BY B. PADMANABHANAIDU AND R. RAMAMURTHY Department of Zoology, Sri Venkateswara University, Tirupati, India. {Received 23 May i960)

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1 J. Exp. Biol. (1961), 38, With 3 text-figures Printed in Great Britain THE INFLUENCE OF SEX AND SIZE ON THE OSMOTIC PRESSURE, THE CHLORIDE AND THE FREE AMINO ACIDS OF THE BLOOD OF THE FRESHWATER FIELD CRAB, PARATELPHUSA SP. AND THE FRESHWATER MUSSEL, LAMELLIDENS MARGINALIS BY B. PADMANABHANAIDU AND R. RAMAMURTHY Department of Zoology, Sri Venkateswara University, Tirupati, India {Received 23 May i960) INTRODUCTION An extensive literature is available on the ionic composition of the blood and osmotic regulation of marine as well as freshwater crustaceans (reviewed by Krogh, 1939; Panikkar, 1941; Prosser et al. 1950; Parry, 1957). But in most of the previous works, except for the very recent valuable contribution of Gilbert (1959 a-c), the influence of sex and size on the blood composition has been overlooked. Gilbert (1959) has shown in Carcinus moenas that the blood conductivity and blood chloride increase steadily to a maximum at a weight of approximately 35 g and then decrease with the increase of body weight in both sexes, and that the females have higher blood chloride throughout the size range. But he has also shown that there is no difference in conductivity between males and females below 35 g., and that the blood conductivity tends to be higher in males than that in females above 35 g. body weight. He reports that the total osmotic pressure of male crabs is higher than that of females at any given body weight. But in both sexes the total osmotic pressure decreases steadily with increase in body weight. Gilbert showed that the curve (regression line) for total osmotic pressure has no peak at the middle of the size range, and he suggested that the discrepancy between ionic composition and the total osmotic pressure must therefore be due to non-electrolytes. It is seen, therefore, that the only detailed study of this nature is confined to a marine crab. It would be of considerable interest to compare this with the conditions to be found in freshwater animals. The main purpose of the present investigation is to extend such a study of the osmotic pressure, blood chloride and free amino acids in the blood to such typical freshwater organisms as the freshwater field crab, Paratelphusa sp. and the freshwater mussel, Lamellidens marginalis. Since the freshwater mussel is a hermaphrodite the osmotic pressure, blood chloride and free amino acids in the blood of this mollusc have been studied in relation to its body size only. 3-z

2 36 B. PADMANABHANAIDU AND R. RAMAMURTHY MATERIALS AND METHODS The crabs were collected in the paddy fields which are located to the southern side of Sri Venkateswara University campus. They were brought to the laboratory as soon as they were collected. The gravid females and injured animals were discarded. The remaining crabs were placed in groups of twelve each in glass troughs containing a small volume of water. The volume of water was adjusted such that the crabs were submerged under water. The initial mortality was relatively great but mortality was low after a few days, by which time the crabs had become adjusted to the laboratory conditions. The dead ones, if there were any, were removed and the water was renewed daily. Once they were adjusted to the laboratory conditions the crabs lived for long periods of time, provided they were fed. They were not fed for one day before the actual commencement of the experiment. This was to eliminate the variation in the blood composition due to differential feeding. The composition of blood varies during the moult cycle (Baumberger & Olmstead, 1928) and there is a considerable increase in the calcium content of the blood (Robertson, 1939) just before moulting. For this reason measurements of osmotic pressure, blood chloride and free amino acids in the blood were confined to animals in the intermoult stage. The animals just after moult were also discarded; these were recognized by the softness and pale colour of the carapace. The crabs were removed from the glass troughs and the excess of water was drained off, since it adds to their weight. Each was weighed in a Pelouze metric balance to the nearest 0-5 g. The sex and body weight of the crab were noted. The blood was drawn through a small incision in the arthrodial membrane at the base of the fourth thoracic appendage. Sufficient blood was obtained by means of a hypodermic syringe for the measurement of osmotic pressure, blood chloride or free amino acids in the blood. For the determination of osmotic pressure an anticoagulant, heparin, was used but not for the estimation of blood chloride and free aminoacids. For the measurement of osmotic pressure, the classical Barger's capillary vapour pressure method modified by Krogh was employed (as described by Indira, i960). The principle is that the osmotically stronger solution grows at the expense of the weaker solution. If the solutions are equally strong there is no increase of the length of either of the droplets. The osmotic pressure of the blood is expressed in terms of percentage sodium chloride. Blood chloride was determined with 1 ml. of the sample and the method employed was that of Sendroy modified by Robertson & Webb (1939). The silver iodate was prepared in the laboratory according to the procedure suggested by Robertson & Webb (1939). To determine the free amino acid content of the blood, the method of Folin and Danielson (Hawk, Oser & Summerson, 1954) was followed. The freshwater mussels were collected from a small enclosed pond called ' Mangala Gunta' which is situated on the way from Tirupati to Alwar Theertham (Kapila Theertham). They were brought to the laboratory and placed in groups of

3 Blood composition of the field crab and the freshwater mussel 37 twelve in glass troughs containing water. While collecting, care was taken to collect the widest size range of the available animals. Before the experiment was begun the animal was weighed entire. One of the shells was removed and the water drained off. Blood was drawn from the heart by means of a hypodermic syringe. Heparin was used for the determination of the osmotic pressure but not for the determination of blood chloride and free amino acids. The weight of the shell was substracted from the total weight so as to get the weight of the soft parts only. RESULTS Blood osmotic pressure. The osmotic pressure was expressed in terms of percentage sodium chloride solution. The values were plotted against the body weight for thirteen males in Fig. 1A and for twelve females in Fig. 1B. From the data it is clear that the percentage sodium chloride value reaches a maximum at about % f r male crabs of 37 g. weight and 0-75 % for female crabs of 31 g. weight , Fig. i. Osmotic pressure, chloride content and free amino acids of the blood of Paratelphusa sp. as a function of sex and body weight. A, Blood osmotic pressure in males; B, blood osmotic pressure in females; C, blood chloride in males; D, blood chloride in females; E, free amino acids in males; F, free amino acids in females. For statistical analysis the data for each sex were divided into two groups. In the case of males, the animals whose weight was below 37 g. formed one group and those above 37 g. a second group. Similarly, the females whose weights were below 31 g. formed one group and those above 31 g. a second group. Statistically, the lines of best fit were obtained by calculating the regression coefficients by the principle of least squares. In the case of male crabs the osmotic pressure rises

4 38 B. PADMANABHANAIDU AND R. RAMAMURTHY steadily with increase in body weight up to 39 g. weight and then falls significantly with further increase in size. But in the case of females the osmotic pressure rises gradually up to 35 g. weight. In general, the males tend to have a higher osmotic pressure than females over the whole size range. A close examination of the Fig. 1A and 1B reveals that the difference in the osmotic pressure between sexes was not significant at their minimum body weights. But this difference is quite significant at the weights corresponding to their maximum osmotic pressure. This difference is still present even in the negative slope of the regression lines; but it is not as great as it is seen at 39 g. and 35 g. weight of males and females respectively. JO Fig. z Fig. 3 Fig. 2. A comparison of the males and females of Paratelphusa sp. in respect of osmotic pressure, chloride and free amino acid content of the blood. Curves taken from Fig. i. Solid lines for males. Broken lines for females. Fig. 3. Osmotic pressure, chloride content and free amino acids of the blood of Lamellidens marginalis as a function of body weight. A, Blood osmotic pressure; B, blood chloride, C, free amino acids of the blood. The values obtained for the blood osmotic pressure of the freshwater mussels are plotted against the body weight for nine animals in Fig. 3 A. From the data it is clear that there is a significant increase in the osmotic pressure with increase in size of the animals. Blood chloride. The blood chloride values in mm./l. were plotted against the body weight for twenty one males in Fig. 1C and for eighteen females in Fig. 1D. The trend of variation in the blood chloride with body weight and sex seemed to be

5 Blood composition of the field crab and the freshwater mussel 39 similar to that of osmotic pressure. From the data it is clear that the maximum blood chloride in males was at 38 g. weight and in females at 30 g. weight with a decrease on either side of these points. As in the case of osmotic pressure the data for each sex have been split up into two groups for statistical analysis, viz. above and below 38 g. weight for males and above and below 30 g. weight for females. The lines of best fit were drawn by calculating the regression coefficients by the method of least squares. From Fig. 1C and 1D it is found in both sexes that the chloride content of the blood increases with increase in weight to a maximum and then gradually decreases. From the calculated lines drawn it is seen that the maximum blood chloride is at about 40 g. in males and 35 g. in females. A close examination of the Fig. 1C and 1D reveals the following facts. It is rather surprising to find that in the positive slope of the regression line the females tend to have a high blood chloride and in the negative slope of the regression line the males tend to have a high blood chloride. The difference in the blood chloride is significant in the smaller crabs, the females having a higher chloride content. In the negative slope of the regression lines the males above 40 g. have higher blood chloride, but this may not be significant. But if the negative slopes of the regression lines of both sexes are extrapolated towards higher weights the differences in blood chloride becomes insignificant, as was found with the blood conductivity in the lower weight ranges of both the sexes in the shore crab Carcinus moenas (Gilbert, 1959a). The blood chloride of the freshwater mussel is plotted against the body weight for twelve specimens in Fig. 3 B. It is clear that there is a slight increase in the blood chloride with increase in size over the size range studied. Free amino acids. The free amino acids present in the blood are expressed as mg./iooml. of blood. The values areplotted against the body weight for twenty-two males in Fig. 1E and for twenty-eight females in Fig. 1F. The amino acid value reaches a maximum at about 32 g. weight in males and at 35 g. weight in females. As above, the data for each sex have been divided into two groups for statistical analysis, viz. above and below 32 g. for males and above and below 35 g. for females. The regression lines for the different groups are fitted as shown in Fig. 1E andi F using the method of least squares. In both sexes the free animo acids increase steadily with increase in size to a maximum and then decrease. In the male crabs the maximum free amino acid is found at about 32 g. and in females at about 35 g. body weight. It is seen from Fig. 1E and 1F that in general the males tend to have a higher free amino acid content in the blood than females at any given weight. But the difference in free amino acids was conspicuous in individuals of 32 g. or less while in individuals above this weight the difference was not found to be significant. The free amino acids in fresh water mussels are plotted against body weight on a double logarithmic graph for nine animals in Fig. 3 C. Though there is a considerable variation in the amino acid values it is clear from the figure that there is a slight increase as the body weight increases.

6 40 B. PADMANABHANAIDU AND R. RAMAMURTHY DISCUSSION The blood composition of thefieldcrab Paratelphusa sp. varies noticeably with body weight and sex, as was described by Gilbert (1959) in the marine shore crab, Carcinus moenas. In the common shore crab (Gilbert, 1959) the total osmotic pressure in both sexes decreased steadily with increase in size and males tended to have a higher osmotic pressure. In the present work the osmotic pressure was found to increase steadily with increase in size up to a maximum and then gradually to decrease. The maximum osmotic pressure in males was at about 40 g. and in females at about 35 g. weight. The males had significantly higher osmotic pressure at any given weight. The pattern of blood chloride was similar to that of the shore crab (Gilbert, 1959) except that the smaller females had higher chloride values, while above 35 g. the males had slightly higher chloride values. Osmotic pressure and blood chloride values when viewed together are of considerable interest. The males have a higher blood chloride. Since the osmotic pressure depends upon the number of free particles present in the blood the above observation is rather puzzling. It is probable that the slightly higher osmotic pressure of the blood of males might be due to the high free amino acid present in the blood of males. In addition, the high blood-chloride in the males in the negative slope of the curve combined with the slightly higher amino acid content in the same range explains the high osmotic pressure difference between males and females larger than 35 g. But the difference in blood osmotic pressure between males and females below 35 g. is relatively reduced on account of the higher blood chloride in females, although in this range the amino acid content of the blood of males is significantly higher than in females. The high amino acids of males in the positive slope of the curve and also the high chloride of males in the negative slope together contribute to the high osmotic pressure in the males. It is seen from the above that the pattern of blood amino acids might play a significant role in keeping the osmotic pressure of males at a higher level than that of females. It has been shown above that the amino acid content of the blood reaches a peak at a smaller size in males than in females. Whether these differences in amino acid content are related to the maturity of the individual in Paratelphusa sp. is not known, since no attempt has been made to study growth in relation to sexual maturity in this crab. But in view of the studies of Gilbert (1959), wherein it was suggested that the variation in non-protein nitrogen of the blood of Carcinus moenas might be related to the sexual maturity of the crabs, it is possible that this may be the case in Paratelphusa sp. In the freshwater mussel, in contrast to the field crab, the blood osmotic pressure, chloride and free amino acids show no maximal value in the middle of the size range. On the other hand, they show a slight but continuing increase with increasing size. In addition, the blood osmotic pressure, chloride and free amino acids were comparatively low in the freshwater mussel.

7 Blood composition of the field crab and the freshwater mussel 41 SUMMARY 1. The influence of sex and body size on the osmotic pressure, chloride and free amino acids in the blood of the freshwater field crab, Paratelphusa sp., and the freshwater mussel, Lamellidens marginalis, were investigated. 2. In the crab the osmotic pressure increased in both the sexes to a maximum at about 40 g. in males and at 35 g. in females and then fell with further increase in weight. Throughout the whole size range the males tended to have a higher osmotic pressure. 3. Blood chloride in the crab also increased with weight in both the sexes to a maximum at about 40 g. in males and 35 g. in females and then steadily decreased as the weight increased. In the positive slope of the regression line the females have a higher blood chloride, but in the negative slope of the curve the males have higher blood chloride. 4. The free amino acid content of the blood reached a maximum at about 32 g. in males and 35 g. in females and then gradually decreased as the weight increased. Over the whole size range the males tended to have higher free amino acid content than the females. 5. In the freshwater mussel both chloride and free amino acid content of the blood showed a small but steady increase with increasing weight. This is also reflected in a significant increase in the blood osmotic pressure with increase in body weight. We are greatly indebted to Prof. Kandula Pampapathi Rao, Head of the Department of Zoology, Sri Venkateswara University, Tirupati, A.P., for suggesting this problem, and for his kind consideration at all times. REFERENCES BAUMBERGER, J. P. & OLMSTEAD, J. M. D. (1928). Changes in osmotic pressure and water content of crabs during moult cycle. Physiol. Zool. 1, GILBERT, A. B. (1959a). The composition of blood of the shore crab Carcinus moenas Pennant in relation to sex and size. I. Blood conductivity and freezing point depressions. J. Exp. Biol. 36, GILBERT, A. B. (19596). The composition of blood of the shore crab Carcinus moenas Pennant in relation to sex and size. II. Blood chloride and sulphate. J. Exp. Biol. 36, GILBERT, A. B. (1959c). The composition of blood of the shore crab Carcinus moenas Pennant in relation to sex and size. III. Blood non-protein nitrogen. J. Exp. Biol. 36, HAWK, P. B., OSER, B. C. & SUMMERSON, W. H. (1954). Practical Physiological Chemistry. McGraw Hill Book Company. New York. INDIRA, T. (i960). Permeability of a parasitic ciliate (Opalina) without a contractile vacuole. J. Anim Morph. Phsyiol. 7, KROGH, A. (1939). Osmotic Regulation in Aquatic Animals. Cambridge University Press. PANIKKAR, N. K. (1941). Osmotic regulation in some palaemonid prawns. J. mar. Biol. Ass. U.K. 2. 3I7-59- PARRY, G. (1957). Osmoregulation in some freshwater prawns. J. Exp. Biol. 34, PROSSER, L. G. et al. (1950). Comparative Animal Physiology. Philadelphia: W. B. Saunders Co. ROBERTSON, J. D. (1937). Some features of the calcium metabolism of the shore crab (Carcinus moenas Pennant). Proc. Roy. Soc. B, 12, ROBERTSON, J. D. & WEBB, D. A. (1939). The microestimation of sodium, potassium, calcium, magnesium, chloride and sulphate in sea water and body fluids of marine animals. J. Exp. Biol. 16,

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