PHYSIOLOGY OF LOW TEMPERATURE ACCLIMATION IN TROPICAL POIKILOTHERMS. BY KANDULA PAldPAPATHI RAO

Transcription

1 PHYSIOLOGY OF LOW TEMPERATURE ACCLIMATION IN TROPICAL POIKILOTHERMS I. Ionic Changes in the Blood of the Freshwater Mussel, Lamellfdens marginalis, and the Earthworm, LampRo mauritii BY KANDULA PAldPAPATHI RAO (Department of Zoology, Sri Venkateswara University, Tirupati, India) Received February 25, 1963 (Communicated by Dr. H. Srinivasa Rao, r.a.sc.) INTRODUCTION SINCE the pioneering studies of Fox (1936, 1939) a wide variety of poikilotherms have been shown to be capable of compensating for changes in temperature in their activity and metabolism (Bullock, 1955; Precht, Christophersen and Hensel, 1955). The mechanism or mechanisms involved in this compensation are not well understood, although changes at the cellular level and in the activity of enzymes have been reported (Precht et al., 1955; Kanungo and Prosser, 1959). Further, it was recently discovered in our laboratory that acclimation to high temperature results in a change in the concentration of some of the ionic constituents of the blood in the freshwater mussel and in a freshwater crab (Pampapathi Rao and Ramachandra, 1961). Since changes in the ratios of ions in the fluid bathing the tissues can have considerable influence on the metabolism of the cells, the problem was pursued further and it was soon discovered that such changes in ions occur not only on warm acclimation but also on cold acclimation and that the changes noticed in laboratory-acclimated animals parallel those that occur with seasonal acclimation in natural populations. It was noticed that these systemic changes are not confined only to the inorganic ions, but are also found with reference to the free amino-acids in the blood, which decrease on cold acclimation. This observation prompted the following detailed investigation, the results of which are reported below in a series, which follows the course of the investigation. These results, as will be discussed later, open up one approach towards an explanation of the phenomena involved in temperature compensation in the metabolism of poikilotherms. Evidence will be presented in the subsequent papers of this series, which shows that the changes in free amino-acids might reflect increased protein synthesis, in acclimation to low temperature, which is triggered by hormonal agents. 290

2 Low Temperature Acclimation in Tropical Poikilotherms--I 291 MATERIALS AND METHODS Freshwater mussels (Lamellidens marginalis) were collected from a stream near Tirupati. In the laboratory they were kept in troughs of tap-water, some at 29 C. (room temperature) and the others at 19 C. (low temperature) in a thermostatically controlled (accurate to ~- 0-5 C.) water-bath. Water was changed daily and the mussels were exposed to normal day and night conditions. They were acclimated to these conditions for four weeks, before any of them were used for analysis. Before drawing the blood for analysis,,ach mussel was weighed to the nearest decigram, after removing the shell (i.e., wet weight of the soft parts). Blood was drawn from the heart directly with a syringe. Special care was taken to avoid contamination due to pericardial fluid. Chloride was estimated in 0" 20 ml. samples using the potentiometric microtitrator (Gallenkamp, England) which is accurate to 1 mm./litre. Free amino-acid content was determined on deproteinized blood by the Folin-Danielson method (Hawk, Oser and Summerson, 1954) using a photoelectric colorimeter. Sodium, potassium and calcium in the blood were determined by flame photometry using a Zeiss Flame Photometer (Model Ill). The solutions were prepared according to the procedures given in Hawk, Oser and Summerson (1954). Earthworms (Lampito mauritii) collected near Tirupati (always from the same locality) were kept in the laboratory partially submerged under water. Sheets of blotting paper were provided between which the worms remained most of the time. Like the mussels, the worms also were kept at two temperatures (29 and 19 C.). The body fluids of the worms were collected in the following manner. The worm was gently blotted to remove the water on its surface and was then cut into pieces and the gut contents were allowed to drop out. These cut pieces were then squeezed by hand in a muslin cloth or fine wire-mesh to force out the coelomic fluid. The fluid thus collected was used for the estimation of chloride, amino-acids, sodium, potassium, calcium and magnesium. The methods used were the same for the mussel, except that magnesium (which was not determined for the mussel blood) was estimated by the method of Hasegawa as given in Milton and Waters (1955). In all eases several determinations were made using animals of a wide size range. RESULTS The freshwater mussel, Lamellidens marginalis.--the results of the analysis are presented in Fig. 1. It is seen that on acclimation to cold there B2

3 292 KANDULA PAMPAPATHI RAO is decrease in the free amino-acids in the blood, as also a decrease in the chloride content. On the other hand the calcium content of the blood shows a remarkable increase on cold acclimation. ~16 o o o C o oo o o o ** o _~1~I 0% 0 o E ~,12 ~ o i 08: D ~6i ~d, t- "0 ""r't~ --QOB -- I tv v I I t ~ I t ~6[ ~..... o oos t- o o ' "~" / ~ 0 _0 0 ~4r oo o ~o o n3~ " d[~ o *...,'..-..'"..'"...,.:.''-- WEIGHT OF THE HUSSEL IN GRAHS FIG. 1. Concentration of ions in the blood of cold (19 C.) acclimated freshwater mussel compared with normal (29 C.) mussels. Closed circles for cold-acclimated individuals and open circles for normal individuals. (A) Blood chloride. (B) Amino-acids. ((2)Potassium. (D) Sodium. (E) Calcium. Potassium and sodium do not show any significant change, although there is a slight tendency for an increase in both these constituents in coldacclimated individuals. The earthworm, Lampito mauritii.--it is seen from Table I that in the earthworm chloride and amino-acid values show a decrease on cold acclimation as in the mussel. But the increase in sodium and potassium on cold acclimation is very distinct, while the increase in calcium, although quite evident, is not as great as in the case of the mussel. Another noteworthy feature is the decrease of magnesium in cold-acclimated worms. Analysis of the body fluids of worms collected in mid-winter (January) and mid-summer (June) showed changes in the concentration of ions paralleling those obtained in laboratory-acclimated worms. That is, there is decrease in calcium, chloride, magnesium and amino-acids in the winter worms and an increase in sodium and potassium (Saroja, 1962).

4 Low Temperature Acclimation in Tropical Poikilotherms--I 293 TABLE I Changes in the concentration of free amino-acids and certain inorganic ions in the body fluids of the earthworm, Lampito mauritii on acclimation to low temperature JAil figures are in rnm./l., except free amino-acids wl-.ich are expressed as rob./100 ml. For each ion (both for normal and cold) not less than 18 individuals were analysed.] Normal Cold Ion estimated 29 C. acclimated 19 C. Free amino-acids Chloride Magnesium Potassium Sodium Calcium t / ~ bl ' :k "642 DISCUSSION The results presented above indicate considerable changes in the concentration of different ions in the blood or body fluids upon acclimation to low temperature and these changes show considerable similarity in the two organisms studied. Similar changes, but in the opposite direction, on warm acclimation have earlier been reported from this laboratory for only certain ions (chloride and amino-acids) in the mussel (Pampapathi Rao and Ramachandra, 1961; Pampapathi Rao, 1962 b) and in a fish (Pampapathi Rao, 1962 a; Selvarajan, 1962). Acclimation to temperature resulting in a change in the rate of metabolism must necessarily depend on organ and cellular changes. Since the changes reported above are occurring in the body fluids, which form the environment of the cells (the cells being bathed in this fluid), such changes would necessarily influence to some degree the metabolism of the cells. It would, therefore, be of interest to discuss the possible effects of such ionic changes on the cells.

5 294 KANDULA PAMPAPATlll RAO The changes in the calcium concentration in the body fluids are of importance since calcium plays an important role in several metabolic processes such as membrane permeability, protein binding (Walser, 1960) and even the regulation of muscle membrane potential in relation to temperature (Apter and Koketsu, 1960). Likewise potassium is also known to have a great influence on muscle metabolism. An increase in potassium in the fluids surrounding muscles results in a noticeable increase in the resting metabolism, and such metabolic effects are of relatively long duration. Recently it has been shown by Kaye and Mommaerts (1960) that the activation of glycolysis of muscle by potassium is modulated by calcium. Therefore, an increase in potassium in the body fluids of cold-acclimated forms will tend to increase muscle metabolism and this effect will be augmented if the calcium concentration also increases, as it does in the cold-acclimated individuals. Comparing different crustaceans, Robertson (1957) has indicated a correlation between the blood magnesium level and the activity of the animal, the more active species having lower blood magnesium levels. It is well known that an increase in magnesiu m in the bathing fluid reduces activity. Hence a decrease in magnesium level in the body fluids of cold-acclimated individuals, as it does occur in the earthworm, is an advantage in as much as it permits increased activity and metabolism. A decrease in chloride level in the body fluids, on cold acclimation and vice versa, has been consistently noticed (Pampapathi Rao and Ramachandra, 1961; Pampapathi Rao, 1962 b; Selvarajan, 1962; Saroja, 1962), with the exception of the crab, Paratelphusa sp. (Pampapathi Rao and Ramachandra, 1961). However the metabolic significance of such a change in chloride is not at present clear. Likewise the significance of the slight increase in the concentration of sodium in cold-acclimated individuals is not very clear. Perhaps this has some relation to the protein binding capacity of the calcium in the fluids (Walser, t960), and might also influence nerve activity. A noteworthy change that has been reported above is the decrease in free amino-acids on cold acclimation. This decrease is quite discrete. It is possible that this decrease indicates an increased incorporation of aminoacids into the tissues, resulting from greater protein synthesis during cold acclimation. This possibility was investigated and the results are presented in a subsequent paper of this series, which presents some evidence for greater accumulation of protein in cells on cold acclimation.

6 Low Temperature Acclimation in Tropical Poikilotherms--I 295 SUMMARY On cold acclimation, mussels show a decrease in the blood chloride and free amino-acids, while blood calcium increases. A slight increase in blood sodium and potassium is also noticeable. Cold-acclimated earthworms also show similar changes except that the increase in Na and K is more conspicuous than in the mussel. In addition to this, magnesium in the body fluids also decreases. These changes are discussed and it is suggested that the ionic changes influence muscle activity and metabolism while the decrease in the amino-acids might be a result of increased degree of protein synthesis. REFEREN CES Apter, J. T. and Koketsu, K. Bullock, T. H. Fox, H. M. Hawk, P. B., Oser, B. C. and Summerson, W.H... Kanungo, M. S. and Prosser,.. C.L. Kaye, L. and Mommaerts, W. F. H. M. Milton, R. F. and Waters, W. A. Pampapathi Rao, K and Ramaehandra, R. "'Temperature studies implicating calcium in regulation of muscle membrane potential," J. Cell. Comp. Physiol., 1960, 56, "'Compensation for temperature in the metabolism and activity of poikilotherms," Biol. Rev., 30, 1955, "The activity and metabolism of poikilothermal animals in different latitudes, I," Proc. Zool. Soc., London, 1936, 106 A, "'The activity and metabolism of poikilothermal animals in different latitudes--v," 1bid., 1939, 109A, Practical Physiological Chemistry, McGraw-Hill Book Company, New York "Physiological and biochemical adaptation of goldfish to cold and warm temperatures. 1I. Oxygen consumrtion of liver homogenate; oxygen consumption and oxidztive phosphorylation of liver mitochondria," J. Cell. Comp. PhysioL, 1959, 54, "The role of calcium ions in the acceleration of resting muscle glycolysis by extracellular potassium," J. Gem PhysioL. 1960, 44, Methods of Quantitative Microanalysi,~, Edwald Arnod, London, "~Adaptive physiology of the fish, Etroplus maculatus," Bull. Biol., 1962 a,l, 1-5. "Metabolism of tropical poikilotlctms witl~ special refier nce to temperature acclimation," Pro('. Stmmler Sch.ol in Zoology, Simla, 1962b (In press). "Effect of acclimatization to high temperature on the blood chloride, free amino-acids and osmotic pressure m tt~e freshwater field crab, Paratelphusa sp., and the freshwater mussel, Lamellidens marginalia,'" J. Exp. Biol., 1961, 311,

7 296 KANDULA PAMPAPATHI RAO Robertson, J, D. Saroja, K. Selvarajan, V. R... "Osmotic and ionic regulation in aquatic inv rtcbrates." In Recent Advances in Invertebrate Physiology," Eugen : University of Oregon Publications, "Studies on the oxygen consumption in earthworms with special reference to temperature acclimation," Ph.D. Thesis, Sri Venkateswara University, Tirupati, "Chloride regulation in a freshwater fish, Cirrhina reba, under heterosmotic conditions and high temperature," Prec. Ind. Acad. Set., 1962, 55B."