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Midgut gland respiration in the estuarine crab, Hemigrapsus nudus (Dana) Hawke, Scott Dransfield 1966

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MIDGUT GLAND RESPIRATION IN THE ESTUARINE CRAB, HEMIGRAPSUS NUDUS (DANA) by S c o t t D r a n s f i e l d Hawke B.S., San Diego State C o l l e g e , 1964  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 standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1966  I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the  requirements  f o r an advanced degree a t tne U n i v e r s i t y o f B r i t i s h Columbia;, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e studyo  and  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f  t h e s i s f o r s c h o l a r l y p u r p o s e s may  be g r a n t e d by the Head o f  Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood that  o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be w i t h o u t my  written permission.  this  my copying allowed  i  ABSTRACT-:  W e i g h t - s p e c i f i c oxygen consumption of midgut g l a n d t i s s u e of Hemigrapsus nudus has been i n v e s t i g a t e d a t three l e v e l s of s a l i n i t y of  (35%> 75% and 125$  experimental temperature  (Warburg) temperatures combinations  sea water), two  levels  (5°C and 20°C) and f o u r acute  (5°, 10°, 15° and 20°C) i n a l l  f o r each season  Metabolic-temperature  (summer and w i n t e r ) .  curves r e v e a l t h a t a t standard  b a s e l i n e c o n d i t i o n s where the animals are h e l d 24 hr a t t h e i r r e s p e c t i v e seasonal temperature  and s a l i n i t y , midgut  gland r e s p i r a t i o n i s h i g h e s t a t a l l acute temperatures the summer animals.  A c u t e l y measured  in  metabolic-temperature  curves f o r midgut g l a n d t i s s u e show t h a t w i n t e r animals a c c l i m a t e d t o t h e i r o p p o s i t e seasonal c o n d i t i o n s of tempera t u r e and s a l i n i t y f o r 10 days demonstrate degree  the g r e a t e s t  of a c c l i m a t i o n .  The e f f e c t of experimental temperature and b i o l o g i c a l l y s i g n i f i c a n t . r a t e i s a t 5°C.  Low  The h i g h e s t r e s p i r a t i o n  temperature  (5°C) may  thermal s t r e s s than a h i g h temperature i n a h i g h e r r a t e of oxygen consumption. temperature response  is statistically  provide a greater  (20°C) r e s u l t i n g Experimental  a l s o i n f l u e n c e s the seasonal r e s p i r a t o r y  of midgut gland t i s s u e to s a l i n i t y .  In summer  animals there i s no c o r r e l a t i o n of midgut gland r e s p i r a t i o n to  s a l i n i t y at 5 ° C  There  i s a increase i n r e s p i r a t i o n  r a t e as the osmotic g r a d i e n t between the blood and medium  ii i n c r e a s e s a t the seasonal b a s e l i n e temperature Winter animals held a t the seasonal baseline  o f 20°C. temperature  o f 5°C d e m o n s t r a t e a " V - s h a p e d " r e l a t i o n s h i p t o s a l i n i t y w i t h t h e lowest r e s p i r a t o r y response  i n 75$ sea w a t e r where  t h e g r a d i e n t b e t w e e n t h e b l o o d a n d medium i s m i n i m a l . Animals in  h e l d a t 20° 0 i n c r e a s e r e s p i r a t i o n w i t h a n i n c r e a s e  salinity. I t i s suggested  that the metabolic a c t i v i t y  o f midgut  g l a n d f r o m summer a n i m a l s may be r e l a t e d t o t h e m a i n t e n a n c e of a osmotic  g r a d i e n t b e t w e e n t h e b l o o d a n d medium o r  a l t e r n a t i v e l y t o t h e e n e r g y demands a s s o c i a t e d w i t h new exoskeleton formation.  The p r o p o s a l i s p u t f o r t h t h a t m i d g u t  g l a n d r e s p i r a t i o n i n w i n t e r a n i m a l s may i n d i c a t e w o r k b e i n g done t o m a i n t a i n t h e o s m o t i c b l o o d a n d medium.  g r a d i e n t between t h e  The p r o d u c t i o n o f a u r i n e h y p o t o n i c t o  t h e b l o o d may a l s o a s s i s t w i n t e r a n i m a l s blood  osmotic  i n regulation of  electrolytes. The  r e g r e s s i o n c o e f f i c i e n t s o f w e i g h t - s p e c i f i c oxygen  c o n s u m p t i o n a s a f u n c t i o n o f body w e i g h t  were n o t s i g n i f i -  c a n t l y d i f f e r e n t f r o m z e r o a t t h e 0.01 p r o b a b i l i t y  level.  TABLE OP CONTENTS Pag INTRODUCTION  1  MATERIALS AND METHODS  5  RESULTS  20  Seasonal Metabolic-Temperature Experiments Main E f f e c t s  20 '  E f f e c t o f Season  (D)  22 22  E f f e c t o f Acute Temperature  (A)  23  E f f e c t o f E x p e r i m e n t a l Temperature (B)  23  E f f e c t o f Experimental S a l i n i t y (C)  25  Interactions  29  First-Order Interactions Acute  29  Temperature-Experimental  Temperature  (AB)  29  Acute Temperature-Season  (AD)  ...29  Acute Temperature-Experimental S a l i n i t y (AC) Experimental Temperature-Experimental S a l i n i t y (BC) Experimental S a l i n i t y - S e a s o n Experimental (BD) Second-Order  (CD)  30 30 30  Temperature-Season 35  Interactions  35  Experimental Temperature-Experimental S a l i n i t y - S e a s o n (BCD)  35  Other Second-Order (ABC,ABD,ACD)  37  Interactions  E f f e c t o f Body Weight  37  iv Page DISCUSSION. Seasonal Metabolic-Temperature  38 Experiments  38  Metabolism-Body Weight R e l a t i o n s h i p  39  E f f e c t of Experimental Temperature  41  E f f e c t o f Experimental S a l i n i t y  43  E f f e c t of Temperature-Salinity-Season Interactions  47  SUMMARY  50  LITERATURE CITED  53  V  LIST' OP FIGURES  F i g u r e No. 1  Page R e l a t i o n s h i p of w e i g h t - s p e c i f i c  oxygen  consumption of midgut g l a n d as a f u n c t i o n of time f o r a 7 g animal ........ 9 2  W e i g h t - s p e c i f i c oxygen consumption of midgut g l a n d f o r animals h e l d 24 h r at  standard seasonal b a s e l i n e  c o n d i t i o n s and 10 days a t o p p o s i t e 21  seasonal c o n d i t i o n s 3  W e i g h t - s p e c i f i c oxygen consumption of midgut gland f o r animals h e l d 10 days i n 35% and 125$ sea water at 5°C  4  24  Experimental temperature-season interaction  (BD) and main e f f e c t o f  experimental temperature (B) on w e i g h t - s p e c i f i c oxygen consumption 26  of midgut gland 5  Experimental s a l i n i t y - s e a s o n  interaction  (CD) and main e f f e c t of experimental salinity  (C) on w e i g h t - s p e c i f i c  oxygen consumption of midgut gland 6  Seasonal comparison of experimental s a l i n i t y and experimental temperature e f f e c t on w e i g h t - s p e c i f i c  oxygen  27  vi F i g u r e No.  Page consumption at  7  of midgut g l a n d , measured 28  10°C acute temperature  E f f e c t of acute temperature-experimental temperature i n t e r a c t i o n (AB) on weights p e c i f i c oxygen consumption of midgut g l a n d  8  31  E f f e c t of acute temperature-experimental s a l i n i t y i n t e r a c t i o n (AC) on weights p e c i f i c oxygen consumption  of  midgut gland 9  32  Acute temperature-season  interaction  (AD) and main e f f e c t of acute temperature  (A) on w e i g h t - s p e c i f i c  oxygen consumption 10  of midgut g l a n d  33  E f f e c t of experimental temperatureexperimental s a l i n i t y i n t e r a c t i o n on w e i g h t - s p e c i f i c oxygen  (BC)  consumption  of midgut g l a n d 11  34-  E f f e c t of experimental temperatureexperimental s a l i n i t y - s e a s o n (BOD)  on w e i g h t - s p e c i f i c  consumption  interaction  oxygen  of midgut g l a n d  36  vii LIST OF TABLES  Table Wo. I  Page Mean v a l u e s of main e f f e c t s f o r weight-specific  oxygen consumption  of midgut gland t i s s u e II  ..11  Mean v a l u e s of i n t e r a c t i o n s f o r weight-specific  oxygen consumption  of midgut gland t i s s u e III  Standard e r r o r o f mean  12  weight-specific  oxygen consumption of midgut gland t i s s u e f o r each experimental combination o f f a c t o r s IV  Analysis  o f v a r i a n c e of  16 weight-specific  oxygen consumption f o r a 4X 2X 3X 2X f a c t o r i a l d e s i g n V  18  Types of seasonal compensation shown for weight-specific  oxygen  consumption o f midgut gland t i s s u e  19  ACKNO WLEDGEMENT S  I w i s h t o t h a n k D r . P. A. e n c o u r a g e m e n t and a d v i c e a n d manuscript.  May  Dehnel  f o r h i s continued  c r i t i c a l a p p r a i s a l of the  I a l s o t h a n k D r . J . R.  Dempster and  p a r t i c u l a r l y M i s s R u t h Hogan f o r h e l p i n g me  i n the  initial  s t a g e s of w r i t i n g a computer program. I e x t e n d my m o s t s i n c e r e a p p r e c i a t i o n t o M i s s M y r n a Young i n a s s i s t i n g me  i n the preparation  of the f i g u r e s .  INTRODUCTION  The m i d g u t g l a n d i s p h y s i o l o g i c a l l y one o f t h e m o s t important organs  i nCrustacea.  I tsecretes digestive  enzymes, a b s o r b s a n d t r a n s f o r m s f o o d a n d i s t h e m a j o r depot for  the storage of m i n e r a l and food r e s e r v e s .  1957) a n d W e e l (1955) h a v e d e m o n s t r a t e d  T r a v i s (1955,  these f u n c t i o n s  i n the s p i n y l o b s t e r , P a n u l l r u s argus, and i n the  brachyuran  c r a b , A t y a s p i n i p e s , by o b s e r v a t i o n s on h i s t o l o g i c a l and h i s t o c h e m i c a l changes o f the g l a n d and c o n n e c t i v e  tissue.  The m i d g u t g l a n d i t s e l f i s composed o f a number o f b l i n d - e n d i n g t u b u l e s s e p e r a t e d f r o m one a n o t h e r b y c o n nective tissue. ducts which,  The t u b u l e s o p e n i n t o s e c o n d a r y  secretion  i n t u r n , open i n t o a p r i m a r y o r c o l l e c t i n g  duct through which midgut ( T r a v i s ,  thedigestive f l u i d  1955; W e e l ,  i s poured  into the  1955).  There a r e v e r y f e w s t u d i e s which have c o n c e n t r a t e d upon a n e x a m i n a t i o n o f m e t a b o l i c a c t i v i t y vertebrate tissue.  Hopkins  (1930) f o u n d  i nexcised i n i n both red and  white muscle o f the p o s t e r i o r adductor o f the clam, mercenaria, a decrease  Venus  i n r e s p i r a t i o n from young t o o l d  c l a m s when r e s p i r a t i o n m e a s u r e m e n t s were made a t 2 7 . 5 ° C . The  body s i z e a n d number o f a n n u a l g r o w t h  r i n g s were (1946)  as c r i t e r i a o f age.  I n a l a t e r paper Hopkins  that i nexcised g i l l  t i s s u e of-Venus m e r c e n a r i a ,  was  h i g h e r i n the cold-adapted animal  t h e warm-adapted a n i m a l  used showed,  respiration  ( b e l o w 20°C) t h a n  (27°C) when m e a s u r e d a t a i n t e r -  2 m e d i a t e t e m p e r a t u r e o f 25° C.  Vernberg  (1956)  demonstrated  a r e l a t i o n s h i p between oxygen consumption of e x c i s e d t i s s u e w i t h h a b i t a t and  activity  d e c a p o d C r u s t a c e a a t 27°C. in not  at a l l i n i s o l a t e d  crab,  of s e v e r a l species of marine  Roberts  muscle t i s s u e only at a h i g h  (1957b) f o u n d a c c l i m a t i o n  sumption of excised g i l l  (23.5°0)  temperature  b r a i n t i s s u e i n the  Pachygrapsus c r a s s l p e s .  gill  striped  Weight-specific  and  rock  oxygen con-  i n Hemigrapsus nudus and  H.  o r e g o n e n s i s showed t h a t t i s s u e f r o m summer a n i m a l s r e s p i r e s at a higher  r a t e than t i s s u e from w i n t e r animals over  p h y s i o l o g i c a l t e m p e r a t u r e r a n g e o f 5°0 McCaughran, 1964).  and in  30$  50$  and  sapidus 10$,  sea w a t e r .  ( b r a c k i s h ) and  i n the  0.  Recently,  t e m p e r a t e zone f i d d l e r  V e r n b e r g and  Kernberg  c r a b s , Uca  sp.,  to  (1966) and  a common t e n d e n c y i n warm-adapted  w e r e made b e t w e e n  10°0  25°0.  R e s p i r a t i o n s t u d i e s on m i d g u t g l a n d more s p a r s e l y d o c u m e n t e d . t h a t t h e r e a r e no m e t a b o l i c  gradients  established for  when c o m p a r i n g t h e a n t e r i o r ,  h i n d p a r t s of the gland.  vary  i n v e r s e f u n c t i o n o f body s i z e regression value  even  i n midgut gland  m e d i a n and  with a negative  t i s s u e are  ejt a l . (1942)  Belding  the k e l p crab, P u g e t t i a producta,  as an  (marine),  b r a i n t i s s u e of t r o p i c a l  a n i m a l s when r a t e d e t e r m i n a t i o n s and  crab,  sapidus  r e s p i r a t i o n m e a s u r e m e n t s t o be h i g h e r  o r 15°C  increase  r e s p e c t i v e l y , when t r a n s f e r e d f r o m 80$  f o u n d i n h e a r t , m u s c l e and  for  t o 20°0 ( D e h n e l  (1965) d e m o n s t r a t e d a n  oxygen consumption of e x c i s e d g i l l  Callinectes by  King  the  The  QQ  2  was  found  (carapace  of -0.285 f o r b o t h  to  length) sexes  3 at  15°C.  Weymouth e t a l .  (1944) d e m o n s t r a t e d a t 15°C i n  t h e m i d g u t g l a n d o f t h e same c r a b a w e i g h t - s p e c i f i c c o n s u m p t i o n r e g r e s s i o n t o b o d y w e i g h t o f -0.203. oxygen  oxygen  The  consumption o f e x c i s e d midgut gland from n i n e s p e c i e s  o f m a r i n e decapod  c r u s t a c e a h a s shown a d i r e c t  correlation  w i t h a c t i v i t y when c o m p a r i n g a n i m a l s w i t h i n a n y one h a b i t a t . ( V e r n b e r g , 1956).  Minamori  (1964) f o u n d t h a t t h e a c t i v i t y  o f h e p a t o p a n c r e a s c a t a l a s e a t 0°C showed a p o s i t i v e b v a l u e t o b o d y w e i g h t o f 0.80 t o 0.81 loach f i s h ,  Cobitis taenia  regression  i nthree races of the  striata.  To a d e q u a t e l y e v a l u a t e e n v i r o n m e n t a l e f f e c t s o n t i s s u e o r w h o l e a n i m a l r e s p i r a t i o n one s h o u l d u s e a m u l t i - f a c t o r i a l a p p r o a c h w h e r e s e v e r a l e n v i r o n m e n t a l f a c t o r s a r e examined simultaneously.  Host s t u d i e s have c o n c e n t r a t e d on a u n i -  f a c t o r i a l a n a l y s i s on w h i c h t o base c o n c l u s i o n s . (1963) p o i n t s o u t , s u c h a n a n a l y s i s may g i v e t h a t h a v e no v a l i d i t y sponds t o t h e whole factors.  ecologically  As Kinne  conclusions  since t h e organism r e -  environment, n o t t o i s o l a t e d  single  The p u r p o s e o f t h e p r e s e n t s t u d y i s t o c o n s i d e r  t h o s e f a c t o r s w h i c h a r e assumed t o be t h e m o s t i m p o r t a n t i n d e f i n i n g t h e r e l a t i o n s h i p o f midgut g l a n d r e s p i r a t i o n t o t h e i n t a c t a n i m a l and i n t u r n t o t h e environment.  A  m u l t i - f a c t o r i a l d e s i g n i s u t i l i z e d t o a c c o m p l i s h t h i s end. The  e f f e c t o f t e m p e r a t u r e , s a l i n i t y and s e a s o n a l changes  i n t h e s e f a c t o r s i s examined  i n terms o f w e i g h t - s p e c i f i c  oxygen consumption o f e x c i s e d midgut g l a n d o f t h e shore c r a b , Hemigrapsus  nudus,  over the p h y s i o l o g i c a l temperature  4 range o f 5°G to 20° C. !  The term "main e f f e c t " w i l l be used to d e s c r i b e of a s i n g l e f a c t o r  (eg. s a l i n i t y ,  effects  temperature) averaged f o r  a l l acute temperatures, experimental temperatures and salinities  and both seasons.  The term " i n t e r a c t i o n " (eg.  s a l i n i t y - t e m p e r a t u r e combination) r e f e r s to the combined e f f e c t of f a c t o r s where d i f f e r e n c e s  i n response t o one f a c t o r  w i t h the l e v e l o f another f a c t o r which i s a p p l i e d t a n e o u s l y ( S t e e l and T o r r i e ,  1960).  of the concept o f i n t e r a c t i o n w i l l The acute temperatures  A further  varies  simul-  discussion  follow.  (5°, 10°, 15° and 20°0) r e f e r  to the Warburg water-bath temperatures to which the t i s s u e samples  are e q u i l i b r a t e d  v a l u e s are r e c o r d e d .  and a t which oxygen  Experimental temperatures  20°C) and experimental s a l i n i t i e s water) are the p h y s i c a l  (5°C and  (35$, 75$ and 125$ sea  parameters to which the animals a r e  a c c l i m a t e d f o r 10 days o r h e l d baseline  consumption  a t f o r 24 h r f o r standard  measurements.  A c c l i m a t i o n , as used i n the context o f t h i s study, w i l l r e f e r to a phenotypic a l t e r a t i o n i n m e t a b o l i c a c t i v i t y due to change i n s a l i n i t y ,  temperature  or s e a s o n a l changes i n  these f a c t o r s when measured over the p h y s i o l o g i c a l range o f 5°C to 20°C.  This d e f i n i t i o n w i l l  include  temperature a l s o the  l i a b i l i t y f o r g e n o t y p i c change as r e f l e c t e d i n a phenotypic a l t e r a t i o n o f metabolism.  Compensation  and a d a p t a t i o n w i l l be  used w i t h the same c o n n o t a t i o n as a c c l i m a t i o n the  concept o f homeostasis.  Homeostasis  and w i l l  include  i s a mechanism by  which the animal p h y s i o l o g i c a l l y m a i n t a i n s i n t e r n a l constancy  5 d e s p i t e changes i n t h e environment. The to  term hepatopancreas  C r u s t a c e a a n d w i l l n o t be u s e d i n t h e t e x t o f t h i s  I n s t e a d , t h e term midgut hepatopancreas. it  i s a m i s n o m e r when a p p l i e d  gland w i l l  study.  be u s e d i n p l a c e o f  B e l d i n g e t a l . (1942) p o i n t o u t , " S i n c e  ( m i d g u t g l a n d ) i s w i t h o u t h o m o l o g y i n t h e mammal, t h e  term  ' "liver"  Because gland"  ' and  1  "hepatopancreas"  i tdevelops from t h e midgut, ' identifies  1  are unjustified.  t h e term  i t w i t h o u t chance  1  "midgut  o f e r r o r a n d w i t h no  misleading implications as t o function." MATERIAL AND METHODS The  c r a b , H e m i g r a p s u s n u d u s , was o b t a i n e d f r o m  S p a n i s h Bank ( L a t . 49° 17'N.j L o n g . British  Columbia.  123° 07'¥.)  Summer a n i m a l s w e r e c o l l e c t e d f r o m J u n e  t h r o u g h August and w i n t e r a n i m a l s were c o l l e c t e d November t h r o u g h M a r c h .  relatively 75$  water.  s t a b l e t e m p e r a t u r e o f 5°C a n d a s a l i n i t y o f  (24&) s e a w a t e r .  the respective The  The a v e r a g e  summer a n d w i n t e r  serve as standard baseline  temper-  conditions  seasons.  s t a n d a r d s e a w a t e r o f 1 0 0 $ (32°^°) u s e d i n t h i s  i s based on a c h l o r i n i t y 31.88%°.  o f 35$  The w i n t e r s e a s o n i s c h a r a c t e r i z e d by a  a t u r e s and s a l i n i t i e s for  from  The summer p e r i o d i s c h a r a c t e r i z e d  by a a v e r a g e t e m p e r a t u r e o f 20°C a n d a s a l i n i t y  (11/4) s e a  Vancouver,  study  o f 17.65^°and a s a l i n i t y o f  The p r o p o r t i o n s o f t h e m a j o r i o n s i n 100$ s e a  w a t e r , a s d e t e r m i n e d by l a b o r a t o r y a n a l y s i s , a r e a s f o l l o w s :  6 Ha: K : Ca: Mg: Cl: The ions a r e complexed sodium.  as c h l o r i d e s , p l u s the s u l f a t e o f  Two experimental temperatures, 5°C and 20°C (i1°C)  and three 125$  433.0 mEq./l. 10.1 '» " 25.6 " " 97.9 " " 497.0 " "  experimental s a l i n i t i e s ,  (40&) sea water  35$ (11#°), 7 5 $ (24%) and  (±1$ sea water) were used i n a l l  combinations f o r each season (summer and w i n t e r ) .  The  animals were h e l d 5, 10 and 15 days a t these experimental combinations to determine the time p e r i o d t h a t r e s u l t e d i n the maximum degree of a c c l i m a t i o n .  A 10 day time p e r i o d  gave the maximal a c c l i m a t i o n response. Only a d u l t i n t e r m o l t male crabs were s e l e c t e d f o r study. They ranged i n weight from 6.0 t o 10.0 g.  The wet weight  of the whole animal a f t e r damp d r y i n g was weighed t o the nearest  0.01 g.  a t e l y placed appropriate aerated  In the l a b o r a t o r y the animals were immedi-  i n p l a s t i c c o n t a i n e r s h o l d i n g 3 . 5 l i t e r s o f the (35$» 75$ o r 125$) sea water.  and t h e c o n t a i n e r s were placed  constant temperature  Sea water was  i n a refrigerator at  (5°C o r 20°C) i n t o t a l darkness.  water was changed d a i l y .  At standard b a s e l i n e  the animals were h e l d 24 h r .  The  conditions  Those animals a c c l i m a t e d i n  the l a b o r a t o r y were h e l d 10 days and not f e d .  Only 10-12  animals were h e l d i n each c o n t a i n e r a t any one time t o minimize m o r t a l i t y due to crowding.  Crabs t h a t molted  during the 10 day a c c l i m a t i o n p e r i o d were d i s c a r d e d . The d i r e c t method of Warburg was used t o measure r e s p i r a t i o n r a t e s of midgut gland (Umbriet, B u r r i s and S t a u f f e r ,  7 1957). was  The G i l s o n M e d i c a l E l e c t r o n i c s  made a v a i l a b l e f o r t h i s work. T i s s u e samples excised  weight from 0.3 to 0.6 g. placed d i r e c t l y  from the animals ranged i n The excised  samples were  The t i s s u e samples were not damp d r i e d  on f i l t e r paper p r i o r to weighing. tubles  tissue  i n pre-weighed aluminum pans and weighed t o  the n e a r e s t 0.1 mg.  the  (Warburg) r e s p i r o m e t e r  The d e l i c a t e nature o f  made i t d i f f i c u l t t o p i c k the gland o f f  filter  paper without fragmenting the t i s s u e w i t h subsequent l o s s of s u b s t r a t e and enzyme f l u i d s .  A f t e r weighing, the t i s s u e  samples were placed i n the Warburg r e a c t i o n 3.0 ml o f p h y s i o l o g i c a l  flasks  containing  s a l i n e prepared as f o l l o w s :  850.0 ml o f 51.0 " " 3 5 . 0 «' " 35.0 " " 17.6 1.3  M  "  M  0.52 0.35 0.35 0.42  M M M M  NaOl MgGlo CaOlp KapSOA  0.38 M H3BO3 0.48 M  pH = 7.8  mOE  °C = - 1 . 6 1  A PP  The  c e n t e r w e l l s o f the r e a c t i o n  of  1 5 $ KOH.  an  ice-bath.  the  During t h e d i s s e c t i o n the f l a s k s were kept i n Three hours and t e n minutes a f t e r e x c i s i o n o f  f i n a l tissue  ice-bath  f l a s k s contained 0 . 2 ml  sample, the f l a s k s were taken out o f the  and attached to the manometers.  The a t t a c h e d  flasks  were then p l a c e d i n the constant temperature water-bath ( * 0 . 1 ° C ) o f the Warburg.  The seventeen t i s s u e samples were  allowed t o e q u i l i b r a t e f o r 1 5 min, were gassed w i t h oxygen f o r 10 min. was  during which time they The r e s p i r a t o r y  rate  measured a t f o u r acute temperatures ( 5 ° » 1 0 ° , 1 5 ° and  20°C) f o r each s e t of experimental c o n d i t i o n s .  An exami-  8  nation could  of Figure 1 reveals  t h a t one set of t i s s u e  samples  r e s p i r e a t two acute temperatures s i n c e the slope of  the w e i g h t - s p e c i f i c shows a s m a l l ,  oxygen consumption r a t e curve w i t h time  but n o n - s i g n i f i c a n t  i n t e r v a l of measurement. between the arrows.  decrease over the time  T h i s i s the p o r t i o n of the curve set o f t i s s u e samples  respired  a t 5°C and 10°C and the second set of t i s s u e samples  respired  a t 15°C  and 20°0.  The f i r s t  Each set of t i s s u e samples r e s p i r e d  90  min a t each o f the two acute temperatures w i t h a 40 min time l a p s e between m e t a b o l i c measurements a t the f i r s t second acute temperatures.  and  I t took 25 min to change the  temperature of the water-bath 5°C and 15 min to e q u i l i b r a t e the t i s s u e s .  The t i s s u e samples were shaken a t a constant  r a t e of 120 o s c i l l a t i o n s / m i n . d i s s e c t i o n were r e q u i r e d  to complete r e s p i r a t i o n measurements  a t two acute temperatures. the  Nine hours from the time of  One  i s j u s t i f i e d i n measuring  r e s p i r a t i o n o f the t i s s u e a t two acute temperatures,  s i n c e measurement of t i s s u e r e s p i r a t i o n i n the r e v e r s e direction  (10° and 5°C o r 20° and 15°C)  does not  significantly  a l t e r the slope o f the metabolic-temperature (M-T) c u r v e s . With the completion o f an experimental run, the t i s s u e samples and s a l i n e were p l a c e d i n pre-weighed  aluminum pans  and d r i e d i n an oven a t 100-106°C  Dry body  f o r 24 h r .  weights were obtained by the same procedure.  The data were  expressed i n u l 0 / g dry g l a n d / h r a t N.T.P. 2  A s t a t i s t i c a l analysis  of the data was performed w i t h  the a i d of the 7040 IBM Computer.  The data were processed  9  Figure  1.  W e i g h t - s p e c i f i c oxygen consumption as a f u n c t i o n  of time f o r midgut gland  of Hemigrapsus nudus.  Each p o i n t  r e p r e s e n t s amount o f o x y g e n consumed d u r i n g a 10 m i n interval.  W e i g h t o f crab, u s e d f o r o x y g e n c o n s u m p t i o n  m e a s u r e m e n t was 7 g.  Gurve i s e y e - f i t t e d .  time  r  CO  (J o  (J °  O in O cvj ro c\j > a: QTZ y j  jf) U J  <  h O) h  o: Q: 3 x x O  UJ UJ <  x UJ*  J  LO  j>j  g I  O  8  tf>  S  O  ->  Jtj /puo|6Ajp B/^Qih  I  LO  1  rsi  I  10  using, t h e F o r t r a n Program. W e i g h t - s p e c i f i c oxygen consumption was p l o t t e d as a f u n c t i o n o f whole body weight on double paper.  l o g a r i t h m i c graph  The data gave a s t r a i g h t l i n e t h a t took the form:  Reduce t o :  l o g O2 = l o g a + b ( l o g W)  where 0- = ulOg/g dry gland/hr, a = i n t e r c e p t , b = slope o f l i n e and W = whole body weight.  The exponent ( b - 1 )  by which body weight i s r a i s e d to a g i v e n v a l u e p r o p o r t i o n a l to metabolism i s r e f e r r e d t o as the r e g r e s s i o n c o e f f i c i e n t . Slope v a l u e s were c o n s i d e r e d s i g n i f i c a n t a t the 0 . 0 1 probability  level.  Using a equal sample s i z e o f twelve, an a n a l y s i s of v a r i a n c e was performed  on the data (see Table I V ) .  The  l e v e l o f s i g n i f i c a n c e a t which the N u l l Hypothesis was accepted t h a t there i s no d i f f e r e n c e between means was the 0 . 0 1 p r o b a b i l i t y l e v e l . sum  treatment  The v a l u e s o f the  o f squares and mean squares. I n Table IV a r e expressed  i n n a t u r a l logarithms. was performed I and I I .  The data upon which the s t a t i s t i c  a r e presented  i n t a b u l a t e d form i n Tables  Only the mean v a l u e s of the main e f f e c t s and  i n t e r a c t i o n s f o r w e i g h t - s p e c i f i c oxygen consumption a r e expressed  i n the t a b l e s .  These mean v a l u e s a r e presented  g r a p h i c a l l y i n the subsequent f i g u r e s .  11  Tab,le I : Mean v a l u e s o f main e f f e c t s f o r w e i g h t - s p e c i f i c oxygen consumption o f midgut gland t i s s u e .  Source o f Variance  Mean (ul0 /g dry gland/hr) 2  A(Acute Temp.° C)  5 10 15 20  427 631  5 20  375 303  35 75 125  351  B(Exp. Temp.°0) C(Exp. S a l i n i t y , $S.W.)  D( Season) Winter Summer  185  259  319 341  344 330  12 Table I I : Mean v a l u e s o f f i r s t and second-order i n t e r a c t i o n s f o r w e i g h t - s p e c i f i c oxygen consumption o f midgut gland t i s s u e .  Source of V a r i a n c e  Mean ( u l 0 / g dry gland/hr) 2  First-Order Interactions  Mlcj. 5 10 15 20  5  AB Bile}.  —  206 295 465 694  5 ,  ,  10 15  T65" 228 392  •  •  20  _20  573 AO:  A(°C')  0(#S.W.)  5  10 15 20 5 10  75  20 5 10  , • 125  190 277 450 643 175 239 404 617  35 ,  15  15  20  TB9 ,  264 429 6J52  BO B(° C)  0(gS.W.) 35 75  5  12J  35 75  20  125  403 329 396  30o~ 310 294  AD A(° C) 5 10 15 20  D(Season) Winter  183 279 421 651  13  Table I I :  Continued  5 10  Summer  15  20 BD B(° C) 5 20 5 20  D(Season) Winter Summer  CD C(#S.tf.) 35 75 125 35 75 125  D(Season) Winter Summer  186 241 433 611  383 309 366 297  350 301  386 353 338 301  Second--Order I n t e r a c t i o n s  A(°C) 5 10 15 20 5 10  ABC  C($S.W.)  5  35  20  35  5  75  15  20 5 10 15  20 5 10  599  20  15  20 5 10  164  75  5  125  20  125  15  20 5 10 15 20  224 324 495 737 162 236 409 561 187 258 404 221  403 635 210 308 502 758 170 226  366 527  14  Table I I :  A(°C)  5 10 15 20 5 10  Continued ABD BC" !?! 5  D( S e a s o n )  5  Winter  20  Winter  5  Summer  20  Summer  15  20 5 10 15 20 5 10 15 20 A(°C)  5 10 15 20 5 10  168  ACD  c(fs7w.)  D(Season)  35  Winter  75  Winter  125  Winter  35  Summer  75  Summer  125  Summer  15  20 5 10 15  20 5 10 15 20 5 10 15  20 5 10 15  20 B(°C)  5  207 317 460 714 162 246 385 593 206 275 470 675  BCD  c(fs7w.) 35 75 125  D(Season) Winter  211 399 552 181 286  443 652 170 223 355 586 199 326 474 723 200 267 457 634 180 245 458 649 150 214 388 553  457 292 422  Table I I :  5 20 20  Continued 35 75 125 35 75  125  35 75 125  Summer Winter Summer  357 369 373  2oT  311 354. 3W  310 244  Table I l l s Standard e r r o r o f mean w e i g h t - s p e c i f i c oxygen consumption g l a n d t i s s u e f o r each e x p e r i m e n t a l c o m b i n a t i o n o f f a c t o r s .  E x p e r i m e n t a l Combination o f F a c t o r s  Mean ( u l 0 / g dry gland/hr) 2  Acute Temp.°C 5 10 15 20 5 10 15 20 5 10 15 20 ' 5 10 15 20 5 10 15 20 5 10 15 20  Exp. Temp.°C  Salinity ($S.W.)  Season  5  35  Winter  5  75  Winter  5  125  Winter  20  35  Winter-  20  75  Winter  20  125  Winter  244 376 551 854 173 237 335 534 210 357 529 794 135 218 356 494 168 230 377 642 189 297 425 659  o f midgut  Standard E r r o r (S^)  129 134 203 188 233 217 268 324 150 153 172 161 278 216 276 310 156 201 301 204 242 236 117 190  Table I I I : 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20  Continued  5  35  Summer  5  75  Summer  5  125  Summer  20  35  Summer  20  75  Summer  20  125  Summer  206 280 444 632 202 281 489 672 210 265 477 725 193 255 470 636 160 213 430 627 154 172 315 422  130 175 200 237 387 136 193 234 133 118 135 155 196 183 331 357 268 289 172 231 172 230 309 423  18  Table IV: A n a l y s i s o f v a r i a n c e o f w e i g h t - s p e c i f i c oxygen consumption f o r the f a c t o r i a l (4X 2X 3X 2X) experiment I n c o r p o r a t i n g f o u r acute temperatures ( A ) , two experimental temperatures ( B ) , t h r e e experimental s a l i n i t i e s (C) and two seasons (D) ( S t e e l and T o r r i e , 1960). The P-values are c o n s i d e r e d s i g n i f i c a n t a t the 0.01 p r o b a b i l i t y l e v e l (**).  Source o f Variance  df  Sum o f Squares  Mean, Squares  F  A (Acute Temp.) B: (Exp. Temp.) 0 (Exp. S a l i n i t y ) D (Season)  3 1 2 1  126.56 6.43 0.91 0.26  42.185 6.433 0.457 0.260  753.3** 114.9** 8.2** 4.6**  AB AC BC AD BD CD  3 6  0.16 0.17 1.72 0.72 0.00 3.35  0.053 0.028 0.859 0.241 0.000 1.674  1.0 0.5 15.3** 4.3** 0.0 29.9**  ABC ABD ACD BCD  6 3 2  0.50 0.03 0.45 4.48  0.083 0.009 0.074 2.238  40.0**  41  145.74  3.555  63.4**  Error  534  29.67  0.056  Total  575  175.41  Total  2  3 1 2  6  Treatment  1.5  0.2 1.3  19  T a b l e V: S e a s o n a l c o m p e n s a t i o n o f w e i g h t - s p e c i f i c oxygen consumption o f midgut g l a n d a t a l l combinations of acute temperature ( A ) , experimental temperature (B), e x p e r i m e n t a l s a l i n i t y (C) a n d s e a s o n ( B ) . The numbers i n t h e body o f t h e t a b l e r e p r e s e n t t h e t y p e of compensation: t y p e 5, i n d i c a t e s t h a t summer a n i m a l s h a v e t h e h i g h e s t r e s p i r a t i o n r a t e ; t y p e 4, s e a s o n a l r a t e s a r e e q u a l ; a n d t y p e 3, w i n t e r a n i m a l s h a v e t h e h i g h e s t r e s p i r a t i o n r a t e ( P r e c h t , 1951).  Experimental  Acute T  Experimental Salinity ($S.¥.)  35  75  125  5  5 10 15 20  3 3 3 3  5 5 5 5  4  20  5 10 15 20  5 5 5 5  3 3 5 3  3 3 3 3  3 3 3  20 RESULTS  Seasonal Metabolic-Temperature Experiments The data for a seasonal comparison of weight-specific oxygen consumption are presented in Table III.  It may be  seen by examining the standard errors that there is a great deal of variability in the respiratory measurements at any one acute temperature.  In Figure 2 is presented a seasonal  comparison of midgut gland respiration.  The metabolic-  temperature (M-T) curves reveal that Hemlgrapsus nudus demonstrates inverse seasonal compensation, type 5 (Precht, 1951).  This is illustrated by the fact that the summer  baseline M-T curve (35$ sea water, 20°C) is higher on the ordinate than the winter at summer baseline M-T curve (acclimated to summer baseline conditions) by 43$ at 5°0, 1 7 $ at 10°0, 32$ at 15°C and 29$ at 20°C acute temperature. Oonversely, the winter baseline M-T curve (75$ sea water, 5°C) is lower on the ordinate than the summer at winter baseline MrT curve (acclimated to winter baseline conditions) by 1 7 $ , 19$, 46$ and 2 6 $ at the same respective acute temperatures. Using Precht's (1951) classification scheme, the types of seasonal compensation are indicated in Table V. Type 5, indicates that summer animals have the highest respiration rate; type 4, seasonal rates are equal; type 3, winter animals have the highest respiration rate.  21  Figure 2.  Seasonal-metabolic temperature curves, acutely  measured, for midgut gland of Hemlgrapsus nudus. Winter (75$ sea water, 5°C) and summer (35$ sea water, 20°0) baseline animals were held 24 hr prior to experimentation. The acclimated animals were held at the opposite seasonal conditions for 10 days prior to experimentation. Each point is the mean of weight-specific oxygen consumption at the respective acute temperatures.  The ratio of the  slopes M'| to; M and ELj to Mg define the degree of ac2  climation shown by the winter and summer animals, respectively.  SEASONAL METABOLIC - TEMR  IO  15 ACUTE TEMR °C  20  22 I n comparing t h e degree t o w h i c h a c c l i m a t i o n h a s been achieved,  t h e compensation temperature c o e f f i c i e n t as f i r s t  suggested  by R o b e r t s  data  (1952) may be a p p l i e d t o t h e p r e s e n t  ( s e e R a o , 1953).  The c o e f f i c i e n t v a l u e  b e t w e e n t w o s l o p e s , M-| a n d Mg.  The s l o p e M  1  i sa r a t i o i s drawn between  t h e h i g h e s t r e s p i r a t i o n r a t e o f t h e w i n t e r a t summer b a s e l i n e M-T. c u r v e a n d t h e l o w e s t b a s e l i n e M-T c u r v e . .  The s l o p e M  The same g r a p h i n g  2  2  i sthe reciprocal  of slope  p r o c e d u r e a p p l i e s t o s l o p e s MJ and  t  M .  r e s p i r a t i o n r a t e o f t h e summer  t  I n t h i s c a s e s l o p e M-j i s d r a w n b e t w e e n t h e h i g h e s t  r e s p i r a t i o n r a t e o f t h e w i n t e r b a s e l i n e M-T c u r v e a n d t h e lowest  r e s p i r a t i o n r a t e o f t h e summer a t w i n t e r b a s e l i n e •  M-T c u r v e . ( P i g . 2).  The s l o p e M  2  i  i sthe reciprocal  of slope  The r a t i o i s l e s s t h a n one i f t h e r e i s a n y  compensation and approaches zero a s t h e degree o f compensation increases. The  c o m p e n s a t i o n c o e f f i c i e n t b e t w e e n w i n t e r a t summer  b a s e l i n e M-T c u r v e a n d summer b a s e l i n e M-T c u r v e  i s 0.601.  The  c o e f f i c i e n t v a l u e b e t w e e n t h e w i n t e r b a s e l i n e M-T  and  summer a t w i n t e r b a s e l i n e M-T c u r v e  comparison of these animals  c o e f f i c i e n t values  i s 0.665.  A  indicates that winter  show t h e g r e a t e s t d e g r e e o f c o m p e n s a t i o n  coefficient value).  curve  (lower  This p o i n t i s brought out i n Table  Main E f f e c t s Effect  o f S e a s o n (D)  The  e f f e c t o f s e a s o n on midgut g l a n d r e s p i r a t i o n i s  V.  23 significant comparing  ( I a b l e I V ) . T h i s e f f e c t was d e t e r m i n e d by  t h e mean o f a l l w i n t e r r e s p i r a t i o n d a t a w i t h t h e  mean o f a l l summer r e s p i r a t i o n d a t a . Midgut gland demonstrates t y p e 5»  inverse seasonal  compensation,  f o r a l l acute temperatures a t standard baseline  conditions of  35$  sea water,  w a t e r , 5°C ( w i n t e r ) . compensation,  t y p e 3,  20°C  (summer) a n d  75$ s e a  E x c e p t f o r two c a s e s , p a r t i a l i s demonstrated  seasonal  at a l lother experi-  mental combinations of a c c l i m a t i o n temperature o r a c c l i m a t i o n salinity Effect  (Table V ) .  o f Acute Temperature  (A)  An e x a m i n a t i o n o f Table IV r e v e a l s t h a t t h e main effect  of acute temperature  was d e r i v e d b y c o m p a r i n g  i s significant.  This  effect  t h e means o f t h e f o u r a c u t e  temper-  a t u r e s a f t e r a v e r a g i n g a l l t h e r e s p i r a t i o n data a t each acute  temperature. Figure 3 i s a t y p i c a l representation o f acute  e f f e c t s on midgut  gland respiration.  r i s e s the rate of tissue  As t h e acute  respiration i s increased.  temperature temperature I t may  be s e e n t h a t t h e a c u t e l y m e a s u r e d m e t a b o l i c - t e m p e r a t u r e c u r v e s a r e i n f l u e n c e d by s e a s o n , e x p e r i m e n t a l t e m p e r a t u r e and e x p e r i m e n t a l s a l i n i t y be e x a m i n e d Effect  interactions.  These e f f e c t s  will  shortly.  o f E x p e r i m e n t a l Temperature  The m a i n e f f e c t  (B)  o f e x p e r i m e n t a l t e m p e r a t u r e was d e t e r m i n e d  24  F i g u r e 3.  M e t a b o l i c - t e m p e r a t u r e c u r v e s , a c u t e l y measured,  f o r midgut g l a n d o f Hemigrapsus nudus.  The summer a n d  w i n t e r a n i m a l s w e r e h e l d 10 d a y s I n 35$ a n d 125$ s e a w a t e r a t 5°0 p r i o r t o e x p e r i m e n t a t i o n .  The p o i n t s on t h e  c u r v e s r e p r e s e n t t h e means o f w e i g h t - s p e c i f i c consumption  f o r each a c u t e  temperature.  oxygen  900-  EXP. T E M R 5 °C  800700600-  — WINTER ©35 %S.W — S U M M E R o 125 % S.W. j  5  i  1  IO 15 A C U T E T E M R °C  L _  20  25 by a v e r a g i n g a l l r e s p i r a t i o n d a t a a t each, e x p e r i m e n t a l temperature  and t h e n comparing t h e means.  This e f f e c t i s  s i g n i f i c a n t (Table I V ) . The main e f f e c t o f e x p e r i m e n t a l temperature to a h i g h e r r e s p i r a t o r y r a t e a t 5°C The f a c t t h a t i n v e r s e ,  i s due  t h a n a t 20°0  (Fig. 4).  t y p e 5, and p a r t i a l , type  3,  compensation i s shown a t 5°C and 20°C i n d i c a t e s t h a t respiratory  response  the  of summer and w i n t e r a n i m a l s i s b e i n g  a l t e r e d by the e x p e r i m e n t a l temperature E f f e c t of Experimental S a l i n i t y  e f f e c t (Table V ) .  (C)  The main e f f e c t of e x p e r i m e n t a l s a l i n i t y was  determined  by comparing the means of the e x p e r i m e n t a l s a l i n i t i e s .  The  means were d e r i v e d by a v e r a g i n g a l l r e s p i r a t i o n d a t a a t each experimental s a l i n i t y .  I t i s shown i n Table IV t h a t  the  main e f f e c t of e x p e r i m e n t a l s a l i n i t y i s s i g n i f i c a n t . The r e s p i r a t o r y  response  of midgut g l a n d shows a  shaped" r e l a t i o n s h i p t o s a l i n i t y .  The l o w e s t  "V-  respiration  r a t e f o r t h e main e f f e c t i s i n 75$ sea water and the h i g h e s t r e s p i r a t i o n r a t e s I n 35$ and  125$ sea water  (Fig. 5).  i s apparent t h a t season and e x p e r i m e n t a l temperature an i n f l u e n c e  on the r e s p i r a t o r y  t i s s u e to experimental s a l i n i t y r a t e may  increase,  response  have  of midgut g l a n d  (Fig. 6).  The  respiratory  d e c r e a s e , show no change o r r e v e a l  "V-shaped" r e l a t i o n s h i p w i t h a change i n s a l i n i t y . e x a m i n a t i o n of t h e s e i n t e r a c t i o n s w i l l  It  follow.  A  a further  26  F i g u r e 4.  Experimental temperature-season  interaction  (BD) and main e f f e c t o f experimental temperature midgut g l a n d o f Hemigrapsus  nudus.  (B) f o r  Each p o i n t on the  experimental temperature-season i n t e r a c t i o n curves i s the mean o f w e i g h t - s p e c i f i c oxygen consumption a t t h a t p a r t i c u l a r experimental temperature-season combination. The main e f f e c t i s the mean o f a l l w e i g h t - s p e c i f i c consumption atures.  oxygen  data a t the r e s p e c t i v e experimental temper-  EXP. T E M R CB) EXR T E M R - S E A S O N INTERACTION (BD)  400  300  EFFECT(B) 200  o SUMMER  EXR T E M R °C  27  F i g u r e 5.  Experimental s a l i n i t y - s e a s o n i n t e r a c t i o n (CD)  and main e f f e c t of experimental s a l i n i t y gland o f Hemigrapsus nudus.  Each p o i n t  (C) f o r midgut on the e x p e r i -  mental s a l i n i t y - s e a s o n i n t e r a c t i o n curves i s the mean o f weight-specific  oxygen consumption a t t h a t p a r t i c u l a r  experimental s a l i n i t y - s e a s o n combination. e f f e c t i s the mean o f a l l w e i g h t - s p e c i f i c sumption data a t the r e s p e c t i v e  The main oxygen con-  experimental  salinities.  EXP. SALINITY (C) EXP. SALINITY - S E A S O N I N T E R A C T I O N CCD) •o o 4 0 0  ©WINTER  «^300 o  £ M A I N E F F E C T CC) 0 1  o SUMMER  200 35  75 EXRSALINITY,%S.  125  28  Figure and  6.  Seasonal  experimental  respiration temperature.  season.  temperature  effects  on m i d g u t  o f H e m l g r a p s u s nudus, m e a s u r e d a t Each p o i n t  weight-specific experimental  comparison of experimental  on t h e  curves  experimental  gland 1 0 ° 0 acute  i s t h e mean o f  oxygen consumption a t t h a t  salinity,  salinity  particular  temperature  and  r  SUMMER 300  200  ©5 °C IO °C ACUTE TEMR  c  °2o°a  o  CD  >^ TJ  cn  WINTER  CM  O 400| 300  200  © 5 °C IO°C ACUTE TEMR o 2 0 °C 35  75 EXR SALINITY. %S.W.  125  29 Interactions S t e e l and T o r r i e  (1960) d e f i n e i n t e r a c t i o n "  a departure of the simple e f f e c t s law o r model based  ( f a c t o r s ) from an  on m a i n e f f e c t s o n l y . "  i n t e r a c t i o n i s one w h e r e t h e f a c t o r s o f one a n o t h e r .  Interactions  First-Order  factors  are  interactions with three interactions.  Interactions Temperature  The means o f w e i g h t - s p e c i f i c a c u t e t e m p e r a t u r e s a n d two  a t 5°0  do n o t a c t i n d e p e n d e n t l y  to as second-order  Acute Temperature-Experimental  plotted  additive  A significant  i n v o l v i n g two  c a l l e d f i r s t - o r d e r i n t e r a c t i o n s and factors are referred  as  i n F i g u r e 7.  (AB)  oxygen consumption a t  experimental temperatures  The m e t a b o l i c - t e m p e r a t u r e (M-T)  i s h i g h e r a t a l l acute temperatures than the  four  are curve M-T  c u r v e a t 20° C. The  i n t e r a c t i o n , however, i s not s i g n i f i c a n t (Table I V ) .  This indicates  that  there i s a great deal of  variability  i n t h e r e s p i r a t i o n m e a s u r e m e n t s a t any one a c u t e experimental temperature Acute Temperature-Season  temperature-  combination. (AD)  The a c u t e t e m p e r a t u r e - s e a s o n  interaction i s significant  (Table I V ) . The means o f t h e r e s p i r a t i o n d a t a a t a l l a c u t e a t u r e s f o r e a c h s e a s o n a r e shown i n F i g u r e 9.  There  temperi s no  30 c o n s i s t e n t t r e n d i n a seasonal response The  d i f f e r e n c e s a r e s m a l l and randomly  Acute  Temperature-Experimental  to acute  distributed.  S a l i n i t y (AC)  This i n t e r a c t i o n i s not s i g n i f i c a n t The  temperature.  (Table I V ) .  means o f t h e r e s p i r a t i o n d a t a a t f o u r a c u t e  temper-  a t u r e s and t h r e e e x p e r i m e n t a l s a l i n i t i e s a r e p l o t t e d i n Figure 8.  The h i g h e s t r e s p i r a t o r y r a t e i s i n 3 5 $ s e a w a t e r  and  the lowest r e s p i r a t i o n rate i s i n 7 5 $ sea water.  The  d i f f e r e n c e s i n t h e magnitude of response  (position of  s l o p e on o r d i n a t e ) a r e s m a l l . Experimental Temperature-Experimental  S a l i n i t y (BC)  This interaction i s s i g n i f i c a n t  (Table I V ) .  I n F i g u r e 10 t h e means o f t h e two e x p e r i m e n t a l  temper-  atures a t three experimental s a l i n i t i e s are graphically represented.  The M-T  c u r v e a t 5°C shows a " V - s h a p e d " s l o p e  w i t h t h e lowest r e s p i r a t o r y r a t e i n 7 5 $ sea water. M-T. c u r v e a t 20°C shows l i t t l e change i n s a l i n i t y Experimental The  from  The  change i n s l o p e w i t h a  3 5 $ t o 125$ sea water.  S a l i n i t y - S e a s o n (CD)  means o f t h e r e s p i r a t i o n v a l u e s f o r e a c h  s a l i n i t y and both  seasons a r e p l o t t e d  r e s p i r a t o r y r a t e f o r t h e w i n t e r M-T  i n Figure 5 .  curve  shows a  experimental The "V-shaped"  r e l a t i o n s h i p to s a l i n i t y with the lowest r e s p i r a t i o n rate i n 7 5 $ sea water.  The summer r e s p i r a t o r y r a t e i s r e p r e s e n t e d  31  F i g u r e 7.  Acute temperature-experimental  temperature  I n t e r a c t i o n (AB) f o r m i d g u t g l a n d o f H e m i g r a p s u s n u d u s . E a c h p o i n t on t h e a c u t e t e m p e r a t u r e - e x p e r i m e n t a l t e m p e r a t u r e i n t e r a c t i o n c u r v e s I s t h e mean o f w e i g h t - s p e c i f i c oxygen consumption  at that p a r t i c u l a r acute  experimental temperature  combination.  temperature-  A C U T E T E M P . - EXP. T E M R I N T E R A C T I O N (AB) 700 600 500 400 300  200 EXP. T E M P . ©5 °C o 20 °C  -J  5  1  1  IO 15 A C U T E T E M R °C  32  Figure  8.  Acute  temperature-experimental  salinity  action  (AC) f o r m i d g u t g l a n d o f H e m i g r a p s u s n u d u s .  p o i n t on t h e a c u t e t e m p e r a t u r e - e x p e r i m e n t a l  interEach  salinity  i n t e r a c t i o n c u r v e s i s t h e mean o f w e i g h t - s p e c i f i c o x y g e n consumption a t that p a r t i c u l a r acute temperature-experimental  salinity  combination.  A C U T E T E M R - E X R SALINITY INTERACTION CAC) 700 600 500 400 300  200  EXR SALINITY ©35 %S.W. o 125 % S.W. A 75 %S.W.  IO  15  A C U T E T E M R °C  20  33  F i g u r e 9.  A c u t e t e m p e r a t u r e - s e a s o n i n t e r a c t i o n (AD)  and m a i n e f f e c t  of acute temperature  g l a n d o f Hemigrapsus nudus.  (A) f o r midgut  Each p o i n t  on t h e a c u t e  t e m p e r a t u r e - s e a s o n i n t e r a c t i o n c u r v e s i s t h e mean o f weight-specific  oxygen consumption  a t that  acute temperature-season combination. i s t h e mean o f a l l w e i g h t - s p e c i f i c data a t the respective  particular  The m a i n  oxygen  acute temperatures.  effect  consumption  A C U T E TEMR (A) ACUTE TEMR-SEASON INTERACTION (AD) 700 600 500 400 300  200 ©WINTER A MAIN E F F E C T (A) o SUMMER  IO 15 A C U T E TEMR °C  20  34  Figure 10.  Experimental temperature-experimental  ty interaction  (BC) f o r m i d g u t g l a n d o f H e m i g r a p s u s  E a c h p o i n t on t h e e x p e r i m e n t a l salinity cific  salini-  temperature-experimental  i n t e r a c t i o n c u r v e s i s t h e mean o f  oxygen consumption a t t h a t p a r t i c u l a r  temperature-experimental  nudus.  salinity  weight-speexperimental  combination.  EXR TEMR - EXR S A L I N I T Y I N T E R A C T I O N CBC) 500 4Q0  300 EXR T E M R ©5 OQ o20°C 200 35 EXR SALINITY, % S  35 by a M-T  curve  i n which the r e s p i r a t o r y  rate  decreases  w i t h an i n c r e a s e i n s a l i n i t y . As  seen  Experimental A  i n Table  Temperature-Season  seasonal  experimental  IV, t h e i n t e r a c t i o n  i s significant.  (BD)  comparison of r e s p i r a t o r y  temperature  response to  i s not s i g n i f i c a n t  I n F i g u r e 4 i t may be s e e n  (Table I V ) .  t h a t t h e means o f t h e  r e s p i r a t i o n v a l u e s f o r each experimental  temperature  o n l y a s m a l l magnitude o f d i f f e r e n c e  seasonally.  rates  t h e summer  o f r e s p i r a t i o n are higher than  r a t e s by a f a c t o r  of  5% a t 5°0 and k% a t 20°C  show  The w i n t e r respiration  experimental  temperature.. Second-Order I n t e r a c t i o n s Experimental This  combination  significant The  Temperature-Experimental  plotted  of factors results  second-order  interaction  means o f a l l t h e r e s p i r a t o r y  mental temperature, i n Figure  interpreted  S a l i n i t y - S e a s o n (BOD)  experimental 11.  i n the only  (Table I V ) . d a t a f o r each e x p e r i -  salinity  The t h r e e f a c t o r  and season a r e  interaction i s  a s a n i n t e r a c t i o n o f t h e i n t e r a c t i o n CD  mental s a l i n i t y - s e a s o n ) with f a c t o r B (experimental ature).  The w i n t e r M-T  curves a t the three  s a l i n i t i e s have s l o p e s w h i c h a r e d i f f e r e n t M-T  curves.  influenced  T h e s e M-T  curves  by e x p e r i m e n t a l  (experitemper-  experimental from  t h e summer  i n t u r n have s l o p e s w h i c h a r e  temperature.  I t i s seen i n  36  Figure 11. ty-season  Experimental temperature-experimental interaction  g r a p s u s nudus.  salini-  (BCD) f o r m i d g u t g l a n d o f H e m l -  E a c h p o i n t on t h e e x p e r i m e n t a l  temper-  ature-experimental s a l i n i t y - s e a s o n i n t e r a c t i o n curves i s t h e mean o f w e i g h t - s p e c i f i c o x y g e n c o n s u m p t i o n a t t h a t particular ty-season  experimental temperature-experimental combination.  salini-  r  EXP. T E M R - E X P . S A L I N I T Y S E A S O N I N T E R A C T I O N CBCD)  500 —  400  c CJ) >«* t_  300  CM  O  200  - W I N T E R ©5 °C o 2 0 °C - S U M M E R ©5 o20  35  1  75 EXP. SALINITY, %S.W.  °C °C  1  125  37  F i g u r e 11 t h a t the slope o f the w i n t e r M-T curve a t 5°C shows a "V-shaped ' r e l a t i o n s h i p to s a l i n i t y . 1  r e s p i r a t i o n r a t e i s i n 7 5 $ sea water. at 20°C  The lowest  The w i n t e r M-T  curve  shows an i n c r e a s e i n slope as the s a l i n i t y i n c r e a s e s .  T i s s u e samples from  summer animals have M-T curves t h a t a l s o  v a r y w i t h the s a l i n i t y due to a change i n experimental temperature.  The summer M-T curve a t 5°C does not show a  d i f f e r e n c e i n the magnitude o f response range of 3 5 $ to 1 2 5 $ sea water.  over t h e s a l i n i t y  A t 20°C, t h e slope of the  M-T curve decreases w i t h a i n c r e a s e i n s a l i n i t y . Other Second-Order I n t e r a c t i o n s (ABC,ABD,ACD) None of these i n t e r a c t i o n s i s s i g n i f i c a n t  (Table IV)«  The d i f f e r e n c e s between the means a r e s m a l l and randomly d i s t r i b u t e d .  The r e l a t i o s h i p s a r e not g r a p h i c a l l y  r e p r e s e n t e d i n the t e x t o f t h i s  study.  E f f e c t of Body Weight The  s l o p e s o f the r e g r e s s i o n l i n e s o f w e i g h t - s p e c i f i c  oxygen consumption a g a i n s t body weight  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 f o r both seasons a t the 0 . 0 1 level.  probability  A t the 0 . 0 5 l e v e l o f s i g n i f i c a n c e , t h r e e of twenty-  f o u r s l o p e s were s i g n i f i c a n t l y d i f f e r e n t from zero f o r , t h e w i n t e r animals and two of twenty-four  s l o p e s were  c a n t l y d i f f e r e n t from zero f o r the summer a n i m a l s .  signifiThese  slope d i f f e r e n c e s were c o n s i d e r e d to be due t o chance alone and t h e r e f o r e were not s t a t i s t i c a l l y confirmed when adjustment  significant.  T h i s was  of the treatment means by the  38  analysis of covariance resulted  i n v e r y s m a l l changes I n  the F-values. DISCUSSION Seasonal Metabolic-Temperature The  Experiments  s e a s o n a l r a t e o f oxygen consumption  g l a n d was  f o r midgut  f o u n d t o be h i g h e r i n summer a n i m a l s a t a l l  t e m p e r a t u r e s f r o m 5°C  t o 20°C ( F i g . 2 ) .  as i n v e r s e s e a s o n a l compensation,  acute  This i s interpreted  type 5 (Precht,  1951).  An i d e n t i c a l p a t t e r n o f r e s p o n s e has been o b s e r v e d f o r excised g i l l animal range.  ( D e h n e l and M c C a u g h r a n , 1964)  tissue  ( D e h n e l , 1960)  and  o v e r t h e same p h y s i o l o g i c a l  V e r n b e r g and V e r n b e r g  whole  temperature  (1966) h a v e r e c e n t l y  found  t h a t t h e m o s t common t y p e o f s e a s o n a l a d a p t a t i o n i n h e a r t , m u s c l e a n d b r a i n t i s s u e o f t e m p e r a t e and t r o p i c a l s p e c i e s o f U c a a t a c u t e t e m p e r a t u r e s f r o m 5°C Inverse  zone  t o 35°C i s  compensation.  P r o s s e r (1958) c o m p e n s a t i o n may to r e f l e c t i n enzymes.  suggests that inverse seasonal  h a v e no a d a p t i v e s i g n i f i c a n c e a n d  appears  a q u a n t i t a t i v e rather than a q u a l i t a t i v e T h a t i s , a p a r t i c u l a r enzyme m i g h t  change  change i n  p r o p o r t i o n t o a n o t h e r enzyme i n p a r a l l e l o r s e r i e s . is  s e e n a s a t r a n s l a t i o n o f t h e summer M-T  left  o r a b o v e t h e w i n t e r M-T  This  curve to the  curve ( F i g . 2).  Prosser's  s u g g e s t i o n h a s f o u n d s u p p o r t i n t h e f i n d i n g s o f Kanungo and P r o s s e r (1959), E k b e r g " a n i m a l , t i s s u e and  (1958) a n d F r e e d (1965) on  enzymes i s o l a t e d f r o m t e m p e r a t u r e  intact ac-  39 climated g o l d f i s h . A l t e r n a t i v e l y , t h e h i g h e r metabolic r a t e o f summer animals may be due t o t h e f a c t t h a t many o f t h e summer animals s e l e c t e d f o r study may have been p h y s i o l o g i c a l l y i n premolt, r a t h e r than i n t e r m o l t .  The r a p i d s y n t h e s i s  of o r g a n i c m a t e r i a l and m o b i l i z a t i o n of i n o r g a n i c i o n s p r i o r to and during exoskeleton f o r m a t i o n could account f o r the h i g h e r r e s p i r a t i o n r a t e of summer animals.  Skinner  (1962) has noted t h a t the s y n t h e s i s of e x p s k e l e t o n d u r i n g late D  2  stage (premolt) i n c r e a s e s the w e i g h t - s p e c i f i c  oxygen consumption  o f the l a n d crab, Gecarcinus l a t e r a l i s ,  by 60$ as compared to stage C4 ( i n t e r m o l t ) . An examination of the types o f compensation  found i n  Table V r e v e a l s t h a t p a r t i a l seasonal compensation predominates  a t s a l i n i t y and temperature  (type 3)  combinations t o  which the animals a r e a c c l i m a t e d ; not a t standard b a s e l i n e conditions.  T h i s i n d i c a t e s t h a t osmotic and/or  temperature  s t r e s s may have a more depressant e f f e c t on the m e t a b o l i c a c t i v i t y o f the w i n t e r animals (see P i g . 3 ) . To o f f s e t t h i s depressant e f f e c t , w i n t e r animals i n c r e a s e m e t a b o l i c a c t i v i t y above t h a t shown i n summer animals. r e l a t e d w i t h the lower compensation  This i s cor-  c o e f f i c i e n t of winter  a c c l i m a t e d animals, i n d i c a t i n g that w i n t e r animals  demonstrate  the g r e a t e s t degree o f a c c l i m a t e d response. Metabolism-Body Weight R e l a t i o n s h i p The r e l a t i o n s h i p o f whole animal and t i s s u e  respiration  40 to body weight has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n i n reviews by Brody Bertalanffy  (1945), Krebs (1950) and Zeuthen  (1953).  (1951) proposed t h a t the r e g r e s s i o n of  metabolism on body weight c o u l d be expressed i n terms o f 2/3,  3/4 o r 1 p r o p o r t i o n a l i t y .  B e r t a l a n f f y suggested t h a t  there Is a s p e c i e s - s p e c i f i c power f u n c t i o n t h a t i s f i x e d . B e r t a l a n f f y and Krywienczyk  (1953) found t h a t the s u r f a c e  law of 2/3 p r o p o r t i o n a l i t y c h a r a c t e r i s t i c of C r u s t a c e a c o u l d be a p p l i e d to the metabolic-weight response of the b r i n e shrimp, Artemia s a l i n a . Zeuthen  Weymouth et a l . ( 1 9 4 4 ) ,  (1953) and Scholander et a l . (1953) have shown i n  o t h e r s p e c i e s o f C r u s t a c e a t h a t the c o r r e l a t i o n between metabolism and body weight tends to assume the 3/4 power function. Hemigrapsus  In the g r a p s o i d crabs, Pachygrapsus  crassipes,  nudus and H. oregonensls, the r e g r e s s i o n  c o e f f i c i e n t s r a r e l y approached the 2/3 o r 3/4 (Roberts 1957a; Dehnel, i 9 6 0 ) .  Dehnel  found i n both s p e c i e s of Hemigrapsus  exponent  (1960) a c t u a l l y a spread i n weight-  s p e c i f i c oxygen consumption r e g r e s s i o n v a l u e s from -0.685 to -0.333. In t h i s study the w e i g h t - s p e c i f i c oxygen  consumption  to body weight r e g r e s s i o n of e x c i s e d midgut g l a n d was to be independent of body weight. was  The same r e l a t i o n s h i p  found by Vernberg and Gray (1953) f o r the QQ  b r a i n t i s s u e of t e l e o s t f i s h .  found  2  of e x c i s e d  The independence of midgut  gland r e s p i r a t i o n from body weight i s i n c o n t r a s t to the mean -O.169 r e g r e s s i o n c o e f f i c i e n t of e x c i s e d g i l l f o r both s p e c i e s of Hemigrapsus  tissue  (Dehnel and McCaughran,  41 1964). I t appears t h a t each t i s s u e has i t s own unique p a t t e r n of response to changes i n "body weight.  These changes have  been r e f l e c t e d as d i f f e r e n c e s i n c e l l u l a r enzyme a c t i v i t y weight r e g r e s s i o n s f o r mammalian t i s s u e s (Rosenthal and Drabkin, 1943; Kunkel and Campbell, 1952; F r i e d and T i p t o n , 1953)•  An attempt to a s s i g n a f i x e d p r o p o r t i o n a l i t y v a l u e  to a whole animal and/or i t s t i s s u e s and enzyme systems i s , therefore, i n v a l i d .  The r e g r e s s i o n c o e f f i c i e n t i s dependent  on the environmental h i s t o r y of the animal, e f f e c t of p h y s i c a l and b i o t i c parameters, and techniques employed measure responses.  to  The f a c t t h a t t i s s u e samples when removed  from the animal are no l o n g e r i n f l u e n c e d by the c e n t r a l nervous system o r by hormones could account i n part f o r the d i s c r e p a n c i e s between t i s s u e and whole animal metabolism to body weight r e g r e s s i o n . E f f e c t of Experimental Temperature Temperature  determines to a g r e a t extent the r a t e s of  chemical r e a c t i o n s and, thus, the r a t e of metabolism and activity.  In t h i s sense temperature i s considered one of  the most important of environmental parameters. Bullock  (1955) and  Prosser  (1955) have  presented  comprehensive reviews of temperature e f f e c t s on r a t e  functions  d e a l i n g w i t h such t h i n g s as thermal l i m i t s of t i s s u e s and whole organisms, oxygen consumption, h e a r t beat and pumping a c t i v i t y .  ciliary  Not only r a t e f u n c t i o n s , but the m e t a b o l i c  42  pathway u t i l i z e d hy an organism may 1958;  Hochachka and Hayes, 1962;  be a l t e r e d  (Ekberg, 1965).  Dean and Vernberg,  There i s a 24$ decrease i n m e t a b o l i c a c t i v i t y of midgut gland w i t h an i n c r e a s e i n experimental temperature from 5°C to 20°C ( P i g . 4 ) .  T h i s percentage decrease h o l d s  f o r both summer and w i n t e r animals. temperature  e f f e c t corresponds to P r e c h t ' s type 3 ( p a r t i a l  compensation).  A similar  effect  i s depicted i n Figure 7  where the experimental temperature acute temperature  i s averaged at each  (5°, 10°, 15° and 20°0) f o r both  and t h r e e experimental s a l i n i t i e s water).  The experimental  (35$, 75$ and  125$  (1943) demonstrated  Emerita t a l p o l d a .  has a l s o  Scholander et a l . (1953) found the same  been demonstrated  P r o s s e r (1959) f o r g o l d f i s h  Partial  compensation  by C l a r k (1955) f o r the t e r -  r e s t r i a l amphipod, T a l i t r u s s y l v a t i c u s ,  s p e c i e s of  Edwards and  t h i s e f f e c t i n the sand crab,  response i n a q u a t i c f i s h and C r u s t a c e a .  by Kanungo and  and by Dehnel (1960) f o r both  Hemigrapsus.  Dehnel and McCaughran (1964) found t h a t excised tissue  sea  T h i s type o f compensation has been documented  e x t e n s i v e l y by a number of i n v e s t i g a t o r s . Irving  seasons  showed no experimental temperature  s p e c i e s o f Hemigrapsus. biologically significant. compensation  gill  e f f e c t f o r both  T h i s e f f e c t was not considered The demonstration o f p a r t i a l  f o r e x c i s e d midgut  s t a t i s t i c a l l y and b i o l o g i c a l l y . of t h i s e f f e c t i s i n t e r p r e t e d  gland i s s i g n i f i c a n t The b i o l o g i c a l  importance  on the grounds t h a t  low  43 t e m p e r a t u r e may p r o v i d e a g r e a t e r t h e r m a l t e m p e r a t u r e (Todd a n d D e h n e l , 1 9 6 0 ) . ature  To o f f s e t t h i s  high temper-  s t r e s s , t h e r a t e o f oxygen consumption i s i n c r e a s e d  above t h a t e x p e r i e n c e d correlated who  s t r e s s than  a t 20°C.  This i n t e r p r e t a t i o n i s  i n part w i t h the observations  e s t a b l i s h e d t h a t cytochrome oxidase  i n the  cold acclimated  o f Freed activity  (1965) increased  (5°C) g o l d f i s h a n d d e c r e a s e d  with  a c c l i m a t i o n t o heat (30°0). E f f e c t of Experimental  Salinity  Salinity, i s t h e other major environmental besides  t e m p e r a t u r e , w h i c h h a s a p r o n o u n c e d e f f e c t on t h e  metabolic their  a c t i v i t y o f a q u a t i c i n v e r t e b r a t e animals and  tissue. The  by  factor,  e f f e c t o f s a l i n i t y may a l t e r t h e m e t a b o l i c  i n c r e a s i n g r e s p i r a t i o n i n sub- and s u p r a - n o r m a l  ties.  salini-  T h i s h a s been d e m o n s t r a t e d by F l e m i s t e r and  F l e m i s t e r (1951) f o r t h e Lofts  response  s a n d c r a b , Ocypode a l b i c a n s , b y  (1956); i n a s a l t m a r s h p o p u l a t i o n o f t h e p r a w n , .  P a l a e m o n e t e s v a r i a n s , a n d b y Rao (1958) f o r t h e b r a c k i s h w a t e r s p e c i e s o f t h e prawn, Metapenseus monoceros.  There  are o t h e r s t u d i e s t h a t have i n d i c a t e d t h a t m e t a b o l i c may  be h i g h e r i n s u b - n o r m a l s a l i n i t i e s .  found i n both  activity  D e h n e l (196O)  s p e c i e s o f Hemigrapsus t h a t t h e r a t e o f  r e s p i r a t i o n was h i g h e s t  i n d i l u t e s e a w a t e r where t h e o s m o t i c  g r a d i e n t b e t w e e n t h e b l o o d a n d medium was g r e a t e s t . (1965) showed t h a t t h e m e t a b o l i c  Lance  r a t e i n 30$ s e a w a t e r was  ^1M' double t h a t i n 100$ A c a r t i a tonsa.  sea water f o r the p l a n k t o n i c copepod,  King  (1965) demonstrated i n the crabs,  Oarclnus medlterraneus and 53$  Increase  O a l l i n e c t e s sapidus, a 33$  and  i n oxygen consumption, r e s p e c t i v e l y , a f t e r  animals were t r a n s f e r e d from 80$ to 50$ c o n e l u s i o n reached by these authors  sea water.  the  The  i s t h a t when the  blood  c o n c e n t r a t i o n i s no longer i s o t o n i c to the medium oxygen consumption i s i n c r e a s e d to m a i n t a i n  the osmotic g r a d i e n t .  S e v e r a l i n v e s t i g a t o r s have r a i s e d o b j e c t i o n s to the p r o p o s a l t h a t i n c r e a s e d oxygen consumption r e f l e c t s osmotic work to m a i n t a i n the osmotic g r a d i e n t between the blood medium.  Gross (1957) has  and  suggested t h a t the i n c r e a s e i n  r e s p i r a t i o n r a t e w i t h an i n c r e a s e i n osmotic g r a d i e n t f o r the rock crab, Pachygrapsus c r a s s i p e s , i s r e l a t e d to increase i n a c t i v i t y .  an  A comparison of r e s p i r a t o r y and  osmoregulatory data i n both s p e c i e s of Hemigrapsus i n d i c a t e s t h a t a i n c r e a s e i n r e s p i r a t i o n r a t e does not n e c e s s a r i l y r e f l e c t osmotic work (Dehnel,  1962).  Dehnel and McOaughran  (1964) found t h a t the r a t e of oxygen consumption f o r e x c i s e d gill any  t i s s u e of w i n t e r s p e c i e s of Hemigrapsus d i d not show c o r r e l a t i o n with s a l i n i t y .  t h a t the e x c i s e d g i l l s of Oarclnus, not  (1965) d i s c o v e r e d  King  an osmoregulator, d i d  show a s i g n i f i c a n t change i n oxygen consumption when  t r a n s f e r e d from 80$ to 50$  sea water.  In Maja, a crab t h a t  remains isoosmotic w i t h the medium, there was in  a 6$  increase  oxygen consumption of e x c i s e d g i l l upon d i l u t i o n of the  suspending medium from 80$  to 50$  sea water.  45 To assess p r o p e r l y the e f f e c t of s a l i n i t y gland r e s p i r a t i o n , one should f i r s t to which the osmotic  on midgut  examine the extent  c o n c e n t r a t i o n of the blood and u r i n e  change w i t h changes i n s a l i n i t y .  The blood c o n c e n t r a t i o n  of w i n t e r and summer Hemigrapsus nudus i s h y p e r t o n i c to the medium over the experimental s a l i n i t y to  1 2 5 $ sea water.  range from 25$  The animals r e g u l a t e t h e i r blood  c o n c e n t r a t i o n i n s a l i n i t i e s from 25$ to 75$ sea water. Beyond t h i s s a l i n i t y  range, r e g u l a t i o n breaks down and the  blood approaches i s o t o n i c i t y w i t h the medium although s t i l l h y p e r t o n i c to i t . The osmotic u r i n e f o r summer animals  c o n c e n t r a t i o n of the  i s equal t o the blood c o n c e n t r a t i o n  over the experimental s a l i n i t y range from 25$ to 1 2 5 $ sea water and h y p e r t o n i c t o the medium.  Winter  animals  have a u r i n e which i s hypotonic to the blood a t a l l experimental s a l i n i t i e s from 25$ to 125$ sea water and a l l experimental temperatures  except  15°0. The u r i n e i s hyper-  t o n i c to the medium below 90$ sea water and hypotonic above t h i s sea water c o n c e n t r a t i o n (Dehnel, Stone,  1962; Dehnel and  1964).  When these osmoregulatory r e s p i r a t o r y response  of e x c i s e d midgut gland t i s s u e , a  seasonal e f f e c t i s noted. decrease  data a r e compared w i t h the  In summer animals there i s a  i n midgut gland r e s p i r a t i o n w i t h a Increase i n  s a l i n i t y from 35$ to 125$ sea water ( F i g . 5 ) .  In 35$ sea  water there i s a l a r g e osmotic g r a d i e n t between the blood and medium.  To m a i n t a i n the blood c o n c e n t r a t i o n h y p e r t o n i c  46  t o t h e medium, a c t i v e a b s o r p t i o n o f i o n s from t h e midgut g l a n d may o c c u r .  Osmotic work would have t o be performed  t o m a i n t a i n t h i s g r a d i e n t and t h e r a t e o f oxygen consumption would be h i g h .  Whole a n i m a l r e s p i r a t i o n o f Hemigrapsus  nudus, however, does n o t support t h i s p r o p o s a l 1962).  (Dehnel,  The b l o o d approaches i s o t o n i c i t y w i t h t h e medium  i n 75$ sea water.  S i n c e t h e osmotic g r a d i e n t i s small,  and t h e a n i m a l i s r e g u l a t i n g i t s b l o o d c o n c e n t r a t i o n t o a m i n i m a l degree, t h e s e f a c t s may account f o r t h e drop i n midgut g l a n d oxygen consumption i n 75$ sea w a t e r .  I n 125$  sea w a t e r osmotic s t r e s s may i n c r e a s e m o r t a l i t y and cause a f u r t h e r drop i n r e s p i r a t i o n .  This i s p l a u s i b l e since  summer a n i m a l s n o r m a l l y do n o t encounter i n the f i e l d .  such a h i g h  salinity  Dehnel and McCaughran (1964) have demonstrated  t h a t t h e g i l l s o f summer Hemigrapsus s p . a l s o appear t o be i m p o r t a n t i n osmotic r e g u l a t i o n when t h e osmotic g r a d i e n t between t h e b l o o d and medium i s maximal (35$ sea w a t e r ) . As has been suggested  e a r l i e r , m e t a b o l i c a c t i v i t y o f midgut  g l a n d from summer a n i m a l s may be r e l a t e d more t o t h e energy demands o f new e x o s k e l e t o n f o r m a t i o n t h a n t o t h e maintenance o f a osmotic g r a d i e n t .  T h i s p r o p o s a l i s v a l i d when i t i s  r e c o g n i z e d t h a t many o f t h e summer a n i m a l s s e l e c t e d f o r study may have been p h y s i o l o g i c a l l y i n p r e m o l t , r a t h e r t h a n intermolt.  I n w i n t e r a n i m a l s t h e r e I s a "V-shaped"  r e l a t i o n s h i p o f midgut g l a n d r e s p i r a t i o n t o s a l i n i t y w i t h t h e l o w e s t r e s p i r a t i o n r a t e i n 75$ sea w a t e r ( P i g . 5 ) . The low m e t a b o l i c response  i n 75$ sea w a t e r corresponds  to the  47 p o i n t where the blood and u r i n e c o n c e n t r a t i o n s approach i s o t o n i c i t y w i t h the medium. i n 35$  sea water may  The h i g h r e s p i r a t o r y  response  i n d i c a t e d work being done to m a i n t a i n  the osmotic g r a d i e n t between the blood and medium. F i g u r e 8 a s i m i l a r p a t t e r n of response  i s found when the  experimental s a l i n i t y e f f e c t i s averaged temperature  (5°, 10°, 15° and  temperatures gland appears  In  a t each acute  20°C) f o r two  experimental  ( 5 0 and 20°C) and both seasons. C  The midgut  to be a c t i v e i n the w i n t e r i n the maintenance  of a osmotic g r a d i e n t although evidence presented by  Gross  (1957) and Dehnel (1962) on whole animalnwould tend to confute t h i s s u g g e s t i o n . t o n i c to the blood may  The p r o d u c t i o n of a u r i n e hypo-  a l s o a s s i s t the midgut g l a n d during  the w i n t e r i n s a l t r e g u l a t i o n of the b l o o d .  P o t t s (1954)  p o i n t s out;, however, t h a t the p r o d u c t i o n of a h y p o t o n i c u r i n e y i e l d s a n e g l i g i b l e saving of osmotic work i n sea water below  50$.  E f f e c t of Temperature-Salinity-Season I n t e r a c t i o n s There are s e v e r a l s t u d i e s which have assessed e f f e c t s of environmental whole animal and t i s s u e .  the  f a c t o r s a c t i n g simultaneously on Panikkar  (1940), Broekema  (1941),  Dehnel (1960) and Todd and Dehnel (1960) have examined the combined e f f e c t s of seasonal changes i n temperature s a l i n i t y on the m o r t a l i t y , m e t a b o l i c a c t i v i t y and  and  thermal  l i m i t s of i n t a c t C r u s t a c e a .  Dehnel and McCaughran (1964)  have determined  temperature,  s a l i n i t y and seasonal e f f e c t s  on e x c i s e d g i l l  t i s s u e i n both s p e c i e s o f Hemigrapsus.  48 (1963,1964)  KInne  has  presented  studies demonstrating temperature  and  differential  experimental  effects  to  a seasonal comparison  of  oxygen consumption of midgut g l a n d f o r the 1O°0  temperature-salinity interaction at  temperature. (Table  response  The  IV).  An  of  salinity.  In F i g u r e 6 i s presented weight-specific  a comprehensive review  interaction  experimental  is statistically temperature  acute  significant  effect  i s evident  when c o m p a r i s o n s a r e made w i t h i n and  between t h e  I n summer a n i m a l s  c o r r e l a t i o n of midgut  gland  a t 5°C  there  i s no  respiration with s a l i n i t y .  temperature w i t h an  of 20°C t h e r e i s a decrease  increase i n s a l i n i t y .  salinity  At the  e f f e c t may  seasons.  summer b a s e l i n e i n oxygen  consumption  This experimental  be r e l a t e d  to osmotic  temperature-  work b e i n g  i n maintenance of a osmotic  g r a d i e n t between t h e  and  demands o f e x p s k e l e t o n  medium o r t o t h e  since  energy  i t i s r e c o g n i z e d t h a t many o f t h e have been i n p r e m o l t .  blood formation,  summer a n i m a l s  in this  s t u d y may  animals  a t t h e i r w i n t e r b a s e l i n e temperature  In the  o f 5°C  r e s p i r a t i o n r a t e I s i n 75$  s e a w a t e r where t h e b l o o d  i s o t o n i c i t y w i t h t h e medium.  a t i o n r a t e i n 35$ i s doing  gradient.  The  s e a w a t e r may  osmotic  the r a t e of midgut g l a n d salinity.  An  lowest  increased  aprespir-  i n d i c a t e t h a t the midgut  work i n r e s p o n s e  winter animals  The  The  a  salinity  gland  i s shown.  used  winter  "V-shaped" r e l a t i o n s h i p t o  proaches  done  to the  osmotic  at 20 C gradually increase 6  r e s p i r a t i o n with an  e x p l a n a t i o n of t h i s response  increase i n  cannot  be  given  49 at  t h i s time.  The same g e n e r a l t r e n d s a r e a l s o  in  F i g u r e 11 w h e r e s e a s o n a l c h a n g e s i n e x p e r i m e n t a l  a t u r e and s a l i n i t y a r e averaged (5°,  f o r a l l acute  evident temper-  temperatures  10°, 15° a n d 2 0 ° C ) . In  a c o m p a r a b l e s t u d y on e x c i s e d g i l l  t i s s u e f o r both  s p e c i e s o f Hemigrapsus , Dehnel and McOaughran (1964) that g i l l in  t i s s u e from  summer a n i m a l s a p p e a r e d t o be a c t i v e  t h e maintenance o f a n osmotic g r a d i e n t between t h e b l o o d  and medium w h i l e t h e g i l l s  of winter animals  showed no c o r -  r e l a t i o n i n r e s p i r a t i o n rate with experimental  salinity.  On a s e a s o n a l b a s i s , t h e s e f i n d i n g s d i f f e r f r o m observed  important  those  f o r midgut g l a n d .  T e m p e r a t u r e and s a l i n i t y b o t h h a v e  biologically  e f f e c t s on s e a s o n a l changes i n midgut g l a n d  respiration.  The m e t a b o l i c a c t i v i t y  summer a n i m a l s may be g e a r e d  o f midgut gland i n  not o n l y t o maintenance o f  an osmotic g r a d i e n t b u t a l s o t o energy  requirements a s -  s o c i a t e d w i t h new e x p s k e l e t o n f o r m a t i o n .  In winter  t h e m i d g u t g l a n d may p l a y a r o l e i n r e g u l a t i n g electrolytes.  This f a c t together w i t h the evidence  p a r t f o r t h e mechanisms o f o s m o t i c  animals.  animals  blood  p r o d u c t i o n o f a u r i n e h y p o t o n i c t o t h e b l o o d may in  found  of the  account  regulation i n winter  50 SUMMARY  1•  Weight-specific oxygen consumption of midgut  gland tissue of Hemigrapsus nudus has been investigated at three levels of salinity (35$, 75$ and 125$ sea water), two levels of experimental temperature (5°0 and 20°0) and four acute (Warburg) temperatures (5°, 10°, 15° and 20°C) in a l l combinations for each season (summer and winter). The data are evaluated and discussed in terms of midgut gland function in the intact animal. 2.  Weight-specific oxygen consumption of midgut  gland from summer animals held 24 hr at seasonal baseline conditions (35$ sea water, 20°0) is higher at a l l acute temperatures of measurement.(5°C to 20°C) than weightspecific oxygen consumption of midgut gland from winter animals held 24 hr at its seasonal baseline conditions (75$ sea water, 5°C). 3.  Acutely measured metabolic-temperature curves  of midgut gland tissue from winter and summer animals held 10 days at their opposite seasonal conditions show that winter animals demonstrate the greatest degree of acclimation. 4.  The effect of experimental temperature is  statistically and biologically significant.  There is a  24$ decrease in metabolic activity of midgut gland with an increase in experimental temperature from 5°C to 20°C. Low temperature may provide a greater thermal stress than a high temperature resulting in a higher rate of oxygen  51 consumption. responds  The e x p e r i m e n t a l t e m p e r a t u r e  effect  t o P r e c h t ' s (1951) t y p e 3 ( p a r t i a l  5.  Experimental temperature  cor-  compensation).  e f f e c t i s noted seasonally  i n t h e r e s p i r a t o r y response o f midgut g l a n d t o s a l i n i t y . Summer a n i m a l s a t 5°0 of  show no c h a n g e i n t h e o r d i n a l  position  t h e m e t a b o l i c - t e m p e r a t u r e c u r v e w i t h a change i n s a l i n i t y .  A t t h e summer b a s e l i n e t e m p e r a t u r e  o f 20°C t h e r e i s a  increase i n r e s p i r a t i o n w i t h a decrease  i n salinity.  Winter animals a t t h e seasonal b a s e l i n e temperature  o f 5°C  demonstrate  The  a "V-shaped" r e l a t i o n s h i p t o s a l i n i t y .  l o w e s t r a t e o f w e i g h t - s p e c i f i c oxygen consumption 75$  sea water.  i s in  The m e t a b o l i c - t e m p e r a t u r e c u r v e o f w i n t e r  a n i m a l s a t 20°C i n c r e a s e s w i t h a n i n c r e a s e i n s a l i n i t y . 6.  I t i s suggested t h a t t h e metabolic a c t i v i t y o f  m i d g u t g l a n d i n summer a n i m a l s may be r e l a t e d of  t o t h e maintenance  a o s m o t i c g r a d i e n t b e t w e e n t h e b l o o d a n d medium.  h i g h e s t r a t e o f oxygen consumption conditions  (35$  The  i s a t summer b a s e l i n e  s e a w a t e r , 20°C) w h e r e t h e o s m o t i c g r a d i e n t  b e t w e e n t h e b l o o d a n d medium i s m a x i m a l . midgut gland r e s p i r a t o r y a c t i v i t y  Alternatively,  may be g e a r e d t o t h e e n e r g y  demands a s s o c i a t e d w i t h new e x o s k e l e t o n f o r m a t i o n . proposal i s v a l i d  This  s i n c e i t i s r e c o g n i z e d t h a t many o f t h e  summer a n i m a l s s e l e c t e d f o r s t u d y may h a v e b e e n p h y s i o l o g i cally  i n premolt.  S i n c e premolt a n i m a l s have a h i g h e r  r e s p i r a t i o n rate than intermolt animals, the higher seasonal summer r a t e s c o u l d be e x p l a i n e d o n t h i s 7. ature  basis.  Winter animals a t t h e i r seasonal b a s e l i n e temper-  (5°C) show a " V - s h a p e d " r e l a t i o n s h i p t o s a l i n i t y .  52 This r e l a t i o n s h i p r e f l e c t s the p o s s i b i l i t y t h a t midgut gland  t i s s u e may  be r e g u l a t i n g b l o o d  r e s p i r a t o r y r e s p o n s e i n 35$ being and  s e a w a t e r may  done t o m a i n t a i n t h e o s m o t i c I n 75$  medium.  the osmotic  a s s i s t winter animals  little  work blood  work would  the g r a d i e n t .  to the blood a l s o  i n maintaining the blood  The  may  concentration  t o t h e medium. The  r e g r e s s i o n of w e i g h t - s p e c i f i c oxygen consumption  as a f u n c t i o n o f body w e i g h t does n o t relationship. different  indicate  g r a d i e n t between the  medium i s a t a minimum and  production of a urine hypotonic  8.  high  g r a d i e n t between the  a p p a r e n t l y h a v e t o be done t o m a i n t a i n  hypertonic  The  s e a w a t e r where t h e r a t e o f o x y g e n  consumption i s minimal, b l o o d and  salts.  The  show a  slope values are not  from zero a t the 0.01  significant  significantly  probability  level.  53 LITERATURE CITED  B e l d i n g , H. S., F i e l d , J . I I and Weymouth, F. W. 194-2. S t u d i e s on t h e m e t a b o l i s m o f m a r i n e i n v e r t e b r a t e tissue. I . R e s p i r a t i o n of the midgut g l a n d of the k e l p c r a b ( P u g e t t i a p r o d u c t a ) . P h y s i o l . Z o o l . , 15: 75-88. v o n B e r t a l a n f f y , L. 1951. M e t a b o l i c t y p e s and t y p e s . 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