Effect of Acclimatization to High Temperature on the Blood Chloride, Free Amino Acids and Osmotic Pressure in the Freshwater Field Crab Paratelphusa Sp. and the Freshwater Mussel Lamellidens Marginalis

It is well established that the metabolism of organisms is size-dependent (Zeuthen, 1955), and that in poikilotherms it is greatly influenced by temperature (Krogh, 1916; Pampapathi-Rao & Bullock, 1954). Further, there is considerable evidence (Bullock 1955) which shows that several poikilotherms can acclimatize to temperature change by suitable compensation in their metabolism. The organ or cellular basis of this homoeostatic mechanism is not well understood, although there is some evidence for compensation at the cellular level and in the activity of enzymes (see Bullock, 1955, for a review).

In homoiosmotic organisms the composition of the blood (or the body fluid as the case may be) is important and is kept relatively constant. It is conceivable that the composition of the internal medium would be an important factor influenced by temperature changes in the environment since temperature would influence the osmotic processes of the organism. There have been few studies on the effect of temperature on osmoregulation and blood composition in invertebrates. One of the earliest studies which show such relationships is that of Loeb & Wasteneys (1912) who showed that Fundulus heteroclitus were better able to withstand osmotic stress at a high temperature if they were acclimatized for some time to that temperature. The studies of Wikgren (1953) are the major contributions in this field. There are some reports which indicate an influence of temperature acclimatization on the blood and blood constituents. For example, the number of red and white corpuscles in some fish is known to change in a regular manner with seasonal change in temperature (Schlicher, 1926; Spoor, 1951). While such studies as the ones cited are not size-controlled and systematic, they indicate sufficiently well that temperature acclimatization involves some changes in the internal medium. The present investigation shows that the osmotic pressure, chloride and free amino acid content of the blood undergo systematic change (under size-controlled conditions) during acclimatization to high temperature in the freshwater field crab, Paratelphusa sp. and the freshwater mussel, Lamellidens marginalis.

Crabs were collected from the paddy fields, south of Tirupati, and were brought to the laboratory as soon as possible, so as to minimize the mortality due to overcrowding. Gravid females and injured animals were discarded. The others were placed in troughs containing water just sufficient to immerse them. The crabs were left in the troughs for 2 or 3 days at the laboratory temperature so that they might become adjusted to the laboratory conditions. In the initial stages the rate of mortality was greater due to the change in the environmental conditions, but gradually the rate of mortality diminished. Once they were adjusted to the laboratory conditions they survived well for considerable lengths of time.

After 2 or 3 days they were transferred in lots of 12 from the laboratory temperature (26±1°C.), to troughs which were gradually heated to the experimental temperature (33 ± 1° C.).

The crabs were kept at the constant temperature of 33 + i° C. for 10 days in order to acclimatize them to that temperature. The water in the trough was changed daily, being replaced with water at the same temperature. At this higher temperature the rate of mortality in the initial stages was high, but gradually it decreased.

The animals were not fed one day prior to experimentation, and it was hoped that variations in their blood composition due to differences in feeding would thus be eliminated (Gilbert, 1959 a, b, c).

After 10 days at 33 ± 1°C., the crabs were used for analysis. Since there is accumulation of calcium just prior to moulting (Robertson, 1937) and since the blood composition varies during the moulting cycle (Baumberger & Olmstead, 1928), measurements of osmotic pressure, etc. were only made on animals in the intermoult stage.

For each experiment crabs were selected so as to cover the maximum possible size range. The crabs were blotted to remove the water adhering to their bodies and were weighed on a Pelouze balance to the nearest 0·5 g. Blood was drawn from them by inserting a heparinized syringe through the arthrodial membrane at the base of one of the legs. The chloride, free amino acids and osmotic pressure of these samples of blood were determined.

Freshwater mussels were collected from a pond nearby, on the eastern side of Tirupati. After they were brought to the laboratory, they received the same treatment as described above for the crabs.

After 12 days at 33 ± 1°C. each mussel was weighed on a Pelouze balance. The wet weight of the soft parts alone was taken into consideration. Blood was drawn from the heart by inserting a syringe into the heart directly, avoiding contamination with the pericardial fluid as far as possible. These blood samples were used for the determination of the chloride and free amino acid contents.

Blood chloride was determined by Sendroy’s method, as modified by Robertson & Webb (1939). One ml. of the sample taken was treated with silver iodate, the displaced iodine was dissolved in potassium iodide and titrated with sodium thiosulphate until two consecutive readings agreed.

Free amino acid content was determined on deproteinized blood by the Folin-Danielson method (Hawk, Oser & Summerson, 1954) using a Duboscq colorimeter. In this method the free amino acids present in the blood react with β-naphtho-quinone-4-sulphonic acid, developing colour.

The osmotic pressure was determined by the well-known Barger’s method (Barger, 1904, as modified by Krogh). The principle is that in an enclosed space solutions of higher concentration grow at the expense of solutions of low concentration.

The blood chloride values are plotted against the body weight on a double logarithmic graph, and these results were compared with the chloride content of the crabs at ordinary temperature, reported by Padmanabhanaidu & Ramamurthy (1961) (Fig. 1 A).

In general, at ordinary temperatures, the females tend to have a higher chloride content than the males. The temperature has a profound effect on the chloride content of the blood. At high temperatures there is an over-all decrease in the chloride content in both the males and females, as compared with the chloride content at ordinary temperatures.

The amino acid values are plotted against the body weight on a double logarithmic graph. A comparison of these values with values obtained from crabs at room temperature shows that on acclimatization to higher temperatures these animals, irrespective of their sex and size, tend to retain a much smaller amino acid content in their blood than at ordinary temperatures (Fig. 1B).

The blood osmotic pressure is expressed as percentage sodium chloride solution. The results are plotted against the body weight on a double logarithmic graph. However, a comparison of these results with measurements made at ordinary temperatures indicates, prima facie, a general tendency for the lowering of the blood osmotic pressure in both the sexes (Fig. 1C).

Fig. 2 shows that the mussel, unlike the crab, shows considerable increase in the chloride and the free amino acids of the blood on acclimatization to high temperature. But the osmotic pressure increases only a little when compared with the values at normal temperatures.

It is seen from the results reported above that acclimatization to high temperature results in a change in the blood composition. The direction of change is not the same in the two species investigated. In the crab the three factors studied show a decrease in concentration, in both the sexes. But the decrease in the total osmotic pressure of the blood does not appear to be as great as would be expected from the combined effects of the decrease in the chloride and in free amino acid content of the blood.

On the other hand there is an increase in all the three factors studied in the freshwater mussel after acclimatization to high temperature. As may be seen from Fig. 2 the increase in free amino acid content of the blood is considerable over the whole size range investigated, while the increase in the chloride content is greater in the larger individuals. However, the total osmotic pressure of the blood does not show any great increase over the normal, and what little increase there is is mainly noticed in the smaller individuals.

It is therefore seen from the above that the changes in the total osmotic pressure with acclimatization to high temperature are not attributable solely to the changes in the chloride and free amino acid content of the blood, irrespective whether these changes involve an increase (as in the mussel) or a decrease (as in the crab). Some other constituents of the blood also appear to be involved in these changes.

Systemic changes in relation to temperature acclimatization have not, hitherto, been investigated in any detail. It is, however, known that in lower vertebrates such as fish even the volume of the red blood cell changes (Kaplan & Crouse, 1956) as well as the number of cells (Schlicher, 1926; Spoor, 1951). Some data also (Cordier & Worbe, 1954) indicate slight increase in permeability in minnows acclimatized to low temperatures. Straub (1957) found a slight shift in the blood dissociation curve of the frog, which might be due to a change in the alkali reserve of the blood. The results presented above from two invertebrates indicate that the systemic changes noticed in the lower vertebrates may be related more basically to changes in the blood composition. While the direction of shift in the blood composition is not the same in the two cases studied above the mechanisms underlying these changes may be the same. The most well-developed hypothesis to account for temperature compensation in the activity of poikilotherms is that of Precht, Christophersen & Hensel (1955). This involves the relation between free and bound water. Increased resistance to thermal stress is readily explained by the reduction of free water. Increased thermal resistance in the active stages of insects is found to be associated with an increased concentration of the haemolymph. The changes in the blood concentration reported here for the mussel and the crab may also be related, although indirectly, to the amount of free-water available in the cells. Since these changes are in the haemolymph, it is apparent that the environment of the cell is also altered as a result of this acclimatization. Therefore, not only the osmotic and ionic relations between the blood and the medium, but also those between the milieu intérieur and the cells are altered. This gradient between the cells and the fluid surrounding them is important in the metabolism of the cells. It is therefore possible that the mechanism of metabolic compensation to temperature involves not only changes in the protein/water relations within the cell itself, but also the ionic and osmotic gradient between the milieu intérieur and the protoplasm of the cells.