The Composition of the Blood of the Shore Crab, Carcinus Moenas (Pennant), in Relation to Sex and Body Size: III. Blood Non-Protein Nitrogen

In a previous communication (Gilbert, 1959a) it was shown that conductivity values for the blood of Carcinus rise with body size until a maximum is reached at a body weight of 35 g. and then steadily fall beyond this weight. Below 35 g. males have a significantly higher conductivity than females. On the other hand, the curve for total osmotic pressure (o.P.) shows no peak at the middle of the range of body size but falls steadily throughout the whole size range, the values for females being consistently lower than for males (Gilbert, 1959 a). Since it was possible to deduce that the relative proportion of the ions remains constant over the whole size range (Gilbert, 1959b), the discrepancy between the ionic composition and the total O.P. must therefore be due to non-electrolyte; the latter should therefore vary inversely with the electrolyte fraction.

The present work reports on the influence of body size and sex on total nonprotein nitrogen (N.P.N.).

Procedures were similar to those already described (Gilbert, 1959a). Blood was removed through a cut in the base of the arthrodial membrane at the base of the large chela using a small glass pipette. Anti-clotting agents were not used.

A semi-micro Kjeldahl method was used for the determination of the N.P.N. Essentially the method was that due to Cole (1944) with some modifications (Shaw & Beadle, 1949). Blood was de-proteinized with tungstic acid, centrifuged, and 2 ml. of supernatant digested in a 6 in. × $34$ in. Pyrex boiling tube in a sand bath at 290 ± 4⁰ C. for 5 hr. The mixture was allowed to cool in a desiccator over concentrated H₂SO₄ to exclude atmospheric ammonia, and the following day the ammonia was distilled from it into O-IN-H₂SO₄ using a micro-burner and condenser. The back titration was carried out with 0·1 N-NaOH using methyl red/brom-cresol green as indicator.

Digestion mixture (1). 150 g. solid K₂SO₄ in 200 ml. distilled water. 400 ml. pure cone. H₂SO₄ was added, followed by 34·4 ml. of a saturated CuSO₄ solution.

Indicator (2). 0·1 % alcoholic solution of methyl red, and 0·1 % aqueous solution of brom-cresol green in the proportion of 1·3 and 0·7 ml. respectively in 100 ml. of the 0·1 N-H₂SO₄.

Sufficient blood could be removed from each crab for two determinations; the mean of these was used in the calculations.

It was unfortunately not possible to investigate the relationship of body size and sexual maturity thoroughly. Note was taken of the dimensions of about 100 berried females which were among the 400-500 females obtained during the course of this work, and the gonads of a random selection of males were examined for traces of mature sperm. The latter examination was unsatisfactory. Accordingly, the male sexual pleopod was removed as close to the base of the limb as possible and weighed after drying in air; in other decapod Crustacea this appendage undergoes a marked change in growth at the onset of sexual maturity (W. Stephenson, personal communication).

The results of the total N.P.N. determinations have been converted to millimoles of nitrogen and plotted against body weight for fifty-nine males in Fig. 1 and fifty-three females in Fig. 2. As expected it can be seen that the curve is roughly the mirror image of that for conductivity, despite the wide scatter of the results. Values for blood N.P.N. fall to a minimum of about 10 mM. for crabs of about 35-40 g. and rise with increasing weight after this region. As in the previous investigation the data for each sex were divided into two groups, above 35 g. and below this weight, and the regression line for each group was calculated by the method of least squares.

The four regression lines, one each for the males and females below 35 g., and one each for the males and females above 35 g., all differ significantly from the horizontal (P < 0·01 for those below 35 g. and P < 0-05 for those above 35 g.). An analysis of covariance showed that the regression coefficients for the lines of negative slope do not differ significantly from each other, nor do those of positive slope (P > 0·05). However, below 35 g. body weight, males have a significantly higher blood N.P.N. than females (P < 0·01); above 35 g. the N.P.N. of males is significantly lower than that of females (P < 0·01).

The data for the berried females is presented as a histogram (Fig. 3). It is clear that the majority of the crabs in berry are to be found in the region 35-55 g. body weight, less than a third of the size-range containing over 80% of the berried females. However, it is also true that a higher proportion of the animals supplied to this laboratory were in this region of the size range. It is clear that the greatest values are obtained for the region 35-55 g. body weight, even when this is allowed for by expressing the results in percentage units for each size interval (Fig. 4). There is an apparent exception for the region 75-85 g., but the values for this size are based on three berried females out of thirteen crabs. I understand that berried females of this body weight are extremely rare so that it seems likely that the higher values in this region are due to chance.

The frequency distribution of berried females shown in the figures could be brought about after the initial onset of sexual activity, either by a variation in the frequency of egg production with size, or by sexual maturity being of a finite duration.

Despite the inadequacies of the method of sampling it does appear that the trough of N.P.N. coincides more or less with the most frequent period of reproductive activity in females.

The data for males is even less complete and gives no more than an indication of the onset of sexual maturity. Due to the shortcomings of the method no figures are presented; however, it did seem that males of 25-35 g. body weight had more mature sperm than those of other weights.

In both cases, therefore, the onset of sexual maturity appears to be in the region of 25-35 g. body weight. Orton (1936) reported that the shore crab reaches sexual maturity at a carapace width of 4-5 cm.; this corresponds to a body weight of 23 g. and the results are in reasonable agreement.

The relationship between log body size and log pleopod weight is shown in Fig. 5. It is clear that in the case of Carcinus there is no change in the growth rate of the pleopod over the size range taken.

It has been shown previously that the ionic content of the blood of the shore crab rises with increasing body weight until a maximum is reached at a weight of about 35 g., and after this decreases with further increase in size (Gilbert, 1959a). Moreover, since it has also been suggested that the relative proportions of the ions must remain constant over the whole size-range (Gilbert, 1959b) it is possible to estimate the total millimolar concentration of the ions at any given weight. By comparison with the results for the freezing-point depression of the blood (Gilbert, 1959a) the millimolar concentration of non-electrolyte can be estimated for any body weight. There is a very close agreement between the calculated figures and those obtained experimentally, except at the two extremes of the size-range. It seems reasonably clear that the concentration of the electrolyte and non-electrolyte in the blood are closely connected. As one fraction increases the other decreases and vice versa, in such a manner that total O.P. remains at a relatively constant level. This suggests that osmotic regulation is important rather than total ionic regulation as postulated by Pantin (1931) and Webb (1940). However, despite the mechanisms for increasing blood electrolyte when the non-electrolyte fraction decreases, the total o.P. is not constant but decreases slightly but steadily with increasing body weight. Moreover, the two sexes differ; females are always hypotonic to the medium and have a significantly lower o.P. than males at any given body weight. At the lower weights males are hypertonic to the medium, at 35 g. they are isotonic, and at higher weights they are hypotonic. Since the control over the electrolyte and nonelectrolyte fractions of the blood seems so subtle and the two mechanisms so closely integrated, it seems remarkable that total O.P. is not maintained constant over the whole size-range. It would be of interest to know what function this variation has.

The frequency distribution of the sort shown in Figs. 3 and 4 could be due to a variety of causes, and we do not know enough about the reproductive physiology of this animal to be able to say anything constructive. But from the present point of view it is likely that, apart from berried females, the females examined in the present work would also show the same frequency distribution of reproductive activity as the sexually mature animals. That is, of the crabs of all sizes whose body fluids were examined, it is likely that a higher proportion of crabs of about 35-55 g. body weight would be about to produce eggs or have already done so. It is the crabs of this body weight that gave lowest values of N.P.N. The same reasoning would apply to males. If this view is correct the curves for the variation of electrolyte and N.P.N. concentration with body weight could be due, at least in part, to the different proportions of reproductive animals at any given weight, and in part to the frequency of egg or sperm production; that is, the peak for the mean electrolyte value and the trough for N.P.N. at 35 g. weight might be due to the relatively high proportion of the animals at this weight with high and low values respectively. But clearly only the study of individual crabs through their growth period could show whether this was so.

1. The influence of sex and body weight on the concentration of the non-protein nitrogen (N.P.N.) in the blood of Carcinus moenas was investigated.

2. Blood N.P.N. decreased with body size in both sexes until a minimum was reached at a body weight of about 35 g. Thereafter it increased with increasing body weight.

3. For body weights less than 35 g. males had higher N.P.N. values than females; above this weight male values were lower. Statistically these differences were highly significant.

4. Frequency distribution of reproductive activity with body size showed peaks which correspond with those for total ionic concentration (Gilbert, 1959a, b) and with the troughs for N.P.N.

5. Results of the present work have been discussed in relation to those reported earlier for conductivity, total o.p., chloride and sulphate (Gilbert, 1959a, b).

I wish to express my thanks to Prof. A. D. Hobson for his consideration and help at all times, and to express my gratitude to Dr C. Ellenby for his untiring encouragement and advice during the course of this work, and for his criticism of this manuscript. I am also indebted to Mr J. Shaw for many suggestions, and to the staff of the Dove Marine Biological Station, Cullercoats.