Biochemical Systematics and Ecology, VoL 14, No. 4, pp. 439-443, 1986. 0305-1978/86 $3.00+0.00 Printed in Great Britain. © 1986 Pergamon Journals Ltd.

Effect of Environmental Acidity and Alkalinity on the Physiology of Tilapia mossambica During Acclimation

M. BHASKAR and S. GOVINDAPPA Fish Physilogy Division, Department of Zoology, S. V. University, Tirupati, 517 502, A.P., India

Key Word Index--Ti/apia rnossambica; carbohydrate metabolism; effect of environmental pH; glycogen; glycolysis; phosphorylase.

Abstract--The pattern of carbohydrate metabolism in the muscle of the freshwater fish, Tilapia mossambica (Peters) varied according to the pH of the environmental medium. On acclimation to a more acidic water, the white muscle showed an elevated glycogen content with suppressed glycolysis. In contrast, the muscle tissue showed an accelerated glycolytic pathway with the accumulation of metabolic acids on acclimation to a basic water. In an acidic medium phosphorylase activity was inhibited with an elevated LDH activity, while the reverse pattern was observed in an alkaline medium. The oxidative pathway was elevated in both. Acclimation to acidic and basic environmental waters leads to an adaptive compensatory mechanisms providing increased resistance capaci~ to the fish under pH stress.

Introduction Freshwater fish encounter alterations in the pH of the water due to influence of environmental pollution, addition of industrial effluents, hot springs, volcanic lakes, mine drainages and geological alterations [1, 2]. These pH changes diminish the wild fish populations in many fresh- water lakes, streams and rivers [3, 4]. In recent years inland water bodies have been encoun- tered with rather extreme changes in hydrogen ion concentrations in different parts of the world including Norway [5], Sweden [3, 6], Canada [7], the United States of America [4] and India [8, 9].

Glycogen metabolism is reported to be highly sensitive to various stress conditions and tissue glycogen reserves are mobilized when animals were subjected to stress conditions [10, 11]. Since H ÷ ion concentration in their water leads to changes in various physiological mechanisms of aquatic animals [12-15] and is recognized as a stress condition, it is likely that the tissue glyco- lytic mechanism might be modulated when the fish was subjected to altered environmental hydrogen ion concentration. Hence the glycogen metabolism has been studied in the white muscle of fish on acclimation to sub-lethal acidic (pH 5.0) and alkaline (pH 9.0) environments.

(Received 21 February 1986)

Results The data obtained from the white muscles of the fish [18-29] are presented in Tables 1 and 2 (see Experimental for details). The white muscle of the fish recorded considerable elevation in glycogen content over the control (-t-20%) on acclimation to the acidic medium (pH 5.0). The activity levels of phosphorylases were depleted (a, -22%; b, -55%; ab, -53%) over the control. The free glucose content was significantly decreased (--26%). The levels of pyruvic acid (--25%) and lactic acid (--20%) were depleted. The activity level of aldolase was highly inhibited (--34%) while that of glucose-6-phosphate

dehydrogenase (G-6-PD) activity was elevated (-I-57%) over the control. The levels of lactate dehydrogenase (NAD-LDH, -I-40%), succinate dehydrogenase (SDH, -t-33%) and malate dehydrogenase (MDH, +65%) activities were significantly elevated in the white muscle on acclimation to pH 5.0.

When the fish was acclimated to the alkaline medium (pH 9.0), the white muscle showed differential pattern of glycogen metabolism from the controls.Glycogen content recorded (--19%) depletion over the control with elevated phosphorylases [active (a), +11%; inactive (b) +37%; and total (ab) +20%] activities. Glucose content (-21%) and aldolase activity (+30%) were depleted. The organic acids, pyruvic acid

439

440 M. BHASKAR AND S. GOVlNDAPPA

TABLE 1. CHANGES IN THE LEVELS OF GLYCOGEN, GLUCOSE, LACTIC ACID, PYRUVlC ACID AND THE ACTIVITY LEVELS OF ENZYMES LIKE PHOS- PHORYLASE a, ab, b AND ALDOLASE IN THE WHITE MUSCLE OF CONTROL AND EXPERIMENTAL FISH

Component Control

Acclimated (15 days)

Acidic Alkaline medium medium (pH 5.0) (pH 9,0)

Glycogen 2.31 2.82 1.92 (mg/g wet wt) _+0.13 _+0,09 _+0.03

( ~ 20%) ( 1 9 % ) P<0.001 P<0.001

Glucose 1.52 1.15 1.24 (mg/g wet wt) ±0.04 ±0.081 ±0.032

( 2 6 % ) ( 2 1 % ) P<~0.001 P-<0.001

Phosphorylase 'a' 2.85 2.23 3.16 ([umol Pi formed/mg protein/h) ±0.21 ±0.26 _+0.22

( 2 1 % ) (~ 11%) P<.0.0Ol P ~-~0.001

Phosphorylase 'b' 12.13 5.49 16.65 (limol Pi formed/mg protein/h) ±0.74 ±0.34 _+ ! .23

( 55%) {! 37%) P<~0.001 P'~0.001

Phosphorylase 'ab' 15.67 7.38 18.82 (p.mol Pi formed/mg protein/h) ±1,12 ±0,32 _+1.13

(-- 53%) ( ~ 20%) P<~0.001 P<~0.O01

Aldolase 35.46 23.44 46,17 (p.rnol of FDP cleaved/mg protein/h) ±2.38 ±2.12 _+2.92

( - 34%) (+ 30%) P<~.0.001 P,<0.001

Pyruvic acid 0.572 0.314 0.714 (p, mol/g wet wt) ± 0.05 ±0.015 _+0.02

(- 25%) (+25%) P<0.001 P<0.001

Lactic acid 2.16 1.73 3.92 (mg/g wet wt) +0.29 ±0.12 +0.16

(-20%) (+81%) P<0.001 P,~0,001

Each value is an average of eight individual observations. Mean + S.D. (percent change respectively over control). 'P" denotes level of statistical significance.

(+25%) and lactic acid (+81%) were increased. G-6-PD activity was inhibited (--30%). NAD-LDH (-11%) and MDH (--9%) activities were inhibited, while the SDH activity was signifi- cantly (+67%) elevated in the white muscle on acclimation to pH 9.0.

Discussion The white muscle had an elevated glycogen content compared with the control on acclima- tion to pH 5.0. The elevated glycogen content might be due to increased glycogenesis or/and

decreased glycogenolysis. The activity levels of phosphorylases were below the control level suggesting that there was inhibition of the pro- cess of glycogenolysis. Such an inhibition of the phosphorylase system could have been partly responsible for sparing of glycogen in the tissue. Consequently, the free glucose was decreased over the control level. This could have been due to decreased formation from the glycogen as revealed by the inhibited phosphorylase activity and/or utilization of glucose towards glycogen synthesis and glycolysis. Since aldolase activity

EFFECT OF ACIDITY AND ALKALINITY ON TILAPIA MOSSAMBICA PHYSIOLOGY 441

TABLE 2. CHANGES IN THE ACTIVITY LEVELS OF ENZYMES LIKE G-6-PD, SDH, NAD-LDH AND MDH IN THE WHITE MUSCLE OF CONTROL AND

EXPERIMENTAL FISH

Component Control

Acclimated (15 days)

Acidic Alkaline medium medium (pH 5.0) (pH 9.0)

G-6-PDH 0.469 0.738 0.33 (pmol formazan formed/mg protein/h) +0.032 :L0.04 +0.012

(+ 57%) (-- 30%) P<0.001 P<0.001

NAD-LDH 0.172 0.24 0.153 (pmol formazan formed/mg protein/h) +0.005 +0.013 +0.004

(+40%) (-- 11%) P<O.001 P<0.001

SDH 0.255 0.339 0.425 (pmol of formazan formed/rag protein/h) +0.022 +0.003 +0.017

(+33%) (+67%) P<O.O01 P<O.O01

MDH 0.023 0.038 0.025 (pmol of forrnazan formed/rag protein/h) +0,002 +0.004 +0.001

(+65%) (+9%) P<O.O01 P<O.001

Each value is an average of eight individual observations. Mean +S.D.; + and - indicate percent increase and decrease respectively over control. 'P' denotes level of statistical significance.

was inhibited, utilization of glucose through glycolysis did not seem possible, and the decreased levels of lactic acid and pyruvic acids in the white muscle support this. Since the activity level of glucose-6-phosphate dehydro- genase (G-6-PD) was significantly elevated, muscle tissue diverted free glucose into the hexose monophosphate pathway which could decrease the formation of intermediary organic acids like pyruvic and lactic acids in the muscle. Elevated activity level of lactate dehydrogenase (NAD-LDH) was suggestive of increased mobilization of lactic acid into oxidative metabolism. Consequent on such changes, there was not only a depleted lactic acid level in the tissue, but also an elevated oxidative phase of metabolism as indicated by succinate and malate dehydrogenase (SDH and MDH) activities. In view of decreased blood pH under acidic stress [30-33], decreased formation of metabolic acids might be an adaptive feature as a result of compensatory changes. Hence this is a clear case of tissue compensation at metabolic level towards the induced acid stress in the medium.

In contrast to the above situation, white muscle glycogen metabolism was quite different when the fish were acclimated to the basic medium (pH 9.0) while muscle glycogen content was depleted, suggesting its active mobilization into tissue metabolism. The depletion of muscle glycogen reflects a state of strenuous activity on the part of the fish [34, 35]. Elevated activities of phosphorylase 'ab' (total), 'a' (active) and 'b' (inactive) in this muscle were suggestive of active glycogenolysis in the tissue. Consequently, the free glucose content should be elevated during the stepped up glyco- genolysis. However the free glucose content was below the normal level suggesting its mobilization into tissue metabolism. Aldolase activity was elevated over the normal level indicating a stepped up hexose diphosphate pathway in the tissue. The levels of pyruvic acid and lactic acid were higher along with inhibited LDH activity, showing an increase in glycolysis to produce organic acids in the body which buffer the alkalinity of the blood under basic stress. G-6-PD activity was inhibited, and a decreased operation of hexose monophosphate

442 M. BHASKAR AND S. GOVIINDAPPA

p a t h w a y m i g h t b e e x p e c t e d . SDH a n d M D H

ac t i v i t i es w e r e e l e v a t e d s u g g e s t i n g the p o s s i b l e

e l e v a t i o n in t he o p e r a t i o n o f t he c i t r ic ac id cycle.

T h e s e m e t a b o l i c t i ssue c o m p e n s a t o r y

m e c h a n i s m s m i g h t lead to a d a p t i v e c h a n g e s

p r o v i d i n g p o s i t i v e su rv i va l v a l u e to the f ish in

a l t e red pH m e d i a .

Experimental Freshwater fish, Tilapia mosambica (Peters) of 12__+1 g weight, were acclimatized to the laboratory conditions in large aquaria with flowing dechlorinated water (25 °, pH 7.0+0.2 and light period of 12 h). They were fed with commercial fish pellets.

Experimentation procedures. The fish were divided into three groups. The first group was maintained in normal tap water at pH 7.0+0.2 (controls). The second and third groups were acclimated to acidic medium (pH 5.0+0.1) and an alkaline medium (pH 9.0_+0.1) for 15 days respectively [16]. Since the fish exhibited stablized oxygen consumption from 12 days onwards, the period of 15 days was taken as the acclimation period [16].

Homogenate preparations. Control and experimental fishes were sacrificed separately and white muscle tissues were isolated from the lateral line of fish at 10 = and kept at 0 °. Tissues were minced and homogenized in 10% trichloroacetic acid for estimation of glycogen, lactic and pyruvic acids and in 80% MeOH for glucose. Phosphorylase homogenate was prepared in a buffer containing 0.1 M NaF and 0.037 M EDTA (pH 6.5) [17]. Homogenates for the estimation of aldolase activity were prepared in H20 and those used for the assay of dehydrogenases in 0.25 M sucrose.

Assay methods. Free glucose [18], glycogen [19], lactic acid [20, 21], soluble proteins [22] and pyruvic acid [23] were estimated in the white muscle tissues of control and experimental fish.

Glycogen phosphorylase (EC 2.4.1.1.) [24] was assayed by the determination of the amount of inorganic phosphate (Pi) formed from glucose-l-phosphate. 0.4 ml of the diluted enzyme was incubated with 2 mg of glycogen for 20 min at 35 °. The reaction was started by the addition of 0.2 ml of 0.016 M glucose-l-phosphate (G-l-P) to one tube (phosphorylase 'a'), and to the other a mixture of 0.2 ml of G-1-P and 0.004 M adenosine-5-monophosphate (phosphorylase 'ab'). The reaction mixture was incubated for 15 min for phosphorylase 'ab' (total) and for 30 min for phosphorylase 'a' (active). The reaction was arrested by the addition of 5 ml of 5 N H2SO 4 and the inorganic Pi was esemated [25].

Aldolase (EC 4.1.2.13) was assayed by the colorimetric method [26], in which the triose phosphates formed were estimated with 2,4-dinitrophenylhydrazine. The incubation mixture in a final volume of 3 ml contained 1.75 ml of collidine-hydrazine buffer (pH 7.4), 0.25 ml of fructose- 1,6-diphosphate (0.1 M, pH 7.4) and 1 ml of enzyme. The mixture was incubated at 37 ~ for 15 min and the reaction was arrested by the addition of 3 ml of cold 10% trichloroacetic acid.

Glucose-6-phosphate dehydrogenase (G-6-PD) (EC 1.1.1.49) [27]: The reaction mixture (2.5 ml) consisted of 20 p.mol of glucose-6-phosphate (disodium salt) 0.5 p, mol of NADP, t0 p, mol of triethanol amine buffer (pH 6.8) 4 p, rnol of INT and 0.5

ml of the enzyme. The reaction mixture was incubated at 37" for 30 min and the reaction was stopped by the addition of 6 ml of HOAc. The formazan formed was extracted into 6 ml of toluene overnight. The activity levels of lactate dehydro- genase (LDH, EC 1.1.1.27) [28] malate dehydrogenase (MDH, EC 1.1.1.37) [28] and succinate dehydrogenase (SDH, EC 1.3.99.1) [29] were estimated similarly.

Acknowledgements--Dr. M. Bhaskar expresses his gratitude to the University Grants Commission, New Delhi, for awarding Research Associateship during the tenure of which the present work was carried out.

References 1. Beamish, R. J., (1972) Trans. Am. Fish. Soc. 101, 355. 2. McKee, J. E. and Wolf, H. W. (1963) Calif Water Quality

Control Board. Pub/. 3A, 41. 3. Almer, B., Dickson, W., Ekstrom, C., Hornstrom, E, and

Miller, U. (1974) Ambio 3, 30. 4. Schofield, C. L. (1975) Lake acidification in the Adirondack

mountains of New York: Causes and consequences. Proc. of the First Int. Symposium on Acid Precip. and Forest ecosystems. Ohio State Uni. Columbus, OH.

5. Jensen, K. W. and Snekvik, E. (1972) Ambio 1, 223 6. Almer, B. (1972) Inf Inst. Fresh Water Res. 12, 47. 7. Beamish., R. J. and Harvey, H. H. (t972) J. Fish Res. Bd Can.

29, 1131. 8. Karuppasamy, P. (1979) Mar. F~sh. Inf. Serv. 7, 11. 9. Pillai, V. K., Ponnaiah, A. G., Vincent, D. and Raj, I. D. (1983)

Mar Fish. Infor. Ser~. 53, 8. 10. Hochachka, P. W., Fields, J. and Mustafa, T. (1973) Am.

Zool. 13, 543. 11. Umminger, B. L. (1970) J. Exp. Zool. 173, 159. 12 Bhaskar. M., Vemananda Reddy, G., Krishna Murthy, V.,

Reddanna, P. and Govindappa, S. (1982) Proc. Ind. Acad. Sci. (Anim. ScL) 91, 235.

13. Daye, P. G. and Garside, E. T. (1976) Can. J. Zoo/. 54, 2140. 14. Hargis, J. R. (1976) J. Exp. Zool. 196, 39. 15. Vaala, S. S. and Mitchill, R. B. (1970) Proc. Pa. Acad. Sci. 44,

41. 16. Murthy, V. K., Reddanna, P. and Govindappa, S. (1981)

Can. J. Zool. 59, 400. 17. Guillory, R. J. and Mommaerts, W. F. H. M. (1982) Biochim.

Biophys. Acta 65, 316 18. Mendel, B., Kemp, A. and Myers, D. K. (1954) Biochem. J.

56, 639. 19. Carroll, N. V., Longlev, R. N. and Roe, J. H. (1956) J. Biol.

Chem. 220, 583. 20. Barker, S. B. and Summerson, W. H. (1941) J. Biol. Chem.

138, 535. 21. Huckabee, W. E. (1956) J. Appl. Physiol. 9, 163. 22. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R.

J. (1951) J. Biol. Chem. 193, 265. 23. Friedemann, T. E. and Haugen, G. E, (1942) J. Biol. Chem.

144, 67. 24. Cori, G. T., Illingworth, B. and Keller, P. G. (1955) in

Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds) Vol 1, p. 200. New York.

25. Tausky, H. M. and Shorr, E. (1953) J. Biol. Chem. 202, 675. 26. Bruns, S. H. and Bergmeyer, H. U. (1965) in Methods of

EFFECT OF ACIDITY AND ALKALINITY ON TILAPIA MOSSAMBICA PHYSIOLOGY 443

Enzymatic Analysis (Bergmeyer, H. U., ed.) p. 724. Academic Press, New York.

27. Georg, W. L. and Waller, H. D. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.) p. 744. Academic Press, New York.

28. Lee, Y. P. and Lardy, H. A. (1965) J. Biol. Chem. 240, 1427. 29. Nachlas, M. M., Margulius, S. P. and Seligman, A. M.

(1965) J. Biol. Chem. 235, 499. 30. Bhaskar, M. (1982) Tissue metabolic profiles of Tilapia

mossambica (Peters) acclimated to sublethal acidic and

alkaline media. Ph.D. Thesis, S.V. University, Tirupati. 31. Dively, J. L., Mudge, J. E., Neff, W. and Anthony, A. (1977)

Comp. Biochem. Physiol. §TA, 347. 32. Neville, C. M. (1979) J. Fish. Res. Bd Can. 36, 84. 33. Packer, R. K. and Dunson, W, A. (1970) J. Exp, Zool. 174,

65. 34. Basha Mohideen, M and Parvatheswara Rao, V. (1972)

Mar. Biol. 16, 68. 35. Black, E. C., Connor, A. R., Lain, K. and Chin, W. (1962) J.

Fish. Res. Bd Can. 19, 409.