The Composition of the Blood of the Shore Crab, Carcinus Moenas Pennant, in Relation to Sex and Body Size: I. Blood Conductivity and Freezing-Point Depressions

Although the literature on the ionic and osmotic regulation of Crustacea is extensive (reviewed by Krogh, 1939; Prosser et al. 1950; Beadle, 1957), in only a few cases has any reference been made to the sex and body size of the animals examined. This is surprising, for it is well known that both of these factors have a profound influence on the general metabolism of animals : their influence on the ionic content and total osmotic pressure (O.P.) of the blood of Carcinus moenas is investigated in the present work.

Crabs were obtained each week from the shore close to the Dove Marine Station, Cullercoats. They arrived in this laboratory within a few hours of being collected. Berried females and injured animals were discarded, and the remainder kept in lots of twelve in aquaria containing 40 1. of well-aerated Cullercoats sea water. Under these conditions the crabs remained alive indefinitely. The animals were allowed to remain in the tanks for at least 24 hr., but prior to the experiments they were transferred in lots of three to similar aquaria, where they remained for 48 hr. in order to reach complete ionic and osmotic equilibrium (Margaria, 1931). Since Bateman (1933) has shown that the vapour pressure of the blood of Carcinus remains constant over the temperature range of 2·3−15·7° C. the aquaria were maintained between 7·11° C. by mains water circulating through coils of polythene tubing.

The animals were not fed while in the laboratory and it was hoped that variations in their blood composition due to differences in feeding would thus be eliminated.

The experiments were begun in 1953, and since it was clear that they would extend over a considerable time they were designed so as to reduce any seasonal effects which might be operating. On any one day the crabs were chosen from the widest size-range possible, including at least one of each sex from the following arbitrary size groups, 0—15, 30—45, 60—75 g-weight. The whole size range was therefore continually covered and any seasonal effect would thus apply equally over each part of the range.

Since the blood composition varies during the moult cycle (Baumberger & Olmstead, 1928), and since particularly just prior to the moult there is a considerable increase in the calcium content of the blood (Robertson 1937), measurements of total O.P. and conductivity were confined to animals in the intermoult stage. This was achieved by discarding crabs obviously about to moult and by only using blood that was pale and clear, since towards the end of the intermoult stage there is an accumulation of red pigment present in the blood.

Crabs were removed from the aquaria, lightly blotted to remove surplus water and weighed to the nearest 0-5 g. on a Tower’s sliding weight balance. Carapace lengths and widths were then measured with vernier callipers.

Blood was removed through a small incision in the arthrodial membrane at the base of the large chela. Using a glass pipette and rubber teat it was possible to obtain sufficient blood from a single crab for both the conductivity and total O.P. determinations. For the O.P. determinations the blood was placed in heparinized tubes to prevent clotting: this was unnecessary for the conductivity measurements since, by reason of the low resistance of whole blood, it had to be used diluted.

A small measured volume of about 0·06–0·07 ml. was removed with a standardized Pyrex capillary pipette from 10 ml. of distilled water; this was replaced with an equal volume of blood, thus diluting the blood about 150-fold. The conductivity was then determined with an EEL conductance bridge and, since dilute blood was used, the conductivity value was compared with that for a standard sodium chloride solution.

There were slight variations in the temperature of the laboratory during the course of the work; this would affect the conductivity values. However, since the whole size range of both sexes was covered each day, this would only influence the variation in the data, but not the essentially comparative nature of the present work.

Freezing-points of 1 ml. samples of heparinized blood were determined by the method of Johlin (1931); the freezing-bath being stirred by hand. Three determinations were made on each sample with a Beckman thermometer reading to ± 0-003° C. and the mean evaluated. Measurements with distilled water were carried out each day to correct for fluctuations in the thermometer zero.

The quantity of heparin used had a barely detectable effect on O.P.

In Fig. 1 carapace width has been plotted against body weight on a double logarithmic grid for 101 female crabs varying in weight from 2-75 to 61·5 g. The relationship between carapace width and body weight remains constant. This was also found to be true for the males and for the relationship between body weight and carapace length of both sexes (see also Shen, 1935). Any of these measurements could therefore be used as a standard of body size. For convenience body weight has been used throughout this work.

Conductivity values expressed as grams of sodium chloride per litre have been plotted against body weight for 81 male crabs in Fig. 2 and 106 females in Fig. 3. Despite the wide scatter of the results, it is clear that in both cases the conductivity is not constant over the whole size range. From a value equivalent to about 31g. NaCl/1. for the smallest animals the conductivity rises steadily and reaches a maximum value of about 33 g. NaCl/1. for crabs of 35 g. body weight. After this there is a slower decrease in the conductivity values until they reach about 31·5 g. NaCl/1. for the largest crabs used.

For the statistical analysis the data for each sex was divided into two groups, viz. for those animals over 35 g. body weight and for those animals less than 35g., and the respective regression coefficients calculated by the method of least squares. Although this treatment is somewhat arbitrary, reference to Figs. 2 and 3 will show that it is not unjustified. As would be expected from the figures, all four regression fines, one each for the males and females below 35 g. body weight, and one each for the males and females above 35 g. body weight, differ significantly from the horizontal (P <0·01 in each case). Moreover, covariance analysis showed that the two regression coefficients for the lines of positive slope for male and female crabs below 35 g. body weight do not differ significantly from each other, nor do the two regression coefficients for the lines of negative slope for males and females above 35 g. body weight (P>0·1 in both cases). There was also no difference in the conductivity for male and female crabs below 35 g. body weight (P > 0·05). However, in the case of animals over 35 g. body weight the conductivity of the blood of the males tended to be higher than that of the females: this difference is highly significant (P < 0·01).

For convenience, values of the freezing-point depressions were converted to a millimolar basis using the relationship that a molar concentration of a non-electro-yte has a freezing-point depression of 1·86° C. These values have been plotted against body weight in Fig. 4 for forty-one males and Fig. 5 for thirty-nine female crabs. In both sexes there is a marked tendency for the values to decrease with increasing body weight, the regression coefficients being −0·283 and −0·202 for the males and females, respectively. With standard errors of +0·10 and +0·061, respectively, both lines differ significantly from the horizontal (P < 0·01 in both cases). There is no significant difference between the regression coefficients for the two sexes p<0·05. However, the O.P. of the males tended to be significantly higher than that of ffie females over the whole size range ; covariance analysis shows that this difference is highly significant (P < 0·01).

It has been shown that the blood composition of the common shore crab varies in the following ways. The O.P. decreases with increasing body weight in both sexes; the male tending to have a higher O.P. than the female. In both sexes the conductivity of the blood of the smallest and the larger crabs is lower than that of crabs of 35 g. body weight. For animals over 35 g. body weight the conductivity values for the blood of the males are significantly higher than those of the females.

These differences in the O.P. of the blood may resolve the controversy as to whether Carcinus is hypotonic, isotonic or hypertonic (Duval, 1925; Nagel, 1934; Schlieper, 1929; Picken, 1936), since the majority of these workers did not record the size or the sex of the animals they used. Moreover, observations of Panikkar (1941) on Leander suggest that a variation of total O.P. may not be true of Carcinus alone.

Clearly, crabs of different size and sex must have different ionic and osmotic relationships with the environment. In the case of the small males the blood is hypertonic, and in the large crabs hypotonic, to the environment, while the females are hypotonic over the whole size range. This may be of importance for the life of a littoral and estuarine animal, and of ecological significance.

Since the conductivity of a solution depends on the charge and the mobility of each ion present, the differences in the blood conductivity could be due either to a variation in the total ions or to a change in the relative proportions of the different ions. Although the observations on the O.P. suggest that the latter is more likely to be correct, the O.P. of the blood is not entirely dependent on the ionic content, and as will be shown in due course the non-electrolyte fraction cannot be disregarded.

It is becoming increasingly apparent that body size is of great importance in zoological investigations. Its effect on oxygen consumption is well known, and recently Parry (1958) has shown that the ability of salmonid fish to withstand dilute sea water is related to body size. Furthermore, both Panikkar (1941) and Bogucki (ref. Beadle 1957) have suggested that some size effect may be operating on the osmotic regulation of Leander and Nereis, respectively. Since size is related to age, the observation of Beadle (1957) on the ‘embryology ‘of osmotic and ionic regulation therefore becomes increasingly significant.

I am indebted to Dr C. Ellenby for his supervision, interest and encouragement throughout this work, and for reading and checking the manuscript for this paper.

I would also like to express my thanks to Prof. A D. Hobson for his consideration and help at all times, to Mr J. Shaw for many useful suggestions and criticisms and to the staff of the Dove Marine Biological Station for the care and attention they paid to the collection of the animals.