g f i s h o f t h e f a m i l y c h a r a c i d a e . I t o c c u r s i n open w a t e r s a n d s m a l l s t r e a m s s c a t t e r e d o v e r n o r t h e r n S o u t h A m e r i c a ( V e n e z e u l a , C o l u m b i a , a n d T r i n i d a d ) a n d i s common t h r o u g h o u t t h e O r i n o c o R i v e r d r a i n a g e . Sexes' a r e ' d i m o r p h i c , m a l e s p o s s e s s i n g e l o n g a t e d d o r s a l , a n a l , a n d c a u d a l f i n s a s w e l l a s an e x t e n s i o n o f t h e o p e r c u l u m u s e d a s b a i t i n m a t i n g d i s p l a y s . E x c l u d i n g t h e c a u d a l f i n e x t e n s i o n , b o t h s e x e s a r e a p p r o x i m a t e l y e q u a l s i z e ( 5 - 6 cm). A l t h o u g h a t t r a c t i v e , t h e s p e c i e s i s n o t common i n t h e a q u a r i u m t r a d e . E x p e r i m e n t a l s t o c k was b r e d f r o m f i r s t g e n e r a t i o n a d u l t s o r i g i n a l l y spawned by D r . N. L i l e y f r o m a T r i n i d a d c o l l e c t i o n . F o u r a d u l t f e m a l e s a n d 2 m a l e s were a l l o w e d t o spawn o v e r 6 d a y s i n a 40 L a q u a r i u m . F r y became f r e e swimming a f t e r 4-6 d a y s a nd were a p p r o x i m a t e l y 4 weeks o l d when e x p e r i m e n t s b e g a n . 68 R i v u l u s h a r t i ( B o u l e n g e r ) Common Name: H a r t ' s R i v u l u s R i v u l u s h a r t i i s a s o l i t a r y c y p r i n o d o n t i n h a b i t i n g d r a i n a g e d i t c h e s , s t a n d i n g p u d d l e s , a n d s m a l l c r e e k s . I t i s f o u n d i n n o r t h e r n S o u t h A m e r i c a i n C o l u m b i a , V e n e z u e l a , Guyana and T r i n i d a d . A l t h o u g h r e l a t i v e l y p e a c e f u l i n t h e company o f o t h e r s p e c i e s , i t i s commonly a g g r e s s i v e t o w a r d c o n s p e c i f i c s . A h a r d y f i s h , t o l e r a t i n g p o o r w a t e r c o n d i t i o n s , i t i s known t o l e a v e w a t e r a n d move a c r o s s wet g r o u n d f r o m one s m a l l p o o l t o a n o t h e r . As a d u l t s , s e x e s a r e d i m o r p h i c ' , f e m a l e s ' d i s t i n g u i s h e d by a l a r g e o c e l l u s on t h e d o r s a l c a u d a l p e d u n c l e . B o t h m ale and f e m a l e r e a c h an a d u l t s i z e o f a p p r o x i m a t e l y 10 cm. R i v u l u s h a r t i i s r a r e l y s t o c k e d i n a q u a r i u m s t o r e s due t o i t s d u l l a p e a r a n c e . E x p e r i m e n t a l s t o c k was o b t a i n e d by s p a w n i n g a n d r e a r i n g t h e f i r s t g e n e r a t i o n o f a p a i r c o l l e c t e d i n T r i n i d a d by D r . N . L i l e y . They were a l l o w e d t o b r e e d o v e r a 2 week p e r i o d i n a 40 L a q u a r i u m . F r y were f r e e swimming a f t e r a p p r o x i m a t e l y 3 weeks and a b o u t 3 weeks o l d e r when e x p e r i m e n t s b e g a n . 69 B r a c h y d a n i o r e r i o ( H a m i l t o n - B u c h a n a n ) Common Name: Z e b r a D a n i o B r a c h y d a n i o r e r i o i s a s m a l l open w a t e r s c h o o l i n g f i s h o f t h e f a m i l y c y p r i n i d a e f o u n d i n open p o r t i o n s o f l a k e s a n d r i v e r s i n e a s t e r n I n d i a . The s t r e n g t h o f t h e s c h o o l i n g h a b i t i s e a s i l y o b s e r v e d i n l a r g e a q u a r i a where t h e y s e l d o m s t r a y f r o m one a n o t h e r ' s p r o x i m i t y , e x c e p t w h i l e f e e d i n g . The s e x e s a r e s l i g h t l y d i m o r p h i c w i t h m a l e s h a v i n g a more y e l l o w c a s t i n c l e a r p o r t i o n s o f t h e a n a l a n d c a u d a l f i n s . F e m a l e s a r e f u l l e r i n g i r t h when r i p e . B o t h s e x e s r e a c h an a d u l t s i z e o f 4-5' cm. B r a c h y d a n i o r e r i o i s one o f t h e most common a q u a r i u m f i s h e s w i t h an a q u a r i u m h i s t o r y d a t i n g t o 1905 ( F r e y 1970) when t h e f i r s t s p e c i m e n s were i m p o r t e d t o E u r o p e . V i r t u a l l y a l l a q u a r i u m s t o c k i s r a i s e d i n c a p t i v i t y a n d e x p e r i m e n t a l s t o c k was o b t a i n e d by b r e e d i n g a d u l t s i m p o r t e d f r o m Hong Kong. S i x f e m a l e s a n d 4 m a l e s were a l l o w e d t o spawn f o r 2 d a y s i n a 60 L a q u a r i u m . F r y were f r e e swimming i n 4 d a y s a n d a p p r o x i m a t e l y 5 weeks o l d when e x p e r i m e n t s s t a r t e d . 70 A s t r o n o t u s o c e l l a t u s ( A g a s s i z ) Common Name: O s c a r A s t r o n o t u s o c e l l a t u s i s a l a r g e t e r r i t o r i a l member o f t h e c i c h l i d a e . T e r r i t o r i a l i t y o n l y d e v e l o p s l a t e r i n l i f e ; y o u n g j u v e n i l e s r e m a i n i n a s c h o o l u n d e r p a r e n t a l c a r e f o r t h e f i r s t few months o f l i f e . I t i s common i n s e c o n d a r y w a t e r s o f l a r g e c e n t r a l S o u t h A m e r i c a n r i v e r s w i t h A t l a n t i c d r a i n a g e ( i e . t h e Amazon, P a r a n a , R i o N e g r o , P a r a g u a y , O r i n o c o , D e m e r e r a , a nd E s q u i b o ) . S e x e s a r e o n l y d i f f e r e n t i a b l e by t h e sha p e o f t h e i r g e n i t a l p a p i l l a i m m e d i a t e l y p r i o r t o " s p a w n i n g . Both* c a n a t t a i n l e n g t h s o f 40 cm a n d w e i g h t s a p p r o a c h i n g one K g . D e s p i t e i t s l a r g e s i z e , A s t r o n o t u s o c e l l a t u s i s p o p u l a r i n t h e a q u a r i u m t r a d e a s a n o v e l t y f i s h . V i r t u a l l y a l l s p e c i m e n s f o u n d i n p e t s t o r e s a r e b r e d i n c a p t i v i t y , u s u a l l y i m p o r t e d f r o m t h e f a r e a s t . Due t o t h e e x p e n s e o f m a t u r e b r e e d i n g a d u l t s , e x p e r i m e n t a l s t o c k was i m p o r t e d f r o m Hong Kong a s s m a l l j u v e n i l e s . B a s e d on s i z e , t h e y were p r o b a b l y 8-10 weeks o l d when e x p e r i m e n t s b e g a n . I t s h o u l d be n o t e d t h a t t h e s e were t h e l a r g e s t f i s h u s e d a n d e x c e e d e d t h e f i n a l w e i g h t o f a l l o t h e r s p e c i e s by more t h a n 3 t i m e s . 71 B e t t a s p l e n d e n s (Regan) Common Name: S i a m e s e F i g h t i n g F i s h B e t t a s p l e n d e n s i s a w i d e l y known and d o m e s t i c a t e d member of t h e a n a b a n t i d a e . M a l e s a r e r e n o w n e d f o r t h e i r t e r r i t o r i a l i t y a n d e x t r e m e p u g n a c i t y t o w a r d c o n s p e c i f i c s . The s p e c i e s o c c u r s n a t u r a l l y t h r o u g h o u t I n d o - C h i n a i n s m a l l s t a n d i n g b o d i e s o f w a t e r a n d c a n t o l e r a t e e x t r e m e l y p o o r w a t e r q u a l i t y , a i d e d a t l e a s t i n p a r t by a u x i l i a r y r e s p i r a t i o n w i t h a l a b y r i n t h o r g a n . A i r b r e a t h i n g i s o b l i g a t e f o r B e t t a s p l e n d e n s w h i c h w i l l l i t e r a l l y d r own i f u n a b l e t o r e a c h t h e s u r f a c e - i n ' water' a t t e m p e r a t u r e s n o r m a l l y o c c u p i e d . The s e x e s , e s p e c i a l l y d o m e s t i c a t e d s t r a i n s , a r e h i g h l y d i m o r p h i c , m a l e s h a v i n g g r e a t l y e x t e n d e d f i n s a n d more i n t e n s e c o l o u r a t i o n . M a l e s a r e s l i g h t l y l a r g e r t h a n f e m a l e s , when m a t u r e , r e a c h i n g 6 a n d 5 cm r e s p e c t i v e l y . The s p e c i e s h a s a l o n g a q u a r i u m h i s t o r y . E u r o p e a n i m p o r t a t i o n s d a t e t o t h e l a t e 1 9 t h c e n t u r y , w i t h e a r l i e r , b u t u n d o c u m e n t e d d o m e s t i c a t i o n i n t h e i r n a t i v e l a n d s . E x p e r i m e n t a l s t o c k was b r e d f r o m one p a i r o f t h e o r n a m e n t a l v a r i e t y . As s u c h t h e y were a d a p t e d t o c l o s e c o n f i n e m e n t i n s t i l l w a t e r . D o m e s t i c a t e d m a l e s a r e n o r m a l l y r a i s e d i s o l a t e d i n one L o r s m a l l e r c o n t a i n e r s . F e m a l e s a r e c r o w d e d a t h i g h d e n s i t y i n s m a l l t a n k s . T h i s i s made p o s s i b l e by t h e s p e c i e s ' t o l e r a n c e of p o o r w a t e r q u a l i t y a nd a u x i l i a r y r e s p i r a t i o n . A d u l t s b r e d f o r t h i s s t u d y were i m p o r t e d f r o m S i n g a p o r e and p r o d u c e d o v e r 500 f r y i n one s p a w n i n g . T h e s e were f r e e swimming w i t h i n 48 h o u r s a n d a p p r o x i m a t e l y 7 weeks o l d when e x p e r i m e n t s b e g a n . 

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