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Aspects of ionic regulation in Cancer magister, dana. Engelhardt, Frank Rainer 1970

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ASPECTS OF IONIC REGULATION IN CANCER MAGISTER, DANA by FRANK RAINER ENGELHARDT B . S c , U n i v e r s i t y of Western O n t a r i o , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA DECEMBER, 1970 In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain shall not be allowed without my written permission. Depa rtment The University of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT R e g u l a t i o n of c h l o r i d e , sodium, potassium, c a l c i u m , and magne-sium i o n s was determined f o r hypo- and h y p e r s a l i n e c o n d i t i o n s i n the crab, Cancer magister, from an e s t u a r i n e environment.-Animals from both summer and wi n t e r were examined. C h l o r i d e r e g u l a t i o n i n the b l o o d was h y p e r t o n i c i n d i l u t e s a l i n i t i e s and h y p o t o n i c i n c o n c e n t r a t e d s a l i n i t i e s , w i t h summer animals m a i n t a i n i n g a g r e a t e r g r a d i e n t i n the former and w i n t e r animals a g r e a t e r g r a d i e n t i n the l a t t e r . Sodium i n the b l o o d i s r e g u l a t e d h y p e r t o n i c a l l y i n a l l experimental s a l i n i t i e s , w i t h summer animals m a i n t a i n i n g a g r e a t e r g r a d i e n t . Blood potassium i s r e g u l a t e d h y p e r t o n i c a l l y i n d i l u t e s a l i n i t i e s , a pproaching i s o t o n i c i t y i n h y p e r s a l i n e media. Summer animals m a i n t a i n a g r e a t e r g r a d i e n t of potassium c o n c e n t r a t i o n . Blood c a l c i u m i s r e g u l a t e d h y p e r t o n i c a l l y i n a l l experimental s a l i n i t i e s , w i t h summer animals m a i n t a i n i n g a g r e a t e r g r a d i e n t i n d i l u t e s a l i n i t i e s and w i n t e r animals a g r e a t e r g r a d i e n t i n co n c e n t r a t e d s a l i n i t i e s . Magnesium i s r e g u l a t e d a t a pronounced hy p o t o n i c l e v e l i n the b l o o d over the e n t i r e e x p e r i m e n t al s a l i n i t y range, w i t h w i n t e r animals m a i n t a i n i n g the g r e a t e r g r a d i e n t . Major changes i n the a d a p t a t i o n of blood i o n i c c o n c e n t r a t i o n s occur w i t h i n a few hours of exposure t o the exp e r i m e n t a l s a l i n i -t i e s , w i t h h a l f of the f i n a l e q u i l i b r a t e d c o n c e n t r a t i o n v a l u e s a t t a i n e d by twelve hours. i i Animal weight was found t o bear no s i g n i f i c a n t r e l a t i o n s h i p t o the i o n i c r e g u l a t o r y a c t i v i t y observed. Renal involvement i n r e g u l a t i o n has been shown f o r a l l the i o n s , w i t h the p r o d u c t i o n of a u r i n e h y p e r t o n i c t o the b l o o d f o r c h l o r i d e and magnesium, and a u r i n e h y p o t o n i c t o the bloo d f o r sodium, potassium, and c a l c i u m . Renal r e g u l a t i o n was g r e a t e r i n w i n t e r animals f o r c h l o r i d e , and g r e a t e r i n summer animals f o r sodium and potassium. I o n i c r e g u l a t i o n by the g i l l s of summer and w i n t e r animals was i n v e s t i g a t e d by p o t e n t i a l d i f f e r e n c e measurements, and was suggested t o occur f o r a l l i o n s . C h l o r i d e may have been r e g u l a t e d by the a b s o r p t i o n from d i l u t e media and e x c r e t i o n i n t o c o n c e n t r a t e d media. Sodium may have been r e g u l a t e d by s e c r e t i o n i n t o d i l u t e media. The involvement of the g i l l i n potassium, c a l c i u m , and magnesium r e g u l a t i o n was i m p l i c a t e d . i i i TABLE OF CONTENTS Page A b s t r a c t i Tabl e of Contents ..... i i i L i s t of Tables v L i s t of F i g u r e s i v i Acknowledgements v i i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods 5 Source of r e s e a r c h animal 5 E x p e r i m e n t a l d e s i g n 7 H o l d i n g c o n d i t i o n s . * 7 Sampling of bloo d and u r i n e 10 Analyses of i o n c o n c e n t r a t i o n s 12 U/B r a t i o s 15 G i l l a c t i v i t y measurements 16 S t a t i s t i c a l a n a l y s i s of data 17 R e s u l t s 19 C h l o r i d e ., 19 Sodium 25 Potassium 27 C a l c i u m 29 Magnesium 31 Animal weight 33 G i l l a c t i v i t y measurements 33 D i s c u s s i o n 41 The e s t u a r i n e environment 41 S i z e 46 TABLE OF CONTENTS iv Page Sex 4 6 C h l o r i d e 47 Sodium 51 Potass ium 54 Ca l c i u m 56 Magnesium 58 Season , 60 Summary 64 L i t e r a t u r e C i t e d 67 • V LIST OF TABLES Tabl e Page 1 F i e l d i o n i c c o n c e n t r a t i o n s i n summer (August 17, 1969) and wi n t e r (February 24, 1970) i n the Roberts Banks area o f f the F r a s e r R i v e r . These v a l u e s a re averages of 12 c o l l e c t i n g s t a t i o n s i n summer and 10 c o l l e c t i n g s t a t i o n s i n w i n t e r (± S.D.) 8 2 Measured i o n i c c o n c e n t r a t i o n s i n exp e r i m e n t a l media 11 v i LIST OF FIGURES Figure Page 1 Map of c o l l e c t i n g area off the south arm of the Fraser River, B r i t i s h Columbia (from Department of Mines and Technological Surveys, Canada; No. 92 G) 6 2 Concentrations of 5 ions, expressed as a percen-tage of the medium, i n blood of summer and winter Cancer magister, as a function of time of exposure, i n hours"! to stock sea water 20 3 Blood chloride concentration, i n mEq/L, of summer and winter Cancer magister, as a function of time of exposure, i n hours, to experimental s a l i n i t i e s of 50 and 125% sea water .21 4 Chloride ion concentration, i n mEq/L, at 96 hours i n blood and urine of summer and winter Cancer  magister, as a function of medium concentration, as expressed i n per cent sea water 23 5 Urine - blood (U/B) r a t i o s of 5 ions of summer and winter Cancer magister, as a function of medium concentration, as expressed i n per cent sea water 24 ^ ^ ] 6 Sodium ion concentration, i n mEq/L, at 96 hours i n blood and urine of summer and winter Cancer  magister, as a function of medium concentration, as expressed i n per cent sea water 26 7 Potassium ion concentration, i n mEq/L, at 96 hours i n blood and urine of summer and winter Cancer  magister, as a function of medium concentration, as expressed i n per cent sea water 28 8 Calcium ion concentration, i n mEq/L, at 96 hours i n blood and urine of summer and winter Cancer  magister, as a function of medium concentration, as expressed i n per cent sea water 30 9 Magnesium ion concentration, in mEq/L, at 96 hours i n blood and urine of summer and winter Cancer magister, as a function of medium concentration, as expressed i n per cent sea water 32 v i i LIST OF FIGURES F i g u r e Page 10 P o t e n t i a l d i f f e r e n c e s , i n mV, of f n v i t r o g i l l p r e p a r a t i o n s of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as ex-pr e s s e d i n per cent sea water. a. In NaCl, where c o n c e n t r a t i o n i s based on sodium i o n . b. In Na2S04, where c o n c e n t r a t i o n i s based on sodium i o n 35 11 P o t e n t i a l d i f f e r e n c e s , i n mV, of i n v i t r o g i l l p r e p a r a t i o n s of summer and winter~C"ancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as ex-pr e s s e d i n per cent sea water. a. In Na-Acetate, where c o n c e n t r a t i o n i s based on' sodium i o n . b. In C h o l i n e - C l , where c o n c e n t r a t i o n i s based on c h l o r i d e i o n 37 12 P o t e n t i a l d i f f e r e n c e s , i n mV,^of i n v i t r o g i l l p r e p a r a t i o n s of summer and w i n t e r T a n c e r magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as ex-p r e s s e d i n per cent sea water. a. In KCI, where c o n c e n t r a t i o n i s based on potassium i o n . b. In CaCl2> where c o n c e n t r a t i o n i s based on c a l c i u m i o n .. 38 13 P o t e n t i a l d i f f e r e n c e s , i n mV, of i n v i t r o g i l l p r e p a r a t i o n s of summer and winter~C*ancer magister, as a f u n c t i o n of magnesium c o n c e n t r a t i o n , as ex-p r e s s e d i n per cent sea water, i n s o l u t i o n s of MgCl 2 40 14 Average p r e c i p i t a t i o n i n inches per month f o r the F r a s e r R i v e r B a s i n , from January 1969 t o August 1970, based on monthly m e t e o r o l o g i c a l r e p o r t s from A b b o t s f o r d , Hope, L y t t o n , Quesnel, Vancouver, and W i l l i a m s Lake, B r i t i s h Columbia 44 v i i i ACKNOWLEDGEMENTS I wish t o expend my thanks t o my s u p e r v i s o r , Dr. Paul A. Dehnel, f o r h i s support and c r i t i c a l e v a l u a t i o n of t h i s study. Thanks ar e a l s o due t o Dr. T.H. C a r e f o o t and Dr. J.E. P h i l l i p s f o r c o n s t r u c t i v e r e a d i n g of t h i s t h e s i s . I am in d e b t e d t o Mr. G. Major, whose v e s s e l s and equipment were used f o r the c o l l e c -t i o n of the r e s e a r c h animals. I wish t o acknowledge the r e c e i p t of f i n a n c i a l support from the N a t i o n a l Research C o u n c i l of Canada. I extend my g r a t i t u d e to my wif e , J e n n i f e r , f o r her enthusiasm and h e l p i n the p r e p a r a t i o n of t h i s t h e s i s . 1 INTRODUCTION Crustaceans i n h a b i t a wide range of environmental s a l i n i t i e s , r a n g i n g from f r e s h water t o g r e a t l y h y p e r s a l i n e c o n d i t i o n s . S i n c e c r u s t a c e a n s have probably evolved i n the sea, and t h e i r t i s s u e s consequently tend t o f u n c t i o n most e f f e c t i v e l y when t h e i r t o t a l i n t e r n a l c o n c e n t r a t i o n i s t h a t of sea water, some r e g u l a -t i o n of body f l u i d s becomes necessary i n a non-marine e n v i r o n -ment such as the e s t u a r i n e c o n d i t i o n . I o n i c c o n c e n t r a t i o n s of body f l u i d s have to be maintained at a l e v e l not too f a r removed from i n t r a c e l l u l a r c o n c e n t r a t i o n s , or e l s e w i t h i n a range where the c e l l s themselves can r e g u l a t e t h e i r c o n c e n t r a t i o n ( F l o r k i n , 1962 a, b; Shaw, 1958 a, b ) . Consequently, the c o n c e n t r a t i o n of these body f l u i d s may be f a r removed from the i o n i c concen-t r a t i o n s found i n the p a r t i c u l a r environmental medium. In the h y p o s a l i n e e s t u a r i n e environment, cr u s t a c e a n s can oppose reduced s a l i n i t i e s by means of r e d u c i n g the. p e r m e a b i l i t y of body membranes, as w e l l as by r e g u l a t i n g the i o n i c c o n c e n t r a t i o n s of body f l u i d s , e s p e c i a l l y those of the b l o o d . An i n c r e a s e d p r o d u c t i o n of u r i n e s e r v e s to r i d the body of excess water tending t o flow i n t o the animal from the h y p o s a l i n e environment. The l o s s of s a l t s accompanying such a u r i n e flow must be compensated f o r by t h e i r a c t i v e r e a b s o r p t i o n ( M a r t i n , 1957). There are s e v e r a l ways i n which r e l a t i v e s t a b i l i t y of body f l u i d i o n i c c o n c e n t r a t i o n i s a c h i e v e d i n an e s t u a r i n e c r u s t a c e a n . 2 - Many r e s e a r c h e r s , past and present, have found the g i l l t o be an i o n r e g u l a t i n g organ. Among these, Webb (1940) found t h a t i n the c r a b C a r c i n u s maenas, i o n i c r e g u l a t i o n i n the blood i s p a r t l y the r e s u l t of a c t i v e a b s o r p t i o n by the g i l l s of potassium, sodium, c a l c i u m , and c h l o r i d e a t a r a t e g r e a t e r than t h a t a t which they are l o s t by d i f f u s i o n . A c t i v e r e g u l a t i o n of sodium a t the g i l l s u r f a c e has a l s o been observed i n the blue crab C a l l i n e c t e s sapidus (Habas, 1965; Habas and P r o s s e r , 1963; Mantel, 1967), and i n the m i t t e n crab E r i o c h i e r s i n e n s i s (Koch, 1953; Koch et a l , 1954). Potassium r e g u l a t i o n has been observed to occur a t the g i l l s u r f a c e of the squat l o b s t e r Galathea  squamifera (Bryan, 1965). D a l l (1965) found that c a l c i u m r e -g u l a t i o n , as w e l l , occurs a t the g i l l s u r f a c e of metapenaeid shrimp. Other r e s e a r c h e r s have used c h l o r i d e i o n r e g u l a t i o n as a rough measure of the e n t i r e i o n t r a n s p o r t o c c u r r i n g i n the g i l l s , both f o r the uptake o f ^ i o n s , as i n Astacus l e p t o d a c -t y l u s and Astacus astacus ( B i e l a w s k i , 1964) and a c t i v e s e c r e t i o n of i o n s , as i n the ghost crab Ocypode a l b i c a n s ( F l e m i s t e r and F l e m i s t e r , 1951). The antennary gland, as an excr_e_t.ory_sys.t.em_c.ombined w i t h the u r i n a r y bladder, i s a b l e to produce a u r i n e h y p e r t o n i c t o the b l o o d w i t h r e g a r d t o such ions as magnesium and s u l p h a t e , and i n t h i s way maintains the blood c o n c e n t r a t i o n s of these ions h y p o t o n i c to the medium. T h i s has been shown, f o r example, i n f i d d l e r crabs, Uca pugnax and Uca p u g i l a t o r by Green, e_t a_l (1959), i n the squat l o b s t e r Galathea squamifera by Bryan (1965), and i n a shore crab, Pachygrapsus c r a s s i p e s by Gross (1959 a, b ) . 3 Two o t h e r s p e c i e s of shore crab, Hemigrapsus nudus and Hemigrap-sus oregonensis have been shown t o r e g u l a t e b l o o d magnesium l e v e l s by s e l e c t i v e u r i n a r y s e c r e t i o n (Dehnel, 1967; Dehnel r e g u l a t e sodium, potassium, and c a l c i u m i n some c r u s t a c e a n s , such as the l o b s t e r Homarus sp. (Burger, 1956 a,b; 1957), the brachyuran crabs Cardisoma sp., Sesarma sp., and Varuna sp. (Gross, e t a l , 1966), and the crab C a r c i n u s maenas ( R i e g e l and Lockwood, 1961; Webb, 1940). Other s i t e s have been found to be i n v o l v e d i n i o n i c r e g u l a t i o n by e s t u a r i n e c r u s t a c e a n s . In the l o b s t e r Homarus sp. (Burger, 1956 a; 1957), the gut i s e f f e c t i v e i n the r e g u l a t i o n of magne-sium and s u l p h a t e . The mid-gut glan d has been suggested by Green, et a l (1959) as having some r e g u l a t o r y f u n c t i o n i n Uca pugnax and Uca p u g i l a t o r . ) Seasonal d i f f e r e n c e s i n the degree of i o n i c r e g u l a t i o n of body f l u i d s have been demonstrated i n s e v e r a l s p e c i e s . For example, r e c e n t work on the shore crabs Hemigrapsus nudus and Hemigrapsus  oregonensis showed an i n t e r r e l a t i o n s h i p of season and c a l c i u m i o n r e g u l a t i o n , these animals being b e t t e r r e g u l a t o r s of t h i s i o n i n w i n t e r a t low s a l i n i t i e s (Dehnel, 1967; Dehnel and Care-f o o t , 1965). Temperature was found by Dehnel and C a r e f o o t (1965) t o a f f e c t the degree of magnesium r e g u l a t i o n by i m p a i r i n g i t a t h i g h temperatures of 25° C. and C a r e f o o t , 1965). The antennary gland a l s o s e r v e s t o T h i s study, i s undertaken to determine the occurrence and extent 4 of i o n i c r e g u l a t i o n i n the body f l u i d s of the crab Cancer magister, taken from an e s t u a r i n e h a b i t a t i n d i f f e r e n t seasons and exposed to a range of hypo- and h y p e r s a l i n e experimental c o n c e n t r a t i o n s . Seasonal d i f f e r e n c e s i n i o n i c r e g u l a t i o n are r e l a t e d to s e a s o n a l c o n c e n t r a t i o n and temperature changes i n the h a b i t a t . The antennary gland system and the g i l l s a r e examined as p o t e n t i a l s i t e s of r e g u l a t o r y a c t i v i t y . 5 MATERIALS AND METHODS Source of Research Animal: The animal used i n t h i s study was the P a c i f i c or common e d i b l e craib, Cancer magister Dana, which ranges from Unalaska, A l a s k a to Magdalena Bay, Lower C a l i f o r n i a (Schmitt, 1921). Male i n t e r m o l t C. magister were o b t a i n e d from an e s t u a r i n e en-vironment, the Roberts Banks area i n t h e ^ S t r a i t of Georgia o f f the mouth of the south arm of the F r a s e r R i v e r ( F i g . 1 ) . T h i s c o l l e c t i n g a r e a i s d e s i g n a t e d as f i s h i n g area 29 A and B by the Department of F i s h e r i e s of Canada. The animals were caught i n s t a n d a r d commercial crab t r a p s , s e t a t depths of 30 to 60 f e e t , mean t i d e l e v e l , u s i n g as b a i t dead f l o u n d e r and other ground f i s h caught i n the same a r e a . The t r a p s were l a i d f o r a d i s t a n c e of about 15 mi l e s a l o n g the coast, and animals were taken from the e n t i r e l e n g t h . C o l l e c t i n g was done i n both summer and w i n t e r i n the same a r e a . Summer animals were c o l l e c t e d on June 6 and August 17, 1969 and J u l y 30, 1970. Winter animals were c o l l e c t e d February 24, 1970. On August 17, 1969 and February 24, 1970, a sample of sea water was taken i n the area a t the depth of each t r a p f o r l a t e r i o n i c c o n c e n t r a t i o n measurements. At the same time, temperature measurements were o b t a i n e d from the same depth of water. Summer temperatures were found to be 14 - 15° C. and w i n t e r temperatures were 7.5 - 8° C. Both f i e l d and experimental c o n c e n t r a t i o n s were expressed as 6 FIGURE 1 Map of c o l l e c t i n g area o f f the south arm of the F r a s e r R i v e r , B r i t i s h Columbia (from Department of Mines and T e c h n o l o g i c a l Surveys, Canada; No. 92 G). S c a l e 1:250,000 * * * * - c o l l e c t i n g s i t e s 7 percentages of sea water, where 100% sea water has been d e t e r -mined i n t h i s l a b o r a t o r y t o have a s a l i n i t y of 31.88 ° / 0 0 and a c h l o r i n i t y of 17.65 ° / Q Q a t 25° C. (Dehnel, 1960, 1962, 1966). C o n c e n t r a t i o n s of the major ions i n 100% sea water, measured i n mEq/L, are then determined t o be 497 f o r c h l o r i d e , 433 f o r sodium, 10.13 f o r potassium, 25.6 f o r ca l c i u m , and 97.9 f o r magnesium. Ion a n a l y s e s of f i e l d sea water showed the c o n c e n t r a t i o n of c h l o r i d e , sodium, potassium, c a l c i u m , and magnesium to be lower i n the summer on August 17, 1969 than i n the w i n t e r on February 24, 1970 (Table 1 ) . A g r e a t e r v a r i a t i o n was presen t i n the summer samples than i n the w i n t e r samples. Co n v e r s i o n of c h l o r i d e c o n c e n t r a t i o n to t o t a l s a l i n i t y by the Knudsen eq u a t i o n ( S t r i c k l a n d and Parsons, 1968, p. 11) g i v e s a v a l u e f o r the summer c o n d i t i o n of 27.3 °/ and f o r the w i n t e r c o n d i t i o n of oo 29.4 ° / 0 0 , both below the s t a n d a r d v a l u e of 31.88 ° / 0 0 . These r e s u l t s a re s i m i l a r t o the a n a l y s e s g i v e n by Waldichuk, e_t a_l (1968 a, b) f o r the F r a s e r R i v e r e s t u a r y i n the Roberts Banks a r e a . E x p e r i m e n t a l Design: H o l d i n g C o n d i t i o n s A f t e r c o l l e c t i o n from the t r a p s , the crabs were t r a n s f e r r e d to h o l d i n g boxes, where they were kept i n damp sea weed (Fucus sp.) 8 TABLE 1 F i e l d i o n i c c o n c e n t r a t i o n s i n summer (August 17, 1969) and wi n t e r (February 24, 1970) i n the Roberts Banks area o f f the F r a s e r R i v e r . These v a l u e s a re averages of 12 c o l l e c t i n g s t a t i o n s i n summer and 10 c o l l e c t i n g s t a t i o n s i n w i n t e r (± S.D.). Ion Summer Winter mEq/L % sea water mEq/L % sea water c i " 426 + 37 86 458 + 14 92 Na+ 370 + 29 85 418 + 5 96 K + 8.2 + 0.9 81 8.3 + 0.2 82 C a + + • 16.1 + 1.8 63 18.3 + 0.4 71 M g + + 83.8 + 6.8 86 94.6 + 3.1 96 9 or i n sea water wetted e x c e l s i o r . They were then t r a n s f e r r e d t o l a b o r a t o r y h o l d i n g tanks, no longer than 3 - 7 hours f o l l o w i n g c o l l e c t i o n . In the l a b o r a t o r y , the crabs were kept i n a c o n t r o l l e d tempera-t u r e environment room, a t 15 ± 1° C. f o r summer and at 7.5 ± 1° C. f o r w i n t e r c o n d i t i o n s . No more than 12 animals were kept i n any one of the covered h o l d i n g tanks. These tanks had a f l o o r o area of 3.14 m and were f i l l e d with a t l e a s t 50 L. of a e r a t e d sea water. The water was changed d a i l y u n t i l a l l d i g e s t e d and u n d i g e s t e d food had been v o i d e d from the animals, then changed every second day. The sea water used f o r the e i g h t - d a y h o l d i n g p e r i o d was from B u r r a r d I n l e t ( F i g . 1). The c o n c e n t r a t i o n s of i o n s i n t h i s s t o c k sea water were found t o be e s s e n t i a l l y the same as those of the summer and w i n t e r f i e l d c o n d i t i o n s i n both r e l a t i v e and a b s o l u t e amounts. This i n d i c a t e d the s u i t a b i l i t y - of s t o c k sea water f o r use d u r i n g the h o l d i n g p e r i o d s . E x p e r i m e n t a l s a l i n i t i e s were determined as f o l l o w s : h y p o s a l i n e media were o b t a i n e d by d i l u t i o n of s t o c k sea water w i t h g l a s s d i s t i l l e d water, w h i l e h y p e r s a l i n e media were o b t a i n e d by the d i l u t i o n of a 200% a r t i f i c i a l sea water (based on 100% sea water of 31.88 ° / 0 0 s a l i n i t y ) . The 200% sea water was made up by the a d d i t i o n of NaCl, Na2S04, KC1, CaCl2, and MgCl2 t o s t o c k sea water (Barnes, 1954). A c o n c e n t r a t i o n range of 50, 75, 100, and 125% sea water was expected, but subsequent measurement showed t h i s t o be the case o n l y f o r c h l o r i d e , sodium, and magnesium. Potassium and c a l c i u m 1 0 v a r i e d from the expected range as a r e s u l t of r e l a t i v e l y lower va l u e s of these two ions i n s t o c k sea water, and, i n the case of c a l c i u m , wetness of the reagent s a l t , t o depress f i n a l e x p e r i -mental s a l i n i t y v a l u e s (Table 2) . A f t e r the eig h t - d a y h o l d i n g p e r i o d , 12 crabs were p l a c e d i n t o each of the experimental media, and t h i s time noted as 0 hours of exposure t o these s a l i n i t i e s . Subsequent unaccountable deaths occurred, but l e f t a t l e a s t 8 animals i n each s a l i n i t y , summer and w i n t e r , which s u r v i v e d the e n t i r e e x p e r i m e n t a l p e r i o d . 5 animals were a l s o used as c o n t r o l s f o r both summer and w i n t e r and were p l a c e d i n t o s t o c k sea water at the same time. Sampling of Blood and U r i n e Blood and u r i n e samples were taken a t 0, 6, 12, 24, 48, 72, and 96 hour time i n t e r v a l s from i n d i v i d u a l animals, p l a c e d a t 0 hours i n t o the experimental s a l i n i t i e s . The c o n t r o l s were sampled the same way to determine that the eig h t - d a y h o l d i n g p e r i o d was of s u f f i c i e n t l e n g t h t o a l l o w the animals t o maintain s t a b l e b l o o d ionic c o n c e n t r a t i o n s , and f u r t h e r , to t e s t any e f f e c t of the sampling o p e r a t i o n on blood i o n i c c o n c e n t r a t i o n s . A group of summer animals was sampled f i r s t , and f o r the a d d i -t i o n a l time p e r i o d s of 120, 144, 168, and 216 hours, to determine the time a t which r e g u l a t o r y a c t i v i t y a c h i e v e d a con s t a n t l e v e l . S i n c e t h i s p o i n t had o c c u r r e d by the 96 hour time i n t e r v a l , w i n t e r animals were sampled subsequently only t o the 96 hour time p e r i o d . 11 TABLE 2 Measured i o n i c c o n c e n t r a t i o n s i n ex p e r i m e n t a l media. Ion E x p e r i m e n t a l S a l i n i t i e s Medium 1 Medium 2 Medium 3 Medium 4 mEq/L % sea mEq/L % sea mEq/L % sea mEq/L % sea water water water water C l " 249 50 373 75 497 100 621 125 N a + 217 50 325 75 433 100 542 125 K + 3.9 38 6.7 66 10.1 100 12.7 125 C a + + 8.8 34 14.3 56 20.8 81 26.8 105 M g + + 48.9 50 73.4 75 97.9 100 122.3 125 12 Bloo d samples of approximately 1.5 ml. were drawn w i t h a No. 22 hypodermic needle and s y r i n g e through the membrane prox i m a l t o the c o x o p o d i t e of the second, t h i r d , or f o u r t h p a i r s of p e r e i o -pods. U r i n e samples were o b t a i n e d by l i f t i n g the operculum c o v e r ->, i n g the u r e t e r from the antennary g l a n d . T h i s l i f t i n g a c t i o n was u s u a l l y s u f f i c i e n t to s t i m u l a t e the flow of u r i n e from the u r e t e r , of which approximately 1.5 ml.were c o l l e c t e d by a s p i r a t i o n . U r i n e samples were drawn at the same time as the blood samples, and from the same ani m a l s . Any contamination of the u r i n e sample w i t h blood, as a r e s u l t of r u p t u r e of the ureter, was r e a d i l y de-termined s i n c e the contaminated sample l o s t i t s c l e a r appearance to take on a t u r b i d i t y due t o the c l o t t i n g of the b l o o d < contaminant. Such samples were d i s c a r d e d . A l i q u o t s of the ex-p e r i m e n t a l sea water were a l s o taken d u r i n g the sampling p e r i o d , to be a n a l y z e d as a check on the s p e c i f i c e x p e r i m e n t a l s a l i n i t i e s . Once a l l blood, u r i n e , and medium sampling a t the p a r t i c u l a r time p e r i o d was completed, i n d i v i d u a l samples were s e a l e d i n g l a s s v i a l s with p a r a f i l m (American Can Company) and r e f r i g e r a t e d a t 1.0 ± 0 . 5 ° C. t o await l a t e r a n a l y s e s . Wet weights of animals used i n the s a l i n i t y experiments were r e c o r d e d a t the end of the experimental p e r i o d . A n a l y s e s of Ion C o n c e n t r a t i o n s : Blood, u r i n e , and medium samples were t r e a t e d u n i f o r m l y i n the 13 a n a l y s e s . The samples were f i r s t a g i t a t e d u s i n g a Vo r t e x mixer, which s e r v e d t o break up the c l o t i n the bloo d samples. Subsequently, they were c e n t r i f u g e d a t 1600 x G. In the time r e q u i r e d f o r these o p e r a t i o n s , the samples a t t a i n e d the room temperatures n e c e s s a r y t o c a r r y out the d i l u t i o n s f o r the i o n a n a l y s e s . C h l o r i d e c o n c e n t r a t i o n s were determined as a measure of the r e g u l a t i o n f o r t h i s i o n , as w e l l as a rough i n d i c a t i o n of the t o t a l i o n i c r e g u l a t i o n o c c u r r i n g i n these animals s i n c e a l a r g e p r o p o r t i o n of the c a t i o n s " i n sea water are i n s o l u t i o n as t h e i r c h l o r i d e s a l t s (Pearse and Gunter, 1957). T o t a l c h l o r i d e con-c e n t r a t i o n was o b t a i n e d by coulometric-amperometric t i t r a t i o n w i t h s i l v e r i ons u s i n g 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 w i t h d i r e c t readout (Buchler Instruments, Inc.) and f o l l o w i n g the "method d e s i g n a t e d by Cotlove, et a_l (1958 a, b) and C o t l o v e (1963). A 100 "lambda a l i q u o t of a 100 mEq/L s o l u t i o n of NaCl was used t o s t a n d a r d i z e the instrument. T h i s standard, as w e l l as 100 lambda a l i q u o t s of 1:4 d i l u t i o n s of blood, u r i n e , and medium samples were t i t r a t e d a t hig h r a t e , .jc.oulometrical.1 y de l i v e r i n g about 0.25 m i c r o e q u i v a l e n t s of s i l v e r per second, i n a 4 ml. s o l u t i o n of 0.1 N HN0 3, 10% g l a c i a l a c e t i c a c i d , and 0.025% g e l a -t i n reagent (Buchler Instruments, I n c . ) . M u l t i p l i c a t i o n of the readout v a l u e by a . f a c t o r of f o u r then gave t o t a l c h l o r i d e c o n c e n t r a t i o n . Fresh p o l i s h i n g of the e l e c t r o d e s , a n a l y s i s of at l e a s t s i x s t a n d a r d samples at the s t a r t of each s e t of c h l o r i d e a n a l y s e s , as w e l l as r e s t a n d a r d i z i n g the instrument every twenty samples, i f r e q u i r e d , and use of i d e n t i c a l volumes of reagent 14, and sample i n each case a s s u r e d minimal v a r i a b i l i t y (± 1%) due to method. C o n c e n t r a t i o n s of sodium, potassium, and c a l c i u m ions were de-termined by flame photometry u s i n g a Z e i s s PF 5 flame photometer, and the method d e s c r i b e d by H o e f e r t (1962). At the s t a r t of the a n a l y s i s f o r each of the three i o n s , the instrument was c a l i b r a t e d u s i n g a blank s o l u t i o n , and a s e r i e s of s t a n d a r d s o l u t i o n s of NaCl, KCI, and CaCl2- Potassium and c a l c i u m c a l i b r a t i o n s o l u t i o n s were c o r r e c t e d f o r flame background by the a d d i t i o n of 1.5 mEq/L of NaCl. The v a l u e s o b t a i n e d f o r each s e t of c a l i b r a t i o n s were p l o t t e d as a c a l i b r a t i o n curve, from which readout v a l u e s of 1:250 d i l u t i o n s of b l o o d and u r i n e , as w e l l as medium, samples y i e l d e d c o n c e n t r a t i o n v a l u e s f o r sodium, potassium, or c a l c i u m i n mEq/L. These were m u l t i p l i e d by a f a c t o r of 250 to g i v e f i n a l i o n i c c o n c e n t r a t i o n s . Magnesium c o n c e n t r a t i o n s were found by d e t e r m i n i n g combined c a l -cium-magnesium c o n c e n t r a t i o n s by the E D T A - t i t r a t i o n method f o r d i v a l e n t c a t i o n s of Schwarzenbach, e_t a_l (1946). 2.0 ml. a l i q u o t s of the same 1:250 d i l u t i o n s used f o r flame photometry were p r e -—5 pared f o r t i t r a t i o n w i t h 5 x 10 M EDTA by the a d d i t i o n of three drops of NH 40H - NH 4C1 b u f f e r (pH 10) and f o u r drops of E r i o -chrome B l a c k T i n d i c a t o r (Hartman Leddon Company). Constant a g i t a t i o n and h e a t i n g of t h i s mixture to 38° C. was found to sharpen the t i t r a t i o n end p o i n t . The c o l o u r change was from a c l e a r magenta t o a c l e a r aquamarine. S i n c e EDTA a t pH 10 combines w i t h both c a l c i u m and magnesium, the volume of EDTA 15 s o l u t i o n used to b r i n g about the t i t r a t i o n end p o i n t i n d i c a t e d the t o t a l amount of both ions p r e s e n t . The c o n c e n t r a t i o n of magnesium alone was determined by s u b t r a c t i n g the c o r r e s p o n d i n g c o n c e n t r a t i o n v a l u e f o r c a l c i u m i n the same sample from the product of the number of ml. of EDTA r e q u i r e d , m u l t i p l i e d by a c o n v e r s i o n f a c t o r of 12.5, based on the 1:250 d i l u t i o n . In a l l of the above a n a l y s e s , o n l y the samples drawn from animals which s u r v i v e d the whole experimental p e r i o d were used. Analyses of blank s o l u t i o n s f o r c h l o r i d e , sodium, potassium, c a l c i u m , and magnesium ions u s i n g the same methods as d e s c r i b e d f o r blood, u r i n e , and sea water samples were c a r r i e d out to determine the l e a c h i n g of these ions from the glassware used i n the experiments. Sodium was found t o c o n t r i b u t e no more than 1% t o the f i n a l e x p e r i m e n t a l c o n c e n t r a t i o n v a l u e s , and thus was i g n o r e d . No i n d i c a t i o n of l e a c h i n g of the f o u r other i o n s c o u l d be found. U/B R a t i o s : Urine t o b l o o d (U/B) r a t i o s were c a l c u l a t e d u s i n g the c o n c e n t r a -t i o n v a l u e s o b t a i n e d by the above methods. U/B r a t i o s g r e a t e r than u n i t y were i n t e r p r e t e d as evidence of r e g u l a t o r y a c t i v i t y by the antennary gland by the p r o d u c t i o n of a h y p e r t o n i c u r i n e . T h i s may have o c c u r r e d by an a c t i v e s e c r e t i o n of ions i n t o the u r i n e , or by the r e a b s o r p t i o n of water from i t . Conversely, 16 U/B r a t i o s l e s s than u n i t y i n d i c a t e a hyp o t o n i c u r i n e , produced by s e l e c t i v e r e a b s o r p t i o n of an i o n from the u r i n e , or an a c t i v e e x c r e t i o n of water. G i l l A c t i v i t y Measurements: Both summer and w i n t e r c o n d i t i o n s were examined, w i t h summer animals c o l l e c t e d on J u l y 30, 1970, and w i n t e r animals c o l l e c t e d on February 24, 1970. These animals r e c e i v e d the same treatment as those used f o r d e t e r m i n a t i o n of blood and u r i n e c o n c e n t r a t i o n s . T h i s i n c l u d e d a h o l d i n g p e r i o d of e i g h t days, and 96 hours of exposure t o the exp e r i m e n t a l c o n c e n t r a t i o n s , at which time the p o t e n t i a l d i f f e r e n c e measurements were c a r r i e d out. I n d i v i d u a l g i l l s , 3 cm. i n le n g t h , measured from the d i s t a l end, of the f o u r t h t o the n i n t h g i l l p a i r s were used. Each g i l l was f l u s h e d and p e r f u s e d w i t h a t o t a l of 10 ml. of a s i n g l e s a l t s o l u t i o n , the c o n c e n t r a t i o n of i t s s p e c i f i c c a t i o n c o r r e s p o n d i n g t o the ex p e r i m e n t a l s a l i n i t y from which the crab had been removed. Sucrose was added t o b r i n g the t o t a l osmotic p r e s s u r e of these s o l u t i o n s e q u a l t o t h a t of the ex p e r i m e n t a l s a l i n i t i e s . The s i n g l e s a l t s o l u t i o n s used f o r both summer and w i n t e r animals were of NaCl, KC1, CaClg, and MgCl2* F u r t h e r , the g i l l s of summer animals were exposed t o s o l u t i o n s of c h o l i n e c h l o r i d e , where the c h l o r i d e c o n c e n t r a t i o n corresponded co the exp e r i m e n t a l s a l i n i t i e s , depending on the treatment g i v e n the c r a b . The g i l l was expected to be impermeable t o the c h o l i n e r a d i c a l (Mantel, 1967). The g i l l s of w i n t e r animals were a d d i t i o n a l l y t r e a t e d 17 w i t h s o l u t i o n s of sodium a c e t a t e and sodium s u l p h a t e . These c p a r t i c u l a r sodium sodium s a l t s were chosen s i n c e the s u l p h a t e and a c e t a t e r a d i c a l s were probably not t r a n s p o r t e d (Mantel, 1967; Shaw, 1960 a ) . In each case, the c o n c e n t r a t i o n of the sodium i o n was equal t o i t s r e s p e c t i v e e x p e r i m e n t a l s a l i n i t y . A l l Of these s o l u t i o n s were b u f f e r e d w i t h 0.2 M T r i s - H C l (Sigma Chemical Company) t o pH 7.4. Once f i l l e d w i t h the experimental s o l u t i o n , the s i n g l e g i l l p r e p a r a t i o n was a t t a c h e d t o a f r i t j u n c t i o n calomel e l e c t r o d e (No. 39071, Beckman Instruments, Inc.) and immersed i n a p p r o x i -mately 50 ml. of the same s o l u t i o n , which was a e r a t e d . The same type of e l e c t r o d e was p l a c e d i n t o t h i s e x t e r n a l s o l u t i o n to a c t as a r e f e r e n c e e l e c t r o d e i n the c i r c u i t . These e l e c t r o d e s were connected t o a pH-meter (Model 1019, Beckman Instruments, Inc.) t o measure p o t e n t i a l d i f f e r e n c e s i n m i l l i v o l t s . Assymetry p o t e n t i a l s measured f o r the e l e c t r o d e s were s u b t r a c t e d from the p o t e n t i a l d i f f e r e n c e s o b t a i n e d w i t h the g i l l p r e p a r a t i o n s . "5 t o 8 s e p a r a t e g i l l p r e p a r a t i o n s were measured f o r each s o l u -t i o n a t each s a l i n i t y and season. * A l l p o t e n t i a l d i f f e r e n c e measurements f o r g i l l s from summer crabs were c a r r i e d out at 15 t 1° C , and wi n t e r crabs a t 7.5 + 1° C. S t a t i s t i c a l A n a l y s i s of Data: A „ l a r g e p a r t of the p r e l i m i n a r y c a l c u l a t i o n of the data was 18 done w i t h the a i d of the computing s e r v i c e s of the B i o l o g y Data Centre a t the U n i v e r s i t y of B r i t i s h Columbia. S t a t i s t i c a l a n a l y s e s were c a r r i e d out u s i n g Student 's t - t e s t t o determine s i g n i f i c a n c e a t a 5% l e v e l (P < 0.05) of blood c o n c e n t r a t i o n s , u r i n e c o n c e n t r a t i o n s , U/B r a t i o s , and p o t e n t i a l d i f f e r e n c e s , as w e l l as of 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 concen-t r a t i o n s , U/B r a t i o s , and p o t e n t i a l d i f f e r e n c e s . 19 RESULTS S i n c e blood c o n c e n t r a t i o n v a l u e s f o r c h l o r i d e , sodium, potassium, c a l c i u m , and magnesium ions a t 0 and 96 hours i n s t o c k sea water were s i g n i f i c a n t l y equal f o r both summer and w i n t e r crabs ( F i g . 2 ) , i t may be assumed that the e i g h t - d a y p r e - e x p e r i m e n t a l h o l d i n g p e r i o d was of s u f f i c i e n t l e n g t h f o r bloo d i o n l e v e l s t o s t a b i l i z e f o l l o w i n g the c o l l e c t i o n of the c r a b s . F u r t h e r , i t may be assumed that the sampling o p e r a t i o n s themselves had no s i g n i f i c a n t e f f e c t on bloo d i o n i c c o n c e n t r a t i o n s . S i n c e b l o o d i o n i c c o n c e n t r a t i o n s of summer C. magister a t t a i n e d a t 96 hours i n a l l s a l i n i t i e s remained s i g n i f i c a n t l y constant when compared wi t h the f i n a l measurement a t 216 hours, i t was assumed t h a t maximum r e g u l a t i o n of each i o n i n the b l o o d was reached by 96 hours. The change i n bloo d c o n c e n t r a t i o n of c h l o r i d e over the 96 hour exposure p e r i o d i n 50 and 125% sea water can be seen i n F i g u r e 3. P l o t t e d curves of s i m i l a r _ s h a p e were observed a l s o i n the c a t i o n s , and i n a l l the experimental s a l i n i t i e s . Blood c o n c e n t r a t i o n s f o r a l l ions had a t t a i n e d about h a l f of t h e i r 96 hour l e v e l of r e g u l a t i o n a f t e r 12 hours of exposure. C h l o r i d e : S i g n i f i c a n t r e g u l a t i o n of bloo d c h l o r i d e o c c u r r e d i n a l l of FIGURE 2 C o n c e n t r a t i o n s of 5 i o n s , expressed as a percentage of the medium, i n b l o o d of summer and w i n t e r Cancer magister, as f u n c t i o n of time of exposure, i n hours, to s t o c k sea water 1 7 5 • • -SUMMER -WINTER Cl No K Co Mg 1 5 0 Q u i 1 2 5 < a* 1 0 0 UJ U z o u 751 o O g 5 5 5 0 1 2 5 h 0 J I I L_ I I L_ 0 6 12 2 4 4 8 7 2 9 6 H O U R S 21 FIGURE 3 Blood c h l o r i d e c o n c e n t r a t i o n , i n mEq/L, of summer and wint e r Cancer magister, as a f u n c t i o n of time of exposure, i n hours, t o e x p e r i m e n t a l s a l i n i t i e s of 50 and 125% sea water. SUMMER WINTER 6 0 0 or LU E 5 5 0 < " 5 0 0 1 u z 8 4 5 0 1 2 4 0 0 1 u a O O 3 5 0 1 00 125% 3 0 0 50% 2 5 0 1 I I i 0 6 12 2 4 4 8 H O U R S 7 2 9 6 22 the e x p e r i m e n t a l s a l i n i t i e s , w i t h h y p e r t o n i c r e g u l a t i o n i n 50 and 75% sea water, and hypo t o n i c r e g u l a t i o n i n 100 and 125% sea water ( F i g . 4 ) , i n both summer and w i n t e r a n i m a l s . Summer and w i n t e r crabs were s i g n i f i c a n t l y d i f f e r e n t i n the degree of c h l o r i d e r e g u l a t i o n they maintained i n 50 and 100% sea water. Summer animals maintained the h i g h e r g r a d i e n t i n 50% — 46% more h y p e r t o n i c than the w i n t e r animals, and w i n t e r animals maintained the g r e a t e r g r a d i e n t i n 100% — 39% more h y p o t o n i c than the summer an i m a l s . S i n c e the c o n c e n t r a t i o n of c h l o r i d e i o n i n the u r i n e of both summer and w i n t e r animals i n 50% sea water was found not to d i f f e r s i g n i f i c a n t l y from t h a t of the c o r r e s p o n d i n g b l o o d c h l o r i d e , r e g u l a t i o n t o a c h i e v e the h y p e r t o n i c b l o o d c h l o r i d e a t t h i s e x p e r i m e n t a l s a l i n i t y i s by an e x t r a - r e n a l r o u t e . A U/B r a t i o e q u a l t o 1.0 a t t h i s s a l i n i t y ( F i g . 5) documents t h i s . When both summer and w i n t e r animals were exposed t o 75, 100, and 125% sea water, however, u r i n e c h l o r i d e was s i g n i f i c a n t l y h i g h e r than t h a t of the blood, and the 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 than 1.0, i n d i c a t i n g the involvement of the antennary g l a n d i n r e g u l a t i o n . I f the measurement of t o t a l c h l o r i d e may be assumed the sum of the r e g u l a t i o n of c h l o r i d e s a l t s , t h i s would i n d i c a t e that r e g u l a t i o n of o v e r a l l b l o o d i o n c o n c e n t r a -t i o n , i n any but the most d i l u t e s a l i n i t y , occurs at l e a s t i n p a r t i n the antennary g l a n d . Summer and w i n t e r animals were not found t o be s i g n i f i c a n t l y d i f f e r e n t i n t h i s , except f o r animals i n 100% sea water, where the summer U/B r a t i o was s i g n i f i c a n t l y l e s s than that of the wi n t e r , i n d i c a t i n g l e s s involvement of the antennary gland, a t l e a s t i n t h i s s a l i n i t y . FIGURE 4 C h l o r i d e i o n c o n c e n t r a t i o n , i n mEq/L, a t 96 hours i n b l o o d and u r i n e of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. 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 ) FIGURE 5 Urine - blood (U/B) r a t i o s of 5 ions of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. 6.0 2 0 " > v-^ -v 1.0 -1.2 h o •—J—I 1 1 I I I i i • 34 38 50 56 66 75 81 100105 125 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 ) 25 T h i s would have c o n t r i b u t e d t o the w i n t e r c h l o r i d e c o n c e n t r a t i o n i n the b l o o d of C. magister being more hypotonic than the summer. Sodium: Summer C. magister was observed t o be a hyper-regulator of sodium i n a l l of the e x p e r i m e n t a l s a l i n i t i e s , hypo- and h y p e r s a l i n e ( F i g . 6 ) . The w i n t e r animals were a l s o observed to r e g u l a t e h y p e r t o n i c a l l y t o a s i g n i f i c a n t degree i n 50, 75, and 125% sea water, w h i l e h y p o t o n i c i n 100%. T h i s l a s t r e s u l t was only b a r e l y s i g n i f i c a n t , and~thus may have been a b e r r a n t , p a r t i c u l a r l y i n view of the h y p e r t o n i c r e g u l a t i o n observed i n w i n t e r crabs a t the 125% s a l i n i t y . Summer crabs maintained a h i g h e r g r a d i e n t of sodium than w i n t e r animals, b e i n g 22, 67, and 38% more hyper-t o n i c i n 50, 75, and 125% sea water, r e s p e c t i v e l y . -Sodium—concentration i n t h e ~ u r i n e of summer animals was found to be s i g n i f i c a n t l y l e s s than t h a t of the c o r r e s p o n d i n g blood sodium i n a l l of the experimental s a l i n i t i e s , except a t 75%, where the r e s u l t s were not s i g n i f i c a n t . A c c o r d i n g l y , U/B r a t i o s ( F i g . 5) i n 50, 100, and 125% sea water were s i g n i f i c a n t -l y l e s s than u n i t y , i n d i c a t i n g a r e n a l involvement i n the r e g u -l a t i o n of h y p e r t o n i c blood sodium. In e x p e r i m e n t a l s a l i n i t i e s of 50 and 75%, the u r i n e sodium c o n c e n t r a t i o n i n w i n t e r animals i n d i c a t e d no s i g n i f i c a n t involvement of the antennary gland i n b l o o d sodium r e g u l a t i o n , and the U/B r a t i o s i n these s a l i n i t i e 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 1.0. At the 100 and 125% s a l i n i t y , however, the u r i n e sodium was lower than t h a t of the FIGURE 6 Sodium i o n c o n c e n t r a t i o n , i n mEq/L, a t 96 hours i n blood and u r i n e of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. 10o« 1 — i 1 J 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 ) 27 b l o o d , and U/B r a t i o s l e s s than 1.0 o c c u r r e d i n w i n t e r animals as w e l l . Thus, a t h i g h e r s a l i n i t i e s , the antennary gland of w i n t e r animals p l a y s a s i g n i f i c a n t r o l e i n the r e g u l a t i o n of bl o o d sodium, but t h i s may be l e s s e f f e c t i v e than t h a t of the summer animals s i n c e the U/B r a t i o of summer animals a t 100% i s s i g n i f i c a n t l y l e s s than t h a t of the w i n t e r animals a t t h i s s a l i n i t y . Potassium: Summer C. magister maintained potassium c o n c e n t r a t i o n s i n the b l o o d s i g n i f i c a n t l y h y p e r t o n i c i n 38, 66, and 100% sea water, becoming i s o t o n i c w i t h the medium i n 125% ( F i g . 7 ) . In w i n t e r c r a b s , b l o o d c o n c e n t r a t i o n s of potassium were h i g h e r than the ex p e r i m e n t a l s a l i n i t y o n l y i n 38 and 66% sea water, and hyper-t o n i c r e g u l a t i o n f a i l e d i n the 100 and 125% s a l i n i t i e s where the b l o o d became i s o t o n i c . In 38, 66, and 125%, the blood potassium of summer animals was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of the w i n t e r a n i m a l s . In 100% sea water, however, summer animals maintained the h y p e r t o n i c g r a d i e n t s i g n i f i c a n t l y h i g h e r , by 160%, than w i n t e r animals, e x e m p l i f y i n g a p o s s i b l y g r e a t e r a b i l i t y f o r h y p e r - r e g u l a t i o n i n the summer animals. Potassium c o n c e n t r a t i o n i n the u r i n e was always s i g n i f i c a n t l y lower than t h a t of the c o r r e s p o n d i n g blood, except f o r w i n t e r crabs a t 38%, when i t was the same. The U/B r a t i o s f o r both summer and w i n t e r animals ( F i g . 5) were s i m i l a r l y l e s s than u n i t y , except f o r w i n t e r animals a t 38%, when the r a t i o was 1.0. 28 FIGURE 7 Potassium i o n c o n c e n t r a t i o n , i n mEq/L, a t 96 hours i n blood and u r i n e of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. 1 7 K * SUMMER WINTER • BLOOD A URINE 0 15 cr LU Z 13 O 5 ii u z o u UJ o Z y 3 Q 1 7 o o 2 CO 38 66 100 125 MEDIUM CONCENTRATION (%SEAWATER) 29 Consequently, the antennary g l a n d was i n v o l v e d i n the h y p e r t o n i c r e g u l a t i o n of bloo d potassium observed. At h i g h e r e x p e r i m e n t a l s a l i n i t i e s , the a c t i v i t y of the antennary gland was not s u f -f i c i e n t t o ma i n t a i n a h y p e r t o n i c s t a t e , and bloo d c o n c e n t r a t i o n s approached i s o t o n i c i t y . Summer animals showed a s i g n i f i c a n t l y lower U/B r a t i o a t 66 and 100% sea water than w i n t e r a n i m a l s . T h i s , combined wi t h the f a c t t h a t the w i n t e r animals showed no antennary g l a n d a c t i v i t y i n 38% sea water w h i l e those of the summer c o n d i t i o n d i d , i n d i c a t e d t h a t the summer antennary g l a n d involvement i n the r e g u l a t i o n of blood potassium l e v e l s i s g r e a t e r i n a l l s a l i n i t i e s except 125% than that of the w i n t e r c o n d i t i o n . Calcium: C a l c i u m i o n i c c o n c e n t r a t i o n i n the b l o o d of both summer and w i n t e r C. magister was r e g u l a t e d s i g n i f i c a n t l y h y p e r t o n i c a l l y 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 i g . 8 ) . At 34%, the summer crabs maintained a s i g n i f i c a n t l y h y p e r t o n i c g r a d i e n t 46% g r e a t e r than t h a t of the w i n t e r animals at t h i s s a l i n i t y . T h i s c o n d i -t i o n became r e v e r s e d i n exp e r i m e n t a l s a l i n i t i e s of h i g h e r c a l c i u m i o n c o n c e n t r a t i o n , where the w i n t e r animals maintained the g r e a t e r g r a d i e n t , s i g n i f i c a n t l y more h y p e r t o n i c by 34% i n 56%, by 77% i n 81%, and by 28% i n 105% sea water. Thus, the w i n t e r animals seemed t o r e g u l a t e t o a g r e a t e r degree at these three h i g h e r s a l i n i t i e s , w h i l e the summer animals were b e t t e r r e g u l a t o r s i n 34% sea water. FIGURE 8 C a l c i u m i o n c o n c e n t r a t i o n , i n mEq/L, a t 96 hours i n blood and u r i n e of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. 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 ) 31 Involvement of the antennary gland i n the maintenance of the h y p e r t o n i c c a l c i u m l e v e l s observed above was i n d i c a t e d by u r i n e c a l c i u m 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 l e s s than those of the bloo d i n 105% f o r summer and 81 and 105% f o r wi n t e r animals, as i n d i -c a t e d by U/B r a t i o s s i g n i f i c a n t l y l e s s than 1.0 a t these s a l i -n i t i e s ( F i g . 5 ) . No e x p l a n a t i o n i s o f f e r e d f o r the a p p a r e n t l y a b e r r a n t h i g h u r i n e c o n c e n t r a t i o n observed i n summer animals a t 81%, r e s u l t i n g i n a U/B r a t i o g r e a t e r than u n i t y . In the two lower e x p e r i m e n t a l s a l i n i t i e s , the U/B r a t i o s were found to be s i g n i f i c a n t l y equal t o u n i t y , i n d i c a t i n g t h a t the anten-nary g l a n d was not i n v o l v e d i n the maintenance of h y p e r t o n i c "blood c a l c i u m a t these s a l i n i t i e s , but t h a t some e x t r a - r e n a l s i t e had t o be a c t i v e . With the e x c e p t i o n of the ab e r r a n t 81% r e s u l t , U/B r a t i o s of summer and w i n t e r animals were not s i g n i -f i c a n t l y d i f f e r e n t , nor, consequently, was the e f f e c t i v e r e g u -l a t o r y a c t i v i t y of t h e i r antennary g l a n d s . Magnesium: R e g u l a t i o n of ma.gnesium i n the blood of C. magister was the s t r o n g e s t of any of the io n s observed, as i l l u s t r a t e d by the l a r g e g r a d i e n t maintained between the blood and the experimental s a l i n i t y ( F i g . 9 ) . Both summer and wint e r animals r e g u l a t e d h y p o t o n i c a l l y , w i t h the w i n t e r animals being s i g n i f i c a n t l y b e t t e r h y p o r e g u l a t o r s f o r t h i s i o n i n a l l of the experimental s a l i n i t i e s . Winter animals kept t h e i r blood magnesium about one t h i r d of the experimental s a l i n i t i e s , w h i l e summer animals maintained t h e i r b l o o d magnesium c o n c e n t r a t i o n at about h a l f 32 FIGURE 9 Magnesium i o n c o n c e n t r a t i o n , i n mEq/L, a t 96 hours i n blood and u r i n e of summer and wi n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. 2 8 0 M g * * SUMMER WINTER • BLOOD V URINE W UJ E Z o z LU U z o u  tt Q z < o g 2 4 0 2 0 0 1 6 0 1 2 0 8 0 4 0 h 5 0 7 5 1 0 0 125 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 ) 33 • o f the e x p e r i m e n t a l s a l i n i t i e s . The g r e a t e s t degree of r e n a l ionic r e g u l a t i o n was f o r magnesium. In both summer and w i n t e r crabs, the u r i n e c o n c e n t r a t i o n remained much h i g h e r than t h a t of the blood, w i t h t h i s g r a d i e n t i n c r e a s -i n g w i t h i n c r e a s i n g s a l i n i t y , so t h a t a t 125% the antennary gland was approximately t h r e e times as a c t i v e i n the e x c r e t i o n of magnesium than a t 50%. The U/B r a t i o , always s i g n i f i c a n t l y g r e a t e r than 1.0, showed a s i m i l a r t r e n d ( F i g . 5 ) . No s i g n i f i -cant d i f f e r e n c e i n the degree of r e n a l magnesium r e g u l a t i o n was determined between summer and w i n t e r animals, which may have been the r e s u l t of the g r e a t e r v a r i a b i l i t y o f the u r i n e magne-sium c o n c e n t r a t i o n v a l u e s , as compared to t h a t of the bloo d v a l u e s . Otherwise, some other s i t e of r e g u l a t i o n was i n d i c a t e d to account f o r the g r e a t e r g r a d i e n t maintained i n w i n t e r animals. Animal Weight: No s i g n i f i c a n t r e l a t i o n s h i p s c o u l d be determined between the weight of i n d i v i d u a l animals and the degree of i o n i c r e g u l a t i o n maintained i n . . t h e i r b l o o d . G i l l A c t i v i t y Measurements: P o t e n t i a l d i f f e r e n c e s a c r o s s a g i l l s u r f a c e in. v i t r o were mea-sure d t o i n v e s t i g a t e the r o l e of the g i l l as an organ of i o n i c r e g u l a t i o n . The r e s u l t s o b t a i n e d by these methods were by no 34 means u n e q u i v o c a l s i n c e the p o t e n t i a l d i f f e r e n c e s observed, i n t e r p r e t e d as an i n d i c a t i o n of i o n movement from on s i d e of the g i l l t o the other, may have been the r e s u l t of exchanges of ions between the c e l l s of the g i l l e p i t h e l i u m and the i n s i d e or o u t s i d e medium, f o r i n s t a n c e . N o t w i t h s t a n d i n g the l a c k of c o n c l u s i v e evidence f o r r e g u l a t o r y involvement by the g i l l , these r e s u l t s a r e p r e s e n t e d as a p o s s i b l e i n d i c a t i o n of such r e g u l a t i o n , and as a p r e l i m i n a r y t o f u r t h e r work i n t h i s a r e a . Winter g i l l p r e p a r a t i o n s of C. magister p l a c e d i n t o s o l u t i o n s of NaCl, c o r r e s p o n d i n g i n s a l i n i t y t o 50, 75, and 100% sea water wi t h r e s p e c t to the sodium i o n , showed a s i g n i f i c a n t n e g a t i v e p o t e n t i a l on the i n s i d e , r e l a t i v e t o the o u t s i d e s o l u t i o n ( F i g . 10 a ) . G i l l s o b t a i n e d from summer animals a l s o showed such a n e g a t i v e p o t e n t i a l , but only a t 50%. In both summer and w i n t e r p r e p a r a t i o n s , the p o t e n t i a l d i f f e r e n c e decreased w i t h i n c r e a s e d sodium c h l o r i d e c o n c e n t r a t i o n , t o a p o i n t at 125%, where no s i g -n i f i c a n t p o t e n t i a l d i f f e r e n c e was observed. The summer and w i n t e r g i l l p r e p a r a t i o n s were not found to d i f f e r s i g n i f i c a n t l y i n t h e i r p o t e n t i a l d i f f e r e n c e r e a d i n g s . A f u r t h e r experiment u s i n g p r e p a r a t i o n s of w i n t e r crab g i l l s and c h l o r i d e - f r e e sodium s a l t s o l u t i o n s r e s u l t e d i n a s i g n i f i -cant n e g a t i v e p o t e n t i a l d i f f e r e n c e , i n s i d e r e l a t i v e t o the out-s i d e , i n s o l u t i o n s of 50, 75, and 100% sea water of Na 2S04. T n e p o t e n t i a l d i f f e r e n c e was not s i g n i f i c a n t l y d i f f e r e n t from z e r o a t 125% ( F i g . 10 b ) . A s i g n i f i c a n t n e g a t i v e p o t e n t i a l was found a l s o u s i n g 50 and 100% s a l i n i t i e s of sodium a c e t a t e , and of the 3 5 FIGURE 10 P o t e n t i a l d i f f e r e n c e s , i n mV, of i r i v i t r o g i l l p r e p a r a t i o n s of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. a. In NaCl, where c o n c e n t r a t i o n i s based on sodium i o n . b. In Na2SC«4, where c o n c e n t r a t i o n i s based on sodium i o n . a) I NaCl -25 -20 -15 -10 ~ -5 SUMMER WINTER P ^  0.05 50 75 100 125 MEDIUM CONCENTRATION (% SEA WATER) 36 same p o l a r i t y as above. The pot e n t i a l difference readings ob-tained f o r 75 and 125% solutions of sodium acetate 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 zero (Fig. 11 a). Using solutions of choline chloride and summer g i l l preparations, a s i g n i f i c a n t negative pot e n t i a l difference, inside r e l a t i v e to the outside, resulted with the 50% s a l i n i t y s o l u t i o n . In 125%, a s i g n i f i c a n t p o s i t i v e p o t e n t i a l difference resulted, insid e r e l a t i v e to the outside. In 75 and 100% s a l i n i t i e s , the potentials were not s i g n i f i c a n t (Fig. 11 b). When s i n g l e s a l t solutions of KCI were used, s i g n i f i c a n t p o t e n t i a l differences, also negative on the inside, were obtained for g i l l preparations of both summer and winter animals, f o r a l l s a l i n i t i e s . Winter p o t e n t i a l differences were s i g n i f i c a n t l y greater i n the 38% s a l i n i t y , but no s i g n i f i c a n t differences were found i n the other concentrations (Fig. 12 a). Si m i l a r l y , s i g n i f i c a n t negative p o t e n t i a l differences were found i n s i n g l e s a l t solutions of CaC^ when the s a l i n i t y f o r calcium was 34% for summer and winter preparations, and 105% for the winter preparation only. No s i g n i f i c a n t differences could be determined between summer and winter preparations (Fig. 12 b). The p o t e n t i a l differences obtained using solutions of MgCl2 were negative on the inside, r e l a t i v e to the outside, and s i g n i -for summer preparations i n 50, 75, and 100% s a l i n i t i e s , and winter preparations i n a l l but 100%. The g i l l a c t i v i t y of summer and winter animals was determined to d i f f e r s i g n i f i c a n t l y only in FIGURE 11 P o t e n t i a l d i f f e r e n c e s , i n mV, of i n v i t r o g i l l p r e p a r a t i o n s of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. a. In Na-Acetate, where c o n c e n t r a t i o n i s based on sodium i o n . b. In C h o l i n e - C l , where c o n c e n t r a t i o n i s based on c h l o r i d e i o n . 1 U l U Z ui ac Ul u_ u_ O a) - 2 5 - 2 0 - 1 5 - 1 0 - 5 0 3 b) Z UJ 5 - 2 0 - 1 5 - 1 0 - 5 0 • 5 h Na-ACETATE CHOLINE-Cl s WINTER P ^ 0.05 5 0 7 5 1 0 0 1 2 5 SUMMER 5 0 7 5 1 0 0 125 MEDIUM CONCENTRATION (% SEA WATER) 38 FIGURE 12 P o t e n t i a l d i f f e r e n c e s , i n mV, of i i i v i t r o g i l l p r e p a r a t i o n s of summer and w i n t e r Cancer magister, as a f u n c t i o n of medium c o n c e n t r a t i o n , as expressed i n per cent sea water. a. In KC1, where c o n c e n t r a t i o n i s based on potassium i o n . b. In CaCl2, where c o n c e n t r a t i o n i s based on c a l c i u m i o n . 3 8 6 6 1 0 0 125 3 4 5 6 81 1 0 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 ) 39 the 75% s a l i n i t y , where t h a t of the w i n t e r was more n e g a t i v e ( F i g . 13). FIGURE 13 P o t e n t i a l d i f f e r e n c e s , i n mV, of i n v i t r o g i l l p r e p a r a t i o n s of summer and w i n t e r Cancer magister, as a f u n c t i o n of magnesium c o n c e n t r a t i o n , as expressed i n per cent sea water, i n s o l u t i o n s of MgCl 2• • 5 1 < • 5 0 7 5 1 0 0 125 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 ) 41 DISCUSSION The E s t u a r i n e Environment: E s t u a r i n e c r u s t a c e a n fauna i s d e r i v e d almost e x c l u s i v e l y from marine s p e c i e s , and as such has a m p l i f i e d g r e a t l y the c a p a b i l i -t i e s f o r i o n i c r e g u l a t i o n of i t s marine p r e c u r s o r , e n a b l i n g the e s t u a r i n e forms t o be e u r y h a l i n e . Although marine c r u s t a c e a n s are s t e n o h a l i n e , l i m i t e d t o a narrow range ofosmotic and i o n i c c o n c e n t r a t i o n s (Gross, 1957; Habas, 1965; Jones, 1941; Kalber and~Costlow, 1968; Mantel, 1967), they are capable of a c t i v e r e g u l a t i o n of those ions c o n t r i b u t i n g to the t o t a l i s o s m o t i c c o n c e n t r a t i o n of the b l o o d . G e n e r a l l y , i n c r e a s e d v a l u e s , r e l a -t i v e t o sea water, of sodium, potassium, and c a l c i u m and lowered v a l u e s of magnesium are found i n the blood. Sodium and c h l o r i d e c o n c e n t r a t i o n s are found t o v a r y l e a s t from the medium, wh i l e -calcium-tends t o b e - r e g u l a t e d - s t r o n g l y h y p e r t o n i c a l l y and magnesium s t r o n g l y h y p o t o n i c a l l y ( P r o s s e r , 1955). Extreme v a r i a -tion"Th" th"e~concentration "of any one i o n away from i t s optimum r e s u l t s i n d e c r e a s e d ^ s u r v i v a l as a r e s u l t of an a l t e r a t i o n of the compensatory e f f e c t s the ions have on each other when present at optimum c o n c e n t r a t i o n s (Pora, 1958, 1960). In a few cases where the b l o o d t o n i c i t i e s do not correspond to the above g e n e r a l scheme, concomitant changes i n the r e g u l a t i o n of other major i o n s o c c u r . For example, i n the l o b s t e r Homarus gammarus and the shrimp Nephrops no r v e g i c u s , potassium i s r e g u l a t e d hypo-t o n i c a l l y t o sea water (Robertson, 1949, 1960). T h i s can be r e l a t e d t o i n c r e a s e d c o n c e n t r a t i o n s of blood sodium, where the 42 h y p e r t o n i c sodium i s i n t u r n a compensation f o r extremely low h y p o t o n i c blood magnesium c o n c e n t r a t i o n s . T h i s same s o r t of s e l e c t i v e r e g u l a t i o n of i o n content i n the b l o o d of marine c r u s -taceans i s enhanced i n t h e i r r e l a t e d e s t u a r i n e s p e c i e s , or i n the e s t u a r i n e p o p u l a t i o n of a s p e c i e s n o r m a l l y c o n s i d e r e d a ma-r i n e form,as i s the case w i t h Cancer magister i n t h i s s t udy. E s t u a r i n e fauna i s a d d i t i o n a l l y m o d i f i e d i n that i t i s e u r y h a l i n e , a b l e to w i t h s t a n d wide environmental s a l i n i t y f l u c t u a t i o n s . I t seems t h a t p h y s i c a l f a c t o r s such as f l u c t u a t i n g osmotic and i o n i c c o n c e n t r a t i o n s are i n v o l v e d i n d e t e r m i n i n g the p o p u l a t i o n dynamics of a s p e c i e s (Kinne, 1967). Those s p e c i e s not a b l e t o compensate f o r the i n c r e a s e d m e t a b o l i c demands of l i f e i n low s a l i n i t i e s cannot s u r v i v e f o r extended p e r i o d s i n an e s t u a r i n e environment. S u r v i v a l depends, of course, not on the average environmental c o n d i t i o n , but on the most extreme c o n d i t i o n , and a d a p t a t i o n t o s e v e r a l extremes i s u s u a l l y i n v o l v e d ( P r o s s e r , 1955). " E s t u a r i e s , as areas of t r a n s i t i o n between the more s t a b l e e n v i -ronments of f r e s h water and the n e i g h b o u r i n g sea, may show r a d i c a l d e p a r t u r e s i n the r e l a t i v e and a b s o l u t e c o n c e n t r a t i o n s of s p e c i f i c ..ions. The F r a s e r R i v e r . i s . a goo.d_example i n t h a t , w h i l e i t has depressed v a l u e s f o r a l l the major sea water i o n s , c a l c i u m and potassium are depressed r e l a t i v e l y more than the other i o n s (Table 1 ) . The c a l c i u m and potassium r e g u l a t o r y a b i l i t i e s of C_. magister a r e consequently s t r e s s e d to a . g r e a t e r degree. Such f l u c t u a t i o n s are dependent on d i v e r s e g e o l o g i c a l f a c t o r s , such as r i v e r bed composition and the t e r r a i n through which the r i v e r flows, p i c k i n g up i t s s p e c i f i c l o a d of i o n s . A b s o l u t e changes i n the s a l i n i t i e s of e s t u a r i e s , as a r e s u l t of d i l u t i o n by f r e s h r i v e r water, may 43, v a r y s e a s o n a l l y as w e l l . P o s i t i v e c o r r e l a t i o n can be found between s e a s o n a l s a l i n i t y changes (Table 1) and p r e c i p i t a t i o n i n the F r a s e r R i v e r B a s i n as shown i n F i g u r e 14 (Vancouver Weather O f f i c e , p e r s o n a l communication). Low s a l i n i t i e s i n the months of June, J u l y , and August are mainly the r e s u l t of the m e l t i n g of h i g h a l t i t u d e i c e and snow, d e p o s i t e d i n the w i n t e r months, c a u s i n g a g r e a t e r f r e s h water d i s c h a r g e by the F r a s e r R i v e r i n t o i t s e s t u a r y . Reduced p r e c i p i t a t i o n i n the months of February and March, as w e l l as reduced m e l t i n g i n the w i n t e r months, all o w s h i g h f i e l d s a l i n i t i e s i n February and March. S e v e r a l processes are i n v o l v e d i n the maintenance of b l o o d i o n s a t c o n c e n t r a t i o n s which d i f f e r from those of the medium. These i n c l u d e : 1) r e d u c t i o n i n the p e r m e a b i l i t y of the body s u r f a c e , 2) t o l e r a n c e a t the c e l l u l a r l e v e l of v a r i a t i o n s i n b l o o d con-c e n t r a t i o n , and 3) enhanced a b i l i t y t o t r a n s p o r t i n o r g a n i c i o n s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t a c r o s s the body s u r f a c e (Lock-wood, 1962). In the lower e s t u a r i n e s a l i n i t y , r e g u l a t i o n by the a c t i v e uptake or s e c r e t i o n of i o n s i s coupled w i t h p h y s i o l o g i c a l adjustment by l o w e r i n g the e f f e c t i v e g r a d i e n t t h a t has t o be maintained. T h i s reduces the m e t a b o l i c i n p u t r e q u i r e d to maintain optimum i o n c o n c e n t r a t i o n (Croghan, 1961). P o t t s (1954) c o n s i d e r s t h i s r e d u c t i o n as the s i n g l e most important means whereby an animal e n t e r i n g b r a c k i s h water can l e s s e n the s t r a i n on i t s r e g u l a t o r y mechanisms. I t i s p r o b a b l y t h i s requirement which becomes the l i m i t i n g f a c t o r to-determine the p e n e t r a t i o n of a s p e c i e s up the e s t u a r y . P e n e t r a t i o n down the e s t u a r y , toward 44 FIGURE 14 Average p r e c i p i t a t i o n i n inches per month f o r the F r a s e r R i v e r B a s i n , from January 1969 t o August 1970, based on monthly m e t e o r o l o g i c a l r e p o r t s from Abbotsford, Hope, L y t t o n , Quesnel, Vancouver, and W i l l i a m s Lake, B r i t i s h Columbia. 5 . 0 h 0 • i i i i . i i i i i » • i i i i i i i i J F M A M J J A S O N D J F M A M J J A 1 9 6 9 1 9 7 0 45 the sea ag a i n , does not seem t o be a l i m i t i n g f a c t o r , a t l e a s t i n some g r a p s o i d crabs (Barnes, 1967), i n d i c a t i n g t h a t e s t u a r i n e c r u s t a c e a n s do not n e c e s s a r i l y l o s e t h e i r a b i l i t y t o l i v e i n s e a water. C. magister r e a c t s s i m i l a r l y , b e i n g a b l e t o s u r v i v e i n 100% sea water. While o f t e n regarded as a s t e n o h a l i n e marine decapod (Jones, 1941), C. magister was found t o be e u r y h a l i n e i n t h i s study, r e -g u l a t i n g the i o n i c c o n c e n t r a t i o n s of i t s b l o o d i n experimental s a l i n i t i e s as low as 34% and as high as 125% sea water. P r e -l i m i n a r y s t u d i e s showed t h a t the s u r v i v a l of C. magister was much reduced i n ex p e r i m e n t a l s a l i n i t i e s of 25% and 150%, i n d i -c a t i n g t h a t the e f f e c t i v e r e g u l a t o r y range of t h i s crab i s between these two extremes. On the b a s i s of t h i s , and u s i n g the c l a s s i f i c a t i o n of e s t u a r i n e fauna proposed by C a r r i k k e r (1967), i t may be concluded t h a t t h i s decapod i s a e u r y h a l i n e marine s p e c i e s , e x t e n d i n g from the sea i n t o the upper d i l u t e reaches of an e s t u a r y , as d i s t i n c t from: 1) s t e n o h a l i n e marine, a b l e t o i n h a b i t o n l y the lower reaches of an est u a r y , such as Cambarus" v i r i l i s ( P r o sser, 1955), 2) e u r y h a l i n e marine migrants, „ a.ble_to move up the e s t u a r y through the e n t i r e m i xohaline range of s a l i n i t i e s b e f o r e r e t u r n i n g t o the sea, such as the b l u e crab C a l l i n e c t e s s apidus (Tan and van Engel, 1966), or 3) t r u e e s t u a -r i n e , i n the middle and lower s a l i n i t i e s of an es t u a r y , spending i t s .whole l i f e t h e r e , but so adapted t o the d i l u t e c o n d i t i o n s , t h a t they a r e not a b l e t o t o l e r a t e marine s a l i n i t i e s . An example o f t h i s i s the brachyuran decapod Rithropanopeus h a r r i s i i (Jones, 1941). 46 S i z e : No s i g n i f i c a n t c o r r e l a t i o n c o u l d be found i n C_. magister between i o n i c r e g u l a t i o n and the s i z e of the animal. T h i s s i t u a t i o n i s s i m i l a r t o t h a t found i n Hemigrapsus nudus and Hemigrapsus  oregonensis (Dehnel, 1959, 1960), and L i g i a o c e a n i c a and L i g i a  g r a n u l o s a (Todd, 1963). The l a c k of c o r r e l a t i o n may not be c o n c l u s i v e s i n c e a r e l a t i o n -s h i p between s i z e and s a l i n i t y optima may e x i s t . T h i s has been p o s t u l a t e d by Broekema (1941) f o r the e s t u a r i n e shrimp Crangon crangon, where on l y the younger animals were a b l e t o t o l e r a t e the most d i l u t e environments. Thus, C. magister i n the c o l l e c -t i n g a r e a may a l l have been of one s i z e range, determined by a p o s s i b l e s i z e dependent t o l e r a n c e of lower s a l i n i t i e s . The f i e l d c rab P a r a t e l p h u s a sp. shows a s i m i l a r r e l a t i o n s h i p (Pad-manabhanaidu and Ramamurthy, 1961). Sex: Although only male C. magister were used i n t h i s study, the r e l a -t i o n s h i p of sex of the animal and i t s body f l u i d r e g u l a t i o n deserves mention. Repeated o b s e r v a t i o n s i n d i c a t e t h a t the females of many s p e c i e s of c r u s t a c e a n s have reduced a b i l i t i e s of i o n i c r e g u l a t i o n . T h i s occurs i n C a r c i n u s maenas ( G i l b e r t , 1959 a ) , i n P a r a t e l p h u s a sp.(Padmanabhanaidu and Ramamurthy, 1961), and a l s o i n C a l l i n e c t e s s apidus (Tan and van Engel, 1966), where the females show reduced sodium r e g u l a t o r y a b i l i t y . T h i s has 47 been extended t o i n c l u d e reduced c h l o r i d e r e g u l a t o r y a b i l i t y i n female c r u s t a c e a n s , as i n C a r c i n u s maenas ( G i l b e r t , 1959 b ) . Examination of female C. magister may r e v e a l d i f f e r e n c e s i n t h e i r i o n r e g u l a t o r y a b i l i t i e s , as compared t o the male animals exa-mined i n t h i s study. C h l o r i d e : R e g u l a t i o n of c h l o r i d e i o n , both hyper- and h y p o t o n i c a l l y i n d i l u t e sea water and h y p o t o n i c a l l y i n c o n c e n t r a t i o n s of 100% sea water and higher, has been observed i n many c r u s t a c e a n s . Marine s p e c i e s m a i n t a i n t h e i r b l o o d c h l o r i d e l e v e l s c l o s e t o t h a t of the medium, r e g u l a t i n g o n l y s l i g h t l y above or below ambient c o n c e n t r a t i o n . Cancer pagurus, f o r i n s t a n c e , has a c h l o r i d e l e v e l 97% of medium c o n c e n t r a t i o n (Robertson, 1939), w h i l e the s p i d e r crabs Hyas araneus and Maia squinado m a i n t a i n a h y p e r t o n i c g r a d i e n t e q u i v a l e n t t o 102% sea water (Robertson, 1953). Measurement of c h l o r i d e c o n c e n t r a t i o n i s an i n d i c a t i o n of the osmotic c o n c e n t r a t i o n i n the animal, and, thus, of osmoregulatory a b i l i t y . T h i s i s t r u e f o r h y p e r s a l i n e and h y p o s a l i n e c o n d i t i o n s down t o about 50% sea water. In Penaeus s e t i f e r u s , Penaeus  az t e c u s , and Trachypenaeus s i m i l i s , f o r i n s t a n c e , the t o t a l i o n c o n c e n t r a t i o n accounts f o r 94 to 97% of the t o t a l osmotic p r e s s u r e i n the above s a l i n i t y range (McFarland and Lee, 1963). At s a l i n i t i e s l e s s than h a l f t h a t of sea water, the r o l e of c h l o r i d e s a l t s i n the maintenance of osmotic e q u i l i b r i u m decreases 48 but i s compensated by the the a d d i t i o n of amino a c i d s , e f f e c t i n g a Donnan e q u i l i b r i u m ( P o t t s and Parry, 1963, pp. 27-32). Thus, the measurement of c h l o r i d e c o n c e n t r a t i o n i n the b l o o d of C. m a g i s t e r i n the e x p e r i m e n t a l s a l i n i t y range was a measure of. t o t a l i o n r e g u l a t i o n of c h l o r i d e s a l t s , as w e l l as of osmoregu-l a t i o n . In s a l i n i t i e s below 100% sea water, C. magister r e g u l a t e s c h l o r i d e h y p e r t o n i c a l l y , as much as 138% of the e x p e r i m e n t a l s a l i n i t y i n 50%. As such, i t i s s i m i l a r t o the decapods Homarus ameri-canus (Burger, 1956 b, 1957) and Hemigrapsus nudus and Hemi-grapsus O r e g o n e n s i s (Dehnel, 1966), the amphipod Corophium  v o l u t a t o r (McLusky, 1968), the is o p o d Mesidotea entomon (Lock-wood and Croghan, 1957), and the stomatopod S q u i l l a empusa (Lee and McFarland, 1962). C o r r e l a t i n g w i t h the c h l o r i d e t o n i c i t y o f t h e i r marine a n c e s t r y , b r a c k i s h water c r u s t a c e a n s w i l l r e g u l a t e c h l o r i d e i n 100% sea water e i t h e r hyper- or h y p o t o n i c a l l y . £. magister does the l a t t e r , r e g u l a t i n g at about 93% of 100% sea water. In t h i s , i t i s s i m i l a r t o the crab Rithropanopeus h a r r i s i i (Smith, 1967) and the e s t u a r i n e shrimp Palaemon s e r r a t u s (Parry, 1954) and Metapenaeus monoceros (Panikkar and Viswanathan, 1948). T h i s c o n d i t i o n i s d i s t i n c t from t h a t observed i n the i n t e r t i d a l b r a c k i s h water c r u s t a c e a n s , such as L i g i a o c e a n i c a (Parry, 1953), Corophium v o l u t a t o r (McLusky, 1968) or Hemigrapsus nudus and Hemigrapsus oregonensis (Dehnel, 1966, 1967), which m a i n t a i n a h y p e r t o n i c b l o o d c h l o r i d e c o n c e n t r a t i o n i n 100% sea water. 49 H y p o t o n i c c h l o r i d e r e g u l a t o r y a b i l i t y i s o f t e n observed i n e s t u a r i n e c r u s t a c e a n s i n h y p e r s a l i n e media. For example, the shrimp Palaemon s e r r a t u s (Parry, 1954) r e a c t s t h i s way. £. ma-g i s t e r behaves s i m i l a r l y , r e g u l a t i n g i t s b l o o d c h l o r i d e hypo-t o n i c a l l y a t about 94% of the experimental s a l i n i t y of 125% sea water. Although Burger (1956 b, 1957) demonstrated t h a t Homarus ameri-canus i s not a b l e t o r e g u l a t e c h l o r i d e by u r i n a r y a c t i v i t y , t h i s a b i l i t y has been demonstrated i n s e v e r a l other c r u s t a c e a n s . In Hemigrapsus nudus, f o r i n s t a n c e , Dehnel (1966) found t h a t the antennary gland can e x c r e t e a h y p e r t o n i c u r i n e , 140% of the b l o o d c h l o r i d e c o n c e n t r a t i o n . The e s t u a r i n e Palaemon s e r r a t u s , as w e l l , produces a h y p e r t o n i c u r i n e (Parry, 1954). These s t u d i e s of C. magister have i l l u s t r a t e d t h a t t h i s crab has a s i m i l a r a b i l i t y t o c o n c e n t r a t e c h l o r i d e i n the u r i n e , at l e a s t i n a c o n c e n t r a t i o n range of 75 t o 125% sea water. E x c r e t i o n of a u r i n e more c o n c e n t r a t e d than the b l o o d i n 125% i s i n s t r u -mental i n keeping the bloo d c h l o r i d e h y p o t o n i c . At 50% sea water, the c o n c e n t r a t i o n of c h l o r i d e i n the u r i n e i s not s u f f i -c i e n t l y d i f f e r e n t from t h a t i n the bloo d t o i n d i c a t e antennary g l a n d a c t i v i t y , but i t a i d s the animal i n m a i n t a i n i n g a hyper-t o n i c b l o o d c h l o r i d e w i t h reduced l o s s of c h l o r i d e by way of the u r i n e . Examination of the f r e s h water c r a y f i s h A u s t r o p o t a -mobius p a l l i p e s and Oronectes v i r i l i s ( R i e g e l , 1963) shows t h a t they produce a h y p o t o n i c u r i n e w i t h r e s p e c t t o blood c h l o r i d e . T h i s may be an e x t e n s i o n of the p r o d u c t i o n of an i s o t o n i c u r i n e observed i n £. magister t o conserve c h l o r i d e f u r t h e r and a i d i n the maintenance of a h y p e r t o n i c b l o o d . In t h i s same paper, 50 R i e g e l a l s o i m p l i c a t e s the b l a d d e r component of the antennary g l a n d as b e i n g the s p e c i f i c t i s s u e i n v o l v e d i n the r e g u l a t i o n of u r i n a r y c h l o r i d e . The l a r g e amounts of u r i n a r y bladder t i s s u e observed i n the body c a v i t y of £. magister may f u n c t i o n s i m i l a r l y . Most of the r e g u l a t i o n of c h l o r i d e has been a t t r i b u t e d to the c r u s t a c e a n g i l l , p a r i c u l a r l y a c t i v e uptake i n d i l u t e s a l i n i t i e s (Burger, 1956 b; F l e m i s t e r and F l e m i s t e r , 1951; Webb, 1940). H i s t o l o g i c a l l y , the g i l l e p i t h e l i u m seems t o be a s e c r e t o r y type ( F l e m i s t e r , 1959), f i l l e d w ith m i t o c h o n d r i a l and osmio-p h i l i c m a t e r i a l s (Copeland, 1963, 1968). Measurements of the p o t e n t i a l d i f f e r e n c e between the i n s i d e of a r e g u l a t i n g g i l l and the o u t s i d e medium may p o s s i b l y suggest an a c t i v e t r a n s p o r t of i o n s i n C_. magister, with an uptake of c h l o r i d e i o n s from the c h o l i n e c h l o r i d e s o l u t i o n s a t d i l u t e s a l i n i t i e s and a p o s s i b l e s e c r e t i o n of c h l o r i d e to the o u t s i d e i n c o n c e n t r a t e d s a l i n i t i e s . E s s e n t i a l l y the same r e s u l t s were o b t a i n e d by Mantel (1967) w i t h f n v i t r o p r e p a r a t i o n s of C a l l i -n e c t e s "sapidus g i l l s . In C. s a p i d u s , c h l o r i d e seems to be t r a n s p o r t e d i n d e p e n d e n t l y of c a t i o n s such as sodium. Shaw (1960 a, b, c) has shown t h i s t o occur a l s o i n the c r a y f i s h A stacus p a l l i p e s , where c h l o r i d e uptake i n t o the g i l l s was i n d e -pendent of and about one t h i r d of the r a t e of sodium uptake. I t may be that shrimp and crabs show a d i f f e r e n t system of c h l o -3fi r i d e e x c r e t i o n , s i n c e C l f l u x measurements i n the shrimp Metapenaeus bennettae i n d i c a t e d c h l o r i d e to be e x c r e t e d e x c l u -s i v e l y by way of the gut, and not the g i l l s ( D a l l , 1967). 51 T h i s i s i n c o n t r a s t t o C. magister e x c r e t i n g c h l o r i d e i n hyper-s a l i n e c o n d i t i o n both by way of the antennary gland and the g i l l s . Croghan (1958 a, b) a l s o invokes the gut as a c h l o r i d e r e g u l a -t o r y organ i n Artemia s a l i n a , i m p l i c a t i n g i t as a s i t e of a c t i v e uptake of c h l o r i d e . T h i s may be o c c u r r i n g i n C_. magister as w e l l t o keep the blood h y p e r t o n i c i n d i l u t e s a l i n i t i e s . Sodium: L i k e most b r a c k i s h water c r u s t a c e a n s , C. magister r e g u l a t e s b l o o d sodium h y p e r t o n i c a l l y i n d i l u t e s a l i n i t i e s . £. magister main-t a i n s a l e v e l of sodium i n the bloo d 156% t h a t of the 50% e x p e r i -mental s a l i n i t y . The i n t e n s i t y of r e g u l a t i o n i s somewhat l e s s than t h a t observed a t t h i s s a l i n i t y f o r the shore crabs Pachy-grapsus c r a s s i p e s (Gross, 1959 a) and Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965). Regula-t i o n of sodium i s g r e a t e r than t h a t i n the e s t u a r i n e prawn P a l a e -mon s e r r a t u s , which r e g u l a t e s sodium at onl y 105% of the e x p e r i -mental 50% s a l i n i t y (Parry, 1954). In 100% sea water, sodium r e g u l a t i o n by C. magister i s much l i k e t h a t of most marine decapods (Burger, 1956 b; Robertson, 1939, 1949), wi t h a maintenance of the bloo d a t about 110% of the environmental c o n c e n t r a t i o n , a t - l e a s t i n animals from the summer c o n d i t i o n . Pachygrapsus c r a s s i p e s (Gross, 1958; P r o s s e r e_t a l , 1955), Hemigrapsus nudus (Dehnel, 1967; Dehnel and C a r e f o o t , 1965), Palaemon s e r r a t u s (Parry, 1954), and the i s o p o d L i g i a  o c e a n i c a (Parry, 1953) are a l l b r a c k i s h water c r u s t a c e a n s 52 e x h i b i t i n g the same s l i g h t degree of h y p e r t o n i c i t y of sodium i n 100% sea water. While C. magister shows agreement wi t h most other b r a c k i s h water s p e c i e s with r e s p e c t t o sodium r e g u l a t i o n i n sea water of 100% or l e s s , i n the h y p e r s a l i n e c o n d i t i o n of 125% i t i s unusual i n t h a t the b l o o d i s s t i l l h y p e r t o n i c at 107% of the medium c o n c e n t r a t i o n . T h i s was not found f o r any of the s p e c i e s above, where blood sodium c o n c e n t r a t i o n e i t h e r approached i s o -t o n i c i t y , as i n Hemigrapsus nudus and O r e g o n e n s i s (Dehnel, 1967; Dehnel and C a r e f o o t , 1965), or where h y p o r e g u l a t i o n of sodium o c c u r r e d , as i n Pachygrapsus c r a s s i p e s (Gross, 1958; P r o s s e r et_ a l , 1955) and Palaemon s e r r a t u s (Parry, 1954). The h y p e r t o n i c i t y of sodium i n C. magister may be c o r r e l a t e d w i t h the e x t e n s i v e degree of h y p o r e g u l a t i o n of magnesium o c c u r r i n g at t h i s s a l i n i t y of 125% sea water ( G i f f o r d , 1962; P r o s s e r , e_t a l , 1955). A s s o c i a t e d w i t h h y p e r t o n i c r e g u l a t i o n of sodium i n C. magister i s the p r o d u c t i o n of a u r i n e h y p o t o n i c t o the blood, p a r t i c u l a r l y at the 100% and 125% s a l i n i t i e s . The antennary gland i s the s i t e most commonly a t t r i b u t e d to c a r r y out sodium r e g u l a t i o n i n c r u s t a c e a n s . Measurement of u r i n e t o n i c i t y w i t h r e f e r e n c e to b l o o d sodium c o n c e n t r a t i o n has r e v e a l e d t h a t e s t u a r i n e c r u s t a c e a n s such as the amphipod Gammarus duebeni (Lockwood, 1961 a, b, 1965; S u t c l i f f e , 1967 a, b) and the shrimp Palaemon  s e r r a t u s (Parry, 1954) show a s i m i l a r c a p a c i t y f o r r e n a l sodium r e g u l a t i o n . I n t e r t i d a l c r u s t a c e a n s a l s o c a r r y out r e n a l r e g u l a t i o n of t h i s i o n , as shown i n Hemigrapsus nudus and orego-53 n e n s i s by Dehnel (1967) and Dehnel and C a r e f o o t (1965), and i n Pachygrapsus c r a s s i p e s by P r o s s e r , e_t a_l (1955). P. c r a s s i p e s shows a h y p o t o n i c u r i n e at h i g h s a l i n i t i e s (170% sea water) and, i n t e r e s t i n g l y , a h y p e r t o n i c u r i n e i n 50% sea water, d i s t i n c t from the antennary gland a c t i v i t y observed i n C. magister. T h i s may be an i n d i c a t i o n of b e t t e r a d a p t a t i o n t o d i l u t e s a l i n i t i e s on the p a r t of Pachygrapsus c r a s s i p e s , which maintains a much lower optimum bloo d sodium l e v e l by the p r o d u c t i o n of t h i s h y p e r t o n i c u r i n e than C. magister. G i l l a c t i v i t y i s commonly a s s o c i a t e d w i t h a h y p e r t o n i c blood sodium. An a c t i v e uptake of sodium by the g i l l epithelium i n d i l u t e media i s found i n the f r e s h water E r i o c h i e r s i n e n s i s (Koch, 1953, 1954; Koch and Evans, 1956; Koch, et a l , 1954). In the e u r y h a l i n e e s t u a r i n e migrant C a l l i n e c t e s s a p i d u s , as w e l l , sodium shows a net i n f l u x i n t o the g i l l i n d i l u t e s a l i n i t i e s . It i s p o s t u l a t e d t h a t i n t h i s animal the c h l o r i d e i n f l u x exceeds t h a t of sodium to c r e a t e the measured n e g a t i v e p o t e n t i a l d i f f e r -ences i n s i d e the g i l l and t o f a c i l i t a t e f u r t h e r sodium i n f l u x i n t h i s way (Habas, 1965; Habas and Prosser, 1963; Mantel, 1967). If the n e g a t i v e p o t e n t i a l d i f f e r e n c e s observed i n C. magister g i l l p r e p a r a t i o n s u s i n g N a 2 S 0 4 and Na-Acetate are i n d i c a t i v e of an outward movement of sodium from the g i l l , sodium r e g u l a -t i o n by the g i l l of t h i s crab would consequently not f i t i n t o the scheme proposed above f o r E r i o c h i e r s i n e n s i s and C a l l i n e c t e s s a p i d u s . F u r t h e r , s i n c e the p o s s i b l e t r a n s p o r t of sodium by the g i l l of C. magister occurs i n the absence of c h l o r i d e and other c a t i o n s , i t may be independent of them. T h i s was a l s o suggested by Shaw (1960 a, b) f o r Astacus p a l l i p e s . He suggested 54 a l s o t h a t when a c e t a t e and s u l p h a t e r a d i c a l s a re used i n the f o r m a t i o n of sodium s a l t s , i t i s p o s s i b l e that sodium i o n s ex-change f o r e i t h e r ammonium or hydrogen i o n s . The h y p o t h e s i s t h a t some s i t e of sodium r e g u l a t i o n other than the g i l l or antennary gland e x i s t s i n cr u s t a c e a n s f i n d s support i n the work of Mantel (1968) on Gec a r c i n u s l a t e r a l i s , where the f o r e g u t of t h i s l a n d crab may be a b l e t o r e g u l a t e sodium, moving i t i n t o and out of the b l o o d as needed. Potassium: Potassium i s h y p e r t o n i c i n C. magister i n 38, 66, and 100% sea water, maintained a t g r a d i e n t s of 174, 144, and 105% of the e x p e r i m e n t a l s a l i n i t i e s , r e s p e c t i v e l y . Pronounced hyper-t o n i c i t y i n t h i s animal at low s a l i n i t i e s can be r e l a t e d t o a r e l a t i v e l y lower potassium l e v e l i n the e s t u a r y (Table 1), s t r e s -s i n g a w e l l - d e v e l o p e d h y p e r t o n i c r e g u l a t i o n of t h i s i o n . The h y p e r t o n i c i t y of b l o o d potassium i n £. magister i s s i m i l a r t o t h a t found i n other e s t u a r i n e or l i t t o r a l c r u s t a c e a n s , such as i n Pachygrapsus c r a s s i p e s (Gross, 1959 a; Pr o s s e r , ert a l , 1955), i n the shrimp Palaemon s e r r a t u s (Parry, 1954), and i n the shore crabs Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965). In 100% sea water, C. magister i s capable of a s l i g h t degree of 55 - h y p e r - r e g u l a t i o n , but t h i s i s not comparable t o the pronounced h y p e r t o n i c i t y observed i n Pachygrapsus c r a s s i p e s (Gross, 1958), i n Palaemon s e r r a t u s (Parry, 1954), or i n s t r i c t l y marine decapods (Robertson, 1939, 1949, 1953). I t would seem t h a t G. magister f a l l s more i n t o a c a t e g o r y of i s o t o n i c i t y i n 100% sea water and above, s i m i l a r t o the shore crabs Hemi- grapsus nudus and oregonensis (Dehnel, 1967; Dehnel and Care-f o o t , 1965). The antennary g l a n d i n C. magister i s s t r o n g l y i n v o l v e d i n potassium r e g u l a t i o n i n a l l but the most d i l u t e s a l i n i t i e s , t o produce a u r i n e h y p o t o n i c to the b l o o d . While not a s > a c t i v e i n h y p e r t o n i c r e g u l a t i o n of the b l o o d as the f r e s h water crab Potamon n i l o t i c u s (Shaw, 1959) or c r a y f i s h ( R i e g e l , 1965), r e n a l involvement i s g r e a t e r than t h a t of marine c r u s t a c e a n s , i n p a r t i c u l a r the decapods Cancer pagurus, Homarus v u l g a r i s (Robertson, 1939, 1949), Homarus americanus (Burger, 1956 b ) , and G a l a t h e a squamifera (Bryan, 1965). I t i s comparable to t h a t shown by b r a c k i s h water c r u s t a c e a n s such as Palaemon s e r r a t u s (Parry, 1954), as w e l l as t h a t shown by the l i t t o r a l crab C a r c i n u s maenas ( R i e g e l and Lockwood, 1961; Webb, 1940). Using the f i n d i n g s of Shaw (1960 c) t h a t the uptake of c h l o r i d e from s o l u t i o n s of KC1 i s s l i g h t , or n o n - e x i s t e n t , t o i n t e r p r e t the n e g a t i v e p o t e n t i a l s observed i n the i r i v i t r o g i l l s of C. magister, an i n d i c a t i o n of an outward movement of potassium through the g i l l epithelium may e x i s t . Potassium t r a n s p o r t , i f t h i s i s what has been observed i n the g i l l p r e p a r a t i o n s , may be independent of the presence of other i o n s , as i n d i c a t e d by 56 • Harvey and Nedergaard (1964) f o r sodium independent a c t i v e t r a n s p o r t of potassium i n the C e c r o p i a sp. midgut. Calcium: H y p e r t o n i c r e g u l a t i o n of b l o o d c a l c i u m i n an e s t u a r i n e c r u s t a -cean such asC. magister may be expected from the reduced c a l c i u m l e v e l s found i n e s t u a r i n e waters, and i s a s s o c i a t e d w i t h the need f o r c a l c i u m i n the s e a s o n a l moult c y c l e (Robertson, 1937). Only i n the i n t e r m o u l t phase i s the c a l c i u m l e v e l a t a l l c o n s t a n t (Hayes, e_t a_l, 1962). Blood c a l c i u m c o n c e n t r a t i o n s i n £ . magister are h y p e r t o n i c i n a l l experimental s a l i n i t i e s , a v e r a g i n g 192, 148, 117, and 113% of medium c a l c i u m c o n c e n t r a t i o n i n e x p e r i m e n t a l s a l i n i t i e s of 34, 56, 81, and 105% sea water, r e s p e c t i v e l y . Calcium tends t o be r e g u l a t e d h y p e r t o n i c a l l y i n " s t r i c t l y marine crabs as w e l l (Robertson, 1939, 1949; Gross, -1964). The a b i l i t y t o r e g u l a t e c a l c i u m demonstrated by C. magister over the e n t i r e experimental s a l i n i t y range corresponds almost e x a c t l y t o t h a t of Palaemon s e r r a t u s (Parry, 1954) and t o t h a t of the shore crabs Pachygrapsus c r a s s i p e s ( P r o s s e r , et a l , 1955) and Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965). H y p e r t o n i c c a l c i u m r e g u l a t i o n would seem t o be a common f e a t u r e of e s t u a r i n e c r u s t a c e a n s . Hypotonic r e g u -l a t i o n i n h i g h s a l i n i t i e s e x i s t s i n some c r u s t a c e a n s , as i n Pachygrapsus c r a s s i p e s ( P r o s s e r , et a l , 1955) and i n the f i d d l e r 57 crabs Uca pugnax and p u g i l a t o r (Green, et a l , 1959). The antennary gland i n C. magister seems t o be i n v o l v e d i n the maintenance of h y p e r t o n i c blood c a l c i u m l e v e l s o n l y i n the 81 and 105% s a l i n i t i e s . Here, a u r i n e h y p o t o n i c t o the b l o o d i s produced. S i m i l a r r e g u l a t i o n of c a l c i u m was observed i n the t e r r e s t r i a l crab Cardisoma armatum (deLeersnyder and H o e s t l a n d t , 1963), i n the l o b s t e r s Homarus americanus (Burger, 1956 a, b) and v u l g a r i s (Robertson, 1939), and i n the e s t u a r i n e shrimp Palaemon s e r r a t u s (Parry, 1954). The l i t t o r a l crabs Pachygrapsus  c r a s s i p e s (Gross, 1959 a) and Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965) do not show any r e n a l involvement i n c a l c i u m r e g u l a t i o n i n t h i s s a l i n i t y range, i n d i -c a t i n g t h a t an e x t r a - r e n a l mechanism must be a c t i v e , p o s s i b l y the g i l l s . These s p e c i e s do show hy p o t o n i c b l o o d r e g u l a t i o n , however, w i t h the p r o d u c t i o n of a h y p e r t o n i c u r i n e , i n h y p e r s a l i n e sea water. No h y p e r t o n i c u r i n e p r o d u c t i o n was observed i n C. magister, but may have been found i f the e x p e r i m e n t a l s a l i n i t y range were extended t o i n c l u d e more of the upper h y p e r s a l i n e c o n c e n t r a t i o n s . F i d d l e r crabs d e f i n i t e l y produce a h y p e r t o n i c u r i n e i n h i g h c a l c i u m c o n c e n t r a t i o n s (Green, e t a l , 1959), which c o r r e l a t e s w i t h their pronounced h y p o t o n i c b l o o d r e g u l a t i o n . S i n c e u r i n e c o n c e n t r a t i o n s of c a l c i u m i n C. magister are equal t o those of the blood i n 34 and 56% e x p e r i m e n t a l s a l i n i t i e s , the antennary g l a n d i s not i n v o l v e d i n the maintenance of hyper-t o n i c blood c a l c i u m a t these s a l i n i t i e s . The source of the c a l c i u m p r o d u c i n g t h i s h y p e r t o n i c i t y i s p r o b a b l y not the c a l c i f i e d 58 exoskeleton, s i n c e i n the l o b s t e r Homarus americanus the source f o r h y p e r t o n i c b l o o d c a l c i u m was found to be not the e x o s k e l e t o n , but d e r i v e d through g i l l a c t i v i t y . The g i l l i n the l o b s t e r a c t i v e l y takes up c a l c i u m when the i n t e r n a l s t a t e becomes hypo-c a l c a e m i c (Hayes, e£ a_l, 1962). I f the p o t e n t i a l d i f f e r e n c e s observed i n the i n v i t r o g i l l p r e -p a r a t i o n s of C. magister are i n d i c a t i v e of c a l c i u m t r a n s p o r t through the g i l l , some involvement of the g i l l i n the r e g u l a t i o n of b l o o d c a l c i u m i s i m p l i e d i n t h i s animal as w e l l , p a r t i c u l a r l y i n d i l u t e s a l i n i t i e s , where the p o t e n t i a l d i f f e r e n c e s were the l a r g e s t . Some other undetermined s i t e may be i n v o l v e d i n the r e g u l a t i o n of a h y p e r t o n i c b l o o d c a l c i u m l e v e l . Burger (1956 b), f o r i n s t a n c e , i n d i c a t e d t h a t d i v a l e n t i o n s entered by way of the stomach i n Homarus americanus. In the amphipod Corophium v o l u t a t o r (McLusky, 1970), as w e l l , a l a r g e p a r t of the i o n uptake i s by way of the gut. F u r t h e r e x p e r i m e n t a t i o n may c l a r i f y the s i t u a t i o n i n C. magister. Magnesium: H y p o r e g u l a t i o n of magnesium i s the most u n i v e r s a l f e a t u r e of i o n i c r e g u l a t i o n i n c r u s t a c e a n blood, A d e f i n i t e c o r r e l a t i o n seems t o e x i s t between the locomotory a c t i v i t y of a p a r t i c u l a r s p e c i e s and i t s b l o o d t o n i c i t y f o r magnesium. Those with low v a l u e s of magnesium are more a c t i v e and capable of f a s t e r move-ment than those w i t h high l e v e l s of magnesium (Lockwood, 1962, 1968 p. 11; McFarland and Lee, 1963; P o t t s and Parry, 1963 pp. 59 100-101; Robertson, 1949, 1953, 1960). T h i s l e a d s to the suppo-s i t i o n t h a t magnesium l e v e l s a r e r e l a t e d t o the speed of neuro-muscular impulse t r a n s m i s s i o n . C_. magister i s c o n s i d e r e d as a f a i r l y a c t i v e c r u s t a c e a n , being a scavenger and moving about r e a d i l y on the sea bottom, and c o r r e s p o n d i n g l y has a magnesium t o n i c i t y h a l f or l e s s than t h a t of the e x p e r i m e n t a l s a l i n i t i e s . C. magister i s s i m i l a r i n t h i s r e s p e c t t o f a i r l y a c t i v e c r u s t a -ceans such as the decapods Cancer pagurus, Homarus v u l g a r i s (Robertson, 1939) and gammarus (Robertson, 1960), which a l s o have magnesium l e v e l s a t l e a s t h a l f of the medium c o n c e n t r a t i o n . By way of comparison, the slower moving s p i d e r crabs Maia  squinado and Hyas araneus have h i g h e r blood magnesium l e v e l s , o n l y s l i g h t l y h y p o t o n i c t o the medium (Robertson, 1953). The antennary gland i s i n v o l v e d i n the r e d u c t i o n of b l o o d magnesium t o n i c i t y i n C. magister. T h i s i s a c h i e v e d by produc-t i o n of a h y p e r t o n i c u r i n e . The pronounced i n c r e a s e i n the t o n i -c i t y of u r i n e at 100 and 125% s a l i n i t y may i n d i c a t e a d i s p r o p o r -t i o n a t e l y g r e a t e r net i n f l u x i n these s a l i n i t i e s . The degree of magnesium r e g u l a t i o n c a r r i e d out by the antennary gland i n 100% sea water i s comparable t o t h a t found i n Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965), w h i l e l a r g e r than t h a t of the marine l o b s t e r s G alathea squamifera (Bryan, 1965) and Homarus v u l g a r i s (Robertson, 1949). I t i s , however, not as l a r g e as i n the e s t u a r i n e shrimp Palaemon s e r -r a t u s (Parry, 1954), which a l s o maintains a lower blood magnesium c o n c e n t r a t i o n than C. magister. I n t e r a c t i o n s of magnesium and sodium are p o s s i b l e , as i n d i c a t e d 60 by G i f f o r d (1962) i n Uca pugnax arid Ocypode a l b i c a n s . A s i m i l a r s i t u a t i o n may e x i s t i n C. magister. While C_. magister r e g u l a t e s f o r a h y p o t o n i c magnesium l e v e l , i t maintains i t s b l o o d sodium h y p e r t o n i c , so t h a t compensatory r e g u l a t i o n may be o c c u r r i n g here. P r o s s e r , et a l (1955) i n d i c a t e t h a t r e g u l a t i o n by the antennary glands t o produce a h y p e r t o n i c u r i n e w i t h r e s p e c t t o magnesium i n some way e f f e c t i v e l y reduces the c o n c e n t r a t i o n of sodium i n the u r i n e t o make i t h y p o t o n i c f o r sodium. Thus, i n c r e a s e d u r i n e h y p e r t o n i c i t y f o r magnesium i n 100 and 125% sea water c o r r e l a t e s with, and may be a f u n c t i o n of, i n c r e a s e d u r i n e h y p o t o n i c i t y of sodium at these s a l i n i t i e s . The g i l l of C. magister may a l s o be i n v o l v e d i n the r e g u l a t i o n of magnesium over the whole experimental s a l i n i t y range, and t h i s p o s s i b l y by an e x t r u s i o n of magnesium i o n s t o lower b l o o d t o n i c i t y . Other organs may c o n t r i b u t e t o the maintenance of h y p o t o n i c b l o o d magnesium. I n d i c a t i o n s of t h i s have been found i n C a l l i n e c t e s  sapidus and Ocypode a l b i c a n s ( G i f f o r d , 1962), as w e l l as the l o b s t e r Homarus americanus• In H. americanus, the c y c l e seems t o i n v o l v e an uptake of magnesium from the gut, w i t h an e x c r e -t i o n by way of the antennary gland (Burger, 1956 a, b ) . Season: The e f f e c t s of season seem to extend t o the r e g u l a t i o n of a l l 61 i o n s s t u d i e d i n £. magister. The process i n v o l v e s a c c l i m a t i o n , where t h i s term i s d e f i n e d as "any demonstrable compensatory change over some l e n g t h of t i m e " ( B u l l o c k , 1955). S a l i n i t y a c c l i m a t i o n i s observed i n C. magister. Temperature a c c l i m a t i o n i s i n v o l v e d as w e l l , s i n c e temperature v a r i e s with season and thus w i l l have a demonstrable e f f e c t on the degree of i o n i c r e g u l a t i o n c a r r i e d out by c r u s t a c e a n s . The e f f e c t s of tempera-t u r e on i o n i c r e g u l a t i o n i n C. magister have not been s t u d i e d independently, but may be i n f e r r e d s i n c e temperature v a r i e d w i t h season. C h l o r i d e i n w i n t e r b l o o d i s lower a t 50% and h i g h e r a t 100% than summer b l o o d . That i s , w i n t e r animals m a i n t a i n a l e s s e r g r a d i e n t at low s a l i n i t i e s , but a h i g h e r one a t h i g h s a l i n i t i e s . T h i s may be c o r r e l a t e d w i t h changes i n c h l o r i d e c o n c e n t r a t i o n s i n the summer and w i n t e r environments, where summer animals become a c c l i m a t e d t o lower s a l i n i t i e s and have a g r e a t e r a b i l i t y "to"maintain" h y p e r t b n i c i t y i n ~ t h e " l o w e s t ~ e x p e r i m e n t a l s a l i n i t y than w i n t e r animals. The r e v e r s e holds f o r the h i g h e r s a l i n i -t i e s . F i g u r e 3 shows t h a t summer animals have s i g n i f i c a n t l y _.lower blood c h l o r i d e c o n c e n t r a t i o n s than__the__aorresponding w i n t e r animals at 0 hours. The p r i n c i p l e i n v o l v e d here may be one suggested by Beadle (1943) f o r f r e s h water animals — the lower the blood c o n c e n t r a t i o n i n i t i a l l y , the lower the con-c e n t r a t i o n of b r a c k i s h water t o which they can be adapted. The converse seems t o h o l d f o r the h y p e r s a l i n e c o n d i t i o n s . Temperature e f f e c t s , as w e l l , may be i n v o l v e d i n c h l o r i d e r e g u l a -t i o n , s i m i l a r to the s i t u a t i o n found i n P o t a m o b i l i s f l u v i a t i l i s , a f r e s h water c r a y f i s h , where low temperatures decreased the 62 a b s o r p t i o n of c h l o r i d e (Wikgren, 1953). The r e s u l t s f o r c h l o r i d e r e g u l a t i o n i n C. magister a re not s i m i l a r t o those o b t a i n e d f o r summer and w i n t e r comparisons of r e g u l a t i o n i n Hemigrapsus  nudus (Dehnel, 1966) or i n C a l l i n e c t e s s apidus ( B a l l a r d and Abbott, 1969), where the wi n t e r b l o o d was maintained h y p e r t o n i c t o the summer blood a t a l l s a l i n i t i e s . S i m i l a r r e s u l t s were ob t a i n e d , however, f o r the c h l o r i d e g r a d i e n t s of u r i n e to blood, a l l showing g r e a t e r antennary gland a c t i v i t y i n the w i n t e r months. The c o n c e n t r a t i o n of sodium i s a l s o h i g h e r i n summer than i n w i n t e r C. m a g i s t e r . Again the s i t u a t i o n i s the r e v e r s e of t h a t found i n Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965). G r e a t e r h y p e r t o n i c i t y of summer sodium i n C. magister can be r e l a t e d d i r e c t l y t o a g r e a t e r degree of r e n a l r e g u l a t i o n of sodium, as i n d i c a t e d by a g r e a t e r g r a d i e n t maintained between b l o o d and u r i n e of summer animals as compared t o w i n t e r . Lower w i n t e r sodium v a l u e s may be r e l a t e d as w e l l t o a s i t u a t i o n found i n A s e l l u s a q u a t i c u s , where a f a l l i n tempera-t u r e caused a decrease i n bloo d sodium as a r e s u l t of decreased uptake (Lockwood, 1960; 1961 a, b; 1962). T h i s may occur a t the antennary gland or some other r e g u l a t o r y s i t e such as the gut. Potassium c o n c e n t r a t i o n i n summer and w i n t e r b l o o d d i f f e r e d only i n the 100% s a l i n i t y , where t h a t of the summer was the g r e a t e r , and the bloo d was h y p e r t o n i c as compared t o i s o t o n i c w i n t e r b l o o d . T h i s r e l a t e d t o g r e a t e r a c t i v i t y of the antennary gland of summer C. magister. 63 With r e g a r d t o the r e g u l a t i o n - o f calcium,—summer blo o d l e v e l s a r e h i g h e r o n l y i n the lowest e x p e r i m e n t a l s a l i n i t y of 34% sea water, w h i l e a t 56, 81, and 105% sea water, the c a l c i u m of w i n t e r animals was r e g u l a t e d more h y p e r t o n i c a l l y . T h i s may be a t t r i b u t a b l e t o a h i g h e r c a l c i u m c o n c e n t r a t i o n i n the w i n t e r f i e l d c o n d i t i o n , r e s u l t i n g i n a c c l i m a t i o n and a h i g h e r optimum b l o o d c a l c i u m l e v e l i n a l l but 34% sea water, where summer animals c a r r y out more e f f e c t i v e r e g u l a t i o n w h i l e t h a t of w i n t e r animals, a c c l i m a t e d to h i g h e r s a l i n i t i e s , breaks down. Higher w i n t e r c a l c i u m l e v e l s a r e a l s o observed i n Hemigrapsus nudus and oregonensis (Dehnel, 1967; Dehnel and C a r e f o o t , 1965). S i n c e 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 are o b t a i n e d f o r the r e g u l a t o r y a c t i v i t y of g i l l or antennary gland, some other s i t e such as the gut may be more e f f e c t i v e i n w i n t e r a n i m a l s . Magnesium r e g u l a t i o n i n C. magister i s s i m i l a r i n t h a t the w i n t e r animals m a i n t a i n a g r e a t e r e f f e c t i v e g r a d i e n t than those of the summer. The g r e a t e r degree of h y p o t o n i c i t y observed f o r the w i n t e r animals may be the r e s u l t of a g r e a t e r l o s s r a t e a t the body s u r f a c e , p o s s i b l y the g i l l . I t c o u l d not be —jde±ermined.as a f u n c t i o n — o f — i n c r e a s e d antennar-y—gland a c t i v i t y . A c c l i m a t i o n i s i n v o l v e d here, as w e l l , w i t h summer animals main-t a i n i n g a lower magnesium g r a d i e n t as a r e s u l t of a c c l i m a t i o n t o a lower environmental s a l i n i t y . The r e v e r s e s i t u a t i o n e x i s t s .for animals from the w i n t e r - c o n d i t i o n . 64 SUMMARY 1) C o n c e n t r a t i o n s of c h l o r i d e , sodium, potassium, c a l c i u m , and magnesium ions i n the blood and u r i n e of Cancer magister have been measured i n f o u r experimental s a l i n i t i e s : c h l o r i d e , sodium, and magnesium i n 50, 75, 100, and 125% sea water; potassium i n 38, 66, 100, and 125% sea water; c a l c i u m i n 34, 56, 81, and 105% sea water. 2) Blood and u r i n e i o n v a l u e s were determined f o r summer animals a t an experimental temperature of 15° C. and f o r w i n t e r o animals a t 7.5 C. 3) Major changes i n the a d a p t a t i o n of bloo d i o n i c c o n c e n t r a -t i o n s t o d i l u t e or c o n c e n t r a t e d s a l i n i t i e s occur w i t h i n a few hours of exposure, h a l f of the f i n a l e q u i l i b r a t e d concen-t r a t i o n a c h i e v e d by twelve hours. 4) Animal weight was found t o bear no s i g n i f i c a n t r e l a t i o n s h i p t o the i o n i c r e g u l a t i o n observed. 5) C h l o r i d e was determined t o be r e g u l a t e d h y p e r t o n i c a l l y i n h y p o s a l i n e media and h y p o t o n i c a l l y i n h y p e r s a l i n e media. Summer animals maintain a g r e a t e r g r a d i e n t i n d i l u t e s a l i n i t i e s , and a l e s s e r g r a d i e n t i n c o n c e n t r a t e d s a l i n i t i e s , than w i n t e r animals. R e g u l a t i o n a t 50% i s e x t r a - r e n a l . At h i g h e r s a l i n i t i e s , the antennary gland a c t i v e l y e x c r e t e s c h l o r i d e by way of a h y p e r t o n i c u r i n e . Renal r e g u l a t i o n i s g r e a t e r i n w i n t e r animals. 65 6) Sodium i s r e g u l a t e d h y p e r t o n i c a l l y i n the b l o o d a t 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 , w i t h summer animals m a i n t a i n i n g the g r e a t e r g r a d i e n t . Renal sodium r e g u l a t i o n occurs a t a l l s a l i -n i t i e s t o produce a h y p o t o n i c u r i n e . Winter animals show l e s s r e n a l a c t i v i t y than summer ani m a l s . 7) Potassium i s maintained h y p e r t o n i c i n d i l u t e s a l i n i t i e s , w ith summer animals m a i n t a i n i n g the g r e a t e r g r a d i e n t . The antennary glands a r e a c t i v e i n r e g u l a t i o n , p r o d u c i n g a hyp o t o n i c u r i n e . Summer animals show g r e a t e r r e n a l r e g u l a t i o n than w i n t e r a n i m a l s . 8 ) C a l c i u m i s r e g u l a t e d h y p e r t o n i c a l l y a t a l l medium concen-t r a t i o n s . Summer animals are the b e t t e r r e g u l a t o r s i n the most d i l u t e medium of 34% sea water, w h i l e w i n t e r animals a r e b e t t e r h y p e r - r e g u l a t o r s i n the h i g h e r s a l i n i t i e s . Except a t 34%, the antennary g l a n d a c t i v e l y r e g u l a t e s c a l c i u m t o produce a h y p o t o n i c u r i n e . 9) R e g u l a t i o n of magnesium i s s t r o n g l y h y p o t o n i c , a t about h a l f the medium c o n c e n t r a t i o n i n summer animals and one t h i r d the medium c o n c e n t r a t i o n i n w i n t e r animals. Renal involvement i n magnesium r e g u l a t i o n i s pronounced w i t h the p r o d u c t i o n of a h y p e r t o n i c u r i n e . Summer and w i n t e r animals showed no d i f f e r e n c e i n t h e i r degree of r e n a l r e g u l a t i o n . 10) Ion r e g u l a t o r y a c t i v i t y by the g i l l s of summer and w i n t e r animals was i n v e s t i g a t e d by p o t e n t i a l d i f f e r e n c e measurements of i n v i t r o g i l l p r e p a r a t i o n s u s i n g s i n g l e s a l t media f o r each 66 of the f i v e i o n s of t h i s study. C h l o r i d e i s suggested t o be r e g u l a t e d by a b s o r p t i o n a t 50% and s e c r e t i o n a t 125%. Sodium may be t r a n s p o r t e d outwards, e s p e c i a l l y i n d i l u t e s a l i n i t i e s . The involvement of the g i l l i n the r e g u l a t i o n of potassium, c a l c i u m , and magnesium i s i m p l i c a t e d . Seasonal d i f f e r e n c e s i n the degree of r e g u l a t o r y a c t i v i t y of the g i l l were determined f o r potassium and magnesium, wit h w i n t e r p r e p a r a t i o n s showing g r e a t e r a c t i v i t y . 11) Seasonal a c c l i m a t i o n i s r e l a t e d t o changes i n the concen-t r a t i o n s of c o n s t i t u e n t i o n s i n summer and w i n t e r of the e s t u a r i n e environment. LITERATURE CITED 67 Beadle, L.C., 1943. Osmotic r e g u l a t i o n and the faunas of i n -land waters. B i o l . Rev. 17:172-183. B a l l a r d , B.S. and W. Abbott, 1969. 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