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Biochemical relations of calcium metabolism in Emerita asiatica
Comp. Biochem. Physiol., 1967, Vol. 20, pp. 629 to 634. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION BIOCHEMICAL RELATIONS OF CALCIUM METABOLISM IN EMERITA ASIATICA P. S I T A R A M A I A H Zoology Research Laboratory, University of Madras, Madras, India (Received 12 April 1966)
Abstract--Calcium content of exuvium and body during moult cycle revealed that 41"8 per cent calcium is excreted and the rest is lost in the exuvium. There are no stores of calcium and seawater (18.37 per cent) and food (81.63 per cent) are the sources of calcium. The low calcium uptake from seawater is related to ecological conditions. INTRODUCTION IT IS well known that hardening of cuticle in crustaceans is by calcification. Travis (1955), H e c h t (1914) and Robertson (1937) indicated the importance of seawater and food as sources of calcium. Robertson (1937) and Maluf (1941) proved calcium absorption occurred only in the soft crab and paper shell crab but not by hard and pillans crabs. T h e calcium absorbed into the body by soft and paper shell crabs when they do not feed and the quantity obtained through food in the hard and pillans crabs were calculated by Robertson (1937), (Hecht, 1914; Robertson, 1937; Travis, 1955). However, variations in the different biochemical constituents during the moult cycle and their role in the calcium metabolism have not been studied. Protein, lipid, chitin, 2 N HC1 solutes and calcium in the cuticle and protein, lipid, calcium and water in the entire body were measured during the four stages of moult cycle of E m e r i t a asiatica. T h e role of protein, lipid and water in the calcium metabolism and the relations of ecological conditions to the degree of calcium absorption and the degree of post-moult calcification are discussed. MATERIAL AND METHODS For the above studies locally-occurring females of Emerita asiatica have been used. The materials were dried at 55 °C to constant weight. Loss in weight was calculated for 100 g wet wt. and is represented as percentage water. Since the percentage water varied considerably during the moult cycle wet weight cannot be taken as an index of weight of the organism. Therefore only dry weight was taken as a basis to calculate all the biochemical constituents. Total nitrogen was estimated with micro-Kjeldahl method using steam distillation apparatus of the Pregl-Pranas-Wagner design (Steyermark, 1951). Lipids were extracted with ether in Soxhiet apparatus (Hawk, Oser & Summerson, 1947). Chitin content of the cuticle was estimated using the procedure of Hackman (1954). The cuticle, after decalcification in 2 N 629
630
P. SITARAMAIAH
HC1, was digested in 5% sodium hydroxide for 3 days at 105°C, the alkali being changed every 12 hr. T h e chitin flakes were centrifuged and washed in distilled water several times, dehydrated in alcohol and dried. T h e materials were dissolved in perchloric acid (Robertson, 1937) or nitric acid (Numanoi, 1937) and the calcium content was estimated by the method of Tisdall (1923) and Krammer & Tisdall (1921) as modified by Clark & Collip (1925) and with the modifications of vanSlyke & Sendroy (1929). The acid solutions were diluted and treated with 1:1 ammonia and compared with phosphate buffer of pH 5'0 using bromcresol purple indicator, before the addition of ammonium oxalate. T h e carbonate and phosphate fractions of calcium were determined by the method given by Numanoi (1937). T h e loss in 2 N HC1 for 12 hr was also estimated (Scudamore, 1947). All the values presented are the averages of more than six organisms of weight ranges indicated. RESULTS
Water, protein, lipid and calcium in the body Water content increased during early pillans, molting, early soft crab and soft crab stages (Table 1 and 2). It was minimal during hard crab and maximum during soft crab stages with an amplitude of 19.625 per cent. The water content of Carcinus maenas (Robertson, 1937) and grapsoid crabs (Baumberger & Olmsted, 1928) are in agreement but the values for Panulirus argus (Travis, 1954) are low compared with the present study. 1.
TABLE 1--CHANGES IN WATER, CALCIUM AND EXUVIAL WEIGHT DURING MOULT CYCLE IN Emerita asiatica*
Moulting stage
Range of calcium Range of Range of (soft dry wt. % water body) (g) (%) (%)
Premoult pillans crab
1"295 2"750
Moulting stage
1-94 2"45
Soft crab 1 hr after moult
1"755 2-870
74 77 (75) 77 77 (77) 77 78 (77)
Range of calcium (entire body) (%)
% Ca
% water
% of body
Ca as % of Ca
7"89 7'42 (7"62) 6"20 5"82 (6-12) 4"364 4"663 (4"43)
25 18 (20) 15 11 (11) 11 9 (10)
29 37 (35) 46 49 (47) 54 44 (48)
29 26 (28) 37 39 (38) 37 44 (38)
94 95 (94) 92 91 (91) 95 96 (95)
0"66 0"73 (0"68) 0.74 0"68 (0'72) 0"325 0"717 (0.515)
Exuvium
* Values in parenthesis indicate averages.
Protein in the soft crab (32.58-36 per cent), increased during paper shell (61 per cent), hard crab (59 per cent), and decreased (39 per cent) by pillans stage (Table 2). There is a decline in protein (from 39 to 34 per cent) from pillans crab to soft crab. Lipids in the body are low (12 per cent) in soft crab decreased (7 per cent) during the paper shell stage and increased (11 per cent) during hard crab
631
BIOCHEMICAL RELATIONS OF CALCIUM METABOLISM IN EMERITA ASIAT1CA
and (22 per cent) in piUans stage (Table 2). The results of protein and lipids are in agreement with the basic pattern recorded in insects (Krishnakumaran, 1962) and in decapods (Scheer, 1957). TABLE 2--WATER,
Stage of moult cycle
CALCIUM, PROTEIN AND LIPIDS IN THE ENTIRE ORGANISMS OF asiatica DURING MOULT CYCLE*
Range of Range of weight water (g) (%)
Soft crab
0"24 2-09
Paper shell crab
0'505 1'890
Hard crab
0"820 3"265
Pillans crab
0"585 3"225
77 83 (81) 70 74 (72) 64 68 (66) 70 77 (72)
Range of calcium (%) 0"31 0"71 (0"46) 1"12 1"58 (1.40) 4"60 10"92 (7"48) 3"11 9"~,4 (6"24)
Range of weight (g) 0-187 2'983 0"730 1"947 0"930 1"023 0-420 2"983
Emerita
Range of protein N x 6.25 (%)
Range of lipids (%)
32 36 (34) 58 66 (61) 55 61 (59) 36 42 (39)
10 13 (12) 6 8 (7) 10 12 (11) 18 25 (22)
* Values in parenthesis are averages. In the premoult pillans crab the percentage calcium content was 0.68 per cent in soft crab body and 7 per cent with the exoskeleton (Table 1). The exuvium contained 94 per cent of total body calcium while the soft body retains only 5 per cent. In moulting stage calcium in the soft body was 0.72 and in the entire body was 6 per cent. The exuvium contained 91 per cent of the total body calcium. In soft crab (1 hr after moult) the percentage calcium in the entire body was 4-43 per cent including exuvium and that in the soft crab body was 0.5 per cent. The exuvium contained 95 per cent of the body calcium. There was 0"46 per cent calcium in the paper shell crab and 7 per cent in the hard crab but this decreased to 6 per cent during the pillans crab stage. This decrease in percentage in calcium content was associated with a 19 per cent fall of protein and an 11 per cent increase of lipids.
2. Protein, lipids, growth, 2 N HC1 solutes and calcium in cuticle The percentage protein in the cuticle of soft crab has a maximum of 28 (Table 3) which decreased gradually to 21 in the paper shell crab, 17 per cent in hard crab, and 11 per cent in the pillans crab. The protein content of the cuticle and the entire body are inversely related. The percentage of lipids in the cuticle increased from 5 per cent in soft crab to 12 per cent (Table 3) in the paper shell crab whilst it decreased from 3 per cent to 1 per cent during the hard crab and the pillans crab stages.
632
P. SITARAMAIAH
The increase in the cuticle from 10 mg to 16 mg (49 per cent) from soft crab to paper shell crab stage was associated with an increase in lipids (5-12 per cent), calcium (1--4 per cent) (Table 4) and chitin decreased (2-•) as did the protein (28-21 per cent). During paper shell to hard crab the increase in cuticle (48 per cent) was associated with a decrease in phosphate of calcium, chitin, protein and an increase in lipids, 2 N HCI solutes, calcium and carbonate calcium. The growth TABLE 3--GROWTH AND BIOCHEMICALCOMPOSITION OF CUTICLE DURING MOULT CYCLE IN Emerita asiatica *
Stage of moult• cycle
Protein Wt. of Chitin in Lipids (N × 6"25) area Range of cuticle in cuticle in cuticle 0.16m 2 wt. (g) (%) (%) (%) (mg)
Soft crab
0"972 2"130
Paper shell crab
0'784 1 '472
Hard crab
0"630
2"162 Pillans crab crab
0'992 2'125
2-63 2"97 (2"74) 1'80 2-20 (1"97) 1 '92 5'60 (4"8) 5'8 8'6 (7.56)
4-89 6-74 (5-29) 11.96 13-49 (12"0) 2-67 5-18 (3-75) 1-05 2"74 (1.66)
26 30 (28) 20 23 (21) 14 20 (17) 10 12 (11)
Increase in wt. of cuticle (mg)
8"0 12'0 (10'75) 15"00 17'00 (16"08) 20.0 25.0 (23"83) 27"0 32"0 (29.92)
Growth O/ (,,o)
10.75
5"33
49.58
7"83
48'31
6-09
25.42
* Values in parenthesis indicate averages. TABLE 4
B I O C H E M I C A L C O M P O S I T I O N OF CUTICLE OF
Stage of moult cycle
Range of dry wts. (g)
Soft crab
0"972 2"130
Paper shell crab
0-784 1"472
Hard crab
0"630 2"162
Pillans crab
0"992 2-125
Range of cuticle calcium (%) 1"21 2"05 (1"66) 2"94 4"52 (3.92) 9"86 12"84 (11"72) 15"42 29-28 (22"54)
* Values in parenthesis indicate averages.
Emerita asiatica
% cuticle calcium (carbonate) (%) 1"00 1"79 (1.41) 2'58 4'13 (3.48) 9"00 11"96 (10"85) 14-46 27"26 (20"79)
DURING MOULT CYCLE*
% cuticle calcium (phosphate) (%) 0"21 0"26 (0"24) 0"36 0"39 (0.37) 0"66 0'88 (0"79) 0"96 1"66 (1 '25)
o,~ loss of wt. in 2 N HCI (%) 32 37 (34) 45 56 (49) 65 76 (69) 68 73 (70)
BIOCHEMICAL RELATIONS OF CALCIUM METABOLISM IN EMERITA ASIATICA
633
(25 per cent) from hard crab to pillans crab was associated with decrease in protein, lipids and an increase in chitin, calcium, 2 N HCI solubles and carbonate calcium. The percentages of 2 N HCI solubles during the moult cycle of Emerita asiatica were: soft crab, 34 per cent; paper shell crab, 49 per cent; hard crab, 69 per cent; pillans crab, 78 per cent. Scudamore (1947) reported 72 per cent in inter-moult carapace, 78 per cent in moulted carapace, 76.9 per cent in the carapace a few days before moult in cray fish. Baumberger & Olmsted (1928) recorded a loss of 79 per cent of carapace, cast or pillans exoskeleton 93 per cent, hard crab 67 per cent; pillans 76 per cent, soft crab or newly moulted 87 per cent, paper shell 82 per cent in grapsoid crabs. The calcium (phosphate) is 14 per cent of the total cuticle calcium of the soft crab which decreased during moult cycle (Table 3). DISCUSSION Variations in the entire body of protein, lipids and calcium during moult cycle revealed the possibility of a dominance of carbohydrates associated with fall in lipids and proteins and formation of chitin in the soft crab (Paul & Sharpe, 1916; Baumberger & Dill, 1928; Scheer, 1957; Travis, 1957; Scheer & Meenakshi, 1961). The increase in water during early pillans and decrease during paper shell crab were associated with high values for lipids and proteins respectively. The decrease in calcium in the exuvium (Table 1) during moulting stages suggest that calcium is resorbed into the body. These results indicate that the percentage of calcium decreased in the soft body due to excretion and the decrease in the entire body including exuvium was due to resorption and excretion. The increase in the percentage calcium in the exuvium compared to the total percentage calcium of the soft crab after moult as well to the previous moulting stage supports the theory that calcium must have been excreted from the body. The apparent rise in the calcium in exuvium and decrease in calcium in the body was due to the addition of organic matter to the soft body through resorption. There are no storage reserves of calcium in the body (Robertson, 1937) and the entire amount of calcium needed for the hardening of new skin is obtained from seawater and food. The calcium obtained through food (81 per cent) during hard and pillans crab and from seawater (18 per cent) during soft and paper shell crab indicate the low percentage of calcium obtained from seawater compared to Cardnus m a ~ a s (Robertson, 1937). This is mainly due to its filterfeeding habit which enables the organism to feed lying stationary in its interstitial habitat, with a lower degree of hardening of limbs and carapace. Carcinus maenas has to move about in search of prey with a greater degree of activity corresponding to a need to hunt and capture prey. Furthermore the interstitial habitat in the intertidal zone does not facilitate continuous submergence under water but only periodic dips with less opportunity in terms of time for absorption of calcium. The results for grapsoid crabs (Baumberger & Olmsted, 1928) clearly show greater loss in soft crab and paper shell crab compared to hard crab and pillans crab compared to Emerita asiatica. The shift in 2 N HC1 solubles indicate greater degree of mineralisation relative to growth of organic matter in soft and paper shell crab stages of grapsoid crabs.
634
P. SITARAMAIAH
REFERENCES BAUMBERGER J. P. & DILL D. B. (1928) A study of the glycogen and sugar and the osmotic pressure of crab during the moult cycle. Physiol. Zool. 1, 545-549. BAUMBm~GERJ. P. & OLMSTED J. M. D. (1928) Changes in the osmotic pressure and water content of crabs during molt cycle. Physiol. Zool. 1, 531-545. CLAI~ E. P. & COt.LIP J. B. (1925) A study of the Tisdall method for the determination of blood serum calcium with a suggested modification, ft. biol. Chem. 63, 461-464. HAWK P. B., OSER B. S. & SUMMmaSON W. H. (1947) Practical Physiological Chemistry. (12th Edn.) Churchill, Lond. HACKMAN R. H. (1954) Studies on chitin I. Enzymatic degradation of chitin and chitin esters. Aust. ft. biol. Sci. 7, 168-178. HECHT S. (1914) Note on the absorption of calcium during the moulting of the blue crab Callinectes sapidus. Science, N. Y. 39, 108. KaAMMma B. & TISDALL F. F. (1921) A simple technique for the determination of Ca and M g in small amounts of serum. J. biol. Chem. 47, 475-481. KRISHNAKUMARANA. (1962) On some biochemical aspects of the moult cycle in Gryllus. Biol. Zb. 81, 529-538. MALUF N. S. R. (1941) T h e uptake of inorganic electrolytes by the cray fish. ft. gen. Physiol., 24, 151-167. NUM~OI H. (1937) Migration of calcium through blood in Legia exotica. Jap.ff. Zool. 7, 241-249. P a u l J. H. & SHARPE J. S. (1916) Studies in calcium metabolism. I. T h e deposition of lime salts in the integument of decapod crustacea..7. Physiol. 50, 183-192. ROBERTSON J. D. (1937) Some features of calcium metabolism of the shore crab (Carcinus maenas) (Prennant). Proc. R. Soc. B124, 162-182. SCUDAMO~ H. H. (1947) T h e influence of sinus glands upon moulting and associated changes in the cray fish. Physiol. Zool. 20, 187-208. ScrmEa B. T. (1957) T h e hormonal control of metabolism in decapoda. In Recent Advances in Invertebrate Physiology, pp. 213-227. University of Oregon Press. SCHEEa B. T. & MEENAKSrIIV. R. (1961) T h e metabolism of carbohydrates in Arthropods. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals. (Edited by Mn_RTIN A. W.) pp. 65-83. University of Washington Press. S~'ZEaMARK A. (1951) Quantitative Organic Micro Analysis. Blakiston, New York. TISDALL F. F. (1923) A note on the K r a m m e r - T i s d a l l method for the determination of calcium in small amounts of serum, ft. biol. Chem. 56, 439-441. TaAvIS D. F. (1954) T h e moulting cycle of the spiny lobster Panulirus argus Latreilte. I. Moulting and growth in laboratory maintained individuals. Biol. Bull., Woods Hole 107, 433-450. TaAVXSD. F. (1955) T h e moulting cycle of the spiny lobster, Panulirus argus (Latreille). III. Physiological changes which occur in the blood and urine during the normal moulting cycle. Biol. Bull., Woods Hole 109, 484--503. TaAVIS D. F. (1957) T h e moulting cycle of spiny lobster Panulirus argus Latreille. IV. Postecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull., Woods Hole 113 (3), 451-471. V A N S L ~ D. D. & SENDaOY J. (1929) Gasometric determination of oxalic acid and calcium and its application to serum analysis, ft. biol. Chem. 84, 217-232.
SHORT COMMUNICATION BIOCHEMICAL RELATIONS OF CALCIUM METABOLISM IN EMERITA ASIATICA P. S I T A R A M A I A H Zoology Research Laboratory, University of Madras, Madras, India (Received 12 April 1966)
Abstract--Calcium content of exuvium and body during moult cycle revealed that 41"8 per cent calcium is excreted and the rest is lost in the exuvium. There are no stores of calcium and seawater (18.37 per cent) and food (81.63 per cent) are the sources of calcium. The low calcium uptake from seawater is related to ecological conditions. INTRODUCTION IT IS well known that hardening of cuticle in crustaceans is by calcification. Travis (1955), H e c h t (1914) and Robertson (1937) indicated the importance of seawater and food as sources of calcium. Robertson (1937) and Maluf (1941) proved calcium absorption occurred only in the soft crab and paper shell crab but not by hard and pillans crabs. T h e calcium absorbed into the body by soft and paper shell crabs when they do not feed and the quantity obtained through food in the hard and pillans crabs were calculated by Robertson (1937), (Hecht, 1914; Robertson, 1937; Travis, 1955). However, variations in the different biochemical constituents during the moult cycle and their role in the calcium metabolism have not been studied. Protein, lipid, chitin, 2 N HC1 solutes and calcium in the cuticle and protein, lipid, calcium and water in the entire body were measured during the four stages of moult cycle of E m e r i t a asiatica. T h e role of protein, lipid and water in the calcium metabolism and the relations of ecological conditions to the degree of calcium absorption and the degree of post-moult calcification are discussed. MATERIAL AND METHODS For the above studies locally-occurring females of Emerita asiatica have been used. The materials were dried at 55 °C to constant weight. Loss in weight was calculated for 100 g wet wt. and is represented as percentage water. Since the percentage water varied considerably during the moult cycle wet weight cannot be taken as an index of weight of the organism. Therefore only dry weight was taken as a basis to calculate all the biochemical constituents. Total nitrogen was estimated with micro-Kjeldahl method using steam distillation apparatus of the Pregl-Pranas-Wagner design (Steyermark, 1951). Lipids were extracted with ether in Soxhiet apparatus (Hawk, Oser & Summerson, 1947). Chitin content of the cuticle was estimated using the procedure of Hackman (1954). The cuticle, after decalcification in 2 N 629
630
P. SITARAMAIAH
HC1, was digested in 5% sodium hydroxide for 3 days at 105°C, the alkali being changed every 12 hr. T h e chitin flakes were centrifuged and washed in distilled water several times, dehydrated in alcohol and dried. T h e materials were dissolved in perchloric acid (Robertson, 1937) or nitric acid (Numanoi, 1937) and the calcium content was estimated by the method of Tisdall (1923) and Krammer & Tisdall (1921) as modified by Clark & Collip (1925) and with the modifications of vanSlyke & Sendroy (1929). The acid solutions were diluted and treated with 1:1 ammonia and compared with phosphate buffer of pH 5'0 using bromcresol purple indicator, before the addition of ammonium oxalate. T h e carbonate and phosphate fractions of calcium were determined by the method given by Numanoi (1937). T h e loss in 2 N HC1 for 12 hr was also estimated (Scudamore, 1947). All the values presented are the averages of more than six organisms of weight ranges indicated. RESULTS
Water, protein, lipid and calcium in the body Water content increased during early pillans, molting, early soft crab and soft crab stages (Table 1 and 2). It was minimal during hard crab and maximum during soft crab stages with an amplitude of 19.625 per cent. The water content of Carcinus maenas (Robertson, 1937) and grapsoid crabs (Baumberger & Olmsted, 1928) are in agreement but the values for Panulirus argus (Travis, 1954) are low compared with the present study. 1.
TABLE 1--CHANGES IN WATER, CALCIUM AND EXUVIAL WEIGHT DURING MOULT CYCLE IN Emerita asiatica*
Moulting stage
Range of calcium Range of Range of (soft dry wt. % water body) (g) (%) (%)
Premoult pillans crab
1"295 2"750
Moulting stage
1-94 2"45
Soft crab 1 hr after moult
1"755 2-870
74 77 (75) 77 77 (77) 77 78 (77)
Range of calcium (entire body) (%)
% Ca
% water
% of body
Ca as % of Ca
7"89 7'42 (7"62) 6"20 5"82 (6-12) 4"364 4"663 (4"43)
25 18 (20) 15 11 (11) 11 9 (10)
29 37 (35) 46 49 (47) 54 44 (48)
29 26 (28) 37 39 (38) 37 44 (38)
94 95 (94) 92 91 (91) 95 96 (95)
0"66 0"73 (0"68) 0.74 0"68 (0'72) 0"325 0"717 (0.515)
Exuvium
* Values in parenthesis indicate averages.
Protein in the soft crab (32.58-36 per cent), increased during paper shell (61 per cent), hard crab (59 per cent), and decreased (39 per cent) by pillans stage (Table 2). There is a decline in protein (from 39 to 34 per cent) from pillans crab to soft crab. Lipids in the body are low (12 per cent) in soft crab decreased (7 per cent) during the paper shell stage and increased (11 per cent) during hard crab
631
BIOCHEMICAL RELATIONS OF CALCIUM METABOLISM IN EMERITA ASIAT1CA
and (22 per cent) in piUans stage (Table 2). The results of protein and lipids are in agreement with the basic pattern recorded in insects (Krishnakumaran, 1962) and in decapods (Scheer, 1957). TABLE 2--WATER,
Stage of moult cycle
CALCIUM, PROTEIN AND LIPIDS IN THE ENTIRE ORGANISMS OF asiatica DURING MOULT CYCLE*
Range of Range of weight water (g) (%)
Soft crab
0"24 2-09
Paper shell crab
0'505 1'890
Hard crab
0"820 3"265
Pillans crab
0"585 3"225
77 83 (81) 70 74 (72) 64 68 (66) 70 77 (72)
Range of calcium (%) 0"31 0"71 (0"46) 1"12 1"58 (1.40) 4"60 10"92 (7"48) 3"11 9"~,4 (6"24)
Range of weight (g) 0-187 2'983 0"730 1"947 0"930 1"023 0-420 2"983
Emerita
Range of protein N x 6.25 (%)
Range of lipids (%)
32 36 (34) 58 66 (61) 55 61 (59) 36 42 (39)
10 13 (12) 6 8 (7) 10 12 (11) 18 25 (22)
* Values in parenthesis are averages. In the premoult pillans crab the percentage calcium content was 0.68 per cent in soft crab body and 7 per cent with the exoskeleton (Table 1). The exuvium contained 94 per cent of total body calcium while the soft body retains only 5 per cent. In moulting stage calcium in the soft body was 0.72 and in the entire body was 6 per cent. The exuvium contained 91 per cent of the total body calcium. In soft crab (1 hr after moult) the percentage calcium in the entire body was 4-43 per cent including exuvium and that in the soft crab body was 0.5 per cent. The exuvium contained 95 per cent of the body calcium. There was 0"46 per cent calcium in the paper shell crab and 7 per cent in the hard crab but this decreased to 6 per cent during the pillans crab stage. This decrease in percentage in calcium content was associated with a 19 per cent fall of protein and an 11 per cent increase of lipids.
2. Protein, lipids, growth, 2 N HC1 solutes and calcium in cuticle The percentage protein in the cuticle of soft crab has a maximum of 28 (Table 3) which decreased gradually to 21 in the paper shell crab, 17 per cent in hard crab, and 11 per cent in the pillans crab. The protein content of the cuticle and the entire body are inversely related. The percentage of lipids in the cuticle increased from 5 per cent in soft crab to 12 per cent (Table 3) in the paper shell crab whilst it decreased from 3 per cent to 1 per cent during the hard crab and the pillans crab stages.
632
P. SITARAMAIAH
The increase in the cuticle from 10 mg to 16 mg (49 per cent) from soft crab to paper shell crab stage was associated with an increase in lipids (5-12 per cent), calcium (1--4 per cent) (Table 4) and chitin decreased (2-•) as did the protein (28-21 per cent). During paper shell to hard crab the increase in cuticle (48 per cent) was associated with a decrease in phosphate of calcium, chitin, protein and an increase in lipids, 2 N HCI solutes, calcium and carbonate calcium. The growth TABLE 3--GROWTH AND BIOCHEMICALCOMPOSITION OF CUTICLE DURING MOULT CYCLE IN Emerita asiatica *
Stage of moult• cycle
Protein Wt. of Chitin in Lipids (N × 6"25) area Range of cuticle in cuticle in cuticle 0.16m 2 wt. (g) (%) (%) (%) (mg)
Soft crab
0"972 2"130
Paper shell crab
0'784 1 '472
Hard crab
0"630
2"162 Pillans crab crab
0'992 2'125
2-63 2"97 (2"74) 1'80 2-20 (1"97) 1 '92 5'60 (4"8) 5'8 8'6 (7.56)
4-89 6-74 (5-29) 11.96 13-49 (12"0) 2-67 5-18 (3-75) 1-05 2"74 (1.66)
26 30 (28) 20 23 (21) 14 20 (17) 10 12 (11)
Increase in wt. of cuticle (mg)
8"0 12'0 (10'75) 15"00 17'00 (16"08) 20.0 25.0 (23"83) 27"0 32"0 (29.92)
Growth O/ (,,o)
10.75
5"33
49.58
7"83
48'31
6-09
25.42
* Values in parenthesis indicate averages. TABLE 4
B I O C H E M I C A L C O M P O S I T I O N OF CUTICLE OF
Stage of moult cycle
Range of dry wts. (g)
Soft crab
0"972 2"130
Paper shell crab
0-784 1"472
Hard crab
0"630 2"162
Pillans crab
0"992 2-125
Range of cuticle calcium (%) 1"21 2"05 (1"66) 2"94 4"52 (3.92) 9"86 12"84 (11"72) 15"42 29-28 (22"54)
* Values in parenthesis indicate averages.
Emerita asiatica
% cuticle calcium (carbonate) (%) 1"00 1"79 (1.41) 2'58 4'13 (3.48) 9"00 11"96 (10"85) 14-46 27"26 (20"79)
DURING MOULT CYCLE*
% cuticle calcium (phosphate) (%) 0"21 0"26 (0"24) 0"36 0"39 (0.37) 0"66 0'88 (0"79) 0"96 1"66 (1 '25)
o,~ loss of wt. in 2 N HCI (%) 32 37 (34) 45 56 (49) 65 76 (69) 68 73 (70)
BIOCHEMICAL RELATIONS OF CALCIUM METABOLISM IN EMERITA ASIATICA
633
(25 per cent) from hard crab to pillans crab was associated with decrease in protein, lipids and an increase in chitin, calcium, 2 N HCI solubles and carbonate calcium. The percentages of 2 N HCI solubles during the moult cycle of Emerita asiatica were: soft crab, 34 per cent; paper shell crab, 49 per cent; hard crab, 69 per cent; pillans crab, 78 per cent. Scudamore (1947) reported 72 per cent in inter-moult carapace, 78 per cent in moulted carapace, 76.9 per cent in the carapace a few days before moult in cray fish. Baumberger & Olmsted (1928) recorded a loss of 79 per cent of carapace, cast or pillans exoskeleton 93 per cent, hard crab 67 per cent; pillans 76 per cent, soft crab or newly moulted 87 per cent, paper shell 82 per cent in grapsoid crabs. The calcium (phosphate) is 14 per cent of the total cuticle calcium of the soft crab which decreased during moult cycle (Table 3). DISCUSSION Variations in the entire body of protein, lipids and calcium during moult cycle revealed the possibility of a dominance of carbohydrates associated with fall in lipids and proteins and formation of chitin in the soft crab (Paul & Sharpe, 1916; Baumberger & Dill, 1928; Scheer, 1957; Travis, 1957; Scheer & Meenakshi, 1961). The increase in water during early pillans and decrease during paper shell crab were associated with high values for lipids and proteins respectively. The decrease in calcium in the exuvium (Table 1) during moulting stages suggest that calcium is resorbed into the body. These results indicate that the percentage of calcium decreased in the soft body due to excretion and the decrease in the entire body including exuvium was due to resorption and excretion. The increase in the percentage calcium in the exuvium compared to the total percentage calcium of the soft crab after moult as well to the previous moulting stage supports the theory that calcium must have been excreted from the body. The apparent rise in the calcium in exuvium and decrease in calcium in the body was due to the addition of organic matter to the soft body through resorption. There are no storage reserves of calcium in the body (Robertson, 1937) and the entire amount of calcium needed for the hardening of new skin is obtained from seawater and food. The calcium obtained through food (81 per cent) during hard and pillans crab and from seawater (18 per cent) during soft and paper shell crab indicate the low percentage of calcium obtained from seawater compared to Cardnus m a ~ a s (Robertson, 1937). This is mainly due to its filterfeeding habit which enables the organism to feed lying stationary in its interstitial habitat, with a lower degree of hardening of limbs and carapace. Carcinus maenas has to move about in search of prey with a greater degree of activity corresponding to a need to hunt and capture prey. Furthermore the interstitial habitat in the intertidal zone does not facilitate continuous submergence under water but only periodic dips with less opportunity in terms of time for absorption of calcium. The results for grapsoid crabs (Baumberger & Olmsted, 1928) clearly show greater loss in soft crab and paper shell crab compared to hard crab and pillans crab compared to Emerita asiatica. The shift in 2 N HC1 solubles indicate greater degree of mineralisation relative to growth of organic matter in soft and paper shell crab stages of grapsoid crabs.
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P. SITARAMAIAH
REFERENCES BAUMBERGER J. P. & DILL D. B. (1928) A study of the glycogen and sugar and the osmotic pressure of crab during the moult cycle. Physiol. Zool. 1, 545-549. BAUMBm~GERJ. P. & OLMSTED J. M. D. (1928) Changes in the osmotic pressure and water content of crabs during molt cycle. Physiol. Zool. 1, 531-545. CLAI~ E. P. & COt.LIP J. B. (1925) A study of the Tisdall method for the determination of blood serum calcium with a suggested modification, ft. biol. Chem. 63, 461-464. HAWK P. B., OSER B. S. & SUMMmaSON W. H. (1947) Practical Physiological Chemistry. (12th Edn.) Churchill, Lond. HACKMAN R. H. (1954) Studies on chitin I. Enzymatic degradation of chitin and chitin esters. Aust. ft. biol. Sci. 7, 168-178. HECHT S. (1914) Note on the absorption of calcium during the moulting of the blue crab Callinectes sapidus. Science, N. Y. 39, 108. KaAMMma B. & TISDALL F. F. (1921) A simple technique for the determination of Ca and M g in small amounts of serum. J. biol. Chem. 47, 475-481. KRISHNAKUMARANA. (1962) On some biochemical aspects of the moult cycle in Gryllus. Biol. Zb. 81, 529-538. MALUF N. S. R. (1941) T h e uptake of inorganic electrolytes by the cray fish. ft. gen. Physiol., 24, 151-167. NUM~OI H. (1937) Migration of calcium through blood in Legia exotica. Jap.ff. Zool. 7, 241-249. P a u l J. H. & SHARPE J. S. (1916) Studies in calcium metabolism. I. T h e deposition of lime salts in the integument of decapod crustacea..7. Physiol. 50, 183-192. ROBERTSON J. D. (1937) Some features of calcium metabolism of the shore crab (Carcinus maenas) (Prennant). Proc. R. Soc. B124, 162-182. SCUDAMO~ H. H. (1947) T h e influence of sinus glands upon moulting and associated changes in the cray fish. Physiol. Zool. 20, 187-208. ScrmEa B. T. (1957) T h e hormonal control of metabolism in decapoda. In Recent Advances in Invertebrate Physiology, pp. 213-227. University of Oregon Press. SCHEEa B. T. & MEENAKSrIIV. R. (1961) T h e metabolism of carbohydrates in Arthropods. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals. (Edited by Mn_RTIN A. W.) pp. 65-83. University of Washington Press. S~'ZEaMARK A. (1951) Quantitative Organic Micro Analysis. Blakiston, New York. TISDALL F. F. (1923) A note on the K r a m m e r - T i s d a l l method for the determination of calcium in small amounts of serum, ft. biol. Chem. 56, 439-441. TaAvIS D. F. (1954) T h e moulting cycle of the spiny lobster Panulirus argus Latreilte. I. Moulting and growth in laboratory maintained individuals. Biol. Bull., Woods Hole 107, 433-450. TaAVXSD. F. (1955) T h e moulting cycle of the spiny lobster, Panulirus argus (Latreille). III. Physiological changes which occur in the blood and urine during the normal moulting cycle. Biol. Bull., Woods Hole 109, 484--503. TaAVIS D. F. (1957) T h e moulting cycle of spiny lobster Panulirus argus Latreille. IV. Postecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull., Woods Hole 113 (3), 451-471. V A N S L ~ D. D. & SENDaOY J. (1929) Gasometric determination of oxalic acid and calcium and its application to serum analysis, ft. biol. Chem. 84, 217-232.