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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 Scott Dransfield Hawke B.S., San Diego State College, 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 thesis as conforming to the required 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 of the requirements f o r an advanced degree at tne U n i v e r s i t y of B r i t i s h Columbia;, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and studyo 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 extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . i ABSTRACT-: Weight-specific oxygen consumption of midgut gland tissue of Hemigrapsus nudus has been investigated at three l e v e l s of s a l i n i t y (35%> 75% and 125$ sea water), two l e v e l s of experimental temperature (5°C and 20°C) and four acute (Warburg) temperatures (5°, 10°, 15° and 20°C) i n a l l combinations f o r each season (summer and winter). Metabolic-temperature curves reveal that at standard baseline conditions where the animals are held 24 hr at t h e i r respective 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 highest at a l l acute temperatures i n the summer animals. Acutely measured metabolic-temperature curves f o r midgut gland tissue show that winter animals acclimated to t h e i r opposite seasonal conditions of temper-ature and s a l i n i t y f o r 10 days demonstrate the greatest degree of acclimation. The effect of experimental temperature i s 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 s i g n i f i c a n t . The highest r e s p i r a t i o n rate i s at 5°C. Low temperature (5°C) may provide a greater thermal stress than a high temperature (20°C) re s u l t i n g i n a higher rate of oxygen consumption. Experimental temperature also influences the seasonal respiratory response of midgut gland tissue 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 rate as the osmotic gradient between the blood and medium i i i n c r e a s e s a t t h e s e a s o n a l b a s e l i n e t e m p e r a t u r e o f 20°C. W i n t e r a n i m a l s h e l d a t t h e s e a s o n a l b a s e l i n e temperature o f 5°C demonstrate 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 w i t h t h e l o w e s t r e s p i r a t o r y r e sponse i n 75$ sea water where the g r a d i e n t between t h e b l o o d and medium i s m i n i m a l . A n i m a l s 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 an i n c r e a s e i n s a l i n i t y . I t i s suggested t h a t t h e 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 t o t h e maintenance o f a o s m o t i c g r a d i e n t between t h e b l o o d and medium o r a l t e r n a t i v e l y t o t h e energy 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 . The p r o p o s a l i s put f o r t h t h a t midgut 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 o s m o t i c work b e i n g done t o m a i n t a i n t h e osmotic g r a d i e n t between t h e b l o o d and medium. 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 i n r e g u l a t i o n o f b l o o d e l e c t r o l y t e s . 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 consumption as a f u n c t i o n o f 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 z e r o a t t h e 0.01 p r o b a b i l i t y l e v e l . TABLE OP CONTENTS Pag INTRODUCTION 1 MATERIALS AND METHODS 5 RESULTS 20 Seasonal Metabolic-Temperature Experiments 20 Main Effects ' 22 Effec t of Season (D) 22 Effec t of Acute Temperature (A) 23 Effec t of Experimental Temperature (B) 23 Effec t of Experimental S a l i n i t y (C) 25 Interactions 29 First-Order Interactions 29 Acute Temperature-Experimental Temperature (AB) 29 Acute Temperature-Season (AD) ...29 Acute Temperature-Experimental S a l i n i t y (AC) 30 Experimental Temperature-Experimental S a l i n i t y (BC) 30 Experimental Salinity-Season (CD) 30 Experimental Temperature-Season (BD) 35 Second-Order Interactions 35 Experimental Temperature-Experimental Salinity-Season (BCD) 35 Other Second-Order Interactions (ABC,ABD,ACD) 37 Effect of Body Weight 37 iv Page DISCUSSION. 38 Seasonal Metabolic-Temperature Experiments 38 Metabolism-Body Weight Relationship 39 Eff e c t of Experimental Temperature 41 Effect of Experimental S a l i n i t y 43 Ef f e c t of Temperature-Salinity-Season Interactions 47 SUMMARY 50 LITERATURE CITED 53 V LIST' OP FIGURES Figure No. Page 1 Relationship of weight-specific oxygen consumption of midgut gland as a function of time f o r a 7 g animal ........ 9 2 Weight-specific oxygen consumption of midgut gland f o r animals held 24 hr at standard seasonal baseline conditions and 10 days at opposite seasonal conditions 21 3 Weight-specific oxygen consumption of midgut gland f o r animals held 10 days i n 35% and 125$ sea water at 5°C 24 4 Experimental temperature-season in t e r a c t i o n (BD) and main effect of experimental temperature (B) on weight-specific oxygen consumption of midgut gland 26 5 Experimental salinity-season i n t e r a c t i o n (CD) and main effect of experimental s a l i n i t y (C) on weight-specific oxygen consumption of midgut gland 27 6 Seasonal comparison of experimental s a l i n i t y and experimental temperature effect on weight-specific oxygen v i Figure No. Page consumption of midgut gland, measured at 10°C acute temperature 28 7 Effect of acute temperature-experimental temperature i n t e r a c t i o n (AB) on weight-s p e c i f i c oxygen consumption of midgut gland 31 8 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 weight-s p e c i f i c oxygen consumption of midgut gland 32 9 Acute temperature-season i n t e r a c t i o n (AD) and main effect of acute temperature (A) on weight-specific oxygen consumption of midgut gland 33 10 E f f e c t of experimental temperature-experimental s a l i n i t y i n t e r a c t i o n (BC) on weight-specific oxygen consumption of midgut gland 34-11 Effect of experimental temperature-experimental salinity-season i n t e r a c t i o n (BOD) on weight-specific oxygen consumption of midgut gland 36 v i i LIST OF TABLES Table Wo. Page I Mean values of main effects f o r weight-specific oxygen consumption of midgut gland tissue ..11 II Mean values of interactions f o r weight-specific oxygen consumption of midgut gland tissue 12 III Standard error of mean weight-specific oxygen consumption of midgut gland tissue f o r each experimental combination of factors 16 IV Analysis of variance of weight-specific oxygen consumption for a 4X 2X 3X 2X f a c t o r i a l design 18 V Types of seasonal compensation shown f o r weight-specific oxygen consumption of midgut gland tissue 19 ACKNO WLEDGEMENT S I w i s h t o thank Dr. P. A. Dehnel f o r h i s c o n t i n u e d encouragement and a d v i c e and c r i t i c a l a p p r a i s a l o f t h e m a n u s c r i p t . May I a l s o thank Dr. J . R. Dempster and p a r t i c u l a r l y M i s s Ruth Hogan f o r h e l p i n g me i n t h e i n i t i a l s t a g e s o f w r i t i n g a computer program. I extend my most 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 Myrna Young i n a s s i s t i n g me i n t h e p r e p a r a t i o n o f t h e f i g u r e s . INTRODUCTION The midgut 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 most i m p o r t a n t organs i n C r u s t a c e a . I t s e c r e t e s d i g e s t i v e enzymes, a b s o r b s and t r a n s f o r m s f o o d and i s t h e major depot f o r t h e s t o r a g e of m i n e r a l and f o o d r e s e r v e s . T r a v i s (1955, 1957) and Weel (1955) have demonstrated t h e s e f u n c t i o n s i n t h e s p i n y l o b s t e r , P a n u l l r u s a r g u s , and i n t h e b r a c h y u r a n 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 t h e g l a n d and c o n n e c t i v e t i s s u e . The midgut 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 from one a n o t h e r by con-n e c t i v e t i s s u e . The t u b u l e s open i n t o secondary s e c r e t i o n d u c t s w h i c h , 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 d u c t t h r o u g h which the d i g e s t i v e f l u i d i s poured i n t o t h e midgut ( T r a v i s , 1955; Weel, 1955). There a r e v e r y few s t u d i e s which have c o n c e n t r a t e d upon an 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 i n e x c i s e d i n -v e r t e b r a t e t i s s u e . Hopkins (1930) found i n b o t h r e d and w h i t e muscle o f t h e p o s t e r i o r a d d u c t o r o f the clam, Venus  m e r c e n a r i a , a dec r e a s e i n r e s p i r a t i o n from young t o o l d clams when r e s p i r a t i o n measurements were made a t 27 . 5°C. The body s i z e and number o f a n n u a l growth r i n g s were used as c r i t e r i a o f age. I n a l a t e r paper Hopkins (1946) showed, t h a t i n e x c i s e d g i l l t i s s u e of-Venus m e r c e n a r i a , r e s p i r a t i o n was h i g h e r i n the c o l d - a d a p t e d a n i m a l (below 20°C) t h a n t h e warm-adapted a n i m a l (27°C) when measured 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 o f e x c i s e d g i l l t i s s u e w i t h h a b i t a t and a c t i v i t y o f s e v e r a l s p e c i e s o f marine decapod Crustacea a t 27°C. R o b e r t s (1957b) f o u n d a c c l i m a t i o n i n muscle t i s s u e o n l y a t a h i g h t emperature (23 . 5°0) and n o t a t a l l i n i s o l a t e d b r a i n t i s s u e i n t h e s t r i p e d r o c k c r a b , Pachygrapsus c r a s s l p e s . W e i g h t - s p e c i f i c oxygen c o n -sumption of e x c i s e d g i l l 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 from summer a n i m a l s r e s p i r e s a t a h i g h e r r a t e t h a n t i s s u e from w i n t e r a n i m a l s o v e r th e p h y s i o l o g i c a l t e m p e r a t u r e range o f 5°0 t o 20°0 (Dehnel and McCaughran, 1964). K i n g (1965) demonstrated an i n c r e a s e i n oxygen consumption o f e x c i s e d g i l l i n the c r a b , C a l l i n e c t e s s a p i d u s ( b r a c k i s h ) and 0. s a p i d u s ( m a r i n e ) , by 30$ and 10$, r e s p e c t i v e l y , when t r a n s f e r e d from 80$ t o 50$ sea w a t e r . R e c e n t l y , Vernberg and K e rnberg (1966) found i n h e a r t , muscle and b r a i n t i s s u e of t r o p i c a l and temperate zone f i d d l e r c r a b s , Uca sp., a common tendency f o r r e s p i r a t i o n measurements t o be h i g h e r i n warm-adapted 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 were made between 10°0 o r 15°C and 25°0. R e s p i r a t i o n s t u d i e s on midgut g l a n d t i s s u e a r e even more s p a r s e l y documented. B e l d i n g ejt a l . (1942) e s t a b l i s h e d t h a t t h e r e a r e no m e t a b o l i c g r a d i e n t s i n midgut g l a n d f o r 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 , when comparing th e a n t e r i o r , median and h i n d p a r t s o f the g l a n d . The QQ 2 was found t o v a r y as an i n v e r s e f u n c t i o n o f body s i z e ( c a r a p a c e l e n g t h ) w i t h a n e g a t i v e r e g r e s s i o n v a l u e o f -0 . 2 8 5 f o r b o t h sexes 3 a t 15°C. Weymouth e t a l . (1944) demonstrated a t 15°C i n th e midgut g l a n d o f the same c r a b a w e i g h t - s p e c i f i c oxygen consumption r e g r e s s i o n t o body w e i g h t o f -0.203. The oxygen consumption o f e x c i s e d midgut g l a n d from n i n e s p e c i e s o f m arine decapod c r u s t a c e a has shown a d i r e c t c o r r e l a t i o n w i t h a c t i v i t y when comparing a n i m a l s w i t h i n any one h a b i t a t . ( V e r n b e r g , 1956). M i n a m o r i (1964) found 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 r e g r e s s i o n b v a l u e t o body w e i g h t o f 0.80 t o 0.81 i n t h r e e r a c e s of t h e l o a c h f i s h , C o b i t i s t a e n i a s t r i a t a . 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 on t i s s u e o r whole a n i m a l r e s p i r a t i o n one s h o u l d use a m u l t i - f a c t o r i a l approach where 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 s i m u l t a n e o u s l y . 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 whi c h t o base c o n c l u s i o n s . As Kinn e (1963) p o i n t s o u t , such an a n a l y s i s may g i v e c o n c l u s i o n s t h a t have no v a l i d i t y e c o l o g i c a l l y s i n c e t h e organism r e -sponds t o t h e whole environment, n o t t o i s o l a t e d s i n g l e f a c t o r s . The purpose o f the p r e s e n t study 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 most 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 he i n t a c t a n i m a l and i n t u r n t o the 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 of w e i g h t - s p e c i f i c oxygen consumption of e x c i s e d midgut g l a n d o f the shore c r a b , Hemigrapsus nudus, o v e r t h e p h y s i o l o g i c a l t e m p e r a t u r e 4 range of 5°G! to 20° C. The term "main e f f e c t " w i l l be used to describe effects of a single f a c t o r (eg. s a l i n i t y , temperature) averaged f o r a l l acute temperatures, experimental temperatures and s a l i n i t i e s and both seasons. The term " i n t e r a c t i o n " (eg. salinity-temperature combination) refers to the combined ef f e c t of factors where differences i n response to one factor varies with the l e v e l of another fa c t o r which i s applied simul-taneously (Steel and Torrie, 1960). A further discussion of the concept of in t e r a c t i o n w i l l follow. The acute temperatures (5°, 10°, 15° and 20°0) r e f e r to the Warburg water-bath temperatures to which the tissue samples are equilibrated and at which oxygen consumption values are recorded. Experimental temperatures (5°C and 20°C) and experimental s a l i n i t i e s (35$, 75$ and 125$ sea water) are the physical parameters to which the animals are acclimated f o r 10 days or held at f o r 24 hr f o r standard baseline measurements. Acclimation, as used i n the context of 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 metabolic a c t i v i t y due to change i n s a l i n i t y , temperature or seasonal changes i n these factors when measured over the physiological temperature range of 5°C to 20°C. This d e f i n i t i o n w i l l include also the l i a b i l i t y f o r genotypic change as re f l e c t e d i n a phenotypic a l t e r a t i o n of metabolism. Compensation and adaptation w i l l be used with the same connotation as acclimation and w i l l include the concept of homeostasis. Homeostasis i s a mechanism by which the animal p h y s i o l o g i c a l l y maintains 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 term h e p a t o p a n c r e a s i s a misnomer when a p p l i e d t o C r u s t a c e a and w i l l n o t be used i n t h e t e x t o f t h i s s t u d y . I n s t e a d , t h e term midgut g l a n d w i l l be used i n p l a c e o f he p a t o p a n c r e a s . B e l d i n g et a l . (1942) p o i n t o u t , " S i n c e i t (midgut g l a n d ) i s w i t h o u t homology i n t h e mammal, t h e term ' " l i v e r " ' and 1 " h e p a t o p a n c r e a s " 1 a r e u n j u s t i f i e d . Because i t d e v e l o p s from t h e midgut, the term 1 "midgut g l a n d " ' i d e n t i f i e s i t w i t h o u t chance o f e r r o r and w i t h no m i s l e a d i n g i m p l i c a t i o n s as t o f u n c t i o n . " MATERIAL AND METHODS The c r a b , Hemigrapsus nudus, was o b t a i n e d from S p a n i s h Bank ( L a t . 49° 17'N.j Long. 123° 07'¥.) Vancouver, B r i t i s h Columbia. Summer a n i m a l s were c o l l e c t e d from June 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 from November t h r o u g h March. 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 average t e m p e r a t u r e o f 20°C and a s a l i n i t y o f 35$ (11/4) sea w a t e r . The w i n t e r season i s c h a r a c t e r i z e d by a r e l a t i v e l y s t a b l e t e m p e r a t u r e o f 5°C and a s a l i n i t y o f 75$ (24&) sea w a t e r . The average summer and w i n t e r temper-a t u r e s and s a l i n i t i e s s e r v e as s t a n d a r d b a s e l i n e c o n d i t i o n s f o r t h e r e s p e c t i v e seasons. The s t a n d a r d sea water o f 100$ (32°^ °) used i n t h i s s t udy i s based on a c h l o r i n i t y o f 17.65^°and a s a l i n i t y o f 31.88%°. The p r o p o r t i o n s o f t h e major i o n s i n 100$ sea wa t e r , as de 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 as f o l l o w s : 6 Ha: 433.0 mEq./l. K : 10.1 '» " Ca: 25.6 " " Mg: 97 .9 " " C l : 497.0 " " The ions are complexed as chlorides, plus the sulfate of sodium. Two experimental temperatures, 5°C and 20°C (i1°C) and three experimental s a l i n i t i e s , 35$ (11#°), 7 5 $ (24%) and 125$ (40&) sea water (±1$ sea water) were used i n a l l combinations f o r each season (summer and winter). The animals were held 5, 10 and 15 days at these experimental combinations to determine the time period that resulted i n the maximum degree of acclimation. A 10 day time period gave the maximal acclimation response. Only adult intermolt male crabs were selected f o r study. They ranged i n weight from 6.0 to 10.0 g. The wet weight of the whole animal a f t e r damp drying was weighed to the nearest 0.01 g. In the laboratory the animals were immedi-ately placed i n p l a s t i c containers holding 3 . 5 l i t e r s of the appropriate (35$» 75$ or 125$) sea water. Sea water was aerated and the containers were placed i n a r e f r i g e r a t o r at constant temperature (5°C or 20°C) i n t o t a l darkness. The water was changed d a i l y . At standard baseline conditions the animals were held 24 hr. Those animals acclimated i n the laboratory were held 10 days and not fed. Only 10-12 animals were held i n each container at any one time to minimize mortality due to crowding. Crabs that molted during the 10 day acclimation period were discarded. The d i r e c t method of Warburg was used to measure r e s p i -r a t i o n rates of midgut gland (Umbriet, Burris and Stauffer, 7 1957). The Gilson Medical Electronics (Warburg) respirometer was made available f o r t h i s work. Tissue samples excised from the animals ranged i n weight from 0.3 to 0.6 g. The excised tissue samples were placed d i r e c t l y i n pre-weighed aluminum pans and weighed to the nearest 0.1 mg. The tissue samples were not damp dried on f i l t e r paper p r i o r to weighing. The delicate nature of the tubles made i t d i f f i c u l t to pick the gland o f f f i l t e r paper without fragmenting the tissue with subsequent loss of substrate and enzyme f l u i d s . A f t e r weighing, the tissue samples were placed i n the Warburg reaction f l a s k s containing 3.0 ml of physiological saline prepared as follows: 850.0 ml of 0.52 M NaOl 5 1 . 0 " 3 5 . 0 «' 3 5 . 0 " 1 7 . 6 1 .3 " " 0.35 M MgGlo " 0.35 M CaOlp " 0.42 M KapSOA M 0.38 M H3BO3 M 0.48 M mOE pH = 7.8 A °C = - 1 . 6 1 PP The center wells of the reaction f l a s k s contained 0 . 2 ml of 1 5 $ KOH. During the dissec t i o n the f l a s k s were kept i n an ice-bath. Three hours and ten minutes a f t e r excision of the f i n a l tissue sample, the flas k s were taken out of the ice-bath and attached to the manometers. The attached fl a s k s were then placed i n the constant temperature water-bath ( * 0 . 1°C) of the Warburg. The seventeen tissue samples were allowed to equilibrate f or 15 min, during which time they were gassed with oxygen f o r 10 min. The respiratory rate was measured at four acute temperatures ( 5 ° » 1 0 ° , 1 5 ° and 20°C) fo r each set of experimental conditions. An exami-8 nation of Figure 1 reveals that one set of tissue samples could respire at two acute temperatures since the slope of the weight-specific oxygen consumption rate curve with time shows a small, but non-significant decrease over the time i n t e r v a l of measurement. This i s the portion of the curve between the arrows. The f i r s t set of tissue samples respired at 5°C and 10°C and the second set of tissue samples respired at 15°C and 20°0. Each set of tissue samples respired 90 min at each of the two acute temperatures with a 40 min time lapse between metabolic measurements at the f i r s t and second acute temperatures. It took 25 min to change the temperature of the water-bath 5°C and 15 min to equilibrate the t i s s u e s . The tissue samples were shaken at a constant rate of 120 osc i l l a t i o n s / m i n . Nine hours from the time of d i s s e c t i o n were required to complete r e s p i r a t i o n measurements at two acute temperatures. One i s j u s t i f i e d i n measuring the r e s p i r a t i o n of the tissue at two acute temperatures, since measurement of tissue r e s p i r a t i o n i n the reverse d i r e c t i o n (10° and 5°C or 20° and 15°C) does not s i g n i f i c a n t l y a l t e r the slope of the metabolic-temperature (M-T) curves. With the completion of an experimental run, the tissue samples and saline were placed i n pre-weighed aluminum pans and dried i n an oven at 100-106°C f o r 24 hr. Dry body weights were obtained by the same procedure. The data were expressed i n u l 0 2 / g dry gland/hr at N.T.P. A s t a t i s t i c a l analysis of the data was performed with the aid of the 7040 IBM Computer. The data were processed 9 F i g u r e 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 o f t i m e f o r midgut g l a n d o f Hemigrapsus nudus. Each p o i n t r e p r e s e n t s amount o f oxygen consumed d u r i n g a 10 min time i n t e r v a l . Weight o f crab, used f o r oxygen consumption measurement was 7 g. Gurve i s e y e - f i t t e d . r CO (J (J o ° O in O cvj ro c\j > a: Q T Z jf) y j < U J h O) h o: Q: 3 x x O UJ UJ < x UJ* J I 8 S I 1 LO O tf> O LO j>j g -> rsi Jtj /puo|6Ajp B/^Qih I 10 using, the Fortran Program. Weight-specific oxygen consumption was plotted as a function of whole body weight on double logarithmic graph paper. The data gave a straight l i n e that took the form: Reduce to: log O2 = log a + b(log W) where 0- = ulOg/g dry gland/hr, a = intercept, b = slope of l i n e and W = whole body weight. The exponent (b - 1 ) by which body weight i s raised to a given value proportional to metabolism i s referred to as the regression c o e f f i c i e n t . Slope values were considered s i g n i f i c a n t at the 0 . 0 1 proba-b i l i t y l e v e l . Using a equal sample size of twelve, an analysis of variance was performed on the data (see Table IV). The l e v e l of significance at which the Null Hypothesis was accepted that there i s no difference between treatment means was the 0 . 0 1 p r o b a b i l i t y l e v e l . The values of the sum of squares and mean squares. In Table IV are expressed i n natural logarithms. The data upon which the s t a t i s t i c was performed are presented i n tabulated form i n Tables I and I I . Only the mean values of the main effects and interactions f o r weight-specific oxygen consumption are expressed i n the tables. These mean values are presented graphically i n the subsequent f i g u r e s . 11 Tab,le I: Mean values of main effects f o r weight-specific oxygen consumption of midgut gland t i s s u e . Source of Variance Mean (u l0 2/g dry gland/hr) A(Acute Temp.° C) 5 185 10 259 15 427 20 631 B(Exp. Temp.°0) 5 375 20 303 C(Exp. S a l i n i t y , $S.W.) 35 351 75 319 125 341 D( Season) Winter 344 Summer 330 12 Table I I : Mean values of f i r s t and second-order i n t e r -actions f o r weight-specific oxygen consumption of midgut gland t i s s u e . Source of Variance Mean (ul0 2/g dry gland/hr) First-Order Interactions AB Mlcj. — B i l e } . 5 206 10 5 295 15 465 20 , , 694 5 • • T65" 10 20 228 15 392 _20 573  AO: A(°C') 0(#S.W.) 5 190 10 35 277 15 450 20 , 643 5 175 10 75 239 15 404 20 , 617 5 • TB9 10 125 264 15 429 20 , 6J52 BO B(° C) 0(gS.W.) 35 403 5 75 329 12J 396 35 30o~ 20 75 310 125 294 AD A(° C) D(Season) 5 183 10 Winter 279 15 421 20 651 13 Table I I : Continued 5 186 10 Summer 241 15 433 20 611 B(° C) BD D(Season) 5 Winter 383 20 309 5 Summer 366 20 297 CD C(#S.tf.) D(Season) 35 350 75 Winter 301 125 386 35 353 75 Summer 338 125 301 Second--Order Interactions ABC A(°C) C($S.W.) 224 5 10 5 35 324 15 495 20 737 5 162 10 20 35 236 15 409 20 561 5 187 10 5 75 258 15 404 20 5 9 9 5 164 10 20 75 221 15 403 20 635 5 210 10 5 125 308 15 502 20 758 5 170 10 20 125 226 15 366 20 527 14 T a b l e I I : C o n t i n u e d A B D A ( ° C ) B C " 5 ! ? ! D( Season) 5 207 10 5 W i n t e r 317 15 460 20 714 5 162 10 20 W i n t e r 246 15 385 20 593 5 206 10 5 Summer 275 15 470 20 675 5 168 10 20 Summer 211 15 399 20 552 A C D A ( ° C ) c(fs7w.) D(Season) 181 5 10 35 W i n t e r 286 15 443 20 652 5 170 10 75 W i n t e r 223 15 355 20 586 5 199 10 125 W i n t e r 326 15 474 20 723 5 200 10 35 Summer 267 15 457 20 634 5 180 10 75 Summer 245 15 458 20 649 5 150 10 125 Summer 214 15 388 20 553 B C D B ( ° C ) c(fs7w.) D(Season) 35 457 5 75 W i n t e r 292 125 422 T a b l e I I : C o n t i n u e d 35 357 5 75 Summer 369 125 373 35 2oT 20 75 W i n t e r 311 125 354. 35 3W 20 75 Summer 310 125 244 Table I l l s Standard error of mean w e i g h t - s p e c i f i c oxygen consumption of midgut gland t i s s u e f o r each experimental combination of f a c t o r s . Experimental Combination of Factors Mean Standard E r r o r ( u l 0 2 / g dry gland/hr) (S^) Acute Exp. S a l i n i t y Season Temp.°C Temp.°C ($S.W.) 5 244 129 10 5 35 Winter 376 134 15 551 203 20 854 188 5 173 233 10 5 75 Winter 237 217 15 335 268 20 534 324 5 210 150 10 5 125 Winter 357 153 15 529 172 20 ' 794 161 5 135 278 10 20 35 Winter- 218 216 15 356 276 20 494 310 5 168 156 10 20 75 Winter 230 201 15 377 301 20 642 204 5 189 242 10 20 125 Winter 297 236 15 425 117 20 659 190 Table I I I : Continued 5 206 130 10 5 35 Summer 280 175 15 444 200 20 632 237 5 202 387 10 5 75 Summer 281 136 15 489 193 20 672 234 5 210 133 10 5 125 Summer 265 118 15 477 135 20 725 155 5 193 196 10 20 35 Summer 255 183 15 470 331 20 636 357 5 160 268 10 20 75 Summer 213 289 15 430 172 20 627 231 5 154 172 10 20 125 Summer 172 230 15 315 309 20 422 423 18 Table IV: Analysis of variance of weight-specific oxygen consumption f o r the f a c t o r i a l (4X 2X 3X 2X) experiment Incorporating four acute temperatures (A), two experimental temperatures (B), three experimental s a l i n i t i e s (C) and two seasons (D) (Steel and Torrie, 1960). The P-values are considered s i g n i f i c a n t at the 0.01 prob a b i l i t y l e v e l (**). Source of Variance df Sum of Mean, F Squares Squares A (Acute Temp.) 3 126.56 42.185 753.3** B: (Exp. Temp.) 1 6.43 6.433 114.9** 0 (Exp. S a l i n i t y ) 2 0.91 0.457 8.2** D (Season) 1 0 . 2 6 0.260 4.6** AB 3 0 . 1 6 0.053 1.0 AC 6 0 . 1 7 0 . 0 2 8 0.5 BC 2 1.72 0.859 15.3** AD 3 0 . 7 2 0.241 4.3** BD 1 0.00 0.000 0.0 CD 2 3.35 1.674 29.9** ABC 6 0.50 0.083 1.5 ABD 3 0.03 0.009 0 . 2 ACD 6 0.45 0.074 1.3 BCD 2 4.48 2.238 40.0** Total Treatment 41 145.74 3.555 63.4** Error 534 29.67 0.056 Total 575 175.41 19 T a b l e V: S e a s o n a l compensation 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 c o m b i n a t i o n s o f a c u t e t e m p e r a t u r e ( A ) , e x p e r i m e n t a l temperature (B), e x p e r i m e n t a l s a l i n i t y (C) and season ( B ) . The numbers i n t h e body o f the t a b l e r e p r e s e n t the type o f 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 have the 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 ; and t y p e 3, w i n t e r a n i m a l s have 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). E x p e r i m e n t a l Acute E x p e r i m e n t a l T S a l i n i t y ($S.¥.) 35 75 125 5 5 10 15 20 3 3 3 3 5 5 5 5 4 3 3 3 20 5 10 15 20 5 5 5 5 3 3 5 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 2 and ELj to Mg define the degree of ac-climation shown by the winter and summer animals, re-spectively. SEASONAL METABOLIC - TEMR IO 15 20 ACUTE TEMR °C 22 I n comparing t h e degree t o which a c c l i m a t i o n has been a c h i e v e d , t h e compensation t e m p e r a t u r e c o e f f i c i e n t as f i r s t s u ggested by R o b e r t s (1952) may be a p p l i e d t o t h e p r e s e n t d a t a (see Rao, 1953). The c o e f f i c i e n t v a l u e i s a r a t i o between two s l o p e s , M-| and Mg. The s l o p e M 1 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. cur v e and t h e l o w e s t r e s p i r a t i o n r a t e o f t h e summer b a s e l i n e M-T c u r v e . The s l o p e M 2 i s t h e r e c i p r o c a l o f s l o p e . The same g r a p h i n g 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 t M 2. I n t h i s case s l o p e M-j 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 b a s e l i n e M-T cur v e and t h e l o w e s t 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 • i M-T c u r v e . The s l o p e M 2 i s t h e r e c i p r o c a l o f s l o p e ( P i g . 2). 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 any compensation and approaches ze r o as t h e degree o f compensation i n c r e a s e s . The compensation c o e f f i c i e n t between w i n t e r a t summer b a s e l i n e M-T cur v e and summer b a s e l i n e M-T cur v e i s 0.601. The c o e f f i c i e n t v a l u e between t h e w i n t e r b a s e l i n e M-T cur v e and summer a t w i n t e r b a s e l i n e M-T cu r v e i s 0.665. A comparison of th e s e c o e f f i c i e n t v a l u e s i n d i c a t e s t h a t w i n t e r a n i m a l s show the g r e a t e s t degree o f compensation ( l o w e r c o e f f i c i e n t v a l u e ) . T h i s p o i n t i s brought out i n Tab l e V. M a i n E f f e c t s E f f e c t o f Season (D) The e f f e c t o f season on midgut g l a n d r e s p i r a t i o n i s 23 s i g n i f i c a n t ( I a b l e I V ) . T h i s e f f e c t was determined by comparing 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 g l a n d demonstrates i n v e r s e s e a s o n a l compensation, t y p e 5» f o r a l l a c u t e t e m p e r a t u r e s a t s t a n d a r d b a s e l i n e c o n d i t i o n s o f 35$ sea w a t e r , 20°C (summer) and 75$ sea w a t e r , 5°C ( w i n t e r ) . Except f o r two c a s e s , p a r t i a l s e a s o n a l compensation, t y p e 3, i s demonstrated a t a l l o t h e r e x p e r i -m e n t a l c o m b i n a t i o n s of a c c l i m a t i o n t e m p e r a t u r e o r a c c l i m a t i o n s a l i n i t y ( T a b l e V ) . E f f e c t o f Acute Temperature (A) An e x a m i n a t i o n o f T a b l e IV r e v e a l s t h a t t h e main e f f e c t o f a c u t e t e m p e r a t u r e i s s i g n i f i c a n t . T h i s e f f e c t was d e r i v e d by comparing 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 d a t a a t each a c u t e t e m p e r a t u r e . F i g u r e 3 i s a t y p i c a l r e p r e s e n t a t i o n o f a c u t e t e m p e r a t u r e e f f e c t s on midgut g l a n d r e s p i r a t i o n . As t h e a c u t e t e m p e r a t u r e r i s e s t h e r a t e o f t i s s u e r e s p i r a t i o n i s i n c r e a s e d . I t may be seen t h a t t h e a c u t e l y measured 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 season, 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 i n t e r a c t i o n s . These e f f e c t s w i l l be examined s h o r t l y . E f f e c t o f E x p e r i m e n t a l Temperature (B) The main e f f e c t 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 de 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 and w i n t e r a n i m a l s were h e l d 10 days I n 35$ and 125$ sea wa 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 oxygen consumption f o r each a c u t e t e m p e r a t u r e . 9 0 0 - EXP. TEMR 5 °C 8 0 0 -7 0 0 -6 0 0 -— WINTER ©35 %S.W — SUMMER o 125 % S.W. j i 1 L _ 5 IO 15 2 0 A C U T E TEMR °C 25 by averaging a l l r e s p i r a t i o n data at each, experimental temperature and then comparing the 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 of experimental temperature i s due to a h i g h e r r e s p i r a t o r y r a t e at 5°C than at 20°0 ( F i g . 4 ) . The f a c t t h a t i n v e r s e , type 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 the r e s p i r a t o r y response of summer and w i n t e r animals i s being a l t e r e d by the experimental temperature e f f e c t (Table V). E f f e c t of Experimental S a l i n i t y (C) The main e f f e c t of experimental s a l i n i t y was determined by comparing the means of the experimental s a l i n i t i e s . The means were derived by averaging a l l r e s p i r a t i o n data at 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 experimental 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 gland 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 . The lowest r e s p i r a t i o n r a t e f o r the main e f f e c t i s i n 75$ sea water and the highest r e s p i r a t i o n r a t e s I n 35$ and 125$ sea water ( F i g . 5 ) . I t i s apparent that season and experimental temperature have an i n f l u e n c e on the r e s p i r a t o r y response of midgut gland t i s s u e to experimental s a l i n i t y ( F i g . 6 ) . The r e s p i r a t o r y r a t e may i n c r e a s e , decrease, show no change or r e v e a l a "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 . A f u r t h e r examination of these i n t e r a c t i o n s w i l l f o l l o w . 26 Figure 4. Experimental temperature-season i n t e r a c t i o n (BD) and main effect of experimental temperature (B) f o r midgut gland of Hemigrapsus nudus. Each point on the experimental temperature-season in t e r a c t i o n curves i s the mean of weight-specific oxygen consumption at that p a r t i c u l a r experimental temperature-season combination. The main effect i s the mean of a l l weight-specific oxygen consumption data at the respective experimental temper-atures. EXP. T E M R CB) EXR T E M R - S E A S O N INTERACTION (BD) 4 0 0 3 0 0 2 0 0 E F F E C T ( B ) o SUMMER EXR TEMR °C 27 Figure 5. Experimental salinity-season i n t e r a c t i o n (CD) and main effect of experimental s a l i n i t y (C) fo r midgut gland of Hemigrapsus nudus. Each point on the experi-mental salinity-season i n t e r a c t i o n curves i s the mean of weight-specific oxygen consumption at that p a r t i c u l a r experimental salinity-season combination. The main effect i s the mean of a l l weight-specific oxygen con-sumption data at the respective experimental s a l i n i t i e s . 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 « ^ 3 0 0 o 0 1 2 0 0 ©WINTER £ M A I N E F F E C T CC) o S U M M E R 35 7 5 E X R S A L I N I T Y , % S . 125 28 F i g u r e 6. Seasonal comparison o f experimental s a l i n i t y and experimental temperature e f f e c t s on midgut gland r e s p i r a t i o n o f Hemlgrapsus nudus, measured at 1 0 ° 0 acute temperature. Each p o i n t on the curves i s the mean of 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 s a l i n i t y , experimental temperature and season. r SUMMER 3 0 0 2 0 0 c o CD >^ T J cn CM O 4 0 0 | 3 0 0 WINTER 2 0 0 ©5 °C ° 2 o ° a IO °C ACUTE TEMR © 5 °C o 2 0 °C IO°C ACUTE TEMR 35 75 EXR SALINITY. %S.W. 125 29 I n t e r a c t i o n s 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 s a d e p a r t u r e o f t h e s i m p l e e f f e c t s ( f a c t o r s ) from an a d d i t i v e law o r model based on main e f f e c t s o n l y . " A s i g n i f i c a n t i n t e r a c t i o n i s one where th e f a c t o r s do n o t a c t i n d e p e n d e n t l y o f one a n o t h e r . I n t e r a c t i o n s i n v o l v i n g two f a c t o r s a r e 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 i n t e r a c t i o n s w i t h t h r e e f a c t o r s a r e r e f e r r e d t o as s e c o n d - o r d e r i n t e r a c t i o n s . F i r s t - O r d e r I n t e r a c t i o n s 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 Temperature (AB) The means of w e i g h t - s p e c i f i c oxygen consumption a t f o u r a c u t e t e m p e r a t u r e s and two e x p e r i m e n t a l t e m p e r a t u r e s a r e p l o t t e d i n F i g u r e 7. The m e t a b o l i c - t e m p e r a t u r e (M-T) c u r v e a t 5°0 i s h i g h e r a t a l l a c u t e t e m p e r a t u r e s t h a n t h e 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 n o t s i g n i f i c a n t ( T a b l e I V ) . T h i s i n d i c a t e s t h a t t h e r e i s a g r e a t d e a l o f v a r i a b i l i t y i n t h e r e s p i r a t i o n measurements a t any one 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 c o m b i n a t i o n . A c u t e Temperature-Season (AD) The 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 i s s i g n i f i c a n t ( T a b l e I V ) . The means of 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 temper-a t u r e s f o r each season a r e shown i n F i g u r e 9. There i s no 30 c o n s i s t e n t t r e n d i n a s e a s o n a l response t o a c u t e t e m p e r a t u r e . The d i f f e r e n c e s a r e s m a l l and randomly d i s t r i b u t e d . 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 S a l i n i t y (AC) T h i s i n t e r a c t i o n i s n o t s i g n i f i c a n t ( T a b l e 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 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 F i g u r e 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 $ sea w a t e r and 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 s i n 7 5 $ sea w a t e r . The d i f f e r e n c e s i n t h e magnitude of response ( p o s i t i o n o f s l o p e on o r d i n a t e ) a r e s m a l l . E x p e r i m e n t a l T e m p e r a t u r e - E x p e r i m e n t a l S a l i n i t y (BC) T h i s i n t e r a c t i o n i s s i g n i f i c a n t ( T a b l e I V ) . I n F i g u r e 10 t h e means of the two e x p e r i m e n t a l temper-a t u r e s a t 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 g r a p h i c a l l y r e p r e s e n t e d . The M-T cur v e a t 5°C shows a "V-shaped" s l o p e w i t h t h e l o w e s t r e s p i r a t o r y r a t e i n 7 5 $ sea w a t e r . The M-T. c u r v e a t 20°C shows l i t t l e change i n s l o p e w i t h a change i n s a l i n i t y from 3 5 $ t o 1 2 5 $ sea w a t e r . E x p e r i m e n t a l S a l i n i t y - S e a s o n (CD) The 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 e x p e r i m e n t a l s a l i n i t y and b o t h seasons a r e p l o t t e d i n F i g u r e 5 . The 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 cur v e shows 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 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 7 5 $ sea w a t e r . 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. 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 (AB) f o r midgut g l a n d o f Hemigrapsus nudus. 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 - e x p e r i m e n t a l temper-a t u r e i n t e r a c t i o n c u r v e s 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 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 c o m b i n a t i o n . A C U T E TEMP. - EXP. T E M R I N T E R A C T I O N (AB) 7 0 0 6 0 0 5 0 0 4 0 0 3 0 0 2 0 0 EXP. TEMP. ©5 °C o 2 0 °C -J 1 1 5 IO 15 A C U T E T E M R °C 32 F i g u r e 8. Acute t e m p e r a t u r e - e x p e r i m e n t a l s a l i n i t y i n t e r -a c t i o n (AC) f o r midgut g l a n d o f Hemigrapsus nudus. 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 - e x p e r i m e n t a l s a l i n i t y 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 a t t h a t p a r t i c u l a r 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 s a l i n i t y c o m b i n a t i o n . ACUTE T E M R - E X R SALINITY INTERACTION CAC) 7 0 0 6 0 0 5 0 0 4 0 0 3 0 0 2 0 0 EXR SALINITY ©35 %S.W. o 125 % S.W. A 75 %S.W. IO 15 ACUTE TEMR °C 2 0 33 F i g u r e 9. Acute 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 main e f f e c t o f a c u t e t e m p e r a t u r e (A) f o r midgut g l a n d o f Hemigrapsus nudus. 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 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 a c u t e t e m p e r a t u r e - s e a s o n c o m b i n a t i o n . The main e f f e c t 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 oxygen consumption d a t a a t t h e r e s p e c t i v e a c u t e t e m p e r a t u r e s . ACUTE TEMR (A) ACUTE T E M R - S E A S O N INTERACTION (AD) 7 0 0 6 0 0 5 0 0 4 0 0 3 0 0 2 0 0 ©WINTER A MAIN E F F E C T (A) o SUMMER IO 15 ACUTE TEMR °C 2 0 34 F i g u r e 1 0 . E x p e r i m e n t a l t e m p e r a t u r e - e x p e r i m e n t a l s a l i n i -t y i n t e r a c t i o n (BC) f o r midgut g l a n d o f Hemigrapsus nudus. Each p o i n t on t h e e x p e r i m e n t a l t e m p e r a t u r e - e x p e r i m e n t a l s a l i n i t y 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 a t t h a t p a r t i c u l a r e x p e r i m e n t a l t e m p e r a t u r e - e x p e r i m e n t a l s a l i n i t y c o m b i n a t i o n . EXR TEMR - EXR S A L I N I T Y I N T E R A C T I O N CBC) 5 0 0 4 Q 0 3 0 0 EXR T E M R ©5 OQ o 2 0 ° C 2 0 0 3 5 EXR SALINITY, % S 35 by a M-T curve i n which the r e s p i r a t o r y r a t e decreases with an i n c r e a s e i n s a l i n i t y . As seen i n Table IV, the i n t e r a c t i o n i s s i g n i f i c a n t . Experimental Temperature-Season (BD) A seasonal comparison of r e s p i r a t o r y response to experimental temperature i s not s i g n i f i c a n t (Table I V ) . In F i g u r e 4 i t may be seen t h a t the means of the r e s p i r a t i o n v a l u e s f o r each experimental temperature show only a s m a l l magnitude of d i f f e r e n c e s e a s o n a l l y . The w i n t e r r a t e s o f r e s p i r a t i o n are h i g h e r than the summer r e s p i r a t i o n 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 experimental temperature.. Second-Order I n t e r a c t i o n s Experimental Temperature-Experimental S a l i n i t y - S e a s o n (BOD) T h i s combination of f a c t o r s r e s u l t s i n the only s i g n i f i c a n t second-order i n t e r a c t i o n (Table I V ) . The means of a l l the r e s p i r a t o r y data f o r each e x p e r i -mental temperature, experimental s a l i n i t y and season are p l o t t e d i n F i g u r e 11. The t h r e e f a c t o r i n t e r a c t i o n i s i n t e r p r e t e d as an i n t e r a c t i o n o f the i n t e r a c t i o n CD ( e x p e r i -mental s a l i n i t y - s e a s o n ) w i t h f a c t o r B (experimental temper-a t u r e ) . The w i n t e r M-T curves a t the th r e e experimental s a l i n i t i e s have slo p e s which are d i f f e r e n t from the summer M-T curves. These M-T curves i n t u r n have slo p e s which are i n f l u e n c e d by experimental temperature. I t i s seen i n 36 F i g u r e 1 1 . E x p e r i m e n t a l t e m p e r a t u r e - e x p e r i m e n t a l 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 (BCD) f o r midgut g l a n d o f Heml-g r a p s u s nudus. Each p o i n t on t h e e x p e r i m e n t a l temper-a t u r e - e x p e r i m e n t a l 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 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 a t t h a t p a r t i c u l a r e x p e r i m e n t a l t e m p e r a t u r e - e x p e r i m e n t a l s a l i n i -t y - s e a s o n c o m b i n a t i o n . r EXP. T E M R - E X P . SALINITY-S E A S O N INTERACTION CBCD) — c CJ) >«* t_ CM O 5 0 0 4 0 0 3 0 0 2 0 0 - W I N T E R ©5 °C o 2 0 °C - S U M M E R ©5 °C o 2 0 °C 1 1 35 75 125 EXP. SALINITY, %S.W. 37 Figure 11 that the slope of the winter M-T curve at 5°C shows a "V-shaped1' re l a t i o n s h i p to s a l i n i t y . The lowest r e s p i r a t i o n rate i s i n 7 5 $ sea water. The winter M-T curve at 2 0 °C shows an increase i n slope as the s a l i n i t y increases. Tissue samples from summer animals have M-T curves that also vary with the s a l i n i t y due to a change i n experimental temperature. The summer M-T curve at 5°C does not show a difference i n the magnitude of response over the s a l i n i t y range of 3 5 $ to 1 2 5 $ sea water. At 20°C, the slope of the M-T curve decreases with a increase i n s a l i n i t y . Other Second-Order Interactions (ABC,ABD,ACD) None of these interactions i s s i g n i f i c a n t (Table IV)« The differences between the means are small 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 are not graphically represented i n the text of t h i s study. Eff e c t of Body Weight The slopes of the regression l i n e s of weight-specific oxygen consumption against 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 at the 0 . 0 1 p r o b a b i l i t y l e v e l . At the 0 . 0 5 l e v e l of significance, three of twenty-four slopes 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 for,the winter animals and two of twenty-four slopes were s i g n i f i -cantly d i f f e r e n t from zero f o r the summer animals. These slope differences were considered to be due to chance alone and therefore were not s t a t i s t i c a l l y s i g n i f i c a n t . This was confirmed when adjustment of the treatment means by the 38 a n a l y s i s o f c o v a r i a n c e r e s u l t e d i n v e r y s m a l l changes I n t h e F - v a l u e s . DISCUSSION S e a s o n a l M e t a b o l i c - T e m p e r a t u r e E x p e r i m e n t s The s e a s o n a l r a t e o f oxygen consumption f o r midgut g l a n d was found t o be h i g h e r i n summer a n i m a l s a t a l l a c u t e t e m p e r a t u r e s from 5°C t o 20°C ( F i g . 2 ) . T h i s i s i n t e r p r e t e d as i n v e r s e s e a s o n a l compensation, t y p e 5 ( P r e c h t , 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 observed f o r e x c i s e d g i l l t i s s u e (Dehnel and McCaughran, 1964) and whole a n i m a l ( D e h n e l , 1960) over t h e same 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 . Vernberg and Vernberg (1966) have r e c e n t l y f o u n d t h a t t h e most common ty 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 , muscle and b r a i n t i s s u e o f temperate and t r o p i c a l zone s p e c i e s o f Uca a t a c u t e t e m p e r a t u r e s from 5°C t o 35°C i s I n v e r s e compensation. P r o s s e r (1958) s u g g e s t s t h a t i n v e r s e s e a s o n a l compensation may have no a d a p t i v e s i g n i f i c a n c e and appears t o r e f l e c t a q u a n t i t a t i v e r a t h e r t h a n a q u a l i t a t i v e change i n enzymes. That i s , a p a r t i c u l a r enzyme might change i n p r o p o r t i o n to 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 . T h i s i s seen as a t r a n s l a t i o n o f t h e summer M-T curve t o t h e l e f t o r above t h e w i n t e r M-T c u r v e ( F i g . 2 ) . P r o s s e r ' s s u g g e s t i o n has found 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 ( 1 9 5 9 ) , Ekberg (1958) and F r e e d (1965) on i n t a c t "animal, t i s s u e and enzymes i s o l a t e d from t e m p e r a t u r e a c -39 climated g o l d f i s h . A l t e r n a t i v e l y , the higher metabolic rate of summer animals may be due to the fact that many of the summer animals selected f o r study may have been ph y s i o l o g i c a l l y i n premolt, rather than intermolt. The rapid synthesis of organic material and mobilization of inorganic ions p r i o r to and during exoskeleton formation could account f o r the higher r e s p i r a t i o n rate of summer animals. Skinner (1962) has noted that the synthesis of expskeleton during la t e D 2 stage (premolt) increases the weight-specific oxygen consumption of the land crab, Gecarcinus l a t e r a l i s , by 60$ as compared to stage C4 (intermolt). An examination of the types of compensation found i n Table V reveals that p a r t i a l seasonal compensation (type 3) predominates at s a l i n i t y and temperature combinations to which the animals are acclimated; not at standard baseline conditions. This indicates that osmotic and/or temperature stress may have a more depressant ef f e c t on the metabolic a c t i v i t y of the winter animals (see Pig. 3). To offset t h i s depressant e f f e c t , winter animals increase metabolic a c t i v i t y above that shown i n summer animals. This i s cor-related with the lower compensation c o e f f i c i e n t of winter acclimated animals, indicating that winter animals demonstrate the greatest degree of acclimated response. Metabolism-Body Weight Relationship The rel a t i o n s h i p of whole animal and tissue r e s p i r a t i o n 40 to body weight has received considerable attention i n reviews by Brody (1945), Krebs (1950) and Zeuthen (1953). Bertalanffy (1951) proposed that the regression of metabolism on body weight could be expressed i n terms of 2/3, 3/4 or 1 proportionality. Bertalanffy suggested that there Is a species-specific power function that i s f i x e d . Bertalanffy and Krywienczyk (1953) found that the surface law of 2/3 proportionality c h a r a c t e r i s t i c of Crustacea could be applied to the metabolic-weight response of the brine shrimp, Artemia s a l i n a . Weymouth et a l . ( 1 9 4 4 ) , Zeuthen (1953) and Scholander et a l . (1953) have shown i n other species of Crustacea that 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. In the grapsoid crabs, Pachygrapsus crassipes, Hemigrapsus nudus and H. oregonensls, the regression c o e f f i c i e n t s r a r e l y approached the 2/3 or 3/4 exponent (Roberts 1957a; Dehnel, i 9 6 0 ) . Dehnel (1960) actually found i n both species of Hemigrapsus a spread i n weight-s p e c i f i c oxygen consumption regression values from -0.685 to -0.333. In t h i s study the weight-specific oxygen consumption to body weight regression of excised midgut gland was found to be independent of body weight. The same rel a t i o n s h i p was found by Vernberg and Gray (1953) f o r the QQ 2 of excised brain tissue of teleost f i s h . The independence of midgut gland r e s p i r a t i o n from body weight i s i n contrast to the mean -O.169 regression c o e f f i c i e n t of excised g i l l tissue f o r both species of Hemigrapsus (Dehnel and McCaughran, 41 1964). It appears that each tissue has i t s own unique pattern of response to changes i n "body weight. These changes have been r e f l e c t e d as differences i n c e l l u l a r enzyme a c t i v i t y -weight regressions f o r mammalian tissues (Rosenthal and Drabkin, 1943; Kunkel and Campbell, 1952; Fr i e d and Tipton, 1953)• An attempt to assign a fixed proportionality value to a whole animal and/or i t s tissues and enzyme systems i s , therefore, i n v a l i d . The regression c o e f f i c i e n t i s dependent on the environmental history of the animal, effect of physical and b i o t i c parameters, and techniques employed to measure responses. The fact that tissue samples when removed from the animal are no longer influenced by the central nervous system or by hormones could account i n part f o r the discrepancies between tissue and whole animal metabolism to body weight regression. E f f e c t of Experimental Temperature Temperature determines to a great extent the rates of chemical reactions and, thus, the rate of metabolism and a c t i v i t y . 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 effects on rate functions dealing with such things as thermal l i m i t s of tissues and whole organisms, oxygen consumption, heart beat and c i l i a r y pumping a c t i v i t y . Not only rate functions, but the metabolic 42 pathway u t i l i z e d hy an organism may be altered (Ekberg, 1958; Hochachka and Hayes, 1962; Dean and Vernberg, 1 9 6 5 ) . There i s a 24$ decrease i n metabolic a c t i v i t y of midgut gland with an increase i n experimental temperature from 5°C to 20°C (Pig. 4). This percentage decrease holds f o r both summer and winter animals. The experimental temperature effect corresponds to Precht's type 3 ( p a r t i a l compensation). A si m i l a r effect i s depicted i n Figure 7 where the experimental temperature i s averaged at each acute temperature (5°, 10°, 15° and 20°0) f o r both seasons and three experimental s a l i n i t i e s (35$, 75$ and 125$ sea water). This type of compensation has been documented extensively by a number of investigators. Edwards and Irving (1943) demonstrated t h i s effect i n the sand crab, Emerita talpolda. Scholander et a l . (1953) found the same response i n aquatic f i s h and Cr u s t a c e a . P a r t i a l compensation has also been demonstrated by Clark (1955) for the t e r -r e s t r i a l amphipod, T a l i t r u s sylvaticus, by Kanungo and Prosser (1959) f o r g o l d f i s h and by Dehnel (1960) f o r both species of Hemigrapsus. Dehnel and McCaughran (1964) found that excised g i l l tissue showed no experimental temperature effect f o r both species of Hemigrapsus. This effect was not considered b i o l o g i c a l l y s i g n i f i c a n t . The demonstration of p a r t i a l compensation f o r excised midgut gland i s s i g n i f i c a n t 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 . The b i o l o g i c a l importance of t h i s e f f e c t i s interpreted on the grounds that 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 s t r e s s t h a n h i g h t e m p e r a t u r e (Todd and Dehnel, 1960). To o f f s e t t h i s temper-a t u r e s t r e s s , t he 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 a t 20°C. T h i s i n t e r p r e t a t i o n i s c o r r e l a t e d i n p a r t w i t h t h e o b s e r v a t i o n s o f Fre e d (1965) who e s t a b l i s h e d t h a t cytochrome o x i d a s e a c t i v i t y i n c r e a s e d i n the c o l d a c c l i m a t e d (5°C) g o l d f i s h and de c r e a s e d w i t h a c c l i m a t i o n t o h e a t (30°0) . E f f e c t o f E x p e r i m e n t a l S a l i n i t y S a l i n i t y , i s t h e o t h e r m ajor e n v i r o n m e n t a l f a c t o r , b e s i d e s t e m p e r a t u r e , w h i c h has a pronounced 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 a q u a t i c i n v e r t e b r a t e a n i m a l s and t h e i r t i s s u e . The 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 response by 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 supra-normal s a l i n i -t i e s . T h i s has been demonstrated by F l e m i s t e r and F l e m i s t e r (1951) f o r the sand c r a b , Ocypode a l b i c a n s , by L o f t s (1956); i n a s a l t marsh p o p u l a t i o n o f the prawn, . Palaemonetes v a r i a n s , and by 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 of t h e prawn, Metapenseus monoceros. There a r e 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 a c t i v i t y may be h i g h e r i n sub-normal s a l i n i t i e s . Dehnel (196O) found i n b o t h 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 sea w a t e r where t h e os m o t i c g r a d i e n t between the b l o o d and medium was g r e a t e s t . Lance (1965) showed t h a t t h e m e t a b o l i c r a t e i n 30$ sea w a t e r was 1^M' double that i n 100$ sea water f o r the planktonic copepod, Acartia tonsa. King (1965) demonstrated i n the crabs, Oarclnus medlterraneus and Oallinectes sapidus, a 33$ and 53$ Increase i n oxygen consumption, respectively, a f t e r the animals were transfered from 80$ to 50$ sea water. The conelusion reached by these authors i s that when the blood concentration i s no longer isotonic to the medium oxygen consumption i s increased to maintain the osmotic gradient. Several investigators have raised objections to the proposal that increased oxygen consumption r e f l e c t s osmotic work to maintain the osmotic gradient between the blood and medium. Gross (1957) has suggested that the increase i n r e s p i r a t i o n rate with an increase i n osmotic gradient f o r the rock crab, Pachygrapsus crassipes, i s related to an increase i n a c t i v i t y . A comparison of respiratory and osmoregulatory data i n both species of Hemigrapsus indicates that a increase i n r e s p i r a t i o n rate does not necessarily r e f l e c t osmotic work (Dehnel, 1962). Dehnel and McOaughran (1964) found that the rate of oxygen consumption f o r excised g i l l tissue of winter species of Hemigrapsus did not show any c o r r e l a t i o n with s a l i n i t y . King (1965) discovered that the excised g i l l s of Oarclnus, an osmoregulator, did not show a s i g n i f i c a n t change i n oxygen consumption when transfered from 80$ to 50$ sea water. In Maja, a crab that remains isoosmotic with the medium, there was a 6$ increase i n oxygen consumption of excised 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 properly the eff e c t of s a l i n i t y on midgut gland r e s p i r a t i o n , one should f i r s t examine the extent to which the osmotic concentration of the blood and urine change with changes i n s a l i n i t y . The blood concentration of winter and summer Hemigrapsus nudus i s hypertonic to the medium over the experimental s a l i n i t y range from 25$ to 1 2 5 $ sea water. The animals regulate t h e i r blood concentration 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, regulation breaks down and the blood approaches i s o t o n i c i t y with the medium although s t i l l hypertonic to i t . The osmotic concentration of the urine f o r summer animals i s equal to the blood concentration over the experimental s a l i n i t y range from 25$ to 1 2 5 $ sea water and hypertonic to the medium. Winter animals have a urine which i s hypotonic to the blood at 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 urine i s hyper-tonic to the medium below 90$ sea water and hypotonic above t h i s sea water concentration (Dehnel, 1962; Dehnel and Stone, 1964). When these osmoregulatory data are compared with the respiratory response of excised midgut gland tissue, a seasonal ef f e c t i s noted. In summer animals there i s a decrease i n midgut gland r e s p i r a t i o n with a Increase i n s a l i n i t y from 35$ to 125$ sea water (Fig. 5 ) . In 35$ sea water there i s a large osmotic gradient between the blood and medium. To maintain the blood concentration hypertonic 46 to the medium, a c t i v e a b s o r p t i o n of ions from the midgut gland may occur. Osmotic work would have to be performed to maintain t h i s gradient and the r a t e of oxygen consumption would be h i g h . Whole animal r e s p i r a t i o n of Hemigrapsus  nudus, however, does not support t h i s proposal (Dehnel, 1962). The blood approaches i s o t o n i c i t y w i t h the medium i n 75$ sea water. Since the osmotic gradient i s small, and the animal i s r e g u l a t i n g i t s blood c o n c e n t r a t i o n to a minimal degree, these f a c t s may account f o r the drop i n midgut gland oxygen consumption i n 75$ sea water. In 125$ sea water osmotic s t r e s s may increase 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 animals normally do not encounter such a hig h s a l i n i t y i n the f i e l d . Dehnel and McCaughran (1964) have demonstrated th a t the g i l l s of summer Hemigrapsus sp. a l s o appear to be important i n osmotic r e g u l a t i o n when the osmotic gradient between the blood and medium i s maximal (35$ sea water). As has been suggested e a r l i e r , metabolic a c t i v i t y of midgut gland from summer animals may be r e l a t e d more to the energy demands of new exoskeleton formation than to the maintenance of a osmotic g r a d i e n t . This proposal i s v a l i d when i t i s recognized that many of the 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 . In wi n t e r animals there I s a "V-shaped" r e l a t i o n s h i p of midgut gland r e s p i r a t i o n to s a l i n i t y w i t h the lowest r e s p i r a t i o n r a t e i n 75$ sea water ( P i g . 5 ) . The low metabolic response i n 75$ sea water corresponds to the 47 point where the blood and urine concentrations approach i s o t o n i c i t y with the medium. The high respiratory response i n 35$ sea water may indicated work being done to maintain the osmotic gradient between the blood and medium. In Figure 8 a si m i l a r pattern of response i s found when the experimental s a l i n i t y effect i s averaged at each acute temperature (5°, 10°, 15° and 20°C) f o r two experimental temperatures (5 C0 and 20°C) and both seasons. The midgut gland appears to be active i n the winter i n the maintenance of a osmotic gradient although evidence presented by Gross (1957) and Dehnel (1962) on whole animalnwould tend to confute t h i s suggestion. The production of a urine hypo-tonic to the blood may also a s s i s t the midgut gland during the winter i n s a l t regulation of the blood. Potts (1954) points out;, however, that the production of a hypotonic urine 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$. Eff e c t of Temperature-Salinity-Season Interactions There are several studies which have assessed the ef f e c t s of environmental factors acting simultaneously on whole animal and t i s s u e . Panikkar (1940), Broekema (1941), Dehnel (1960) and Todd and Dehnel (1960) have examined the combined effects of seasonal changes i n temperature and s a l i n i t y on the mortality, metabolic a c t i v i t y and thermal l i m i t s of intact C rustacea. Dehnel and McCaughran (1964) have determined temperature, s a l i n i t y and seasonal effects on excised g i l l tissue i n both species of Hemigrapsus. 48 KInne (1963,1964) has presented a comprehensive review of s t u d i e s demonstrating d i f f e r e n t i a l response e f f e c t s to temperature and s a l i n i t y . In F i g u r e 6 i s presented a seasonal comparison of w e i g h t - s p e c i f i c oxygen consumption of midgut glan d f o r the experimental t e m p e r a t u r e - s a l i n i t y i n t e r a c t i o n a t 1O°0 acute temperature. The i n t e r a c t i o n i s s t a t i s t i c a l l y s i g n i f i c a n t (Table I V ) . An experimental temperature e f f e c t i s e v i d e n t when comparisons are made w i t h i n and between the seasons. In summer animals a t 5°C 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 w i t h s a l i n i t y . At the summer b a s e l i n e temperature of 20°C there i s a decrease i n oxygen consumption wi t h an i n c r e a s e i n s a l i n i t y . T h i s experimental temperature-s a l i n i t y e f f e c t may be r e l a t e d to osmotic work being done i n maintenance of a osmotic g r a d i e n t between the blood and medium or to the energy demands of expskeleton f o r m a t i o n , s i n c e i t i s r e c o g n i z e d t h a t many of the summer animals used i n t h i s study may have been i n premolt. In the w i n t e r animals a t t h e i r w i n t e r b a s e l i n e temperature of 5°C a "V-shaped" r e l a t i o n s h i p to s a l i n i t y i s shown. The lowest r e s p i r a t i o n r a t e Is i n 75$ sea water where the blood ap-proaches i s o t o n i c i t y w i t h the medium. The i n c r e a s e d r e s p i r -a t i o n r a t e i n 35$ sea water may i n d i c a t e t h a t the midgut gland i s doing osmotic work i n response to the osmotic g r a d i e n t . The w i n t e r animals a t 2 0 6 C g r a d u a l l y i n c r e a s e the r a t e of midgut glan d r e s p i r a t i o n w i t h an i n c r e a s e i n s a l i n i t y . An e x p l a n a t i o n of t h i s response cannot be g i v e n 49 a t t h i s t i m e . The same g e n e r a l t r e n d s a r e a l s o e v i d e n t i n F i g u r e 11 where s e a s o n a l changes i n e x p e r i m e n t a l temper-a t u r e and s a l i n i t y a r e averaged f o r a l l a c u t e t e m p e r a t u r e s (5°, 10°, 15° and 20°C). I n a comparable 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 bo t h s p e c i e s o f Hemigrapsus, Dehnel and McOaughran (1964) found t h a t g i l l t i s s u e from summer a n i m a l s appeared t o be a c t i v e i n t h e maintenance of an o s m o t i c 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 o f w i n t e r a n i m a l s 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 r a t e w i t h e x p e r i m e n t a l s a l i n i t y . 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 from t h o s e observed f o r midgut g l a n d . Temperature and s a l i n i t y b o t h have b i o l o g i c a l l y i m p o r t a n t 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 r e s p i r a t i o n . The 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 i n summer a n i m a l s may be gea r e d n o t o n l y t o maintenance o f an o s m o t i c g r a d i e n t but a l s o t o energy r e q u i r e m e n t s 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 . I n w i n t e r a n i m a l s t h e midgut 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 b l o o d e l e c t r o l y t e s . T h i s f a c t t o g e t h e r w i t h t h e e v i d e n c e of 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 ac c o u n t i n p a r t f o r the mechanisms of o s m o t i c r e g u l a t i o n i n w i n t e r a n i m a l s . 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 all 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 all acute temperatures of measurement.(5°C to 20°C) than weight-specific 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. The e x p e r i m e n t a l t e m p e r a t u r e e f f e c t c o r -responds t o P r e c h t ' s (1951) t y p e 3 ( p a r t i a l c o m p e n s a t i o n ) . 5. E x p e r i m e n t a l t e m p e r a t u r e e f f e c t i s n o t e d s e a s o n a l l y i n t h e r e s p i r a t o r y r e s ponse 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 show no change i n t h e o r d i n a l p o s i t i o n o f t h e m e t a b o l i c - t e m p e r a t u r e curve 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 i n c r e a s e i n r e s p i r a t i o n w i t h a d e c r e a s e i n s a l i n i t y . W i n t e r a n i m a l s a t t h e s e a s o n a l b a s e l i n e t e m p e r a t u r e o f 5°C demonstrate 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 . The 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 i s i n 75$ sea w a t e r . 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 an 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 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 i n summer a n i m a l s may be r e l a t e d t o the maintenance o f a o s m o t i c g r a d i e n t between the b l o o d and medium. The h i g h e s t r a t e o f oxygen consumption i s a t summer b a s e l i n e c o n d i t i o n s (35$ sea w a t e r , 20°C) where t h e os m o t i c g r a d i e n t between t h e b l o o d and medium i s maximal. A l t e r n a t i v e l y , midgut g l a n d r e s p i r a t o r y a c t i v i t y may be geared t o t h e energy 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 . T h i s p r o p o s a l i s v a l i d 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 st u d y 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 . S i n c e p r e m o l t 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 r a t e t h a n i n t e r m o l t a n i m a l s , t h e h i g h e r s e a s o n a l summer r a t e s c o u l d be e x p l a i n e d on t h i s b a s i s . 7. W i n t e r a n i m a l s a t t h e i r s e a s o n a l b a s e l i n e temper-a t u r e (5°C) show 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 . 52 T h i s r e l a t i o n s h i p r e f l e c t s t h e p o s s i b i l i t y t h a t midgut g l a n d t i s s u e may be r e g u l a t i n g b l o o d s a l t s . The h i g h r e s p i r a t o r y r e s ponse i n 35$ sea w a t e r may i n d i c a t e work 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 g r a d i e n t between th e b l o o d and medium. I n 75$ sea w a t e r where the r a t e o f oxygen consumption i s m i n i m a l , 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 a t a minimum and l i t t l e work would a p p a r e n t l y have to be done t o m a i n t a i n the g r a d i e n t . 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 the b l o o d a l s o may a s s i s t w i n t e r a n i m a l s i n m a i n t a i n i n g t h e b l o o d c o n c e n t r a t i o n h y p e r t o n i c t o t h e medium. 8 . The r e g r e s s i o n o f 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 show a s i g n i f i c a n t r e l a t i o n s h i p . The s l o p e v a l u e s a r e 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 from z e r o a t t h e 0 . 0 1 p r o b a b i l i t y l e v e l . 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. 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