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Chloride ion and water balance in the prosobranch gastropod Collisella persona Clark, Geoffrey A. 1982

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CHLORIDE ION AND WATER BALANCE IN THE PROSOBRANCH GASTROPOD COLLI SELLA PERSONA by GEOFFREY A. CLARK B.Sc. U n i v e r s i t y Of B r i t i s h Columbia 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA 20 August 1982 © G e o f f r e y A. C l a r k , 1982 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 at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree t h a t permission f o r extensive 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 that 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 permission. Department of 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 i i ABSTRACT The c h l o r i d e i o n c o n c e n t r a t i o n o f b l o o d , u r i n e , and f o o t m u s c l e , b l o o d and m u s c l e water c o n t e n t , and b l o o d and u r i n e o s m o t i c p r e s s u r e , were measured i n summer and w i n t e r a d a p t e d p o p u l a t i o n s of t h e p r o s o b r a n c h l i m p e t C o l l i s e l l a p e r s o n a , o v e r a r a n g e o f s a l i n i t i e s from 25% t o 125% SW (100% SW = 480 mEq C l " / L ) . B l o o d , u r i n e , and f o o t m u s c l e C I " were n o t r e g u l a t e d i n a l l s a l i n i t i e s t e s t e d . B l o o d C I " was g e n e r a l l y h y p o i o n i c and u r i n e i s o i o n i c t o t h e e x p e r i m e n t a l s e a water c o n c e n t r a t i o n s . M u s c l e C I " d i d n o t r e a c h a l e v e l g r e a t e r t h a n o n e - h a l f o f e q u i v a l e n t b l o o d o r s e a water v a l u e s i n a l l s a l i n i t i e s t e s t e d . However, m u s c l e and b l o o d v a l u e s were s i m i l a r , i f m u s c l e C l " were e x p r e s s e d i n t e r m s of e x t r a c e l l u l a r w a t e r . E x t r a c e l l u l a r volume r e m a i n e d r e l a t i v e l y u n c h a n g e d and showed o n l y a 7% i n c r e a s e i n s a l i n i t i e s r a n g i n g f r o m 50% t o 125% SW. S e a s o n a l d i f f e r e n c e s were a p p a r e n t i n m u s c l e C l " , w i t h w i n t e r v a l u e s h i g h e r t h a n summer v a l u e s by 20-25 mEq/kg t i s s u e , i n s a l i n i t i e s r a n g i n g f r o m 75% t o 125% SW. However, t h i s c o u l d be a c c o u n t e d f o r by t h e s l i g h t l y l a r g e r (4-8%) e x t r a c e l l u l a r s p a c e r e c o r d e d f r o m w i n t e r a n i m a l s . Summer and w i n t e r a d a p t e d l i m p e t s were o s m o c o n f o r m e r s i n s a l i n i t i e s r a n g i n g from 25% t o 125% SW. Summer a n i m a l s had b l o o d h y p e r o s m o t i c , and u r i n e i s o s m o t i c t o t h e e x p e r i m e n t a l s e a wa t e r c o n c e n t r a t i o n s . W i n t e r l i m p e t s had b l o o d h y p e r o s m o t i c t o 75% SW, and i s o s m o t i c i n 25% and 125% SW. U r i n e was h y p o s m o t i c t o 25% and 75% SW, and i s o s m o t i c i n 125% SW. Summer b l o o d was h y p e r o s m o t i c t o w i n t e r b l o o d , a l t h o u g h summer and w i n t e r u r i n e r e m a i n e d i s o s m o t i c . In a d d i t i o n , f o r b o t h summer and w i n t e r a n i m a l s , b l o o d was h y p e r o s m o t i c t o u r i n e . The f o o t m u s c l e water c o n t e n t of w i n t e r a d a p t e d l i m p e t s r e t u r n e d t o t h e c o n t r o l v a l u e i n 50% t o 125% SW, between 48 h o u r s and 1 week. Over t h e same t i m e p e r i o d , summer a n i m a l s o n l y r e g u l a t e d m u s c l e w a t e r i n 50% and 75% SW. B l o o d water c o n t e n t r e m a i n e d r e l a t i v e l y u n changed f o r b o t h summer and w i n t e r a d a p t e d l i m p e t s , and showed a 4% d e c r e a s e o v e r a s a l i n i t y r a n g e from 25% t o 125% SW. T h e r e were no s i g n i f i c a n t s e a s o n a l d i f f e r e n c e s i n b l o o d w ater c o n t e n t . S e a s o n a l d i f f e r e n c e s r e c o r d e d d u r i n g t h e p r e s e n t s t u d y , i n p a r t i c u l a r t h e c o m p a r a t i v e l y l a r g e v a r i a t i o n i n measurements from summer a n i m a l s , were a t t r i b u t e d t o s e a s o n a l c h a n g e s i n s a l t and w ater p e r m e a b i l i t y , o r m e t a b o l i c r a t e . In a d d i t i o n , an a t t e m p t was made t o r e l a t e t h e p h y s i o l o g i c a l r e s p o n s e s r e c o r d e d from l i m p e t s i n t h e l a b o r a t o r y , t o t h e s u r v i v a l o f l i m p e t s i n t h e f i e l d . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x I n t r o d u c t i o n 1 M a t e r i a l and Methods 12 C o l l e c t i o n and Measurement of Samples 17 Bl o o d Samples 17 U r i n e Samples . . 18 T i s s u e Samples 18 E x t r a c e l l u l a r Volume 19 S t a t i s t i c a l A n a l y s i s 21 R e s u l t s 22 I . C h l o r i d e Ion Response of B l o o d , U r i n e , and Muscle T i s s u e 22 Response of B l o o d and U r i n e C l " 22 Response of Foot Muscle C l " 38 I I . E x t r a c e l l u l a r Volume of Foot Muscle 49 I I I . B l o o d and U r i n e Osmotic P r e s s u r e 52 IV. Water B a l a n c e 55 Response of Foot Muscle Water 55 Response of B l o o d Water 66 D i s c u s s i o n 70 I . The E f f e c t of S a l i n i t y on C h l o r i d e Ion Ba l a n c e .* 70 B l o o d and U r i n e C h l o r i d e 70 F o o t M u s c l e T i s s u e C h l o r i d e 74 I I . E x t r a c e l l u l a r Volume of F o o t M u s c l e T i s s u e 77 I I I . B l o o d and U r i n e O s m o t i c P r e s s u r e 80 IV. The E f f e c t of S a l i n i t y on Water B a l a n c e 83 F o o t M u s c l e T i s s u e Water C o n t e n t 83 B l o o d Water C o n t e n t 86 V. S e a s o n a l V a r i a t i o n 87 V I . E c o l o g i c a l I m p l i c a t i o n s 90 Summary 94 L i t e r a t u r e C i t e d 97 v i L I S T OF TABLES T a b l e 1. E x p e r i m e n t a l S a l i n i t i e s 15 T a b l e 2. E f f e c t o f t i m e and s a l i n i t y on b l o o d and u r i n e C l " c o n c e n t r a t i o n s 28 T a b l e 3. E f f e c t of s a l i n i t y on p o o l e d b l o o d and u r i n e C l " v a l u e s 35 T a b l e 4. E f f e c t of s a l i n i t y on p o o l e d f o o t m u s c l e C l " v a l u e s 44 T a b l e 5.. E f f e c t of s a l i n i t y on b l o o d and f o o t m u s c l e C l " c o n c e n t r a t i o n s 47 v i i L I S T OF FIGURES F i g u r e 1. L i g h t h o u s e p a r k t e m p e r a t u r e and s a l i n i t y measurements 13 F i g u r e 2. E f f e c t of t i m e and s a l i n i t y on b l o o d and u r i n e C l " f r o m summer l i m p e t s 23 F i g u r e 3. E f f e c t o f t i m e and s a l i n i t y on b l o o d and u r i n e C l " f r o m w i n t e r l i m p e t s 26 F i g u r e 4. E f f e c t of s a l i n i t y on b l o o d and u r i n e C l " from summer l i m p e t s . . 31 F i g u r e 5. E f f e c t o f s a l i n i t y on b l o o d and u r i n e C l " f r o m w i n t e r l i m p e t s 33 F i g u r e 6. E f f e c t o f t i m e and s a l i n i t y on f o o t m u s c l e C l " .. 39 F i g u r e 7. E f f e c t o f s a l i n i t y on m u s c l e and b l o o d C l " ...... 42 F i g u r e 8. E f f e c t of s a l i n i t y on f o o t m u s c l e e x t r a c e l l u l a r volume 50 F i g u r e 9. E f f e c t o f s a l i n i t y on b l o o d and u r i n e o s m o t i c p r e s s u r e 53 F i g u r e 10. E f f e c t o f t i m e and s a l i n i t y on t h e m u s c l e w a t e r c o n t e n t o f summer l i m p e t s 56 F i g u r e 11. E f f e c t o f t i m e and s a l i n i t y on t h e m u s c l e w a t e r c o n t e n t o f w i n t e r l i m p e t s 58 F i g u r e 12. M u s c l e wet w e i g h t r e g r e s s e d a g a i n s t d r y w e i g h t f o r summer l i m p e t s 61 F i g u r e 13. M u s c l e wet w e i g h t r e g r e s s e d a g a i n s t d r y w e i g h t f o r w i n t e r l i m p e t s 63 v i i i F i g u r e 14. E f f e c t o f s a l i n i t y on f o o t m u s c l e and b l o o d water c o n t e n t s 67 i x ACKNOWLEDGEMENTS I w i s h t o thank my s u p e r v i s o r D r . P.A. D e h n e l f o r h i s c o n t i n u a l s u p p o r t and h e l p f u l d i s c u s s i o n t h r o u g h o u t t h e c o u r s e of t h i s s t u d y . The t i m e he d o n a t e d t o w a r d s t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t was g r e a t l y a p p r e c i a t e d . In a d d i t o n , I w i s h t o thank D r . D. Z i t t i n f o r h i s a s s i s t a n c e w i t h t h e s t a t i s t i c a l a n a l y s i s , and D r s . J . E . P h i l l i p s , A.G. L e w i s , and T. G r i g l i a t t i f o r t h e i r p r o f i t a b l e e d i t o r i a l comments. I a l s o e x t e n d t h a n k s t o t h e M u n i c i p a l i t y o f West V a n c o u v e r , f o r a l l o w i n g me t o c o l l e c t l i m p e t s . f r o m L i g h t h o u s e P a r k . F i n a l l y , I w i s h t o thank my w i f e , M a r l e n e , w i t h o u t whose c o n t i n u a l p a t i e n c e and f i n a n c i a l s u p p o r t t h i s t h e s i s would n o t have been c o m p l e t e d . T h i s s t u d y was f u n d e d by a g r a n t f r o m t h e N a t i o n a l S c i e n c e and E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada t o D r . P.A. D e h n e l . 1 INTRODUCTION P r o s o b r a n c h g a s t r o p o d s o f t h e genus C o l l i s e l l a , and r e l a t e d l i m p e t g e n e r a , Acmaea, and Notoacmaea, a r e among t h e most commonly d i s t r i b u t e d m a r i n e i n t e r t i d a l f a u n a a l o n g t h e P a c i f i c C o a s t o f N o r t h A m e r i c a . C o l l i s e l l a p e r s o n a , w h i c h r a n g e s from A l a s k a t o C a l i f o r n i a ( T e s t , 1945), i s f o u n d a b u n d a n t l y a l o n g t h e s o u t h e r n c o a s t o f B r i t i s h C o l u m b i a . The a r e a a r o u n d V a n c o u v e r , B r i t i s h C o l u m b i a i s e s t u a r i n e , w i t h s u r f a c e t e m p e r a t u r e s a n d s a l i n i t i e s r a n g i n g s e a s o n a l l y f r o m 5°C and 24% 0 d u r i n g w i n t e r , t o 20°C and 8%» i n summer ( D e h n e l , 1979). In a d d i t i o n t o s e a s o n a l t e m p e r a t u r e and s a l i n i t y s t r e s s , h i g h i n t e r t i d a l l i m p e t s i n c l u d i n g C. p e r s o n a , a r e a f f e c t e d by e v a p o r a t i v e d e s i c c a t i o n and f r e s h w ater r u n o f f d u r i n g low t i d e more o f t e n t h a n a r e l o w e r i n t e r t i d a l o r g a n i s m s . In t h i s s t u d y s e a s o n a l c h a n g e s i n b l o o d , u r i n e , and f o o t m u s c l e C l " , b l o o d and m u s c l e w a t e r , and b l o o d and u r i n e o s m o t i c p r e s s u r e , w i l l be i n v e s t i g a t e d by p e r f o r m i n g e x p e r i m e n t s on b o t h summer and w i n t e r a d a p t e d C. p e r s o n a . I n a d d i t i o n , t h e i n f o r m a t i o n g a i n e d from t h e s e s t u d i e s w i l l be i n t e g r a t e d w i t h e c o l o g i c a l d a t a from t h e l i t e r a t u r e , t o d e t e r m i n e how t h e c h l o r i d e and water b a l a n c e of C. p e r s o n a i s r e l a t e d t o i t s s u r v i v a l i n t h e f i e l d . The a b i l i t y t o r e g u l a t e t h e volume o f body f l u i d s i s o f g r e a t i m p o r t a n c e t o an i n v e r t e b r a t e i n h a b i t i n g an e n v i r o n m e n t where t h e s a l i n i t y i s c o n s t a n t l y f l u c t u a t i n g , s u c h as an e s t u a r y ( K i n n e , 1971; Hoyaux e t a l , 1976; G i l l e s and J e u n i a u x , 1979; O g l e s b y , 1981). Volume r e g u l a t o r s , when p l a c e d i n d i l u t e o r 2. c o n c e n t r a t e d media, i n i t i a l l y g a i n o r l o s e w ater but e v e n t u a l l y r e t u r n t o t h e i r o r i g i n a l weights... Volume- c o n f ormerS'-show no, such.> t e n d e n c y t o r e t u r n t o . t h e i r i n i t i a l w e i g h t , even a f t e r an e x t e n d e d p e r i o d of t i m e i n h y p e r - o r h y p o t o n i c media (Ramsay, 1954; R o b e r t s o n , 1.964; P o t t s and P a r r y , 1964; O g l e s b y , 1981). i Volume r e g u l a t o r y mechanisms i n c l u d e : a c t i v e c h a n g e s i n t h e c o n c e n t r a t i o n s of o r g a n i c o r i n o r g a n i c o s m o t i c a l l y a c t i v e s u b s t a n c e s , b e h a v i o r a l r e s p o n s e s s u c h as s h e l l c l o s u r e or c l a m p i n g down t o t h e s u b s t r a t e , mucous s e c r e t i o n , and r e d u c t i o n i n body w a l l p e r m e a b i l i t y t o w a t e r o r an i n c r e a s e i n p e r m e a b i l i t y t o s a l t s ( M c A l i s t e r and F i s h e r , 1968; B o y l e , 1969; K i n n e , 1971; Lange, 1972; Shumway, 1977a and b; G i l l e s and J e u n i a u x , 1979; Moran and T u l l i s , 1980; O g l e s b y , 1981). A l l a q u a t i c i n v e r t e b r a t e s have some d e g r e e of i o n r e g u l a t i o n , r a n g i n g f r o m a K + a c c u m u l a t i o n i n e c h i n o d e r m s ( 3 % t o 160% o f s e a w ater K + ) t o t h e r e g u l a t i o n of a l m o s t a l l i o n s i n t h e c e p h a l o p o d s and most of t h e C r u s t a c e a ( R o b e r t s o n , 1949; 1953; 1964; P o t t s and P a r r y , 1964; D e h n e l , 1966; 1979; T u c k e r , 1970;. Moran and T u l l i s , 1980). Among t h e m o l l u s c s , slow moving b i v a l v e s and g a s t r o p o d s have l e s s e f f i c i e n t i o n r e g u l a t o r y a b i l i t i e s t h a n more a c t i v e c e p h a l o p o d s . B i v a l v e s and g a s t r o p o d s a c c u m u l a t e s m a l l amounts of K + and C a + 2 and e x c r e t e S 0 3 ~ 2 ; N a + , M g + 2 , and C l " c o n c e n t r a t i o n s r e m a i n i s o i o n i c w i t h t h e ambient medium ( R o b e r t s o n , 1949; 1953; 1964; B e a d l e , 1957; P o t t s and P a r r y , 1964; Webber, .1966; Webber and D e h n e l , 1968a; K i n n e , 1971). F r e s h w a t e r a n n e l i d s , b i v a l v e s , g a s t r o p o d s , and c r u s t a c e a n s a l l show an i n c r e a s e i n i o n r e g u l a t o r y a b i l i t y , when 3 compared w i t h r e l a t e d m a r i n e s p e c i e s ( P o t t s and P a r r y , 1964; R o b e r t s o n , 1964; O g l e s b y , 1981). S m i t h (1970a; 1976), w o r k i n g w i t h N e r e i s d i v e r s i c o l o r , f o u n d t h a t body w a l l p e r m e a b i l i t y t o N a + and C l " d e c r e a s e d f r o m s e a w a t e r t o f r e s h w a t e r ; i n f a c t , t h e s e i o n s were a c t i v e l y t a k e n up a t low s a l i n i t i e s . The b r a c k i s h w ater c r a b R h i t h r o p a n o p e u s h a r r i s i showed h y p e r -r e g u l a t i o n of C l " below 60-70% SW, and a s l i g h t h y p o r e g u l a t i o n i n h i g h e r s a l i n i t i e s ( S m i t h , 1967). In a d d i t i o n , R. h a r r i s i was a b l e t o d e c r e a s e body w a l l w a t e r p e r m e a b i l i t y i n l o w e r s a l i n i t i e s ( S m i t h , 1967). Sodium and c h l o r i d e i o n s were a l s o a c t i v e l y r e g u l a t e d by t h e f r e s h w a t e r m u s s e l L i g u m i a s u b r o s t r a t a ( D i e t z and B r a n t o n , 1975). M a r i n e b i v a l v e s i n c l u d i n g M y t i l u s  e d u l i s , S c r o b i c u l a r i a p l a n a , and G l y c y m e r i s g l y c y m e r i s , and t h e g a s t r o p o d s L i t t o r i n a l i t t o r e a , and P u r p u r a l a p i l l u s , had b l o o d C l " c o n c e n t r a t i o n s i s o i o n i , c t o s e a w a t e r (Hoyaux e t a l , 1976). Hand an d S t i c k l e (1977) measured t h e b l o o d C l " l e v e l of C r a s s o s t r e a v i r g i n i c a o v e r a t i d a l f l u c t u a t i o n o f 65-30-65% SW, and f o u n d t h a t b l o o d was i s o i o n i c w i t h t h e a m bient m e d i a . Moran and T u l l i s (1980) showed t h a t t h e b l o o d C l " c o n c e n t r a t i o n of t h e c h i t o n M o p a l i a muscosa r e m a i n e d i s o i o n i c w i t h s e a w a t e r o v e r a s a l i n i t y r a n g e o f 60-120% SW. One p u r p o s e of t h e p r e s e n t s t u d y was t o d e t e r m i n e t h e d e g r e e o f C l " r e g u l a t i o n i n C. p e r s o n a . T h i s i n v e s t i g a t i o n i n c l u d e d t h e d e t e r m i n a t i o n o f C l " c o n c e n t r a t i o n i n b l o o d and u r i n e , and f o o t m u s c l e , o v e r a r a n g e of s a l i n i t i e s f r o m 25-125% SW. Samples were t a k e n o v e r a 1 week p e r i o d i n o r d e r t o measure t h e c h a n g e s i n C l " c o n c e n t r a t i o n i n t h e v a r i o u s t i s s u e s w i t h 4 t i m e . The r e s u l t s s h o u l d i n d i c a t e w h ether t h i s l i m p e t i s c a p a b l e of C l " r e g u l a t i o n , u n l i k e o t h e r r e l a t e d g a s t r o p o d s . Most b i v a l v e s and g a s t r o p o d s , e x c l u d i n g f r e s h w a t e r f o r m s , a r e o s m o c o n f o r m e r s w i t h an i n t e r n a l s o l u t e c o n c e n t r a t i o n s i m i l a r t o t h a t o f t h e a m bient medium ( R o b e r t s o n , 1964; K i n n e , 1.971; Lange, 1972; Hoyaux e t a l , 1976; G i l l e s and J e u n i a u x , 1979; O g l e s b y , 1981). T u r g e o n (1976) f o u n d t h a t U r o s a l p i n x c i n e r e a d i d n o t o s m o r e g u l a t e i n s a l i n i t i e s r a n g i n g from 30-130% SW. S e p t u s  b r e v i c u l u s was p o i k i l o s m o t i c o v e r t h e r a n g e o f 75-115% SW ( T u c k e r , 1970). B o y l e (1969) r e p o r t e d t h a t t h e c h i t o n S y p h a r o c h i t o n p e l l i s e r p e n t i s had a p e r i c a r d i a l f l u i d o s m o t i c c o n c e n t r a t i o n t h a t a d j u s t e d t o t h e medium o v e r a s a l i n i t y r a n g e of 50-150% SW. He a l s o f o u n d t h a t h i g h t e m p e r a t u r e s i n c r e a s e d t h e r a t e o f o s m o t i c a d j u s t m e n t , and c o n s e q u e n t l y t h e m o r t a l i t y r a t e , a t l o w e r s a l i n i t i e s . I n t h e p r e s e n t s t u d y , t h e o s m o t i c c o n c e n t r a t i o n of b l o o d and u r i n e from C. p e r s o n a was m e a s u r e d i n c o n t r o l s a l i n i t i e s and i n 25% and 125% SW. The p u r p o s e was t o d e t e r m i n e i f t h i s l i m p e t were c a p a b l e o f o s m o r e g u l a t i o n . Many i n v e r t e b r a t e s a r e c a p a b l e of i s o s o m o t i c r e g u l a t i o n , w h i c h i s r e g u l a t i o n between i n t r a c e l l u l a r a n d e x t r a c e l l u l a r s p a c e s ( R o b e r t s o n , 1964; L a n g e , 1972; Hoyaux e t a l , 1976; D e h n e l , 1979; G i l l e s and J e u n i a u x , 1979; O g l e s b y , 1 9 8 1 ) . I n v e r t e b r a t e s w i t h an open c i r c u l a t o r y s y s t e m have an e x t r a c e l l u l a r s p a c e c o n t i n u o u s w i t h t h e b l o o d s p a c e , u n l i k e t h e s i t u a t i o n i n v e r t e b r a t e s ( D e h n e l , 1979). S p a a r g a r e n (1972) s t a t e d t h a t t h e e x t r a c e l l u l a r volume o r s p a c e may a c t as an 5 o s m o t i c b u f f e r , r e d u c i n g c h a n g e s i n c e l l u l a r c o n c e n t r a t i o n , and p a r t l y a l l e v i a t i n g s h o r t t e r m o s m o t i c s t r e s s . T h i s i s i m p o r t a n t t o e s t u a r i n e o r g a n i s m s e x p o s e d t o s a l i n i t y f l u c t u a t i o n . E x t r a c e l l u l a r volume has been e s t i m a t e d u s i n g a v a r i e t y o f methods. The d i l u t i o n method, u s i n g s m a l l m o l e c u l e s i n c l u d i n g i n u l i n , t h i o s u l p h a t e , a m a r a n t h , o r s u l p h a t e , y i e l d s v a r i a b l e r e s u l t s ( S p a a r g a r e n , 1972). S i e b e r s and L u c u (1973; c i t e d i n D e h n e l , 1979) f o u n d t h a t t h e e x t r a c e l l u l a r s p a c e o f t h e c r a b , C a r e i n u s maenas, measured u s i n g a m a r a n t h was 33.1% o f t h e body w e i g h t , w h i l e t h e i n u l i n s p a c e was 17.9%. Manery (1954) c a l c u l a t e d e x t r a c e l l u l a r s p a c e b a s e d on t h e c o n c e n t r a t i o n o f b l o o d and t i s s u e C l " , and b l o o d w a t e r . In d o i n g so however she made t h e a s s u m p t i o n t h a t a l l C l " was e x t r a c e l l u l a r . T h i s a s s u m p t i o n has been p r o v e n i n c o r r e c t by G a y t o n and H i n k e ( 1 9 6 8 ) , who r e p o r t e d m y o p l a s m i c C l " c o n c e n t r a t i o n s o f 44.3 and 34.5 mM/kg f i b r e water i n m u s c l e c e l l s of t h e b a r n a c l e B a l a n u s  n u b i l u s . B e r g e r e t a l (1978) m e n t i o n e d t h a t t h e e x t r a c e l l u l a r volume of o s m o c o n f o r m i n g g a s t r o p o d s o f t h e genus L i t t o r i n a d e c r e a s e d i n h y p o s m o t i c c o n d i t i o n s . I f an a n i m a l were c a p a b l e of c e l l volume r e g u l a t i o n t h e r e l a t i o n s h i p between c e l l u l a r and e x t r a c e l l u l a r volume would be r e s t o r e d . In t h e p r e s e n t s t u d y , t h e e x t r a c e l l u l a r volume o f C. p e r s o n a was e s t i m a t e d u s i n g C l " f o r t h e c a l c u l a t i o n , i n s a l i n i t i e s r a n g i n g f r o m 25-125% SW. I t was e x p e c t e d t h a t a h i g h i n t e r t i d a l e s t u a r i n e l i m p e t would show a h i g h d e g r e e o f volume r e g u l a t i o n . Many i n v e r t e b r a t e s use amino a c i d s and o t h e r o r g a n i c 6 s u b s t a n c e s t o reduce the osmotic p r e s s u r e d i f f e r e n c e s between i n t r a and e x t r a c e l l u l a r s p a c e s , t h a t d e v e l o p when a n i m a l s a re t r a n s f e r r e d t o d i l u t e or c o n c e n t r a t e d media (Ramsay, 1954). C l a r k (1968b) demonstrated t h a t when v a r i o u s p o l y c h a e t e s were t r a n s f e r r e d t o d i l u t e sea water amino a c i d s and o t h e r n i n h y d r i n -p o s i t i v e s u b s t a n c e s were a c t i v e l y t r a n s p o r t e d out of the c e l l . Moran and T u l l i s (1980) r e p o r t e d a s i m i l a r a n i s o s m o t i c r e g u l a t i o n of f r e e amino a c i d s i n the c h i t o n M o p a l i a muscosa. T h i s phenomenon a l s o o c c u r s i n many o t h e r m o l l u s c s i n c l u d i n g M o d i o l u s ( P i e r c e and Greenburg, 1971; 1972; 1973; 1976; P i e r c e and Amende, 1981; B i s h o p e t a l , 1981), M y t i l u s e d u l l s , S c r o b i c u l a r i a - p l a n a , G l y c y m e r i s g l y c y m e r i s , L i t t o r i n a l i t t o r e a , P u r p u r a l a p i l l u s , P a t e l l a v u l g a t a (Hoyaux e t a l , 1976), and U r o s a l p i n x c i n e r e a (Turgeon,1976). I s o s m o t i c amino a c i d r e g u l a t i o n a l s o t a k e s p l a c e i n the C r u s t a c e a ( P o t t s and P a r r y , 1964; Lange, 1972). G i l l e s (1979) f o r m u l a t e d a p o s s i b l e mechanism f o r the i n t r a c e l l u l a r r e g u l a t i o n of amino a c i d s i n c r u s t a c e a n s . In summary the h y p o t h e s i s was as f o l l o w s : c e l l s s w e l l when p l a c e d i n hyposmotic media, and the s t r e t c h i n g of the c e l l membrane i n t r o d u c e s c o n f o r m a t i o n a l changes which p e r m i t the e f f l u x of amino a c i d s . O s m o t i c a l l y o b l i g a t e d water i s thus removed from the c e l l , l o w e r i n g the i n t r a c e l l u l a r o s m o t i c p r e s s u r e . In a d d i t i o n , an i n t r a c e l l u l a r c a t a b o l i s m of amino a c i d s t a k e s p l a c e m ediated by a r e d u c t i o n i n the c o n c e n t r a t i o n of an i n t r a c e l l u l a r i o n , p r o b a b l y C l " . Under hyposmotic s t r e s s the e f f l u x of amino a c i d s may be more s i g n i f i c a n t than t h e i r breakdown. In 7 hyperosmotic media the r i s i n g i n t r a c e l l u l a r C l " concentration reduces., amino acid- catabo.li.smv resulting,,., in a., build up, of. i n t r a c e l l u l a r solutes. There i s some influx of amino acids in hyperosmotic media, but i t is r e l a t i v e l y minor ( G i l l e s , 1979). Most crustaceans tend to regulate body volume more e f f e c t i v e l y under hyposmotic than hyperosmotic conditions (Prosser, 1973). G i l l e s (1979) stated that t h i s may be because under hyposmotic conditions two processes act to remove amino acids from the c e l l , while only one process (anabolism of amino acids) takes place during hyperosmotic stress. Therefore, in addition to acting as an important osmotic e f f e c t e r , the presence of C l " i n t r a c e l l u l a r l y may be important to the process of c e l l volume regulation (Rorive and G i l l e s , 1979). Zurburg and DeZwaan (1981) mentioned that amino acid regulation in molluscs may be similar to that described by G i l l e s (1979) for crustaceans. They found that in Modiolus, anaerobic mechanisms were involved in amino acid synthesis under hyperosmotic stress. They postulated that under hyposmotic conditions most amino acids extruded from c e l l s would be moved elsewhere in the organism for deamination. Pierce and Amende (1981) discovered physical evidence of membrane conformational changes under hyposmotic stress. Working on blood c e l l s from the clam, Noet ia ponderosa, they found a decrease in surface membrane p a r t i c l e d i s t r i b u t i o n upon d i l u t i o n . Under hyperosmotic stress the c e l l membrane p a r t i c l e density increased. This may have indicated a stretching or shrinking of the c e l l membrane, or a removal of p a r t i c l e s from one area of the membrane to 8 another (Pierce and Amende, 1981). Pierce and Amende (1981) did not report, an. excretion. o& ammonia, or any. other.:. indie ation> ofv amino acid catabolism. However, a loss of amino acids and an increase in ammonia excretion under hyposmotic conditions, has been reported for Scrobicularia plana and Mya arenaria (Shumway et a l , 1977). It i s not unreasonable to assume that c e l l permeability to C l " may be important to c e l l volume regulation in molluscs as i t is in crustaceans. However, Pierce and Greenburg (1971) reported that s e l e c t i v e l y deleting C l " from the ambient sea water did not result in amino acid efflux from isolated Modiolus hearts. There is evidence that marine forms are less capable of tissue water regulation than estuarine forms and, of the estuarine animals, species found in the high i n t e r t i d a l are better regulators. (Lange, 1972; Oglesby, 1981). Water permeability of Nereis d i v e r s i c o l o r dropped 50% when thi s polychaete was transferred from sea water to fresh water (Smith, 1970b). Berger et a l (1978) reported that L i t t o r i n a l i t t o r e a and L. s a x a t i l i s found in 16-32% SW were able to regulate tissue water p a r t i a l l y , while L. obtusata (not found in s a l i n i t i e s below 42% SW) was unable to do so. However, Moran and T u l l i s (1980) discovered that the marine chiton, Mopalia muscosa, was capable of foot muscle water content regulation in s a l i n i t i e s ranging from 60-120% SW. Water permeability also decreases in estuarine forms, from the low to the high i n t e r t i d a l zones. Mayes (1962) reported that snails of the genus L i t t o r i n a , showed an increased tendency to 9 g a i n w e i g h t i n h y p o t o n i c SW, i n t h e f o l l o w i n g o r d e r : L. n e r e t o i d e s , L. s a x a t i l i s , L. l i t t o r e a , L . l i t t o r a l i s . In a d d i t i o n , t h e t i d a l h e i g h t l e v e l f o r t h e s e a n i m a l s i n c r e a s e d i n t h e r e v e r s e o r d e r . L i t t o r i n a n e r e t o i d e s , i n h a b i t i n g t h e s p l a s h z o n e , was t h e b e s t e q u i p p e d f o r d e a l i n g w i t h s a l i n i t y e x t r e m e s . P r u s c h and H a l l (1978) d e s i g n e d an e x p e r i m e n t w h i c h showed t h a t w a t e r p e r m e a b i l i t y was r e l a t e d t o i n t e r t i d a l h e i g h t . They m e a s u r e d t h e d i f f u s i o n a l water p e r m e a b i l i t y a c r o s s t h e i s o l a t e d m a n t l e membrane o f a v a r i e t y of b i v a l v e s , u s i n g t r i t i a t e d w a t e r . Water p e r m e a b i l i t y d e c r e a s e d f r o m P l a c o p e c t e n m a g e l l e n i c u s ( s u b t i d a l ) t o M o d i o l u s d e m i s s u s ( h i g h i n t e r t i d a l ) . W i t h i n one s p e c i e s , M y t i l u s e d u l i s , w a ter p e r m e a b i l i t y d e c r e a s e d f r o m low t o h i g h p o p u l a t i o n s . P r u s c h and H a l l (1978) a l s o p r o v i d e d e v i d e n c e t h a t t h e d e g r e e of w a t e r p e r m e a b i l i t y was under p h y s i o l o g i c a l c o n t r o l . T r a n s p l a n t e d M. e d u l i s q u i c k l y a d o p t e d t h e w a t e r p e r m e a b i l i t y c h a r a c t e r i s t i c of o t h e r members of t h e same s p e c i e s a t a s i m i l a r i n t e r t i d a l h e i g h t ( P r u s c h and H a l l , 1 978). The a b i l i t y t o a l t e r w a t e r p e r m e a b i l i t y may e n a b l e e s t u a r i n e o r g a n i s m s t o r e g u l a t e t i s s u e w a t e r c o n t e n t more e f f i c i e n t l y t h a n w h o l l y m a r i n e f o r m s . I n f o r m a t i o n a v a i l a b l e on l i m p e t s i n d i c a t e s t h a t s a l i n i t y f l u c t u a t i o n e f f e c t s t i s s u e w a t e r c o n t e n t i n a manner s i m i l a r t o t h a t o f o t h e r g a s t r o p o d s . K o z l i t i n a (1972) r e p o r t e d an i n c r e a s e d t o l e r a n c e t o w a r d a d e c r e a s i n g s a l i n i t y , from t h e s u b l i t t o r a l Acmaea p a l l i d a , t o t h e h i g h i n t e r t i d a l C o l l i s e l l a r a d i a t a and C. d o r s u o s a . W i t h i n a s i n g l e s p e c i e s . P a t e l l a v u l g a t a , D a v i e s (1969) f o u n d t h a t under d e s i c c a t i n g c o n d i t i o n s t h e median l e t h a l 10 water l o s s was 50-55% f o r low l e v e l , and 60-65% f o r h i g h l e v e l limpets... He a l s o . r,e.cor.ded a, ;median lethal-wa«te?r* lo.ss™ of 3-0-35%>.», f o r P. a s p e r a l o c a t e d l o w e r i n t h e i n t e r t i d a l zone t h a n low l e v e l P. v u l g a t a . F i n a l l y , T a r r (1977) r e p o r t e d t h a t h i g h i n t e r t i d a l C. d i g i t a l i s n e i t h e r g a i n e d nor l o s t as much w e i g h t as l o w e r i n t e r t i d a l C. scutum, o v e r a r a n g e o f s a l i n i t i e s from 50-150% SW. In t h e p r e s e n t s t u d y , t h e w a t e r c o n t e n t of f o o t m u s c l e and b l o o d f r o m C. p e r s o n a was measured i n s a l i n i t i e s r a n g i n g from 25-125% SW. The p u r p o s e was t o d e t e r m i n e whether t h i s l i m p e t was c a p a b l e o f t h e d e g r e e o f b l o o d o r t i s s u e w a ter r e g u l a t i o n shown by o t h e r h i g h i n t e r t i d a l g a s t r o p o d s . When a b i v a l v e i s p l a c e d i n media more d i l u t e or c o n c e n t r a t e d t h a n f u l l s t r e n g t h s e a water ( 3 2 % 0 ) , i t r e a c t s w i t h immediate v a l v e c l o s u r e . T h i s has been o b s e r v e d i n M y t i l u s  e d u l i s (Hoyaux e t a l , 1976; Shumway, 1977a and b; D a v e n p o r t , 1979), S c r o b i c u l a r i a p l a n a (Freeman and R i g l e r , 1957; Hoyaux e t a l , 1976; Shumway, 1977a and b ) , G l y c y m e r i s g l y c y m e r i s (Hoyaux e t a l , 1976), C r a s s o s t r e a (Hand and S t i c k l e , 1977; Shumway, 1977a and b ) , M o d i o l u s m o d i o l u s , Chlamys o p e r c u l a r i s , and Mya  a r e n a r i a (Shumway, 1977a and b ) . G a s t r o p o d s d e m o n s t r a t e s i m i l a r r e s p o n s e s i n c l u d i n g , w i t h d r a w l i n t o t h e s h e l l i n L i t t o r i n a (Todd, 1964a; Avens and S l e i g h , 1965; B e r g e r e t a l , 1978), and H y d r o b i a u l v a e (Avens, 1965), and c l a m p i n g t o t h e s u b s t r a t e i n t h e l i m p e t s C o l l i s e l l a d o r s u o s a , C. r a d i a t a ( K o z l i t i n a , 1972; Y a r o s l a v t s e v a and K a r p e n k o , 1980), and P a t e l l a v u l g a t a (Hoyaux e t a l , 1976). By w i t h d r a w i n g i n t o t h e s h e l l , t h e s e m o l l u s c s a r e 1.1 a b l e t o t e m p o r a r i l y i s o l a t e t h e m s e l v e s from t h e a m bient medium, t h u s a v o i d i n g o s m o t i c s h o c k . T h e s e s h e l l c l o s u r e o r c l a m p i n g r e s p o n s e s c o m b i n e d w i t h wide s a l i n i t y t o l e r a n c e s , e n a b l e o s m o c o n f o r m i n g i n t e r t i d a l m o l l u s c s t o s u r v i v e i n a r e a s where s a l i n i t y f l u c t u a t e s w i d e l y ( K i n n e , 1971). 12 MATERIAL AND METHODS L i m p e t s , C o l l i s e l l a p e r s o n a , u s e d i n t h i s s t u d y were c o l l e c t e d f r o m t h e i n t e r t i d a l r e g i o n o f L i g h t h o u s e P a r k , West V a n c o u v e r , B r i t i s h C o l u m b i a . T h i s a r e a i s an e s t u a r i n e h a b i t a t w i t h sea water t e m p e r a t u r e s and s a l i n i t i e s r e c o r d e d d u r i n g t h i s s t u d y , r a n g i n g from 6°C and 29%© d u r i n g t h e w i n t e r months, t o ,21 °C and 11%o d u r i n g summer ( F i g . 1 ) . L i m p e t s w i t h s h e l l l e n g t h s between 2 cm. and 5 cm. were c o l l e c t e d f r o m an a r e a between 3.5 m and 4.5 m above t h e z e r o t i d e l e v e l . In t h e l a b o r a t o r y , a n i m a l s were k e p t i n 7 L p l a s t i c c o n t a i n e r s (15 l i m p e t s p e r c o n t a i n e r ) i n a t e m p e r a t u r e c o n t r o l l e d i n c u b a t o r . Summer a n i m a l s were h e l d i n 50% ( 1 6 % 0 ) s e a water a t 17°C; w i n t e r l i m p e t s i n 75% ( 2 3 % 0 ) sea water a t 5°C. A n i m a l s were k e p t i n d a r k n e s s and n o t f e d . C. p e r s o n a u s e d i n t h e s e e x p e r i m e n t s were m a i n t a i n e d under h o l d i n g c o n d i t i o n s f o r no l o n g e r t h a n 2 weeks. E x p e r i m e n t s were c o n d u c t e d a t 5°C d u r i n g t h e w i n t e r and 17°C d u r i n g summer. The e x p e r i m e n t a l s a l i n i t i e s u s e d were 25%, 50%, 75%, 100%, and 125% s e a w a t e r ( T a b l e 1 ) . T h e s e c o n c e n t r a t i o n s were b a s e d on a s t a n d a r d 100% sea water w i t h a s a l i n i t y of 31%© and a c h l o r i d e i o n c o n t e n t of 480 mEq/L. S a l i n i t i e s l o w e r t h a n 100% were o b t a i n e d by a d d i n g g l a s s d i s t i l l e d w a t e r t o a s t o c k s o l u t i o n o f 100% s e a w a t e r . One h u n d r e d and t w e n t y - f i v e p e r c e n t s e a w a t e r was made by a d d i n g s e a s a l t , o b t a i n e d by a i r e v a p o r a t i o n a t 20°C from a r e c i r c u l a t i n g s e a w a t e r s y s t e m , t o a 100% s t o c k s o l u t i o n . Samples were t a k e n from a n i m a l s e x p o s e d t o e x p e r i m e n t a l sea water c o n c e n t r a t i o n s 13 F i g u r e 1: T e m p e r a t u r e ( c l o s e d c i r c l e s ) , and s a l i n i t y (open c i r c l e s ) measurements from L i g h t h o u s e P a r k , B r i t i s h C o l u m b i a . T h e s e were t a k e n between J u n e , 1979, and O c t o b e r , 1981. 15 T a b l e 1. E x p e r i m e n t a l s e a w a t e r c o n c e n t r a t i o n s : e x p r e s s e d as a p e r c e n t a g e , c h l o r i d e c o n c e n t r a t i o n i n m i l l i e q u i v a l e n t s p e r l i t r e , and s a l i n i t y a s p a r t s p e r t h o u s a n d . TABLE 1 E x p e r i m e n t a l Sea Water C o n c e n t r a t i o n s P e r c e n t C h l o r i d e S a l i n i t y Sea Water C o n c e n t r a t i o n 25 120 8 50 240 16 75 360 23 100 480 31 125 600 39 1 7 f o r 3, 6, 12, 24, 48, 72, and 144 h o u r s , and 1 week. B l o o d and u r i n e s a m p l e s f o r o s m o t i c p r e s s u r e and b l o o d w ater d e t e r m i n a t i o n , were t a k e n from a n i m a l s e x p o s e d t o e a c h s a l i n i t y f o r o n l y 1 week. C o l l e c t i o n and Measurement of Samples B l o o d Samples B l o o d s a m p l e s were t a k e n f r o m t h e p e d a l s i n u s t h r o u g h an i n c i s i o n i n t h e f o o t m u s c l e . B l o o d was drawn i n t o 25 nL c a p i l l a r y t u b e s , s e a l e d w i t h " s e a l - e a s e " c l a y and f r o z e n ; t h e s e s a m p l e s r e m a i n e d v i a b l e f o r up t o s i x months. S t a t i s t i c a l a n a l y s i s showed no s i g n i f i c a n t d i f f e r e n c e between t h e c h l o r i d e c o n c e n t r a t i o n s o f f r o z e n and f r e s h l y drawn b l o o d s a m p l e s ( t = 0.5; P = 0.05; d . f . = 9 ) . F o r c h l o r i d e i o n d e t e r m i n a t i o n s , 5 nL o f b l o o d were p l a c e d i n 4 mL of 0.1N HN0 3 and 10% a c e t i c a c i d , and r u n on t h e h i g h r a t e o f a B u c h l e r - C o t l o v e C h l o r i d o m e t e r . B l o o d w a t e r c o n t e n t was d e t e r m i n e d by p l a c i n g a p p r o x i m a t e l y 10 iiL o f b l o o d i n p r e w e i g h e d aluminum d i s h e s , and w e i g h i n g t o t h e n e a r e s t 0.01 mg. on a M e t t l e r b a l a n c e . Samples were p l a c e d i n an oven a t 100°C, d r i e d f o r 48 h o u r s and r e w e i g h e d . F o r b l o o d o s m o t i c p r e s s u r e d e t e r m i n a t i o n , s e p a r a t e b l o o d samples o f 10 n a n o l i t r e s e a c h were p l a c e d under i m m e r s i o n o i l i n a p l a t i n u m sample p l a t e , and run on a C l i f t o n Osmometer. 18 U r i n e Samples U r i n e samples were o b t a i n e d by t h e f o l l o w i n g p r o c e d u r e : a n i m a l s were removed from e x p e r i m e n t a l s ea water s o l u t i o n s and damp d r i e d w i t h l e n s t i s s u e p a p e r . T a c t i l e s t i m u l a t i o n c a u s e d t h e m a n t l e t o r e t r a c t and water t h a t d i d not f l o w f r o m t h e m a n t l e and n u c h a l c a v i t i e s was removed by a s p i r a t i o n . U r i n e f l o w was i n i t i a t e d by p r e s s i n g g e n t l y on t h e f o o t . I t was o b s e r v e d t h a t most i f n o t a l l o f t h e u r i n e f l o w e d f r o m t h e r i g h t n e p h r i d i o p o r e . T h i s u r i n e was removed from t h e m a n t l e c a v i t y by a s p i r a t i o n and d i s c a r d e d . The f o o t was g e n t l y d e p r e s s e d a g a i n and u r i n e was c o l l e c t e d i n a 25 *«L g l a s s c a p i l l a r y t u b e and . f r o z e n . C h l o r i d e i o n c o n t e n t of f r o z e n u r i n e samples was not s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f f r e s h u r i n e ( t = 1.4; P = 0.05; d . f . = 9 ) . T h i s method i n s u r e d a p u r e sample, as any r e s i d u a l s e a water was p r e v i o u s l y washed out of t h e m a n t l e c a v i t y w i t h u r i n e . The methods u s e d t o d e t e r m i n e u r i n e C l " and o s m o t i c p r e s s u r e were t h e same as t h a t f o r b l o o d . T i s s u e Samples S m a l l s a m p l e s of f o o t m u s c l e t i s s u e (10-20 mg) were removed from t h e l i m p e t s , w i t h d i s s e c t i n g s c i s s o r s , and damp d r i e d on l e n s t i s s u e p a p e r . The method of e x t r a c t i n g C l " i o n s from m u s c l e t i s s u e was s i m i l a r t o t h a t e mployed by C o t l o v e ( 1 9 6 3 ) . T i s s u e s a m p l e s were p l a c e d i n 4 mL o f d i s t i l l e d w a t e r and i o n s were e x t r a c t e d f o r 48 h r s . C h l o r i d e i o n c o n t e n t was d e t e r m i n e d by a d d i n g 0.5 mL o f 1.5 N HN0 3 and 50% a c e t i c a c i d t o d u p l i c a t e 2 19 mL a l i q u o t s o f t h e e x t r a c t a n t , and r u n n i n g t h e samples on t h e : low r a t e o f a Buchler-Cofclov.es:•Ghl-orvi;d'ome»fee.r.. Foe. t i s s u e ; water., c o n t e n t d e t e r m i n a t i o n , a p p r o x i m a t e l y 20 mg of m u s c l e t i s s u e was p l a c e d on p r e w e i g h e d aluminum d i s h e s and w e i g h e d t o t h e n e a r e s t 0.1 mg. Samples were d r i e d and r e w e i g h e d f o l l o w i n g t h e p r o c e e d u r e u s e d f o r b l o o d w a t e r s a m p l e s . E x t r a c e l l u l a r Volume C a l c u l a t e d e x t r a c e l l u l a r volume v a l u e s were o b t a i n e d f o l l o w i n g t h e p r o c e d u r e u s e d by Manery ( 1 9 5 4 ) . C a l c u l a t i o n s were b a s e d on b l o o d and m u s c l e c h l o r i d e s , and b l o o d w a t e r c o n t e n t s , of summer and w i n t e r a n i m a l s h e l d f o r 1 week a t e a c h of t h e e x p e r i m e n t a l s a l i n i t i e s . The f o l l o w i n g e q u a t i o n s were b a s e d on two m a j o r a s s u m p t i o n s : ( 1 ) , t h a t a l l f u n c t i o n a l c h l o r i d e i s e x t r a c e l l u l a r ( M a n e r y , 1 9 5 4 ) ; and ( 2 ) , t h e a p p a r e n t l a c k o f a n a t o m i c a l or p h y s i o l o g i c a l i s o l a t i o n between t h e b l o o d and e x t r a c e l l u l a r s p a c e s o f i n v e r t e b r a t e s ( D e h n e l , 1 9 7 9 ) . The e q u a t i o n s a r e as f o l l o w s : [ A ] E = [ A ] s / r A 1. [ A ] E = ( A ) s / r A x 1 0 0 0 / ( H 2 O ) S The volume of e x t r a c e l l u l a r water i n a k i l o g r a m of t i s s u e u s i n g c h l o r i d e f o r t h e c a l c u l a t i o n i s : 20 (H 20)|' = ( C 1 ) T / [ C 1 ] E x 1000 S u b s t i t u t i n g f o r [ C 1 ] E from e q u a t i o n 1 i n . w h i c h A i s r e p l a c e d by C l : 2. (H 20)|' = ( C l ) T / ( C l ) s x ( H 2 0 ) s x r C l Where A = an a n i o n ; r = G i b b s - D o n n a n d i s t r i b u t i o n r a t i o a s s u m i n g t h e f l u i d d e r i v e d f r o m t h e p l a s m a c o n t a i n s 1% p r o t e i n . F o r i n v e r t e b r a t e s , r i s t a k e n t o e q u a l 0.977; [ C 1 ] E = m i l l i e q u i v a l e n t s o f c h l o r i d e p e r k i l o g r a m of e x t r a c e l l u l a r w a t e r ; (H 2Q.) E' = grams o f e x t r a c e l l u l a r water p e r k i l o g r a m o f t i s s u e ; ( C 1 ) T = m i l l i e q u i v a l e n t s of c h l o r i d e p e r k i l o g r a m o f t i s s u e ; ( C l ) s = m i l l i e q u i v a l e n t s o f c h l o r i d e p e r k i l o g r a m o f b l o o d ; ( H 2 0 ) s = grams o f w a t e r p e r k i l o g r a m o f b l o o d . 21 S t a t i s t i c a l A n a l y s i s A t h r e e - w a y a n a l y s i s of v a r i a n c e (Anova) was p e r f o r m e d on summer and w i n t e r b l o o d and u r i n e c h l o r i d e v a l u e s a t a l l s a l i n i t i e s t e s t e d . B l o o d and u r i n e o s m o t i c p r e s s u r e d a t a were s u b j e c t e d t o a s i m i l a r a n a l y s i s f o r o n l y 25% and 125% s e a w a t e r . S e a s o n a l t i s s u e c h l o r i d e , t i s s u e w a t e r , and b l o o d water measurements, were a n a l y s e d u s i n g a two-way a n a l y s i s of v a r i a n c e ( A n o v a ) . A Duncan's m u l t i p l e r a n g e t e s t (DMRT) was a l s o c o n d u c t e d on a l l t h e above d a t a . B l o o d , u r i n e , and m u s c l e C l " v a l u e s were t e s t e d a g a i n s t e x p e r i m e n t a l s a l i n i t y C l " c o n c e n t r a t i o n s , u s i n g t w o - t a i l e d S t u d e n t ' s t - t e s t s ( t - t e s t ) . An a n a l y s i s o f c o v a r i a n c e (Ancov) was p e r f o r m e d on summer and w i n t e r e x t r a c e l l u l a r volume d a t a , and on t h e r e g r e s s i o n s o b t a i n e d from wet and d r y m u s c l e w e i g h t v a l u e s . A n a l y s e s were c o n d u c t e d on p o o l e d d a t a a s w e l l a s d a t a o b t a i n e d from a n i m a l s k e p t i n e x p e r i m e n t a l s a l i n i t i e s f o r 1 week o n l y . T w o - t a i l e d S t u d e n t ' s t - t e s t s were p e r f o r m e d on f r o z e n and u n f r o z e n b l o o d and u r i n e s a m p l e s . The p r o b a b i l i t y l e v e l f o r a l l t e s t i n g was P = 0.05. 22 RESULTS I_. C h l o r i d e Ion Response o f B l o o d , U r i n e , and M u s c l e T i s s u e To d e t e r m i n e t h e e f f e c t o f s a l i n i t y change on b l o o d and u r i n e C l " c o n c e n t r a t i o n , l i m p e t s were c o l l e c t e d f r o m L i g h t h o u s e P a r k and p l a c e d i n 25%, 50%, 75%, 100%, and 125% s e a w a t e r , f o r t i m e p e r i o d s o f 3, 6, 12, 24, 48, 72, 144, and 168 h o u r s (1 week). E x p e r i m e n t s were p e r f o r m e d on summer and w i n t e r a n i m a l s . Samples were c o l l e c t e d f r o m 10 a n i m a l s a t e a c h t i m e p e r i o d and s a l i n i t y . R e s p o n s e o f B l o o d and U r i n e C l ~ The e f f e c t o f t i m e and s a l i n i t y on t h e b l o o d and u r i n e C l " c o n c e n t r a t i o n o f summer l i m p e t s i s shown i n F i g u r e 2 and T a b l e 2. T a b l e 2 a l s o shows s t a t i s t i c a l c o m p a r i s o n s between i n d i v i d u a l b l o o d and u r i n e C l " means, and t h e r e s p e c t i v e e x p e r i m e n t a l s e a w a t e r c o n c e n t r a t i o n s . In g e n e r a l , b l o o d and u r i n e C l " l e v e l s i n c r e a s e d i n 75%, 100%, and 125% s e a w a t e r , d e c r e a s e d i n 25% SW, and r e m a i n e d unchanged ( t - t e s t ) i n 50% SW ( F i g . 2 ) . Maximum l e v e l s f o r b l o o d and u r i n e C l " were r e a c h e d a f t e r 12 h o u r s i n 75%, 100%, and 125% SW, w i t h C l " v a l u e s 10-20% h i g h e r t h a n c o r r e s p o n d i n g s e a w ater v a l u e s . T h e s e d i f f e r e n c e s were s i g n i f i c a n t ( T a b l e 2 ) . Between 12 and 24 h o u r s , b l o o d and u r i n e C l " d e c r e a s e d t o r e l a t i v e l y c o n s t a n t l e v e l s t h a t d i f f e r e d no more t h a n 6% f r o m e q u i v a l e n t s e a w ater v a l u e s ( t - t e s t ) , f o r t h e r e m a i n d e r o f t h e week ( T a b l e 2 ) . 23 F i g u r e 2: R e s p o n s e o f b l o o d ( c l o s e d c i r c l e s ) , and u r i n e (open c i r c l e s ) C l " c o n c e n t r a t i o n s f r o m summer a d a p t e d C o l l i s e l l a p e r s o n a t o c h a n g e s i n e x t e r n a l s a l i n i t y . Eaph p o i n t r e p r e s e n t s t h e mean o f measurements on 10 a n i m a l s . 01——J 1 i I I l I l I I I I 10 20 30 40 50 60 70 80 150 160 170 TIME (hours) 25 In 25% SW, c o n s t a n t l e v e l s f o r b l o o d and u r i n e C l " ( t - t e s t ) were r e a c h e d between 72 and 144 h o u r s , w i t h b l o o d C l " 24% below sea water C l " , and u r i n e 8% above s e a water ( F i g . 2 ) . The e f f e c t o f t i m e and s a l i n i t y on t h e b l o o d and u r i n e C l " c o n c e n t r a t i o n o f w i n t e r a n i m a l s i s shown i n F i g u r e 3 and T a b l e 2. As w i t h summer C l " , w i n t e r b l o o d and u r i n e C l " l e v e l s i n c r e a s e d i n h i g h e r s a l i n i t i e s (100% and 125% SW), and d e c r e a s e d i n l o w e r s a l i n i t i e s (25% and 50% S W ) ( F i g . 3 ) . However, u n l i k e summer C l " l e v e l s , w i n t e r v a l u e s r e a c h e d e q u i l i b r i u m between 6 and 12 h o u r s i n 50%, 75%, 100%, and 125% SW, and between 24 and 48 h o u r s i n 25% SW ( t - t e s t ) ( F i g . 3 ) . E q u i l i b r a t e d v a l u e s v a r i e d no more t h a n 6% from r e s p e c t i v e s e a water v a l u e s i n 75%, -100%, and 125% SW ( T a b l e 2 ) . In 50% SW, b l o o d and u r i n e C l " r e m a i n e d 10-14% below SW C l " , f r o m 12 h o u r s t o 1 week ( F i g . 3, T a b l e 2 ) . T h e s e d i f f e r e n c e s were s i g n i f i c a n t ( t - t e s t ) . B l o o d and u r i n e C l " c o n c e n t r a t i o n s d e c r e a s e d s l o w l y but c o n t i n u o u s l y f r o m 12 h o u r s t o 1 week i n 25% SW ( F i g . 3 ) . B l o o d C l " was s l i g h t l y h y p o t o n i c (4-13%) t o s e a w ater C l " between 12 and 48 h o u r s , but become s i g n i f i c a n t l y h y p o t o n i c (34%) a f t e r 1 week ( F i g . 3 , T a b l e 2 ) . U r i n e C l " was 4-13% h i g h e r t h a n s e a w a t e r C l " between 12 and 48 h o u r s , but a l s o became s i g n i f i c a n t l y h y p o t o n i c (16%) a f t e r 1 week ( F i g . 3, T a b l e 2 ) . B l o o d C l " v a l u e s were g e n e r a l l y l o w e r t h a n e q u i v a l e n t u r i n e v a l u e s a t a l l s a l i n i t i e s t e s t e d , o v e r t h e e n t i r e e x p e r i m e n t a l p e r i o d ( F i g s . 2, and 3 ) . The e f f e c t o f s a l i n i t y on t h e b l o o d and u r i n e C l " 26 F i g u r e 3: R e s p o n s e o f b l o o d ( c l o s e d c i r c l e s ) , and u r i n e (open c i r c l e s ) C l " c o n c e n t r a t i o n s from w i n t e r a d a p t e d C o l l i s e l l a p e r s o n a t o c h a n g e s i n e x t e r n a l s a l i n i t y . E a c h p o i n t r e p r e s e n t s t h e mean of d e t e r m i n a t i o n s on 10 a n i m a l s . BLOOD AND URINE CHLORIDE (mequiv/L) — rv> OJ -P> cn CD ->j o o o o o o o o o o o o o o o 28 T a b l e 2. E f f e c t o f s e a s o n , s a l i n i t y , and t i m e on b l o o d and u r i n e c h l o r i d e c o n c e n t r a t i o n s (mEq/L). B l o o d and u r i n e C l " were compared w i t h s e a w a t e r C l " , u s i n g t h e S t u d e n t ' s t - t e s t . * i n d i c a t e s s i g n i f i c a n c e a t t h e 0.05 l e v e l . Sea water c o n c e n t r a t i o n s i n mEq/L C l " a r e g i v e n i n p a r e n t h e s e s . E a c h v a l u e r e p r e s e n t s t h e mean o f d e t e r m i n a t i o n s on 10 a n i m a l s . 29 TABLE 2 Time i n Hou r s S a l i n i t y ' 3 6 12 24 48 1 week 25%(120) b l o o d 154. 0* 146. 0* 1 66. 0* 147. 0* 129. 0* 91 . 3 * summer u r i n e 201 . 7 * 150. 0* 127. 0 1 67. 0* 1 52. 0* 130 .0 b l o o d 234. 3* 198. 8* 116. 0 116. 0 105. 0 78 . 9 * w i n t e r u r i n e 267. 8* 187. 0* 1 32. 0 1 35. 0 1 22. 0 101 .4 * 50%(240) b l o o d 237. 0 240. 4 239. 0 237. 3 238. 9 236 .4 summer "ur i n e 238. 7 242. 5 232. 2* 237. 5 245. 0 244 . 5 * b l o o d 245. 0 240. 0 216. 0* 212. 5* 211. 0* 206 . 7 * w i n t e r u r i n e 267. 5* 221 . 3* •205. 7 * 207. 5* 212. 0* 216 . 3 * 75%(360) b l o o d 320. 0* 378. g* 435. 0* 348. 0* 358. 0 351 . 2 * summer ur i n e 436. 0* 441 . 3* 444. 5* 381 . 0* 374. 4* 368 .8 b l o o d 354. 3 376. 3* 373. 3* 380. 0* 371 . 4* 361 . 1 w i n t e r u r i n e 392. 5* 383. 3* 383. 5* 377. 5* 381 . 1* 373 . 8 * 100%(480) b l o o d 482. 2 475. 6 532. 5* 468. 8 469. 0 468 .0 summer u r i n e 495. 0 545. 7 * 575. 0* 494. 0* 480. 0 480 .0 b l o o d 481 . 3 502. 0 481 . 3 484. 0 494. 4 468 . 8 * w i n t e r u r i n e 505. 0* 503. 8 508. 0* 486. 3 490. 0 488 .0 125%(600) b l o o d 581 . 1* 588. 6 720. 0* 585. 0* 602. 2 575 . 0 * summer u r i n e 615. 0* 695. 0 665. 7 * 630. 0* 605. 0 61 1 .3 b l o o d 558. 9 * 596. o • 594. 0 593 .8 w i n t e r u r i n e 576. 0* 587. 5* 595. 0 607 . 8 * 30 c o n c e n t r a t i o n o f summer and w i n t e r l i m p e t s sampled a f t e r 1 week, i s shown i n F i g u r e s 4 and 5, r e s p e c t i v e l y . B l o o d C l " c o n c e n t r a t i o n s of a n i m a l s i n 25%, 50%, 75%, 100%, and 125% SW were s i g n i f i c a n t l y d i f f e r e n t from one a n o t h e r ; t h e same was t r u e f o r u r i n e ( F i g s . 4, and 5 ) ( A n o v a , DMRT). The l i n e s f o r summer b l o o d and u r i n e C l " i n s a l i n i t i e s r a n g i n g from 25% t o 125% SW had p o s i t i v e s l o p e s and were p a r a l l e l t o t h e i s o i o n i c l i n e f o r s e a w a t e r C l " ( F i g . 4 ) . B l o o d v a l u e s were l o w e r t h a n e q u i v a l e n t sea water v a l u e s by 10-30 mEq/L C l " , b u t were o n l y s i g n i f i c a n t l y l o w e r . i n 25%, 75%, and 125% SW ( t - t e s t ) ( F i g . 4 ) . U r i n e v a l u e s were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m c o r r e s p o n d i n g s e a w a t e r v a l u e s ( t - t e s t ) , and d i f f e r e d no more t h a n 10 mEq/L C l " from sea water ( F i g . 4 ) . B l o o d and u r i n e C l " v a l u e s i n 50%, 75%, 100%, and 125% SW, were p o o l e d ( t o r e d u c e sample v a r i a b i l i t y ) f r o m 24 h o u r s t o 1 week (144 h o u r s t o 1 week f o r 25% SW) i n summer, and from 12 h o u r s t o 1 week (48 h o u r s t o 1 week f o r 25% SW) i n w i n t e r ( T a b l e 3 ) . P o o l e d summer b l o o d C l " v a l u e s were s i g n i f i c a n t l y l o w e r t h a n e q u i v a l e n t s e a water v a l u e s by 5-25 mEq/L ( t - t e s t ) ( T a b l e 3 ) . U r i n e v a l u e s were up t o 22 mEq/L C l " h i g h e r t h a n e q u i v a l e n t s e a wa t e r v a l u e s , b u t were o n l y s i g n i f i c a n t l y h i g h e r i n 25%, 75%, and 125% SW ( t - t e s t ) ( T a b l e 3 ) . W i n t e r b l o o d and u r i n e C l " v a l u e s f o l l o w e d a t r e n d s i m i l a r t o summer v a l u e s ; t h e l i n e s f o r b l o o d and u r i n e C l " i n s a l i n i t i e s r a n g i n g from 25% t o 125% SW were p a r a l l e l t o t h e l i n e o f i s o t o n i c i t y f o r s e a w a t e r C l " ( F i g . 5 ) . B l o o d C l " was s i g n i f i c a n t l y h y p o t o n i c t o s e a w a t e r C l " by 35 and 45 mEq/L, i n u r e 4: B l o o d ( c l o s e d c i r c l e s ) , and u r i n e (open c i r c l e s ) C l " c o n c e n t r a t i o n s from summer a d a p t e d l i m p e t s a s a f u n c t i o n of s a l i n i t y . E a c h p o i n t r e p r e s e n t s t h e mean o f measurements on 10 a n i m a l s a f t e r e x p o s u r e t o e x p e r i m e n t a l s a l i n i t i e s f o r 1 week. The s o l i d l i n e r e p r e s e n t s t h e i s o i o n i c l i n e f o r s e a w a t e r . 32 33 F i g u r e 5: B l o o d ( c l o s e d c i r c l e s ) , and u r i n e (open c i r c l e s ) C l " c o n c e n t r a t i o n s from w i n t e r a d a p t e d l i m p e t s a s a f u n c t i o n of s a l i n i t y . E a c h p o i n t r e p r e s e n t s t h e mean o f measurements on 10 a n i m a l s a f t e r e x p o s u r e t o e x p e r i m e n t a l s a l i n i t i e s f o r 1 week. The s o l i d l i n e r e p r e s e n t s t h e i s o i o n i c l i n e f o r s e a w a t e r . 34 700 M E D I U M C O N C E N T R A T I O N ( % S E A W A T E R ) 35 T a b l e 3. P o o l e d b l o o d and u r i n e C l " v a l u e s f r o m 25%, 50%, 75%, 100%, and 125% SW. U/B r a t i o s were t e s t e d ^ f o r s i g n i f i c a n c e u s i n g t h e S t u d e n t ' s t - t e s t . * i n d i c a t e s s i g n i f i c a n c e a t t h e 0.05 l e v e l . U/B r a t i o s shown i n s q u a r e b r a c k e t s , were c a l c u l a t e d u s i n g b l o o d and u r i n e v a l u e s o b t a i n e d f r o m l i m p e t s k e p t i n e x p e r i m e n t a l s a l i n i t i e s f o r 1 week (N=10). T h e s e r a t i o s were t e s t e d f o r s i g n i f i c a n c e u s i n g t h e Duncan's M u l t i p l e Range T e s t . 36 TABLE 3 Sea Water C o n c e n t r a t i o n (mEq/L C h l o r i d e ) 120 240 360 480 600 (25%) (50%) (75%) (100%) (125%) Summer b l o o d 94.6 235.8 353.3 465.3 582.1 u r i n e 141.5 241.3 371.0 480.9 613.1 N 20 50 50 50 50 W i n t e r b l o o d 100.2 213.6 370.8 482.0 590.5 u r i n e 111.7 214.1 377.5 488.1 601.3 N 20 40 40 40 30 U r i n e / B l o o d summer 1.5* 1.02* 1.05* 1.03* 1.05* R a t i o [1.42*] [1.03] [1.05] [1.03] [1.06*] w i n t e r 1.11* 1.0 1.02* 1.01 1.02* [1.29*] [1.05] [1.04] [1.04] [1.02] 37 50% and 25% SW, r e s p e c t i v e l y ( t - t e s t ) ( F i g . 5 ) . U r i n e C l " was 25 mEq/L l o w e r t h a n s e a water C l " i n 50% SW, and 20 mEq/L l o w e r i n 25% SW ( F i g . 5 ) . Th e s e v a l u e s were s i g n i f i c a n t ( t - t e s t ) . A t h i g h e r s a l i n i t i e s ( 7 5 % , 100%, and 125% SW) b l o o d v a l u e s were o n l y up t o 10 mEq/L C l " l o w e r t h a n e q u i v a l e n t s e a water v a l u e s ; u r i n e v a l u e s were from 5-15 mEq/L C l " h i g h e r t h a n s e a water v a l u e s ( F i g . 5 ) . Th e s e d i f f e r e n c e s were n o t s i g n i f i c a n t ( t -t e s t ) . P o o l e d w i n t e r b l o o d v a l u e s i n 25%, 50%, and 125% SW, were s i g n i f i c a n t l y l o w e r t h a n s e a w a t e r v a l u e s by 10-27 mEq/L C l " ( t -t e s t ) ( T a b l e 3 ) . In 75% SW, b l o o d C l " was s i g n i f i c a n t l y g r e a t e r t h a n s e a water C l " by 11 mEq/L ( t - t e s t ) ; b l o o d C l " i n 100% SW was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m s e a . w a t e r C l " ( t - t e s t ) . W i n t e r u r i n e C l " i n 25% and 50% SW was s i g n i f i c a n t l y l o w e r t h a n e q u i v a l e n t s e a water C l " by 8-26 mEq/L ( t - t e s t ) ( T a b l e 3 ) . I n 75% SW, u r i n e C l " was s i g n i f i c a n t l y h i g h e r t h a n s e a water C l " by 18 mEq/L ( t - t e s t ) ; u r i n e C l " i n 100% and 125% SW was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m s e a water C l " ( t - t e s t ) . S e a s o n a l d i f f e r e n c e s between b l o o d and u r i n e C l " were o n l y a p p a r e n t a t l o w e r s a l i n i t i e s ( 2 5 % and 50% S W ) ( F i g s . 4, and 5 ) . T h e r e were no s i g n i f i c a n t s e a s o n a l d i f f e r e n c e s i n b l o o d C l " c o n c e n t r a t i o n i n 25% SW (DMRT). However, u r i n e v a l u e s were s i g n i f i c a n t l y h i g h e r i n summer t h a n i n w i n t e r by 30 mEq/L C l " (DMRT). I n 50% SW summer b l o o d and u r i n e C l " were s i g n i f i c a n t l y h i g h e r t h a n w i n t e r b l o o d and u r i n e v a l u e s by 25 mEq/L (DMRT). Summer and w i n t e r b l o o d C l " c o n c e n t r a t i o n s were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m one a n o t h e r i n 75%, 100%, and 125% 38 SW (Anova, DMRT); u r i n e v a l u e s showed t h e same t r e n d a s b l o o d (Anova, DMRT). C a l c u l a t e d u r i n e / b l o o d r a t i o s (U/B) f o r summer a n d . w i n t e r l i m p e t s h e l d i n s a l i n i t i e s r a n g i n g f r o m 25% t o 125% SW f o r 1 week, a r e shown i n T a b l e 3. U/B r a t i o s f o r summer a n i m a l s r a n g e d from 1.03 i n 50% and 100% SW, t o 1.42 i n 25% SW ( T a b l e 3 ) . U/B r a t i o s were o n l y s i g n i f i c a n t l y g r e a t e r t h a n one f o r 25% and 125% SW (DMRT). W i n t e r U/B r a t i o s r a n g e d from 1.02 i n 125% SW, t o 1.29 i n 25% SW ( T a b l e 3 ) . As w i t h summer r a t i o s , a l l w i n t e r r a t i o s a r e g r e a t e r t h a n one. However, o n l y t h e U/B r a t i o f o r 25% SW i s s i g n i f i c a n t l y g r e a t e r t h a n one (DMRT). U/B r a t i o s c a l c u l a t e d f r o m p o o l e d summer b l o o d and u r i n e C l " c o n t e n t s r a n g e d from 1.02 i n 50% SW, t o 1.5 i n 25% SW ( T a b l e 3 ) ; a l l r a t i o s were s i g n i f i c a n t l y g r e a t e r t h a n one ( t - t e s t ) . W i n t e r U/B r a t i o s r a n g e d from 1.0 i n 50% SW, t o 1.11 i n 25% SW ( T a b l e 3 ) . U/B r a t i o s were s i g n i f i c a n t l y g r e a t e r t h a n one i n 25%, 75%, and 125% SW ( t - t e s t ) . R e sponse of F o o t M u s c l e C l " The e f f e c t o f t i m e and s a l i n i t y on t h e f o o t m u s c l e C l " c o n c e n t r a t i o n o f summer and w i n t e r l i m p e t s i s shown i n F i g u r e 6. In g e n e r a l , a s w i t h b l o o d and u r i n e , t h e C l " c o n c e n t r a t i o n o f f o o t m u s c l e f o r b o t h summer and w i n t e r a n i m a l s i n c r e a s e d i n s a l i n i t i e s h i g h e r t h a n t h e r e s p e c t i v e c o n t r o l s a l i n i t i e s , and d e c r e a s e d i n l o w e r s a l i n i t i e s . Summer m u s c l e t i s s u e C l " c o n c e n t r a t i o n s showed g r e a t e r v a r i a t i o n t h a n w i n t e r v a l u e s e x c e p t i n 100% SW. S i m i l a r l y , summer b l o o d and u r i n e C l " showed 39 F i g u r e 6: Response o f f o o t m u s c l e t i s s u e C l " c o n c e n t r a t i o n of summer ( c l o s e d c i r c l e s ) , and w i n t e r (open c i r c l e s ) a d a p t e d l i m p e t s t o c h a n g e s i n e x t e r n a l s a l i n i t y . E a c h p o i n t r e p r e s e n t s t h e mean v a l u e o f measurements on 10 a n i m a l s . MUSCLE TISSUE CHLORIDE (mequiv/kg wet weight) 0*7 41 more v a r i a t i o n t h a n w i n t e r v a l u e s . The r e s p o n s e c u r v e of f o o t m u s c l e C l " f o r summer l i m p e t s a t e a c h e x p e r i m e n t a l s a l i n i t y o v e r t i m e l e v e l e d o f f a f t e r 12 h o u r s i n 25% and 50% SW, 6 h o u r s i n 75% and 100% SW, and a f t e r 24 h o u r s i n 125% SW ( F i g . 6 ) . A l t h o u g h v a r i a t i o n between p o i n t s r e m a i n e d h i g h a f t e r t h e c u r v e s had l e v e l e d o f f (as much as 70 mEq C l " / k g m u s c l e between 48 and 72 h o u r s i n 125% SW), o n l y 1 v a l u e was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e r e s t a t any i n d i v i d u a l s a l i n i t y ( t - t e s t ) . S i m i l a r l y , w i n t e r m u s c l e C l " l e v e l e d o f f a f t e r 144 h o u r s i n 25% and 50% SW, 3 h o u r s i n 100% SW, and 12 h o u r s i n 125% SW ( t - t e s t ) ; v a l u e s i n 75% SW r e m a i n e d r e l a t i v e l y l e v e l o v e r t h e e n t i r e week ( t - t e s t ) ( F i g . 6 ) . A f t e r 1 week i n e x p e r i m e n t a l s a l i n i t i e s , summer and w i n t e r f o o t m u s c l e C l " c o n c e n t r a t i o n s r e m a i n e d 41% t o 53% below e q u i v a l e n t sea water c o n c e n t r a t i o n s ( F i g . 7 ) . Th e s e d i f f e r e n c e s were s i g n i f i c a n t ( t - t e s t ) . S e a s o n a l d i f f e r e n c e s i n f o o t m u s c l e C l " a f t e r 1 week were n o t a p p a r e n t a t l o w e r s a l i n i t i e s . A l t h o u g h summer v a l u e s were 1-3 mEq C l " / k g t i s s u e h i g h e r t h a n w i n t e r v a l u e s i n 25% and 50% SW, t h i s d i f f e r e n c e was n o t s i g n i f i c a n t (DMRT) ( F i g . 7, T a b l e 4 ) . However, i n 75%, 100%, and 125% SW, w i n t e r v a l u e s were s i g n i f i c a n t y h i g h e r t h a n summer v a l u e s by 20-25 mEq C l " / k g t i s s u e (Anova, D M R T ) ( F i g . 7, T a b l e 4 ) . To d e t e r m i n e i f s e a s o n a l d i f f e r e n c e s were due t o a g r e a t e r v a r i a b i l i t y o f summer d a t a , v a r i a t i o n was r e d u c e d by p o o l i n g f o o t m u s c l e C l " v a l u e s f r o m 12 h o u r s t o 1 week i n 25% and 50% SW, 6 h o u r s t o 1 week i n 75% and 100% SW, and f r o m 24 h o u r s t o 1 42 F i g u r e 7: E f f e c t o f s a l i n i t y on t h e C l " c o n c e n t r a t i o n of b l o o d , and f o o t m u s c l e t i s s u e f r o m summer, and w i n t e r a d a p t e d l i m p e t s . E a c h p o i n t r e p r e s e n t s t h e mean o f measurements from 10 a n i m a l s a f t e r e x p o s u r e t o e x p e r i m e n t a l s a l i n i t i e s f o r 1. week. The s o l i d l i n e i s th e i s o i o n i c l i n e f o r s e a w a t e r . 43 2 5 5 0 7 5 1 0 0 1 2 5 M E D I U M C O N C E N T R A T I O N (% S E A W A T E R ) 44 T a b l e 4. P o o l e d summer and w i n t e r t i s s u e C l " c o n c e n t r a t i o n s f r o m 25%, 50%, 75%, 100%, and 125% SW. S e a s o n a l r a t i o s f o r f o o t m u s c l e C l " were t e s t e d f o r s i g n i f i c a n c e u s i n g t h e S t u d e n t ' s t - t e s t . * i n d i c a t e s s i g n i f i c a n c e a t t h e 0.05 l e v e l . S e a s o n a l r a t i o s i n s q u a r e b r a c k e t s were c a l c u l a t e d u s i n g summer and w i n t e r t i s s u e C l " v a l u e s o b t a i n e d f r o m l i m p e t s k e p t i n e x p e r i m e n t a l s a l i n i t e s f o r 1 week (N=10). T h e s e r a t i o s were t e s t e d f o r s i g n i f i c a n c e u s i n g t h e Duncan's M u l t i p l e Range T e s t . 45 TABLE 4 Sea Water C o n c e n t r a t i o n (mEq/L C h l o r i d e ) 1 20 (25%) 240 (50%) 360 (75%) 480 (100%) 600 (125%) Summer M u s c l e 60.5 1 26.8 198.2 255.9 355.6 N 60 60 70 70 50 W i n t e r M u s c l e 55.4 117.7 212.4 279.2 363.8 N 20 20 90 80 50 Summer/Winter M u s c l e R a t i o 1 .09 [ 1 .03] 1 .08 [1.03] 0.93* [0.89*] 0.92* [0.91*] 0.98 [0.94*] 46 week i n 125% SW ( T a b l e 4 ) . W i n t e r v a l u e s were p o o l e d from 144 h o u r s t o 1 week i n 25% and 50% SW, 3 h o u r s t o 1 week i n 75% and 100% SW, and from 12 h o u r s t o 1 week i n 125% SW ( T a b l e 4 ) . As-w i t h u n p o o l e d d a t a , p o o l e d v a l u e s were s i g n i f i c a n t l y h y p o i o n i c t o e q u i v a l e n t s e a water v a l u e s by 39% t o 54% ( T a b l e 4 ) . T h e r e were no s i g n i f i c a n t s e a s o n a l d i f f e r e n c e s i n f o o t m u s c l e C l " i n 25% and 50% SW, a l t h o u g h summer v a l u e s were 5-11 mEq C l " / k g t i s s u e h i g h e r t h a n w i n t e r v a l u e s ( t - t e s t ) ( T a b l e 4 ) . In 125% SW, summer m u s c l e was 8 mEq C l " / k g t i s s u e l o w e r t h a n w i n t e r m u s c l e ( T a b l e 4 ) . T h i s d i f f e r e n c e was not s i g n i f i c a n t ( t - t e s t ) . However, i n 75% and 100% SW, w i n t e r m u s c l e C l " was s i g n i f i c a n t l y h i g h e r t h a n summer m u s c l e C l " by 14-24 mEq C l " / k g t i s s u e ( T a b l e 4) . F o o t m u s c l e and b l o o d C l " were s i g n i f i c a n t l y h y p o i o n i c t o e q u i v a l e n t s e a water C l " a t a l l s a l i n i t i e s t e s t e d ( t - t e s t ) ( F i g . 7, T a b l e 5 ) . T h i s r e l a t i o n s h i p was c o n s i s t a n t f o r b o t h summer and w i n t e r a d a p t e d l i m p e t s . In summer a d a p t e d l i m p e t s b l o o d C l " was g r e a t e r t h a n f o o t m u s c l e C l " by 35% i n 25% SW, 47% i n 50% SW, 46% i n 75% SW and 100% SW, and by 40% i n 125% SW ( F i g . 7, T a b l e 5 ) . Th e s e d i f f e r e n c e s were s i g n i f i c a n t ( t - t e s t ) . W i n t e r a d a p t e d l i m p e t s had b l o o d C l " c o n c e n t r a t i o n s s i g n i f i c a n t l y h i g h e r * t h a n m u s c l e v a l u e s by 26% i n 25% SW, 42% i n 50% and 75% SW, a n d by 38% i n 100% SW a n d 125% SW ( t - t e s t ) ( F i g . 7, T a b l e 5 ) . In a d d i t i o n , t h e d i f f e r e n c e between b l o o d and m u s c l e C l " was 3-9% g r e a t e r i n summer t h a n i n w i n t e r a t a l l s a l i n i t i e s t e s t e d ( T a b l e 5 ) . The s l o p e s o f t h e l i n e s f o r b l o o d were g r e a t e r t h a n t h e s l o p e s f o r m u s c l e , s u g g e s t i n g a more e f f i c i e n t r e g u l a t i o n o f 47 T a b l e 5. C h l o r i d e c o n c e n t r a t i o n o f b l o o d , e x t r a c e l l u l a r f l u i d , and f o o t m u s c l e (mEq/kg), and b l o o d w a t e r c o n t e n t (g/kg of b l o o d ) , from a n i m a l s h e l d f o r 1 week i n e x p e r i m e n t a l s a l i n i t i e s (N=10). E x t r a c e l l u l a r f l u i d v a l u e s were c a l c u l a t e d by t h e method u s e d by Manery (1954) . 48 TABLE 5 Sea Water B l o o d E x t r a c e l l u l a r M u s c l e B l o o d C h l o r i d e F l u i d C h l o r i d e C h l o r i d e Water 25% summer 87.7 89.2 57.4 961.0 w i n t e r 75.5 77.1 55.9 957.0 50% summer 221.3 231.0 117.2 936.0 w i n t e r 198.2 201.9 114.1 959.0 75% summer 328.7 337.3 178.0 952.0 w i n t e r 344.9 352.8 200.5 955.0 100% summer 445.5 467.5 242.3 931.0 w i n t e r 438.3 458.0 267.1 935.0 125% summer 539.4 561.8 323.5 925.0 w i n t e r 549.3 580.0 343.8 925.0 49 C l " between b l o o d and m u s c l e , t h a n between b l o o d and s e a water ( F i g . 7 ) . 11. E x t r a c e l l u l a r Volume o f F o o t M u s c l e F o o t m u s c l e e x t r a c e l l u l a r v o l u m e s were c a l c u l a t e d u s i n g t h e b l o o d and m u s c l e C l " c o n c e n t r a t i o n s , and b l o o d w ater v a l u e s , shown i n T a b l e 5. The e x t r a c e l l u l a r volume o f f o o t m u s c l e t i s s u e from w i n t e r l i m p e t s was 4-11% h i g h e r t h a n t h a t of summer l i m p e t s a t a l l s a l i n i t i e s t e s t e d ( F i g . 8 ) . L i n e a r r e g r e s s i o n a n a l y s i s o f e x t r a c e l l u l a r volume a g a i n s t s a l i n i t y r e s u l t e d i n p o s i t i v e s l o p e s f o r summer (0.66) and w i n t e r ( 0 . 3 7 ) , t h a t were n o t s i g n i f i c a n t l y d i f f e r e n t ( A n c o v ) ( F i g . 8 ) . B o t h s l o p e s , however, were s i g n i f i c a n t l y g r e a t e r t h a n z e r o ( A n c o v ) . The y - i n t e r c e p t f o r w i n t e r v a l u e s was s i g n i f i c a n t l y h i g h e r t h a n t h a t f o r summer v a l u e s by 13% ( A n c o v ) ( F i g . 8 ) . The c a l c u l a t e d summer and w i n t e r e x t r a c e l l u l a r v olumes from 25% SW were a t l e a s t 11% h i g h e r t h a n v a l u e s f r o m o t h e r s a l i n i t i e s , and were n o t i n c l u d e d i n t h e a n a l y s i s . T w e n t y - f i v e p e r c e n t s e a w a t e r may have been beyond t h e t o l e r a n c e r a n g e o f l i m p e t s u s e d i n t h i s s t u d y ; c e l l damage o r r u p t u r e may have c a u s e d t h e i n c o n s i s t e n t l y h i g h v a l u e s . C a l c u l a t e d e x t r a c e l l u l a r f l u i d C l " c o n c e n t r a t i o n s a r e shown i n T a b l e 5. T h e s e v a l u e s were no more t h a n 2-6% g r e a t e r t h a n e q u i l i v a l e n t b l o o d C l " v a l u e s r e g a r d l e s s of s a l i n i t y o r s e a s o n ( T a b l e 5 ) . T h i s s u p p o r t s t h e s u g g e s t i o n t h a t b l o o d and 50 F i g u r e 8: R e g r e s s i o n o f c a l c u l a t e d e x t r a c e l l u l a r volume b a s e d on C l " c o n c e n t r a t i o n , as a f u n c t i o n o f s a l i n i t y . E a c h p o i n t r e p r e s e n t s a c a l c u l a t i o n b a s e d on d a t a from 10 a n i m a l s k e p t i n e x p e r i m e n t a l s a l i n i t i e s f o r 1 week. Open c i r c l e s r e p r e s e n t w i n t e r v a l u e s ; c l o s e d c i r c l e s r e p r e s e n t summer v a l u e s . V a l u e s f o r 25% sea water have been e x c l u d e d from t h e a n a l y s i s . The r e g r e s s i o n e q u a t i o n s a r e as f o l l o w s : summer y = 0.66x + 449 w i n t e r y = 0.37x + 519 51 M E D I U M C O N C E N T R A T I O N (% S E A W A T E R ) 52 e x t r a c e l l u l a r f l u i d s p a c e s a r e c o n t i n u o u s ( D e h n e l , 1979). I I I . B l o o d and U r i n e O s m o t i c P r e s s u r e To d e t e r m i n e t h e e f f e c t of s a l i n i t y on b l o o d and u r i n e o s m o t i c p r e s s u r e , l i m p e t s were p l a c e d i n 25%, 50%, 75%, and 125% SW, and sampled a f t e r 1 week. Summer a n i m a l s were measured i n 25%, 50%, and 125% SW, w i n t e r l i m p e t s i n 25%, 75%, and 125% SW ( F i g . 9 ) . B l o o d o s m o t i c p r e s s u r e v a l u e s f o r b o t h summer and w i n t e r l i m p e t s were s i g n i f i c a n t l y d i f f e r e n t f r o m one a n o t h e r r e g a r d l e s s o f s a l i n i t y (DMRT); u r i n e v a l u e s f o l l o w e d t h e same t r e n d ( D M R T ) ( F i g . 9 ) . Summer b l o o d v a l u e s were s i g n i f i c a n t l y h i g h e r t h a n e q u i v a l e n t s e a w a t e r v a l u e s by 11-23% ( t - t e s t ) ( F i g . 9 ) . W i n t e r b l o o d r a n g e d from 12% below (25% SW), t o 12% above (75% SW) e q u i v a l e n t s e a water v a l u e s , b u t was o n l y s i g n i f i c a n t l y h i g h e r i n 75% SW ( t - t e s t ) ( F i g . 9 ) . Summer u r i n e o s m o t i c p r e s s u r e s were 4-10% below e q u i v a l e n t sea water o s m o t i c p r e s s u r e s , b u t n o t s i g n i f i c a n t l y d i f f e r e n t ( t - t e s t ) ( F i g . 9 ) . W i n t e r u r i n e was s i g n i f i c a n t l y l o w e r t h a n sea water by 16-35%, i n 25% and 75% SW r e s p e c t i v e l y ( t - t e s t ) ; u r i n e i n 125% SW was o n l y 3% below s e a wa t e r o s m o t i c p r e s s u r e and n o t s i g n i f i c a n t l y d i f f e r e n t ( t -t e s t ) ( F i g . 9 ) . B l o o d o s m o t i c p r e s s u r e was s i g n i f i c a n t l y h i g h e r t h a n u r i n e o s m o t i c p r e s s u r e i n summer a d a p t e d l i m p e t s by 17-27%, a t a l l s a l i n i t i e s t e s t e d ( t - t e s t ) ( F i g . 9 ) . W i n t e r b l o o d o s m o t i c p r e s s u r e was 7-26% h i g h e r t h a n u r i n e o s m o t i c p r e s s u r e a t a l l 53 F i g u r e 9: O s m o t i c p r e s s u r e o f b l o o d ( c i r c l e s ) , and u r i n e ( t r i a n g l e s ) a s a f u n c t i o n o f s a l i n i t y . Open sym b o l s r e p r e s e n t d a t a from w i n t e r l i m p e t s ; c l o s e d symbols d a t a from summer l i m p e t s . E a c h p o i n t r e p r e s e n t s a mean d e t e r m i n e d from measurements on 10 a n i m a l s , a f t e r e x p o s u r e t o e x p e r i m e n t a l s a l i n i t i e s f o r 1 week. Sea w a ter o s m o t i c p r e s s u r e i s r e p r e s e n t e d by a s o l i d l i n e . 54 0 I J 1 I I L_ 2 5 5 0 7 5 1 0 0 1 2 5 M E D I U M C O N C E N T R A T I O N (% S E A W A T E R ) 55 s a l i n i t i e s t e s t e d ( F i g . 9 ) . However, t h i s d i f f e r e n c e was o n l y s i g n i f i c a n t i n 75% SW ( t - t e s t ) . The b l o o d o s m o t i c p r e s s u r e o f summer l i m p e t s was s i g n i f i c a n t l y h i g h e r t h a n t h a t o f w i n t e r a n i m a l s by 10% i n 125% SW ( t - t e s t ) ( F i g . 9 ) . Summer and w i n t e r b l o o d v a l u e s i n 25% SW were not s i g n i f i c a n t l y d i f f e r e n t , a l t h o u g h summer b l o o d was 29% h i g h e r t h a n w i n t e r b l o o d ( t - t e s t ) ( F i g . 9 ) . No s i g n i f i c a n t s e a s o n a l d i f f e r e n c e s were a p p a r e n t i n u r i n e v a l u e s , a l t h o u g h summer u r i n e r a n g e d from 1% (125% SW) below t o 28% ( 2 5 % SW) above w i n t e r u r i n e ( t - t e s t ) ( F i g . 9 ) . IV. Water B a l a n c e R esponse o f F o o t M u s c l e Water The e f f e c t o f t i m e and s a l i n i t y on t h e f o o t m u s c l e w a t e r c o n t e n t o f summer and w i n t e r l i m p e t s i s shown i n F i g u r e s 10, and 11, r e s p e c t i v e l y . In g e n e r a l , m u s c l e w a t e r c o n t e n t of summer l i m p e t s i n c r e a s e d o v e r t i m e i n 25% SW, r e m a i n e d r e l a t i v e l y l e v e l i n 50% and 75% SW, and d e c r e a s e d i n 100% and 125% SW. As w i t h b l o o d , u r i n e , and f o o t m u s c l e C l " , f o o t m u s c l e w a t e r showed more v a r i a t i o n i n summer t h a n w i n t e r ( F i g s . 10, and 1 1 ) . The r e s p o n s e c u r v e o f f o o t m u s c l e water c o n t e n t f o r summer l i m p e t s a t e a c h e x p e r i m e n t a l s a l i n i t y o v e r t i m e l e v e l e d o f f a f t e r 3 h o u r s i n 25% and 125% SW, 72 h o u r s i n 100% SW, and r e m a i n e d r e l a t i v e l y l e v e l o v e r t h e e n t i r e week i n 50% and 75% SW ( F i g . 1 0 ) . A l t h o u g h v a r i a t i o n between p o i n t s r e m a i n e d h i g h a f t e r 56 F i g u r e 10: Respo n s e of f o o t m u s c l e w a t e r c o n t e n t o f summer a d a p t e d l i m p e t s t o 25% ( c l o s e d c i r c l e s ) , 50% (open c i r c l e s ) , 75% ( c l o s e d t r i a n g l e s ) , 100% (open t r i a n g l e s ) , and 125% (x) sea w a t e r . E a c h p o i n t i s t h e mean o f d e t e r m i n a t i o n s on 10 a n i m a l s . 58 F i g u r e 11: R e s p o n s e o f f o o t m u s c l e water c o n t e n t o f w i n t e r a d a p t e d l i m p e t s t o 25% ( c l o s e d c i r c l e s ) , 50% (open c i r c l e s ) , 75% ( c l o s e d t r i a n g l e s ) , 100% (open t r i a n g l e s ) , a nd 125% (x) s e a w a t e r . E a c h p o i n t r e p r e s e n t s t h e mean o f measurements from 10 a n i m a l s . 59 60 t h e c u r v e s had l e v e l e d o f f (as much as 4% between 12 and 144 h o u r s i n 25% SW), o n l y 1 v a l u e was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e r e s t i n any i n d i v i d u a l s a l i n i t y ( t - t e s t ) ( F i g . 1 0 ) . The f o o t m u s c l e water c o n t e n t of w i n t e r a n i m a l s i n i t i a l l y i n c r e a s e d i n 25% and 50% SW, d e c r e a s e d i n 100% and 125% SW, and r e m a i n e d r e l a t i v e l y l e v e l i n 75% SW ( t - t e s t ) ( F i g . 11). In 100% and 125% SW, m u s c l e w a t e r r e a c h e d a minimum v a l u e a t 12 h o u r s , t h e n i n c r e a s e d t o t h e c o n t r o l l e v e l (850 g/kg) between 12 h o u r s and 1 week ( F i g . 11). M u s c l e w a t e r c o n t e n t i n 25% SW r e a c h e d a maximum v a l u e a t 48 h o u r s , t h e n d e c r e a s e d t o a v a l u e 4% above t h e c o n t r o l l e v e l a f t e r 1 week ( F i g . 1 1 ) . In 50% SW, m u s c l e w a t e r l e v e l e d o f f a f t e r 3 h o u r s ( t - t e s t ) , and r e m a i n e d 2% h i g h e r t h a n t h e c o n t r o l l e v e l f o r t h e r e m a i n d e r of..the week ( F i g . 1 1 ) . T h i s d i f f e r e n c e was n o t s i g n i f i c a n t (DMRT). A l i n e a r r e g r e s s i o n a n a l y s i s o f wet w e i g h t o f f o o t m u s c l e t i s s u e a g a i n s t d r y w e i g h t f o r summer l i m p e t s , a t t h e end o f 1 week i n e x p e r i m e n t a l s a l i n i t i e s , i s shown i n F i g u r e 12. The s l o p e f o r 25% SW was 53% g r e a t e r t h a n t h e s l o p e s f o r e i t h e r 100% o r 125% SW ( A n c o v ) ( F i g . 1 2 ) . No o t h e r s l o p e d i f f e r e n c e s were s i g n i f i c a n t ( A n c o v ) . The i n t e r c e p t f o r 25% SW was s i g n i f i c a n t l y l o w e r t h a n i n t e r c e p t s f o r e i t h e r 75%, 100%, o r 125%, by 0.1 mg ( 7 5 % SW) t o 1.8 mg (100% SW) wet w e i g h t o f t i s s u e ( A n c o v ) ( F i g . 1 2 ) . The i n t e r c e p t f o r 50% SW was s i g n i f i c a n t l y l o w e r t h a n t h a t f o r 125% SW by 0.3 mg wet w e i g h t o f t i s s u e ( A n c o v ) ( F i g . 1 2 ) . No o t h e r i n t e r c e p t d i f f e r e n c e s were s i g n i f i c a n t ( A n c o v ) . A s i m i l a r l i n e a r r e g r e s s i o n a n a l y s i s o f wet w e i g h t o f f o o t m u s c l e t i s s u e a g a i n s t d r y w e i g h t was p e r f o r m e d u s i n g d a t a f r o m 61 F i g u r e 12: R e g r e s s i o n o f wet w e i g h t as a f u n c t i o n o f d r y w e i g h t f o r summer a d a p t e d l i m p e t s k e p t i n 25% ( c l o s e d c i r c l e s ) , 50% (open c i r c l e s ) , 75% ( c l o s e d t r i a n g l e s ) , 100% (open t r i a n g l e s ) , and 125% (x) s e a w a t e r , f o r 1 week. R e g r e s s i o n e q u a t i o n s and c o r r e l a t i o n c o e f f i c i e n t s ( r ) a r e as f o l l o w s : 25% S.W. y = 9. 2x - 1 . 2 r = = 0.93 50% S.W. y = 6. 1x + 0. 15 r = = 0.91 75% S.W. y = 6. 7x - 1 . 1 r -= 0 .91 1 00% S.W. y = 4. 3x 0. 66 r = = 0.85 1 25% S.W. y = 4. 4x + 0. 49 r = = 0.91 62 JL 0.5 1.0 1.5 2.0 DRY WEIGHT OF MUSCLE TISSUE (mg) 63 F i g u r e 13: R e g r e s s i o n o f wet w e i g h t as a f u n c t i o n o f d r y w e i g h t f o r w i n t e r a d a p t e d l i m p e t s k ept i n 25% ( c l o s e d c i r c l e s ) , 50% (open c i r c l e s ) , 75% ( c l o s e d t r i a n g l e s ) , 100% (open t r i a n g l e s ) , and 125% (x) s e a w a t e r , f o r 1 week. R e g r e s s i o n e q u a t i o n s and c o r r e l a t i o n c o e f f i c i e n t s ( r ) a r e as f o l l o w s : 25% S.W. y = 5.7x + 4.0 r = = 0.74 50% S.W. y = 7.7x - 0.3 r = = 0.94 75% S.W. y = 4.3x + 3.7 r = = 0.69 1 00% S.W. y "= 3.9x + 7.8 r = = 0.92 125% S.W. y = 3.4x + 6.3 r = = 0.69 64 65 w i n t e r l i m p e t s ( F i g . 1 3 ) . The s l o p e f o r 50% SW was s i g n i f i c a n t l y h i g h e r t h a n s l o p e s f o r e i t h e r 100% o r 125% SW, by 49-56% ( A n c o v ) ( F i g . 1 3 ) . No o t h e r s l o p e d i f f e r e n c e s were s i g n i f i c a n t ( A n c o v ) . The y - i n t e r c e p t s were n o t s i g n i f i c a n t l y d i f f e r e n t , f o r a l l e x p e r i m e n t a l s a l i n i t i e s , a l t h o u g h t h e y r a n g e d between -0.3 mg (50% SW) and 7.8 mg (100% SW) wet w e i g h t of t i s s u e ( A n c o v ) ( F i g . 1 3 ) . S e a s o n a l c o m p a r i s o n s between l i n e s f o r e a c h e x p e r i m e n t a l s a l i n i t y r e v e a l e d no s i g n i f i c a n t s l o p e d i f f e r e n c e s ( A n c o v ) . However, s i g n i f i c a n t y - i n t e r c e p t d i f f e r e n c e s r e s u l t e d between summer and w i n t e r l i n e s f o r 100% (7.1 mg), and 125% SW (5.8 mg wet w e i g h t o f t i s s u e ) ( A n c o v ) . The e f f e c t of s a l i n i t y on t h e wa t e r c o n t e n t o f f o o t m u s c l e t i s s u e and b l o o d from summer and w i n t e r l i m p e t s a f t e r 1 week i n e x p e r i m e n t a l s a l i n i t i e s , i s shown i n F i g u r e 14. Summer l i m p e t s i n 25% SW had a m u s c l e w a t e r c o n t e n t 4-6% h i g h e r t h a n t h a t o f a n i m a l s i n 50% o r 75% SW, and 9% h i g h e r t h a n t h a t o f l i m p e t s i n 100% o r 125% SW ( F i g . 1 4 ) . Th e s e d i f f e r e n c e s were s i g n i f i c a n t (DMRT). The f o o t m u s c l e w a t e r c o n t e n t o f l i m p e t s i n 50% and 75% SW was n o t s i g n i f i c a n t l y d i f f e r e n t (DMRT); n e i t h e r was m u s c l e water c o n t e n t i n 100% and 125% SW ( D M R T ) ( F i g . 1 4 ) . The m u s c l e water c o n t e n t of l i m p e t s i n 50% and 75% SW, was s i g n i f i c a n t l y h i g h e r t h a n t h a t o f a n i m a l s i n 100% o r 125% SW, by 4-6% ( D M R T ) ( F i g . 1 4 ) . A f t e r 1 week i n e x p e r i m e n t a l s a l i n i t i e s t h e m u s c l e water c o n t e n t o f w i n t e r l i m p e t s i n 25% SW was s i g n i f i c a n t l y h i g h e r t h a n t h a t o f l i m p e t s i n 125% SW, by 4% ( D M R T ) ( F i g . 1 4 ) . No o t h e r 66 s a l i n i t y r e l a t e d d i f f e r e n c e s i n f o o t m u s c l e w a t e r were s i g n i f i c a n t (DMRT). S e a s o n a l d i f f e r e n c e s i n m u s c l e water c o n t e n t were n o t s i g n i f i c a n t i n 25% and 50% SW (DRMT), a l t h o u g h summer v a l u e s were 1-3% l o w e r t h e n w i n t e r v a l u e s ( F i g . 1 4 ) . However, i n 75%, 100%, and 125% SW, summer v a l u e s were s i g n i f i c a n t l y l o w e r t h a n w i n t e r v a l u e s by 4-8% ( D M R T ) ( F i g . 1 4 ) . Response of B l o o d Water Summer b l o o d water c o n t e n t s r a n g e d from 925 mg/kg b l o o d t o 961 mg/kg b l o o d , a d i f f e r e n c e o f o n l y 4% ( F i g . 1 4 ) . B l o o d w ater v a l u e s i n 25% and 75% SW, were s i g n i f i c a n t l y g r e a t e r t h a n b l o o d v a l u e s i n 50%, 100%, and 125% SW, by 2-4% ( D M R T ) ( F i g . 1 4 ) . No o t h e r s a l i n i t y r e l a t e d d i f f e r e n c e s i n b l o o d water c o n t e n t were s i g n i f i c a n t (DMRT). W i n t e r b l o o d w ater r a n g e d f r o m 925 mg/kg b l o o d t o 957 mg/kg b l o o d , a d i f f e r e n c e of 4% ( F i g . 1 4 ) . B l o o d water v a l u e s i n 25%, 50%, and 75% SW, were s i g n i f i c a n t l y h i g h e r t h a n b l o o d v a l u e s i n 100% and 125% SW, by 2-4% ( D M R T ) ( F i g . 14). O t h e r s a l i n i t y r e l a t e d b l o o d w ater d i f f e r e n c e s were n o t s i g n i f i c a n t (DMRT). T h e r e were no s i g n i f i c a n t s e a s o n a l d i f f e r e n c e s i n b l o o d w a ter c o n t e n t e x c e p t i n 50% SW, where w i n t e r b l o o d w a t e r was h i g h e r t h a n summer by 2% ( D M R T ) ( F i g . 1 4 ) . B l o o d w a t e r v a l u e s were h i g h e r t h a n m u s c l e v a l u e s by 9% (25% SW) t o 15% (125% SW) f o r summer l i m p e t s , and by 8% ( 2 5 % and 125% SW) t o 10% ( 7 5 % SW) f o r w i n t e r a n i m a l s ( F i g . 1 4 ) . Th e s e d i f f e r e n c e s were s i g n i f i c a n t ( t - t e s t ) . 67 F i g u r e 14: E f f e c t of s a l i n i t y on t h e w a t e r c o n t e n t o f b l o o d and f o o t m u s c l e t i s s u e from summer and w i n t e r a d a p t e d l i m p e t s , t o c h a n g e s i n e x t e r n a l s a l i n i t y . E a c h p o i n t r e p r e s e n t s t h e mean of d e t e r m i n a t i o n s on 10 a n i m a l s , a f t e r e x p o s u r e t o e x p e r i m e n t a l s a l i n i t i e s f o r 1 week. 68 MEDIUM CONCENTRATION (% SEA WATER) 69 D u r i n g the c o u r s e of t h i s s tudy i t became e v i d e n t t h a t C. persona showed s e a s o n a l s u r v i v a l d i f f e r e n c e s i n 25% and 125% SW. Limpets s u r v i v e d d u r i n g summer and w i n t e r f o r more than 2 weeks w i t h o u t m o r t a l i t y i n 50%, 75%, and 100% SW. Win t e r a n i m a l s s u r v i v e d i n 25% and 125% SW f o r 1 week. Summer a n i m a l s , however, showed between 50% and 75% m o r t a l i t y when h e l d i n 25% SW or 125% SW f o r more than 48 h o u r s . T w e n t y - f i v e p e r c e n t and 125% SW may be lower than and h i g h e r t h a n , r e s p e c t i v e l y , the l e t h a l s a l i n i t y l i m i t s of the s e l i m p e t s . 70 DISCUSSION I_. The E f f e c t o f S a l i n i t y on C h l o r i d e Ion B a l a n c e B l o o d and U r i n e C h l o r i d e Summer and w i n t e r p o p u l a t i o n s of t h e l i m p e t C o l l i s e l l a  p e r s o n a d i d n o t r e g u l a t e b l o o d o r u r i n e C l " o v e r a s a l i n i t y r a n g e of 25-125% SW. I n v e s t i g a t i o n i n t o t h e e f f e c t of s a l i n i t y on t h e b l o o d C l " of o t h e r o s m o c o n f o r m i n g g a s t r o p o d s has shown t h a t t h e s e a n i m a l s do n o t r e g u l a t e C l " ( R o b e r t s o n , 1949; 1953; P o t t s and P a r r y , 1964; K i n n e , 1971; P i e r c e and G r e e n b u r g , 1976). L i t t l e (1967) d i s c o v e r e d t h a t t h e b l o o d C l " c o n c e n t r a t i o n o f Strombus g i g a s was i s o i o n i c t o sea w a t e r . The same a u t h o r f o u n d a s i m i l a r r e l a t i o n s h i p between b l o o d and sea w a t e r C l " i n t h e g a s t r o p o d N e r i t a f u l g u r a n s ( L i t t l e , 1972). B l o o d and s e a water C l " were a l s o i n a p a s s i v e e q u i l i b r i u m i n P a t e l l a v u l g a t a (Hoyaux e t a l , 1976). The C l " l e v e l i n b l o o d and u r i n e of summer a d a p t e d C. p e r s o n a r e a c h e d e q u i l i b r i u m a f t e r 24 h o u r s i n 75%, 100%, and 125% SW ( F i g . 2 ) . W i n t e r a n i m a l s , however, r e a c h e d e q u i l i b r i u m w i t h i n 12 h o u r s a t t h e same s a l i n i t i e s ( F i g . 3 ) . S i m i l a r s e a s o n a l d i f f e r e n c e s were a p p a r e n t i n t h e l o w e s t s a l i n i t y t e s t e d , 25% SW. Summer a n i m a l s r e a c h e d e q u i l i b r i u m i n 144 h o u r s ( F i g . 2 ) , and w i n t e r a n i m a l s w i t h i n 48 h o u r s ( F i g . 3 ) . F u r t h e r m o r e , t h e b l o o d and u r i n e C l " c o n c e n t r a t i o n s of summer a d a p t e d l i m p e t s a f t e r 12 h o u r s i n 75%, 100%, and 125% SW were 10-20% h i g h e r t h a n e q u i v a l e n t s e a water v a l u e s ( F i g . 2 ) . T h i s 71 r e s p o n s e d i d n o t o c c u r i n w i n t e r a d a p t e d l i m p e t s under t h e same c o n d i t i o n s . . Summer CL" va-l,ues; a l s o , show.ed.a g.r>eafcear.- v a r i a b i l i t y * , t h a n w i n t e r v a l u e s . B l o o d and u r i n e C l " l e v e l s , t h e r e f o r e , r e a c h e d e q u i l i b r i u m d i f f e r e n t l y i n summer, t h a n i n w i n t e r a d a p t e d l i m p e t s . O g l e s b y ( 1 9 8 1 ) , m e n t i o n e d t h a t p e r m e a b i l i t y t o s a l t s i n c l u d i n g N a + and C l " , was r e d u c e d i n f r e s h and b r a c k i s h w ater i n v e r t e b r a t e s , when compared t o t h e same o r r e l a t e d s p e c i e s i n f u l l s t r e n g t h s e a w a t e r . I t i s n o t u n r e a s o n a b l e t o assume t h a t s a l t and water p e r m e a b i l i t y o f e s t u a r i n e i n t e r t i d a l i n v e r t e b r a t e s may a l s o change w i t h s e a s o n . S e a s o n a l p e r m e a b i l i t y d i f f e r e n c e s may have a c c o u n t e d f o r t h e l o n g e r t i m e p e r i o d r e q u i r e d t o r e a c h e q u i l i b r i u m i n summer a d a p t e d C. p e r s o n a , as w e l l a s t h e h i g h b l o o d and u r i n e C l " l e v e l s r e c o r d e d a t 12 h o u r s i n 75-125% SW. In a d d i t i o n , summer and w i n t e r a d a p t e d l i m p e t s i n 25% SW may have been l e s s p e r m e a b l e t o s a l t s and w a t e r t h a n l i m p e t s i n h i g h e r s a l i n i t i e s . A l t h o u g h b l o o d and u r i n e C l " from C. p e r s o n a r e a c h e d e q u i l i b r i u m w i t h i n 1 week i n a l l e x p e r i m e n t a l s a l i n i t i e s , v a l u e s were n o t n e c e s s a r i l y e q u a l t o r e s p e c t i v e s e a water c o n c e n t r a t i o n s . A s i m i l a r s i t u a t i o n has been f o u n d i n o t h e r i n v e r t e b r a t e s . O g l e s b y (1973) r e p o r t e d t h a t t h e b l o o d C l " c o n c e n t r a t i o n of t h e lugworm, A b a r e n i c o l a pac i f i c a , f o l l o w e d c h a n g e s i n e x p e r i m e n t a l s a l i n i t y . However, b l o o d C l " was h y p o i o n i c t o s e a water by 10-50 mM C l " o v e r a s a l i n i t y r a n g e of 150-550 mM N a C l . S m i t h (1967) r e p o r t e d t h a t t h e b l o o d C l " o f t h e c r a b , R h i t h r o p a n o p e u s ha r r i s i , was h y p o i o n i c t o u r i n e . He 72 a t t r i b u t e d t h e d i f f e r e n c e t o t h e p r e s e n c e o f p r o t e i n and a l o w e r w a t e r c o n t e n t i n t h e b l o o d . (Smith., 1967). Rumsey (1973) f o u n d t h a t t h e b l o o d C l " of L i t t o r . i n a 1 i t t o r e a was h y p e r i o n i c t o sea w a ter below 40% SW, and i s o i o n i c a b o v e . Summer and w i n t e r a d a p t e d C. p e r s o n a had b l o o d C l " c o n c e n t r a t i o n s c o n s i s t a n t l y l o w e r t h a n e q u i v a l e n t s e a water or u r i n e C l " , by up t o 30 mEq/L C l " r e g a r d l e s s of s a l i n i t y ( T a b l e 2 ) . Webber ( 1 9 6 6 ) , and Webber and D e h n e l (1968a) f o u n d a s i m i l a r d i f f e r e n c e between b l o o d and u r i n e C l " f o r Acmaea (= C o l l i s e l l a ) scutum i n 50% SW, a t t i m e p e r i o d s o f 1.5, 6, and 12 h o u r s . A t a l l o t h e r t i m e s and s a l i n i t i e s b l o o d and u r i n e C l " c o n c e n t r a t i o n s were not s i g n i f i c a n t l y d i f f e r e n t from one a n o t h e r . However, T a r r (1977) showed t h a t b l o o d C l " of Notoacmaea (= C o l l i s e l l a ) scutum, and C o l l i s e l l a d i g i t a l i s was s i g n i f i c a n t l y l o w e r t h a n s e a w ater C l " by 25-100 mEq/L, o v e r a s a l i n i t y r a n g e of 50-150% SW. U r i n e v a l u e s were not measured but were p r o b a b l y s i m i l a r t o sea water C l " as was u r i n e from A. scutum (Webber, 1966) and C. p e r s o n a . W o l c o t t (1973) d i s c o v e r e d b l o o d C l " v a l u e s h y p o i o n i c t o e x t r a c o r p o r e a l w a t e r ( u r i n e and m a n t l e w a t e r ) f r o m d e s i c c a t e d A. d i g i t a l i s . W o l c o t t (1973) and T a r r (1977) m e n t i o n e d t h a t t h e d i f f e r e n c e between b l o o d and u r i n e ( o r e x t r a c o r p o r e a l w a t e r ) C l " was p r o b a b l y due t o o r g a n i c m a t e r i a l p r e s e n t i n t h e b l o o d and, c o n s e q u e n t l y , a Donnan e q u i l i b r i u m . L i t t l e (1965) f o u n d t h a t t h e f r e s h w a ter s n a i l V i v i p a r u s v i v p a r u s had a b l o o d C l " c o n c e n t r a t o n h y p e r i o n i c t o sea w ater below 40 mM/L N a C l and h y p o i o n i c above, i n s a l i n i t i e s r a n g i n g from 1-20% SW. The d i f f e r e n c e between b l o o d and s e a w ater was a t t r i b u t e d t o t h e 73 p r e s e n c e of b i c a r b o n a t e i o n r a t h e r t h a n o r g a n i c components i n t h e b l o o d ( L i t t l e , 1965). K i n n e (1971) m e n t i o n e d t h a t m a r i n e i n v e r t e b r a t e s may have a p p r e c i a b l e q u a n t i t i e s o f p r o t e i n i n t h e i r b l o o d . In a d d i t i o n , R o b e r t s o n (1953) r e p o r t e d t h a t a l t h o u g h c o e l e n t e r a t e s , e c h i n o d e r m s , p o l y c h a e t e s , and l a m e l l i b r a n c h s have b l o o d p r o t e i n c o n t e n t s below 1 g/L, p r o s o b r a n c h g a s t r o p o d s may have p r o t e i n l e v e l s h i g h e r t h a n t h i s . L i t t l e (1967) r e p o r t e d t h a t t h e b l o o d of Strombus g i g a s had a p r o t e i n c o n t e n t of 3.37% wet w e i g h t of b l o o d . I o n s b i n d i n g t o p r o t e i n i n b l o o d c an r e s u l t i n a Donnan e q u i l i b r i u m , w i t h b l o o d i o n c o n c e n t r a t i o n s s l i g h t l y l o w e r t h a n s e a w a t e r ( P o t t s and P a r r y , 1964). T h e r e i s some e v i d e n c e t h a t even s m a l l m o n o v a l e n t i o n s i n c l u d i n g C l " can b i n d t o p r o t e i n . G a y t o n and H i n k e (1968) m e n t i o n e d t h a t many p r o t e i n s a r e c a p a b l e o f b i n d i n g C l " i o n s . D i c k (1959; c i t e d i n P o t t s and P a r r y , 1964) f o u n d t h a t t h e p r o t e i n a l b u m i n was c a p a b l e o f c o m b i n i n g up t o 10 C l " i o n s p e r a l b u m i n m o l e c u l e . P r o s s e r (1973) r e p o r t e d t h a t Donnan e f f e c t s i n c r e a s e i n t h e f o l l o w i n g s e r i e s : m a n t l e water ( s e a w a t e r ) , p e r i c a r d i a l f l u i d , b l o o d . Fynn (1976) f o u n d t h a t t h e low p r o t e i n c o n c e n t r a t i o n i n th e p e r i c a r d i a l f l u i d o f t h e b r a c k i s h water c l a m , R a n g i a c u n e a t a , s u g g e s t e d a v e r y s m a l l G i b b s - D o n n a n e f f e c t . However, t h i s was not enough t o a c c o u n t f o r t h e b l o o d h y p e r o s m o t i c i t y r e c o r d e d f r o m R. c u n e a t a ( F y h n , 1976). A l t h o u g h d i a l y s i s was n o t p e r f o r m e d on t h e b l o o d of C. p e r s o n a i t was not u n r e a s o n a b l e t o assume t h a t t h e h y p o i o n i c 74 b l o o d C l " was due t o t h e p r e s e n c e of o r g a n i c m a t e r i a l i n t h e b l o o d , and a Donnan e f f e c t . . G e n e r a l l y , f o r summer and w i n t e r a d a p t e d C. p e r s o n a u r i n e C l " was i s o i o n i c t o a l l e x p e r i m e n t a l s a l i n i t i e s . However, w i n t e r l i m p e t s h e l d i n 25% and 50% SW f o r 1 week e x c r e t e d u r i n e h y p o i o n i c t o s e a w a t e r . Todd (1964b) r e p o r t e d t h a t a n o t h e r e s t u a r i n e s n a i l , P o tamopyrgus j e n k i n s i , m a i n t a i n e d o s m o t i c b a l a n c e by p r o d u c i n g a h y p o s m o t i c u r i n e . In a d d i t i o n , many f r e s h w a t e r m o l l u s c s a r e c a p a b l e o f e x c r e t i n g a h y p o s m o t i c u r i n e ( B a r n e s , 1974), i n c l u d i n g L i g u m i a s u b r o s t r a t a (Murphy and D i e t z , 1976). However, h y p o r e g u l a t i o n o f u r i n e C l " by C. p e r s o n a was u n l i k e l y b e c a u s e , a l t h o u g h u r i n e was h y p o i o n i c t o s e a w ater i t was not h y p o i o n i c t o b l o o d . The r e l a t i o n s h i p between b l o o d and u r i n e i n 25% and 50% SW was c o n s i s t a n t w i t h t h a t shown i n h i g h e r s a l i n i t i e s . In a d d i t i o n , C l " r e g u l a t i o n o f b l o o d o r u r i n e was n o t i n d i c a t e d i n any of t h e o t h e r d a t a . F o o t M u s c l e T i s s u e C h l o r i d e As w i t h b l o o d and u r i n e C l " , t h e t i s s u e C l " c o n c e n t r a t i o n of summer and w i n t e r a d a p t e d C. p e r s o n a was not r e g u l a t e d o v e r a range of s a l i n i t i e s f r o m 25-125% SW. However, t i s s u e C l " d i d n o t a p p r o a c h s e a water C l " , but r e a c h e d a c o n s t a n t l e v e l a p p r o x i m a t e l y o n e - h a l f of e q u i v a l e n t s e a water v a l u e s . The time, r e q u i r e d t o r e a c h e q u i l i b r i u m was s i m i l a r f o r b l o o d , u r i n e , and m u s c l e C l " i n a l l e x p e r i m e n t a l s a l i n i t i e s f o r b o t h summer and w i n t e r a d a p t e d a n i m a l s . However, w i n t e r t i s s u e C l " v a l u e s l e v e l e d o f f more r a p i d l y i n h i g h e r s a l i n i t i e s and more s l o w l y i n 75 25% and 50% SW, t h a n summer v a l u e s . In a d d i t i o n , summer t i s s u e C l " l e v e l s . showed .more.., v a r i a t i o n , thanu-wi.nter l e v e l s in-, a- manner, s i m i l a r t o t h e v a r i a t i o n shown by summer b l o o d C l " . T h i s may have been due t o s e a s o n a l d i f f e r e n c e s i n p e r m e a b i l i t y t o water and s a l t s . A f t e r 1 week i n e x p e r i m e n t a l s a l i n i t i e s s e a s o n a l d i f f e r e n c e s were a p p a r e n t i n 75%, 100%, and 125% SW, w i t h w i n t e r t i s s u e C l " c o n c e n t r a t i o n s h i g h e r t h a n summer v a l u e s by 20-25 mEq C l " / k g t i s s u e ( F i g . 7 ) . S e a s o n a l d i f f e r e n c e s i n t i s s u e C l " c o u l d be a c c o u n t e d f o r by t h e r e l a t i v e l y l a r g e r t i s s u e water c o n t e n t and e x t r a c e l l u l a r s p a c e , or a g r e a t e r i n t r a c e l l u l a r C l " c o n c e n t r a t i o n , i n w i n t e r a d a p t e d C. p e r s o n a . However, t h e more l i k e l y e x p l a n a t i o n i s t h e p r e s e n c e o f a s l i g h t y l a r g e r e x t r a c e l l u l a r s p a c e i n w i n t e r l i m p e t s . T i s s u e C l " v a l u e s were l e s s t h a n e q u i v a l e n t b l o o d v a l u e s by 35-47% i n summer a d a p t e d a n i m a l s a n d by 26-42% i n w i n t e r l i m p e t s , o v e r a range o f s a l i n i t i e s f r o m 25-125% SW. A l t h o u g h t h e s e d i f f e r e n c e s were s i g n i f i c a n t , i f t i s s u e C l " was e x p r e s s e d i n terms of e x t r a c e l l u l a r water (mEq/kg e x t r a c e l l u l a r w a t e r ) r a t h e r t h a n m u s c l e wet w e i g h t , t h e C l " v a l u e s of b l o o d and m u s c l e were s i m i l a r ( T a b l e 5 ) . F o r example, i n 25% SW t h e b l o o d C l " c o n c e n t r a t i o n of summer a n i m a l s was 87.7 mEq/kg b l o o d , w h i l e t i s s u e C l " was 57.4 mEq/kg. E x p r e s s e d i n t e r m s of e x t r a c e l l u l a r w a t e r , t h e m u s c l e C l " v a l u e was 89.2 mEq/kg; a v a l u e v e r y s i m i l a r t o t h e e q u i v a l e n t b l o o d v a l u e ( T a b l e 5 ) . The f a c t t h a t c a l c u l a t e d C l " v a l u e s f o r e x t r a c e l l u l a r w a t e r were a l w a y s l a r g e r t h a n b l o o d v a l u e s may have i n d i c a t e d t h e p r e s e n c e of a low 76 c o n c e n t r a t i o n o f i n t r a c e l l u l a r C l " . O t h e r g a s t r o p o d s show t i s s u e C l " r e s u l t s , s i m i l a r t o those.-., o b t a i n e d d u r i n g t h e p r e s e n t s t u d y f o r C. p e r s o n a . T u r g e o n (1976) d i s c o v e r e d t h a t U r o s a l p i n x c i n e r e a d i d n o t r e g u l a t e t o t a l t i s s u e C l " i n s a l i n i t i e s r a n g i n g f r o m 32-129% SW. However, as w i t h C. p e r s o n a , t h e t i s s u e C l " c o n c e n t r a t i o n was much l e s s t h a n e q u i v a l e n t sea w ater C l * . In 97% SW t h e v a l u e f o r t i s s u e C l " was 235 mM/kg ( T u r g e o n , 1976), w h i l e a v a l u e o f 242 mEq/kg was r e c o r d e d f r o m C. p e r s o n a i n 100% SW. B o t h v a l u e s were a p p r o x i m a t e l y o n e - h a l f e q u i v a l e n t sea w ater v a l u e s . Webber (1966) f o u n d t h a t t h e l i m p e t A. scutum d i d n o t r e g u l a t e t i s s u e C l " o v e r a range of s a l i n i t i e s from 50-125% SW. T i s s u e C l " v a l u e s r e a c h e d a c o n s t a n t l e v e l w i t h i n 12 h o u r s a t a l l s a l i n i t i e s t e s t e d . T h e s e r e s u l t s were s i m i l a r t o t h o s e o b t a i n e d from C. p e r s o n a d u r i n g t h e p r e s e n t s t u d y , e x c e p t t h a t t i s s u e C l " f r o m w i n t e r a d a p t e d l i m p e t s r e q u i r e d a l o n g e r p e r i o d of t i m e t o r e a c h a c o n s t a n t l e v e l i n 50% SW. T i s s u e C l " was l o w e r t h a n sea w a ter C l " by 60-71% i n A. scutum (Webber, 1966), and by 41-53% i n C. p e r s o n a . However, c a l c u l a t i n g t i s s u e C l " i n terms of e x t r a c e l l u l a r w a ter f o r A. scutum r e s u l t e d i n v a l u e s s i m i l a r t o t h o s e f o r b l o o d o r sea w a t e r , a t a l l s a l i n i t i e s t e s t e d . In a d d i t i o n , a l t h o u g h t h e r e was l e s s t h a n a 3% d i f f e r e n c e between se a w a t e r c o n c e n t r a t i o n s u s e d i n Webber's (1966) s t u d y and t h e p r e s e n t s t u d y , t i s s u e C l " was l o w e r i n A. scutum t h a n i n C. p e r s o n a by 41% i n 50% SW, 36% i n 75% SW, and by 25% i n 125% SW. T h i s m i g h t be u n e x p e c t e d , as t h e two s p e c i e s a r e v e r y s i m i l a r and r e c e n t l y have beert p l a c e d i n t h e same genus. 77 However, A. scutum i s .found i n t h e l o w e r i n t e r t i d a l .zone, w h i l e C. p e r s o n a i s a h i g h i n t e r t i d a l LimpeA-.XUes-t,,- 1945). The method us e d t o measure t i s s u e C l " v i a e x t r a c t i o n w i t h d i s t i l l e d w a ter c o u l d not s e p a r a t e e x t r a c e l l u l a r f r o m i n t r a c e l l u l a r C l " . T h e r e f o r e , d i f f e r e n c e s r e c o r d e d i n t i s s u e C l " from Webber's (1966) s t u d y and t h e p r e s e n t s t u d y c o u l d have been due t o a g r e a t e r i n t r a c e l l u l a r C l " c o n c e n t r a t i o n , o r a l a r g e r e x t r a c e l l u l a r s p a c e i n C. p e r s o n a . 11. E x t r a c e l l u l a r Volume o f F o o t M u s c l e T i s s u e In s a l i n i t i e s r a n g i n g from 50-125% SW, t h e e x t r a c e l l u l a r volume of b o t h w i n t e r and summer a d a p t e d C. p e r s o n a i n c r e a s e d by an a v e r a g e of 7% ( F i g . 8 ) . A 7% i n c r e a s e i n e x t r a c e l l u l a r volume o r d e c r e a s e i n c e l l volume o v e r a 150% i n c r e a s e i n s a l i n i t y , i n d i c a t e d t h a t C. p e r s o n a was a b l e t o r e g u l a t e c e l l volume a t a r e l a t i v e l y c o n s t a n t l e v e l . In a l l s a l i n i t i e s , however, t h e w i n t e r v a l u e s were an a v e r a g e o f 7% h i g h e r t h a n summer v a l u e s . T h i s r e f l e c t e d t h e r e l a t i v e l y g r e a t e r m u s c l e C l " and water v a l u e s o b t a i n e d from w i n t e r C. p e r s o n a . Webber (1966) s t a t e d t h a t t h e v a r i a t i o n i n water and m u s c l e i o n s i n A. scutum was p r o b a b l y due t o c h a n g i n g l e v e l s o f s t o r e d m e t a b o l i c p r o d u c t s s u c h as l i p i d , p r o t e i n , and g l y c o g e n , r e s u l t i n g i n c h a n g e s i n e x t r a c e l l u l a r volume. The s e a s o n a l c h a n g e s i n e x t r a c e l l u l a r volume shown by C. p e r s o n a may have been due t o a s i m i l a r phenomenon. L i m p e t s may s t o r e m e t a b o l i c p r o d u c t s d u r i n g t h e s p r i n g , and u t i l i z e them d u r i n g summer when t h e f o o d s u p p l y i s 78 low. The p r e s e n c e o f t h e s e s t o r a g e p r o d u c t s i n s i d e m u s c l e c e l l s w o uld r e s u l t i n a .hyperosmotic.. c o n d i t i o n , . , Osmotica-ll-v- o b l i g a t e d ^ w a t e r would move i n t o t h e c e l l s , a nd c e l l u l a r s w e l l i n g would t e n d t o r e d u c e t h e e x t r a c e l l u l a r s p a c e between m u s c l e f i b r e s . I f t h e b l o o d and e x t r a c e l l u l a r s p a c e s a r e c o n t i n o u s , t h e n a r e d u c t i o n of e x t r a c e l l u l a r volume would i m p l y a d e c r e a s e i n t h e b l o o d volume of m u s c l e t i s s u e . Thus i f m u s c l e C l " and water were e x p r e s s e d p e r u n i t w e i g h t , t h e n l i m p e t s w i t h a l a r g e r f o o t m u s c l e e x t r a c e l l u l a r volume would have h i g h e r m u s c l e C l " and wa t e r v a l u e s . The r e c o r d e d e x t r a c e l l u l a r volume from l i m p e t s i n 25% SW was 11% h i g h e r t h a n a t any o t h e r s a l i n i t y , r e g a r d l e s s of s e a s o n ( F i g . 8 ) . However, t h e 7% d i f f e r e n c e between w i n t e r and summer v a l u e s was c o n s i s t a n t , w i t h w i n t e r h i g h e r t h a n summer. W i n t e r l i m p e t s s u r v i v e d i n 25% SW f o r t h e e n t i r e e x p e r i m e n t a l p e r i o d and s u f f e r e d no m o r t a l i t y . Most summer l i m p e t s , however, s u r v i v e d no l o n g e r t h a n 72 h o u r s i n 25% SW. C e l l r u p t u r e may have r e s u l t e d i n t h e r e l a t i v e l y l a r g e e x t r a c e l l u l a r volume measurements, and would have a c c o u n t e d f o r t h e m o r t a l i t y of summer a n i m a l s . I t would n o t , however, e x p l a i n t h e s u r v i v a l of w i n t e r a d a p t e d l i m p e t s . P e r h a p s d u r i n g t h e w i n t e r months c e l l volume was r e g u l a t e d , and e x c e s s w ater was s h u n t e d i n t o t h e e x t r a c e l l u l a r s p a c e . D u r i n g summer t h i s mechanism may have b r o k e n down. V e r n b e r g and S i l v e r t h o r n (1979) r e p o r t e d t h a t many o r g a n i s m s s u r v i v e d d i l u t e s e a w a t e r b e t t e r a t l o w e r t e m p e r a t u r e s i n c l u d i n g t h e g a s t r o p o d N a s s a r i u s r e t i c u l a t u s , and t h e b i v a l v e s A e q u i p e c t e n i r r a d i a n s , and M o d i o l u s m o d i o l u s . 79 ! i D e h n e l (1979) measured t h e e x t r a c e l l u l a r volume of t h e c r a b , H e m i g r a p s u s nudus , o v e r a, s a l i n i t y . - , range. ..of- 6-1 75:%% SW^ u s i n g C l " f o r t h e c a l c u l a t i o n . A l i n e a r r e g r e s s i o n a n a l y s i s o f . t h e d a t a r e s u l t e d i n i n s i g n i f i c a n t s l o p e d i f f e r e n c e s , and s i g n i f i c a n t i n t e r c e p t d i f f e r e n c e s . Summer a d a p t e d c r a b s had l a r g e r e x t r a c e l l u l a r s p a c e s v a l u e s t h a n w i n t e r c r a b s . In C. p e r s o n a , w i n t e r a d a p t e d l i m p e t s had t h e l a r g e r e x t r a c e l l u l a r volume measurements. Webber ( 1 9 6 6 ) , and Webber and D e h n e l (1968a) measured t h e e x t r a c e l l u l a r volume of A. scutum u s i n g C l " and i n u l i n , o v e r a r a n g e of s a l i n i t i e s from 50-125% SW. They r e p o r t e d t h a t t h e C l " s p a c e was l a r g e r t h a n t h e i n u l i n s p a c e by 30% i n 50% SW, 24% i n 75% SW, and 4% i n 125% SW. T h i s may have i n d i c a t e d t h e p r e s e n c e of i n t r a c e l l u l a r C l " . However, even t h o u g h t h e p r e s e n c e of C l " i n t r a c e l l u l a r l y i n t r o d u c e d e r r o r i n t o t h e C l " method, t h i s c o n c e n t r a t i o n was s m a l l and would have n e g l i g i b l e e f f e c t on t h e c a l c u l a t i o n s ( D e h n e l , 1979). Webber (1966) b e l i e v e d t h a t t h e i n u l i n method was t h e b e t t e r e s t i m a t e of e x t r a c e l l u l a r s p a c e . R o r i v e (1975; c i t e d i n R o r i v e a n d G i l l e s , 1979) a l s o m e n t i o n e d t h a t t h e i n u l i n method was t h e b e s t e x p e r i m e n t a l a p p r o a c h t o m e a s u r i n g e x t r a c e l l u l a r s p a c e . The e x t r a c e l l u l a r s p a c e of C. p e r s o n a was l a r g e r t h a n t h a t o f A. scutum by 43% i n 50% SW, 38% i n 75% SW, and by 30% i n 125% SW. As m e n t i o n e d p r e v i o u s l y , t h e i n c r e a s e i n e x t r a c e l l u l a r s p a c e of m u s c l e t i s s u e f o r summer o r w i n t e r a d a p t e d C. p e r s o n a i n 50-125% SW a v e r a g e d 7%. The i n c r e a s e i n e x t r a c e l l u l a r s p a c e o v e r t h e same s a l i n i t y range f o r A. scutum was a p p r o x i m a t e l y 28% 80 (Webber, 1966; Webber and D e h n e l , 1968a). I t was a p p a r e n t t h a t C. p e r s o n a , a h i g h i n t e r t i d a l l i m p e t , was c a p a b l e of a more, e f f i c i e n t c e l l volume r e g u l a t i o n t h a n the : l o w e r i n t e r t i d a l A. scutum. However t h e l i m p e t s t h a t Webber (1966) c o l l e c t e d , were members of a summer a d a p t e d m a r i n e , a n d not e s t u a r i n e , p o p u l a t i o n . A. scutum c o l l e c t e d f r o m an e s t u a r y may have a more e f f i c i e n t volume r e g u l a t o r y a b i l i t y . B e r g e r e t a l (1978) r e p o r t e d t h a t g a s t r o p o d s o f t h e genus L i t t o r i n a showed d i f f e r e n c e s i n c e l l volume r e g u l a t o r y a b i l i t y between m a r i n e and e s t u a r i n e p o p u l a t i o n s . They f o u n d t h a t t h e e s t u a r i n e L. l i t t o r e a and L. s a x a t i l i s were p a r t i a l l y a b l e t o r e g u l a t e c e l l volume, w h i l e t h e m a r i n e L. o b t u s a t a was u n a b l e . The l e s s e r e u r y h a l i n i t y of L. o b t u s a t a was p r o b a b l y due t o t h e a b s e n c e of a volume r e g u l a t o r y mechanism ( B e r g e r e t a l , 1978). I I I . B l o o d and U r i n e O s m o t i c P r e s s u r e The l i m p e t C. p e r s o n a d i d n o t r e g u l a t e t h e o s m o t i c c o n c e n t r a t i o n of b l o o d o r u r i n e o v e r a r a n g e of s a l i n i t i e s f r o m 25-125% SW. T h i s i n f o r m a t i o n i s i n a c c o r d a n c e w i t h t h a t d e r i v e d from o t h e r p r o s o b r a n c h g a s t r o p o d s . Webber (1963) r e p o r t e d t h a t t h e l i m p e t A. scutum d i d not o s m o r e g u l a t e i n 25-125% SW. W o l c o t t (1973) d i s c o v e r e d t h a t t h e haemolymph of d e s i c c a t e d A. d i g i t a l i s r e m a i n e d i n o s m o t i c e q u i l i b r i u m w i t h t h e e x t r a c o r p o r e a l w ater ( m a n t l e c a v i t y w a ter and u r i n e ) . S e g a l and D e h n e l (1962) f o u n d t h a t t h e l i m p e t A. 1 i m a t u l a d i d n o t o s m o r e g u l a t e i n s a l i n i t i e s 81 r a n g i n g from 25-150% SW. They a l s o r e p o r t e d t h a t t h e e x t r a -v i s c e r a l w a ter a c t e d . a-Si. an , o s m o t i c and- temp.e.r.a.ture:, b.u#frerv under., c o n d i t i o n s of e x p o s u r e . I f a p o r t i o n of t h e e x t r a - v i s c e r a l w a t e r was removed from e x p o s e d l i m p e t s , t h e o s m o t i c c o n c e n t r a t i o n o f haemolymph and r e m a i n i n g e x t r a - v i s c e r a l w ater i n c r e a s e d , when compared t o u n d i s t u r b e d l i m p e t s . In t h e p r e s e n t s t u d y , t h e haemolymph o f summer a d a p t e d C. p e r s o n a was h y p e r o s m o t i c t o sea w a t e r , and u r i n e i s o s m o t i c , a t a l l s a l i n i t i e s t e s t e d ( F i g . 9 ) . W i n t e r l i m p e t s had b l o o d c o n c e n t r a t i o n s i s o s m o t i c i n 25% and 125% SW, and h y p e r o s m o t i c t o 75% SW. U r i n e was h y p o s m o t i c t o 25% and 75% SW, and i s o s m o t i c i n 125% SW ( F i g . 9 ) . The b l o o d h y p e r o s m o t i c i t y shown by C. p e r s o n a c o r r e s p o n d s t o d a t a o b t a i n e d from o t h e r g a s t r o p o d s . Todd (1964b) r e p o r t e d t h a t t h e s n a i l H y d r o b i a u l v a e was s l i g h l t y h y p e r o s m o t i c i n 50-100% SW, becoming i n c r e a s i n g l y h y p e r o s m o t i c from 50-25% SW. L i t t l e (1965) f o u n d t h a t V i v i p a r u s v i v p a r u s , a f r e s h w ater s n a i l , had a b l o o d o s m o t i c p r e s s u r e h y p e r o s m o t i c t o t h e media below 8.5% SW, and i s o s m o t i c a b o v e . The Red Whelk Neptunea  a n t i q u a was an osmoconformer i n s a l i n i t i e s r a n g i n g from 30-115% SW, w i t h a hemolymph o s m o c o n c e n t r a t i o n 25 mOsm above t h e ambient s e a w a ter ( S t a a l a n d , 1972). L i t t l e and Andrews (1977) r e p o r t e d t h a t t h e s n a i l A s s i m i n e a g r a y a n a was an osmoconformer from 50-200% SW, but was h y p e r o s m o t i c t o t h e ambient medium by 32-67 mM/L N a C l . P i e r c e (1970) f o u n d t h a t M o d i o l u s d e m i s s u s , M. g r a n o s s i s i m u s , M. m o d i o l u s , and M. squamosous, were a l l o s m o c o n f o r m e r s , but were s l i g h t l y h y p e r o s m o t i c t o sea water by 10-20 mOsm. He a l s o s t a t e d t h a t any a n i m a l h a v i n g p r o t e i n i n t h e 8.2 e x t r a c e l l u l a r water and p e r m e a b l e e x t e r n a l membranes must be h y p e r o s m o t i c S n a i l s of the. genus. L . i t t p r i n a ( L. 1 i t t o r . e a , L. l i t t o r a l i s , and L. s a x a t i l i s ) were i s o s m o t i c i n s a l i n i t i e s r a n g i n g f r o m 50-150% SW, and i n c r e a s i n g l y h y p e r s o m o t i c from 50-25% SW (Todd, 1964a). Rumsey (1973) o b t a i n e d s i m i l a r r e s u l t s from L. l i t t o r e a . A l t h o u g h Todd (1964a) and Rumsey (1973) o b s e r v e d c l o s u r e of t h e o p e r c u l u m i n s a l i n i t i e s below 50% SW, t h e y assumed t h a t t h e s h e l l was n o t c o m p l e t e l y s e a l e d . G i l l e s (1972) r e p o r t e d t h a t a l t h o u g h t h e b i v a l v e s G l y c y m e r i s g l y c y m e r i s and Myt i l u s e d u l i s were o s m o c o n f o r m e r s , s h e l l c l o s u r e m a i n t a i n e d h y p e r o s m o t i c i t y f o r up t o 96 h o u r s when a n i m a l s were p l a c e d f r o m f u l l s t r e n g t h sea water i n t o 25% SW. Slow s t e p w i s e a c c l i m a t i o n t o r e d u c e d s a l i n i t y removed t h e s h e l l c l o s u r e r e s p o n s e and r e s u l t a n t b l o o d h y p e r o s m o t i c i t y i n b i v a l v e s ( G i l l e s , 1972). F o r summer and w i n t e r a d a p t e d C. p e r s o n a , t h e o s m o t i c c o n c e n t r a t i o n of b l o o d was g r e a t e r t h a n t h a t o f u r i n e . T h i s t r e n d was t h e r e v e r s e o f b l o o d and u r i n e C l " c o n c e n t r a t i o n s . The C l " c o n c e n t r a t i o n of t h e b l o o d was l o w e r t h a n t h a t o f u r i n e o r sea w a t e r a t a l l s a l i n i t i e s t e s t e d . The h y p e r o s m o t i c i t y of t h e b l o o d must t h e r e f o r e be due t o t h e p r e s e n c e of o t h e r compounds, p o s s i b l y o r g a n i c . T h e r e i s some e v i d e n c e t h a t t h e s e compounds may be amino a c i d s o r p r o t e i n s p r e s e n t i n t h e haemolymph and n o t p r e s e n t i n u r i n e ( P o t t s and P a r r y , 1964; P i e r c e and G r e e n b u r g , 1971; 1972; 1973; 1976; Lange, ' 1972; Hoyaux e t a l , 1976; T u r g e o n , 1976; G i l l e s , 1979; Moran and T u l l i s , 1980; P i e r c e and Amende, 1981). In t h e p r e s e n t s t u d y , s e a s o n a l d i f f e r e n c e s i n b l o o d o s m o t i c c o n c e n t r a t i o n may have been t h e r e s u l t of h i g h e r 83 amino a c i d l e v e l s i n t h e b l o o d of summer a d a p t e d l i m p e t s . T h e r e was no e v i d e n c e of t h i s , however, as. amino, a c i d l e v e l s , were not measu r e d . I t i s u n l i k e l y t h a t t h e h y p o s o m o t i c u r i n e r e p o r t e d f o r w i n t e r a d a p t e d l i m p e t s i n 25% and 75% SW was due t o h y p o r e g u l a t i o n . As m e n t i o n e d p r e v i o u s l y , t h e h y p o i o n i c u r i n e from w i n t e r l i m p e t s i n 25% and 50% SW was p r o b a b l y n ot due t o h y p o r e g u l a t i o n , a s i t was n o t r e f l e c t e d i n t h e b l o o d o r t i s s u e C l " d a t a . U r i n e C l " from w i n t e r l i m p e t s i n 75% SW was i s o i o n i c t o t h e ambient medium, w h i l e t h e u r i n e o s m o t i c p r e s s u r e was h y p o s m o t i c . U r i n e o s m o t i c p r e s s u r e was not measured i n 50% SW f o r w i n t e r l i m p e t s and, t h e r e f o r e , a c o m p a r i s o n w i t h u r i n e C l " c o u l d n o t be made. IV. The E f f e c t of S a l i n i t y on Water B a l a n c e F o o t M u s c l e T i s s u e Water C o n t e n t The m u s c l e water c o n t e n t of summer and w i n t e r a d a p t e d C. p e r s o n a was measured o v e r a s a l i n i t y r a n g e o f 25-125% SW ( F i g . 1 4 ) . Summer a d a p t e d l i m p e t s were a b l e t o r e g u l a t e m u s c l e water i n 50% and 75% SW, but n o t i n 25%, 100%, o r 125% SW. T i s s u e w ater c o n t e n t d e c r e a s e d i n 125% SW by 5%, and i n c r e a s e d i n 25% SW by t h e same amount. In a d d i t i o n , t w e n t y - f i v e p e r c e n t SW was o n l y h a l f a s c o n c e n t r a t e d a s t h e c o n t r o l s a l i n i t y , w h i l e 125% was 1.5 t i m e s more c o n c e n t r a t e d . T h i s i n d i c a t e d t h a t summer a d a p t e d a n i m a l s were b e t t e r a b l e t o s u r v i v e h i g h e r t h a n l o w e r 84 s a l i n i t i e s . A l t h o u g h l i m p e t s i n 25% SW and 125% SW showed h i g h m o r t a l i t y r a t e s a f t e r - 48- hour.s-., 50%, o f the?-ani-ma-1 s ii,n.. 1 25%:.SWv were d e a d compared t o 80% . i n .25% SW. In a d d i t i o n , l i m p e t s i n 100% SW showed no m o r t a l i t y o v e r t h e e n t i r e e x p e r i m e n t a l p e r i o d . W i n t e r a d a p t e d l i m p e t s u n l i k e summer a n i m a l s r e g u l a t e d t i s s u e w a t e r c o n t e n t c o m p l e t e l y i n 50-125% SW. A l t h o u g h some r e g u l a t i o n o c c u r e d i n 25% SW, i t was i n c o m p l e t e . S e a s o n a l d i f f e r e n c e s were a p p a r e n t i n 75-125% SW, w i t h w i n t e r v a l u e s 4-8% h i g h e r t h a n summer v a l u e s . I t s h o u l d be n o t e d t h a t even i n summer a d a p t e d a n i m a l s t h e t o t a l d i f f e r e n c e i n m u s c l e t i s s u e water c o n t e n t i n 25-125% SW was a t t h e most 12%. In w i n t e r a d a p t e d a n i m a l s t h i s d i f f e r e n c e was r e d u c e d t o 4%. S i m i l a r s e a s o n a l d i f f e r e n c e s i n t i s s u e w a ter c o n t e n t were r e p o r t e d by S t a a l a n d (1972) f o r N eptunea a n t i q u a . He d i s c o v e r e d t h a t t h e w a t e r c o n t e n t o f autumn w h e l k s was 2-4% h i g h e r t h a n t h a t of s p r i n g a n i m a l s o v e r a s a l i n i t y range of 40-110% SW. Webber ( 1 9 6 6 ) , and Webber and D e h n e l (1968b) f o u n d t h a t members of a m a r i n e p o p u l a t i o n o f A. scutum were u n a b l e t o r e g u l a t e f o o t m u s c l e water c o n t e n t i n 50-125% SW o v e r a 1 week e x p e r i m e n t a l p e r i o d . E s t u a r i n e l i m p e t s , however, were c a p a b l e of t i s s u e w a t e r r e g u l a t i o n under t h e same e x p e r i m e n t a l c o n d i t i o n s (Webber, 1966). These l i m p e t s were c o l l e c t e d d u r i n g t h e w i n t e r , t h e r e f o r e , no c o m p a r i s o n c o u l d be made w i t h summer a d a p t e d C. p e r s o n a i n t h e p r e s e n t s t u d y . I t i s unknown whether t h e r e g u l a t i o n shown by w i n t e r a d a p t e d A. scutum would s t i l l p e r s i s t i n summer a n i m a l s . M u s c l e w a t e r v a l u e s f o r e s t u a r i n e A. scutum were 84% and 82%, i n 50% SW and 125% SW, r e s p e c t i v e l y (Webber, 85 1966). However, m u s c l e v a l u e s f o r w i n t e r a d a p t e d C. p e r s o n a were 86.5% i n 50% SW., and 85.%» i-nv 125%. SW.. The* f a c t . that.;, vad.ue.s,.. f.o.r.,, C. p e r s o n a were l a r g e r t h a n e q u i v a l e n t v a l u e s f o r A. scutum, may have been due t o t h e l a r g e r e x t r a c e l l u l a r s p a c e r e p o r t e d i n t h e p r e s e n t s t u d y f o r C. p e r s o n a . T a r r (1977) measured t h e t i s s u e h y d r a t i o n o f C. d i g i t a l i s o v e r a s a l i n i t y r a n g e of 50-150% SW. I t i s unknown whether T a r r c o l l e c t e d t h e s e l i m p e t s d u r i n g t h e summer or w i n t e r months. However, t h e h o l d i n g t e m p e r a t u r e o f 13°C u s e d by T a r r ( 1 9 7 7 ) , was c l o s e r t o t h a t u s e d f o r summer a n i m a l s (17°C) t h a n w i n t e r l i m p e t s (5°C) i n t h e p r e s e n t s t u d y . The r e s u l t s o b t a i n e d were s i m i l a r t o t h o s e r e c o r d e d from summer a d a p t e d C. p e r s o n a . In 50-125% SW, t i s s u e h y d r a t i o n d e c r e a s e d from 84.5% t o 79% i n C. d i g i t a l i s ( T a r r , 1977), and from 84% t o 79% f o r C. p e r s o n a . T a r r (1977) d i d not measure t i s s u e w a t e r c o n t e n t i n 25% SW f o r Notoacmaea scutum or C. d i g i t a l i s , t h e r e f o r e , no c o m p a r i s o n can be made w i t h C. p e r s o n a i n t h e p r e s e n t s t u d y . However, T a r r (1977) s t a t e d t h a t volume r e g u l a t i o n i n C. d i g i t a l i s and N. scutum b r o k e down i n low s a l i n i t i e s . The water c o n t e n t of f o o t m u s c l e t i s s u e f r o m C. p e r s o n a showed a l a r g e amount of v a r i a t i o n even a f t e r a r e l a t i v e l y c o n s t a n t v a l u e had been r e a c h e d ( F i g s . 10, and 1 1 ) . T h i s v a r i a t i o n was more a p p a r e n t i n summer t h a n w i n t e r a d a p t e d l i m p e t s . A p o s s i b l e e x p l a n a t i o n f o r t h i s e f f e c t c o u l d i n c l u d e t h e movement of b l o o d back and f o r t h f r o m t h e f o o t m u s c l e t o t h e v i s c e r a . Brown (1964) r e p o r t e d t h a t i n t h e s n a i l , B u l l i a , b l o o d i s p a s s e d from t h e p e d a l s i n u s t o t h e v i s c e r a l s i n u s d u r i n g 86 r e t r a c t i o n o f t h e f o o t . A s i m i l a r mechanism i n C. p e r s o n a c o u l d have a l t e r e d t h e b l o o d volume o f t h e f o o t , m u s c l e enough, t o i n c r e a s e w ater c o n t e n t v a r i a t i o n . L i m p e t s removed from e x p e r i m e n t a l s a l i n i t i e s g e n e r a l l y showed c o n t r a c t i o n of t h e f o o t m u s c l e b e f o r e t i s s u e s amples were t a k e n . B l o o d Water C o n t e n t B l o o d w ater c o n t e n t i n C. p e r s o n a was r e l a t i v e l y c o n s t a n t , r a n g i n g from 961 g/kg b l o o d i n 25% SW t o 925 g/kg b l o o d i n 125% SW. T h e r e was no s i g n i f i c a n t s e a s o n a l d i f f e r e n c e i n b l o o d w ater c o n t e n t , a l t h o u g h w i n t e r v a l u e s were up t o 2.5% h i g h e r t h a n summer v a l u e s . Webber (1966) r e p o r t e d a b l o o d w ater c o n t e n t of 980 g/L of b l o o d f o r A. scutum. However, he gave no i n d i c a t i o n how t h e v a l u e was d e r i v e d , nor t o w h i c h s a l i n i t y i t was r e l a t e d . D e h n e l (1979) gave b l o o d w a t e r v a l u e s f o r t h e c r a b H e m i g r a p s u s  nudus of 935-880 g/kg i n s a l i n i t i e s r a n g i n g from 6-175% SW. He a l s o f o u n d t h a t b l o o d w ater c o n t e n t o f summer a d a p t e d c r a b s was s i g n i f i c a n t l y h i g h e r t h a n t h a t o f w i n t e r c r a b s . In t h e p r e s e n t s t u d y , t h e i n c r e a s e o f e x t r a c e l l u l a r volume w i t h s a l i n i t y d i s c u s s e d e a r l i e r was n o t r e f l e c t e d by t h e b l o o d w a ter r e s u l t s , a l t h o u g h b l o o d w a t e r d e c r e a s e d s l i g h t l y i n 100% and 125% SW. D e h n e l (1979) r e p o r t e d a s i m i l a r s i t u a t i o n i n H. nudus and p r o p o s e d t h e f o l l o w i n g e x p l a n a t i o n . In H. nudus, as s a l i n i t y was l o w e r e d and i o n c o n c e n t r a t i o n of m u s c l e and b l o o d d e c r e a s e d , t h e r e was a s t r o n g r e g u l a t i o n o f i o n s i n b o t h t i s s u e s . Water t h e r e f o r e moved from t h e e x t r a c e l l u l a r s p a c e i n t o 87 t h e m u s c l e t i s s u e . The f a c t t h a t t h e r e d u c t i o n o f w ater i n t h e e x t r a c e l l u l a r space-,.was,, not. r e f l e c t e d by. the. b l o o d , w ater may be a c h a r a c t e r i s t i c of a n i m a l s w i t h an open c i r c u l a t o r y s y s t e m . Even t h o u g h b l o o d movement i s a c c o m p l i s h e d v i a m u s c l e movement and v i c e v e r s a (Chapman, 1958), t h e m u s c l e and b l o o d s i n u s e s a r e s t i l l p h y s i o l o g i c a l l y s e p a r a t e d and t o t a l m i x i n g may not o c c u r ( D e h n e l , 1979). D e h n e l (1979, page 530) a l s o s t a t e d t h a t "... t h e r e may be a t i m e l a g between p e r i p h e r a l a r e a s and t h e main s i n u s e s of t h e c i r c u l a t o r y s y s t e m . " In t h e p r e s e n t s t u d y , C. p e r s o n a showed no r e g u l a t i o n of C l " i n b l o o d o r m u s c l e t i s s u e . However, as t h e c o n c e n t r a t i o n o f i n o r g a n i c i o n s i n t h e b l o o d d e c r e a s e d under h y p o s o m o t i c c o n d i t i o n s , t h e c o n c e n t r a t i o n of o r g a n i c s u b s t a n c e s may have i n c r e a s e d . Thus b l o o d water c o n t e n t w o u l d r e m a i n r e l a t i v e l y u nchanged. V. S e a s o n a l V a r i a t i o n In t h e e s t u a r i n e e n v i r o n m e n t n e a r V a n c o u v e r , B.C., t e m p e r a t u r e i n c r e a s e s and s a l i n i t y d e c r e a s e s d u r i n g t h e summer months, w h i l e t h e r e v e r s e t a k e s p l a c e d u r i n g w i n t e r . V e r n b e r g and S i l v e r t h o r n (1979) r e p o r t e d t h a t t h e o s m o r e g u l a t o r y a b i l i t y o f e s t u a r i n e i n v e r t e b r a t e s d e c r e a s e s under c o n d i t i o n s of h i g h t e m p e r a t u r e , as does t h e i r r ange of s a l i n i t y t o l e r a n c e . Under c o n d i t i o n s o f low s a l i n i t y and h i g h t e m p e r a t u r e , t h e r e f o r e , t h e s e a n i m a l s a r e s e v e r e l y s t r e s s e d . T e m p e r a t u r e a l s o i n f l u e n c e s m e t a b o l i c r a t e ; i n c r e a s i n g t e m p e r a t u r e r e s u l t s i n an i n c r e a s e i n m e t a b o l i c r a t e and v i c e v e r s a . I f m e t a b o l i c r a t e and 88 c o n s e q u e n t l y t h e r a t e of o s m o r e g u l a t i o n i n c r e a s e s , m a r i n e i n v e r t e b r a t e s ^ exposed, t o low s a l i n i t i e s may d i e b e f o r e . . n o r m a l s a l i n i t y c o n d i t i o n s r e t u r n . In an e s t u a r y i t would be a d v a n t a g e o u s f o r h i g h i n t e r t i d a l forms t o have l o w e r m e t a b o l i c r a t e s , t h u s , r e t a r d i n g t h e e f f e c t s o f low s a l i n i t y . D a v i e s (1966; 1967) r e p o r t e d t h a t h i g h l e v e l P a t e l l a v u l g a t a showed lower r e s p i r a t i o n r a t e s d u r i n g t h e summer months t h a n e i t h e r low l e v e l P.. v u l g a t a or P. a s p e r a . The same was t r u e f o r g r o w t h r a t e ( D a v i e s , 1967). T r a n s p l a n t e x p e r i m e n t s showed t h a t t h e m e t a b o l i c r a t e r e d u c t i o n was p h e n o t y p i c and n o t g e n o t y p i c . H i g h l e v e l P. v u l g a t a showed an i n c r e a s e i n m e t a b o l i c r a t e when moved t o a l o w e r t i d a l l e v e l d u r i n g t h e summer. S e g a l (1956a) d i s c o v e r e d s i m i l a r m e t a b o l i c r a t e r e d u c t i o n i n t h e l i m p e t Acmaea 1 i m a t u l a . L i m p e t s from t h e low i n t e r t i d a l had f a s t e r h e a r t r a t e s t h a n h i g h l e v e l l i m p e t s , a l t h o u g h a n i m a l s f r o m b o t h a r e a s r e d u c e d h e a r t r a t e d u r i n g t h e summer months. T r a n s p l a n t i n g low l e v e l l i m p e t s t o t h e upper i n t e r t i d a l r e s u l t e d i n a l o w e r i n g of h e a r t r a t e t o t h a t of o t h e r h i g h l e v e l l i m p e t s w i t h i n 29 d a y s . D a v i e s (1967) p r o p o s e d t h a t a l o w e r m e t a b o l i c r a t e d u r i n g summer would be a d v a n t a g e o u s t o a h i g h l e v e l l i m p e t b e c a u s e , f o o d r e q u i r e m e n t s would be r e d u c e d a t a t i m e when t h e r e was l e s s a l g a e p r e s e n t . He e x p l a i n e d t h a t t h e i n a b i l i t y of P. a s p e r a t o r e d u c e m e t a b o l i c r a t e may be a f a c t o r t h a t r e s t r i c t s t h i s l i m p e t t o t h e l o w e r i n t e r t i d a l e n v i r o n m e n t ( D a v i e s , 1967). A l t h o u g h no measurements of m e t a b o l i c r a t e were p e r f o r m e d on C. p e r s o n a d u r i n g t h e p r e s e n t s t u d y , i t i s not u n r e a s o n a b l e t o assume t h a t t h i s l i m p e t would show an a l t e r a t i o n i n m e t a b o l i c a c t i v i t y s i m i l a r t o t h a t 89 r e p o r t e d f o r o t h e r l i m p e t s . D u r i n g t h e summer a low m e t a b o l i c r a t e would r e d u c e f o o d r e q u i r e m e n t s , when a l g a e > was, less*, a v a i l a b l e i n t h e h i g h i n t e r t i d a l z o n e . In a d d i t i o n , a low m e t a b o l i c r a t e would e n a b l e t h e a n i m a l s t o r e s i s t t h e r a p i d c h a n g e s of s a l i n i t y p e r s i s t a n t d u r i n g t h e summer months. D u r i n g t h e p r e s e n t s t u d y , t h e p o p u l a t i o n of l i m p e t s i n t h e f i e l d d e c r e a s e d by a p p r o x i m a t e l y 50% o v e r t h e summer months. The o n l y i n d i v i d u a l s t h a t r e m a i n e d were r e l a t i v e l y s m a l l ; l e s s t h a n 3 cm. L a r g e i n d i v i d u a l s were o n l y p r e s e n t d u r i n g t h e w i n t e r months. A l a r g e number of empty s h e l l s i n t h e c o l l e c t i n g a r e a i n d i c a t e d t h a t l i m p e t m o r t a l i t y was p a r t i a l l y r e s p o n s i b l e f o r t h e d e c l i n e i n t h e summer p o p u l a t i o n . However, t h e r e i s a g r e a t d e a l o f e v i d e n c e t h a t i n d i c a t e s t h a t l i m p e t s m i g r a t e s e a s o n a l l y up and down t h e i n t e r t i d a l z o n e . L e w i s (1954) f o u n d t h a t t h e d i s a p p e a r a n c e o f h i g h l e v e l P. v u l g a t a i n June was due m a i n l y t o m o r t a l i t y , a l t h o u g h some l i m p e t s m i g r a t e d downward. B r e e n (1972) r e p o r t e d a h i g h summer m o r t a l i t y i n A. d i g i t a l i s . In a d d i t i o n , s e a s o n a l m i g r a t i o n s i n t h i s l i m p e t were upward i n f a l l , and downward d u r i n g s p r i n g and summer. Seapy and Hoppe (1973) f o u n d t h a t A. s t r i g a t e l l a m i g r a t e d f o u r t i m e s p e r y e a r , upwards i n s p r i n g and f a l l , and downwards d u r i n g summer and w i n t e r . In t h e p r e s e n t s t u d y , t h e p r e s e n c e o f many l o o s e b o u l d e r s a t t h e c o l l e c t i n g s i t e made i t d i f f i c u l t t o o b s e r v e any m i g r a t i n g b e h a v i o r i n C. p e r s o n a . However, c o n s i d e r i n g t h e n e g a t i v e l y p h o t o t a t i c b e h a v i o r o f t h i s s p e c i e s ( T e s t , 1945), t h e l a r g e r i n d i v i d u a l s p r o b a b l y moved d e e p e r under t h e r o c k s t o a v o i d h i g h t e m p e r a t u r e s i n summer. Due t o t h e p r e s e n c e of a 90 r e l a t i v e l y l a r g e e x t r a v i s c e r a l s p a c e , s m a l l l i m p e t s . s u r v i v e d e x p o s u r e n o t t o l e r a t e d by l a r g e r i n d i v i d u a l s . Im a d d i t i o n , , t h e s e a s o n a l d i f f e r e n c e s i n e x t r a c e l l u l a r volume, m u s c l e C l " and water c o n t e n t , and b l o o d and u r i n e o s m o t i c p r e s s u r e r e c o r d e d from C. p e r s o n a d u r i n g t h e p r e s e n t s t u d y , e m p h a s i z e t h e i m p o r t a n c e o f c o n d u c t i n g p h y s i o l o g i c a l s t u d i e s on a s e a s o n a l o r y e a r l y b a s i s . V I . E c o l o g i c a l I m p l i c a t i o n s The p h y s i o l o g i c a l r e s p o n s e s d i s c u s s e d i n t h e p r e s e n t s t u d y on C. p e r s o n a a r e p r o b a b l y n o t e f f e c t i v e enough t o e n a b l e t h i s l i m p e t t o s u r v i v e s h o r t t e r m s a l i n i t y f l u c t u a t i o n , a l t h o u g h t h e y would be more e f f e c t i v e on a l o n g t e r m b a s i s . C. p e r s o n a r e g u l a t e d t i s s u e water between 48 h o u r s and 1 week d u r i n g t h e w i n t e r months. However, d u r i n g a 12 hour t i d a l c y c l e l i m p e t s a r e e x p o s e d t o d e s i c c a t i n g c o n d i t i o n s d u r i n g one p a r t , and t o d i l u t e sea w ater f o r t h e o t h e r p a r t , e s p e c i a l l y i n summer. T h e r e f o r e , t h e r e must be p h y s i c a l o r b e h a v i o r a l mechanisms t h a t e n a b l e C. p e r s o n a t o s u r v i v e s h o r t t e r m (12 h o u r ) s a l i n i t y s t r e s s . A r n o l d (1957) r e p o r t e d t h a t t h e l i m p e t P a t e l l a v u l g a t a c l a m p e d i t s s h e l l t o t h e s u b s t r a t e when s p l a s h e d w i t h f r e s h w a t e r , and l i f t e d i t when s p l a s h e d w i t h sea w a t e r . He a l s o m e n t i o n e d t h a t t h e r e was a g r e a t e r t o l e r a n c e t o s p l a s h and r e d u c e d s a l i n i t y i n h i g h l e v e l l i m p e t s , t h a n low i n t e r t i d a l l i m p e t s . A r n o l d (1957; 1959) p r o p o s e d t h a t s a l i n i t y p e r c e p t i o n was by C l " r e c e p t o r s i n t h e c e p h a l i c t e n t a c l e s and m a n t l e f r i n g e 91 o f P. v u l g a t a . K o z l i t i n a ( 1 9 7 2 ) , and Y a r s l a v t s e v a and K a r p e n k o ( 1980) r e p o r t e d t h a t C. d o r s u o s a • and G-.» r a d i a t a , . c lamped to- the; s u b s t r a t e and s e a l e d t h e m a n t l e c a v i t y when e x p o s e d t o h i g h o r low s a l i n i t i e s . They a l s o f o u n d t h a t t h e h i g h i n t e r t i d a l C. d o r s u o s a was b e t t e r a b l e t o r e s i s t s i g n i f i c a n t s a l i n i t y r e d u c t i o n t h a n low l e v e l C. r a d i a t a • M c A l i s t e r and F i s h e r (1968) r e p o r t e d t h a t a l t h o u g h t h e f a l s e l i m p e t S i p h o n a r i a p e c t i n a t a was an o smoconformer, i t c o u l d s u r v i v e a s a l i n i t y r a n g e o f 30-130% SW due t o i t s a b i l i t y t o c l a m p t o t h e s u b s t r a t e . A n i m a l s t h a t d i d n o t a t t a c h o n l y s u r v i v e d a 65-130% SW s a l i n i t y r a n g e . S. p e c t i n a t a was u n a b l e t o s u r v i v e below 30% SW b e c a u s e t h e f o o t was so s w o l l e n i t c o u l d n o t g r i p t h e s u b s t r a t e p r o p e r l y ( M c A l i s t e r and F i s h e r , 1968). W o l c o t t (1973) s t u d i e d t h e e c o l o g y of s e v e r a l s p e c i e s of l i m p e t s i n c l u d i n g C. p e r s o n a . He f o u n d t h a t t h e h i g h l e v e l l i m p e t s A. d i g i t a l i s , A. p e r s o n a , and A. s c a b r a were c a p a b l e of f o r m i n g a mucous s h e e t between t h e s h e l l m a r g i n and t h e r o c k s u r f a c e t o r e d u c e d e s i c c a t i o n . T h i s r e s p o n s e was not seen i n t h e low e r l e v e l l i m p e t s A. scutum and A. p e l t a . A l l s p e c i e s showed a l i f t i n g o f t h e s h e l l a s a mechanism of e v a p o r a t i v e c o o l i n g when e x p o s e d a t low t i d e . T h i s r e s p o n s e d i s a p p e a r e d under t h e a p p l i c a t i o n of m i l d d r y i n g c o n d i t i o n s . T h e r e f o r e , under d e s i c c a t i n g c o n d i t i o n s l i m p e t s t e n d e d t o c o n s e r v e water r a t h e r t h a n use i t t o r e g u l a t e t e m p e r a t u r e ( W o l c o t t , 1973). M c A l i s t e r and F i s h e r (1968) f o u n d t h a t t h e f a l s e l i m p e t , S i p h o n a r i a  p e c t i n a t a , c o u l d s u r v i v e d e s i c c a t i o n f o r up t o 72 h o u r s i f t h e a n i m a l s were f i r m l y a t t a c h e d t o t h e s u b s t r a t e . S e g a l and D e h n e l 92 (1962) r e p o r t e d t h a t A. 1 i m a t u l a r a i s e d i t s s h e l l f r o m t h e s u b s t r a t e i n r e s p o n s e .tev.increased* ••t-empe*s;a.tur.,e.s-.,.Ev;apo.riation-.,of e x t r a v i s c e r a l w ater from t h e m a n t l e and n u c h a l c a v i t i e s p r o b a b l y s e r v e d t o c o o l l i m p e t s e x p o s e d o n . r o c k s u r f a c e s d u r i n g t h e day. However, p r o l o n g e d e v a p o r a t i o n o f e x t r a v i s c e r a l w ater r e s u l t e d i n e x t r e m e d e s i c c a t i o n , and e v e n t u a l d e a t h . S e g a l (1956b) p o s t u l a t e d t h a t t h e p e r c e n t a g e o f water l o s s o v e r t i m e would be l e s s i n l i m p e t s ( A. 1 i m a t u l a ) w i t h l a r g e e x t r a v i s c e r a l s p a c e s . D e h n e l (1978) f o u n d t h a t h i g h l e v e l C. p e r s o n a and C. p e l t a had l a r g e r e x t r a v i s c e r a l s p a c e s t h a n low l e v e l C. scutum . He a l s o r e p o r t e d t h a t s m a l l e r i n d i v i d u a l s had r e l a t i v e l y g r e a t e r e x t r a v i s c e r a l s p a c e s t h a n l a r g e r i n d i v i d u a l s . S h o t w e l l (1950) d i s c o v e r e d t h e same r e l a t i o n s h i p between e x t r a v i s c e r a l w ater ( s t o r a g e w a t e r ) and a n i m a l s i z e i n t h e l i m p e t s A. scutum, A. p e l t a , A. m i t r a , and A. d i g i t a l i s . In t h e p r e s e n t s t u d y , C. p e r s o n a was not o b s e r v e d c l a m p i n g f i r m l y t o t h e s u b s t r a t e i n any of t h e e x p e r i m e n t a l s a l i n i t i e s . Summer a n i m a l s were so d e h y d r a t e d i n 125% SW, and so s w o l l e n i n 25% SW, t h a t t h e y c o u l d n o t f a s t e n . p r o p e r l y t o t h e p l a s t i c c o n t a i n e r s . T h e s e l i m p e t s showed a h i g h m o r t a l i t y p o s s i b l y b e c a u s e t h e y were u n a b l e t o i s o l a t e t h e m s e l v e s t e m p o r a r i l y from t h e e n v i r o n m e n t . A slow s t e p w i s e a c c l i m a t i o n t o 25% and 125% SW may have r e d u c e d s a l i n i t y s h o c k , and t h e r e s u l t i n g m o r t a l i t y r a t e . Summer a n i m a l s i n 5 0 % - l 0 0 % SW and w i n t e r l i m p e t s i n a l l s a l i n i t i e s , a l t h o u g h a t t a c h e d f i r m l y t o t h e s u b s t r a t e , showed gaps between t h e s h e l l m a r g i n and t h e p l a s t i c c o n t a i n e r . A l l t h e s e a n i m a l s were t h u s . e x p o s e d d i r e c t l y t o s e a water and showed 93 no i l l e f f e c t s . In c o n c l u s i o n . , C o l l i s e l l a:.,, pe r son a i s an o s m o c o n f o r m e r and. does n o t r e g u l a t e C l " , as i s t y p i c a l , of o t h e r p r o s o b r a n c h g a s t r o p o d s . However, as w i t h o t h e r h i g h i n t e r t i d a l i n v e r t e b r a t e s t h i s l i m p e t i s c a p a b l e o f a r e m a r k a b l e d e g r e e o f t i s s u e and b l o o d w a t e r r e g u l a t i o n . In t h e f i e l d l i m p e t s p r o b a b l y s u r v i v e s h o r t t e r m s a l i n i t y f l u c t u a t i o n by c l a m p i n g t o t h e r o c k s u r f a c e , and l o n g t e r m s e a s o n a l change by r e g u l a t i n g t i s s u e water c o n t e n t . W o l c o t t (1973) r e p o r t e d t h a t A. p e r s o n a c o u l d s u r v i v e f o r up t o 42 h o u r s i n f r e s h w a t e r , a t i m e p e r i o d much l o n g e r t h a n a n o r m a l t i d a l c y c l e . T e s t (1945) and W o l c o t t (1973) f o u n d t h a t A. p e r s o n a was n e g a t i v e l y p h o t o t a c t i c , h i d i n g i n c r a n n i e s and u nder l e d g e s d u r i n g t h e day and f e e d i n g o n l y a t n i g h t . T h i s b e h a v i o r , t h e c l a m p i n g r e s p o n s e , p l u s t h e a b i l i t y t o s e a l t h e s h e l l w i t h mucous when e x p o s e d a t low t i d e , p r o b a b l y c o n t r i b u t e t o t h e a b i l i t y of C. p e r s o n a t o s u r v i v e an e s t u a r i n e h i g h i n t e r t i d a l h a b i t a t . 94 SUMMARY 1. The c h l o r i d e i o n c o n c e n t r a t i o n o f b l o o d , u r i n e , and f o o t m u s c l e t i s s u e f r o m C o l l i s e l l a p e r s o n a , has been m e a s u r e d i n s a l i n i t i e s r a n g i n g f r o m 25% t o 125% s e a water (100% SW = 480 mEq C l " / L ) . B l o o d and f o o t m u s c l e water c o n t e n t were d e t e r m i n e d o v e r t h e same s a l i n i t y r a n g e . In a d d i t i o n , b l o o d and u r i n e o s m o t i c p r e s s u r e was measured i n t h e c o n t r o l s a l i n i t i e s , 25% SW, and 125% SW. 2. B l o o d and u r i n e C l " were not r e g u l a t e d , b u t c o n f o r m e d t o t h e ambient s a l i n i t i e s , f o r b o t h summer and w i n t e r a d a p t e d l i m p e t s . B l o o d C l " was g e n e r a l l y h y p o i o n i c t o u r i n e and s e a wa t e r i n a l l s a l i n i t i e s t e s t e d . U r i n e C l " was i s o i o n i c w i t h s e a w a t e r , e x c e p t f o r w i n t e r a d a p t e d l i m p e t s i n 25% and 50% SW, where u r i n e was h y p o i o n i c . 3. F o o t m u s c l e water C l " was n o t r e g u l a t e d , but r e a c h e d a c o n s t a n t l e v e l a p p r o x i m a t e l y o n e - h a l f o f e q u i v a l e n t s e a w a t e r o r b l o o d C l " . I f m u s c l e C l " was e x p r e s s e d a s mEq/kg e x t r a c e l l u l a r w a t e r i n s t e a d o f mEq/kg m u s c l e t i s s u e , t h e n v a l u e s f o r b l o o d and m u s c l e C l " were s i m i l a r . S e a s o n a l d i f f e r e n c e s were a p p a r e n t i n 75%, 100%, and 125% SW, w i t h w i n t e r v a l u e s g r e a t e r t h a n summer v a l u e s by 20-25 mEq C l " / k g m u s c l e t i s s u e . 4. F o o t m u s c l e e x t r a c e l l u l a r volume was c a l c u l a t e d u s i n g b l o o d and m u s c l e C l " , and b l o o d w a t e r v a l u e s . E x t r a c e l l u l a r volume showed o n l y a 7% i n c r e a s e o v e r a s a l i n i t y r a n g e o f 50% t o 95 125% SW, f o r b o t h summer and w i n t e r a d a p t e d l i m p e t s . W i n t e r v a l u e s were an a v e r a g e of 7% g r e a t e r t h a n summer v a l u e s a t a l l s a l i n i t i e s t e s t e d . In 25% SW, e x t r a c e l l u l a r s p a c e was 11% h i g h e r t h a n t h a t c a l c u l a t e d f o r o t h e r s a l i n i t i e s . 5. The o s m o t i c p r e s s u r e o f b l o o d and u r i n e from summer and w i n t e r a d a p t e d l i m p e t s c o n f o r m e d t o s e a water o s m o t i c p r e s s u r e . Summer a n i m a l s had b l o o d s l i g h t l y h y p e r o s m o t i c , and u r i n e i s o s m o t i c t o s e a w a t e r . W i n t e r l i m p e t s had b l o o d h y p e r o s m o t i c t o 75% SW, and i o s o m o t i c i n 25% and 125% SW. U r i n e was h y p o s m o t i c t o 25% and 75% SW, and i s o s m o t i c i n 125% SW. F o r b o t h summer and w i n t e r l i m p e t s b l o o d was h y p e r o s m o t i c t o u r i n e . 6. The f o o t m u s c l e t i s s u e w a t e r c o n t e n t o f w i n t e r a d a p t e d l i m p e t s was c o m p l e t e l y r e g u l a t e d i n 50% t o 125% SW, and p a r t i a l l y r e g u l a t e d i n 25% SW,' a f t e r 1 week i n e x p e r i m e n t a l s a l i n i t i e s . Summer a n i m a l s o n l y r e g u l a t e d m u s c l e water i n 50% and 75% SW. W i n t e r a n i m a l s had a g r e a t e r f o o t m u s c l e w a t e r c o n t e n t t h a n summer a n i m a l s . 7. B l o o d water c o n t e n t was a l m o s t c o m p l e t e l y r e g u l a t e d , and showed o n l y a 4% d e c r e a s e o v e r a s a l i n i t y r a n g e from 25% t o 125% SW. T h e r e was no s i g n i f i c a n t s e a s o n a l d i f f e r e n c e i n b l o o d w a t e r c o n t e n t , a l t h o u g h w i n t e r v a l u e s were s l i g h t l y h i g h e r t h a n summer v a l u e s . 8. The r e l a t i o n s h i p o f t h e p h y s i o l o g y o f C o l l i s e l l a p e r s o n a 96 t o i t s e c o l o g y i s d i s c u s s e d . 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