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Effects of temperature on Drosophila—VII. Glutamate-aspartate transaminase activity
Comp. Biochem. Physiol., 1970, Vol. 37, pp. 251 to 256. PergamonPress. Printed in Great Britain
E F F E C T S OF T E M P E R A T U R E ON D R O S O P H I L A - - V I I . GLUTAMATE-ASPARTATE TRANSAMINASE ACTIVITY MARY JO BURR and ALICE S. HUNTER Physiology Laboratory, Civil Aeromedical Research Institute, Oklahoma City, Oklahoma (Received 4 May 1970) A b s t r a c t - - 1 . Glutamate-aspartate transaminase activity in homogenates of young adults is reported for stocks of Drosophila melanogaster, D. immigrans, D. pseudoobscura and D. willistoni grown at 15 and 25°C. 2. Female D. melanogaster acclimated at 25°C, have lower glutamateaspartate transaminase activity, measured at 20°C, than do those grown at the "normal" temperature of 15 °C. 3. Glutamate-aspartate transaminase activity of young adult males and females of D. immigrans acclimated at 25°C is lower than that of flies grown at 15 °C when measured at 20°C. 4. Transaminase activity does not vary with acclimation temperature in young adults of the two stenothermal species, D. pseudoobscura and D. willistoni. 5. The data reported support the theory that eurythermal species of Drosophila have greater capacity for physiological adaptation than do stenothermal species. It is theorized that respiratory control in Drosophila may be related to rates of amino acid conversions and the level of substrates entering the Krebs cycle.
INTRODUCTION IT HAS been suggested that eurythermal species of Drosophila (those distributed widely with respect to temperature) have a superior capacity for metabolic adaptation to different temperatures than do stenothermal species (Hunter, 1964). Females of D. melanogaster and both sexes of D. hydei and D. immigrans have a lower rate of respiration when acclimated to a temperature higher than normal (Hunter, 1964, 1965, 1968). These species are widely distributed in regions of both high and low temperature and are considered eurythermal. On the other hand, the stenothermal species studied, D. pseudoobscura, D. viracochi and D. willistoni, do not show adaptive changes in oxygen consumption after acclimation to different temperature (Hunter, 1965, 1966). Attempts to explain the presence or lack of respiratory adaptation on the basis of differences in enzyme activity were unsuccessful. In other poikilotherms examples of changes in enzymes of carbohydrate metabolism with adaptation to different temperatures exist (see, for example, Kanungo & Prosser, 1959; Ekberg, 1962; Hochachka, 1963; Freed, 1965). Of the Krebs cycle reactions studied in Drosophila, however, the variations of oxidative enzyme activities with temperature did not parallel the respiratory changes (Hunter & Cediel, 1970). 251
252
MARY Jo BURRAND ALICE S. HUNTER
Anders et al. (1964) have demonstrated the level of free amino acids to be inversely related to the environmental temperature in D. melanogaster. T h e concentration of glutamic acid, however, decreases with decreasing temperatures. It was therefore felt that,a study of enzymes in amino acid metabolism might yield information on temperature adaptation on the enzyme level in Drosophila and might also help clarify the basis of respiratory adaptation in eurythermal species. T h e present paper reports the activity of glutamate-aspartate transaminase in crude homogenate fractions of two eurythermal species, D. melanogaster and D. immigrans, and of two stenothermal species, D. pseudoobscura and D. willistoni. MATERIALS AND METHODS Descriptions and maintenance of the Drosophila stocks have been reported (Hunter, 1964, 1965, 1968). Young adult flies of the same age as those used in respiration studies were chosen for the enzyme experiments. The flies grown at 25°C were collected at 29-3I days after the parents had been set up, while those at 15°C were used at 50-60 days. Each temperature stock had been growing for several generations since the original collection and no attempt was made to distinguish environmentally induced short-term adaptations from genetic adaptations. The transaminase system employed (Cohen, I955) contained 0'045 mg pyridoxal phosphate, 60/zM DL-aspartic acid, 30/zM ketoglutarate and 1"5 ml of a supernatant fraction of homogenate made up to a total volume of 4"8 ml 0'05 M phosphate buffer, pH 7"6. The appearance of oxaloacetate was determined by the increase in optical density at 280 m/z. The reaction was carried out at 20°C in a Beckman DU Spectrophotometer and was followed for 30 min with readings taken at 5 min intervals. The homogenate was prepared from approximately 24 mg fresh weight of flies in 6 ml of the buffer and was centrifuged 1"5 min to a maximum of 400 g. The resultant supernatant fraction used for the enzyme assay contained 40-50 per cent of the protein of the original homogenate and approximately 70 per cent of the glutamate-aspartate transaminase activity present in the whole homogenate. Similar supernatant fractions were prepared for protein analyses from more concentrated homogenates of sibling flies (70 mg fresh wt./6 ml buffer). The protein fraction obtained after precipitation with perchloric acid was solubilized in hot 1 N NaOH, and the concentration of protein was determined by the Biuret method (Layne, 1957). RESULTS T h e glutamate-aspartate transaminase activity in /xmoles of oxaloacetate p r o d u c e d / m g fresh fly wt. per hour is reported in Table 1 for homogenates of D. melanogaster and D. immigrans and in Table 2 for D. pseucloobscura and D. willistoni. I n general, activity of the enzyme is higher in D. melanogaster than in D. pseudoobscura and D. immigrans. D. willistoni possesses an intermediate level of the transaminase. In the eurythermal species, D. melanogaster, there is no statistically significant difference in the glutamate-aspartate transaminase activity expressed/mg wet wt. of males grown at the different temperatures. Females acclimated at 25°C have a lower rate of transamination than do those grown at 15°C when measured at 20°C (P = 0.02, P = 0-10). Within the two D. melanogaster stocks growing at 15°C there are no significant differences in transaminase activity between the 10pair and the 120-fly stocks.
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I n b o t h sexes o f D. immigrans y o u n g a d u l t s o f t h e 25°C stock have l o w e r activities o f g l u t a m a t e - a s p a r t a t e t r a n s a m i n a s e t h a n d o t h e c o r r e s p o n d i n g 15°C flies ( P = 0.01). A s seen in T a b l e 2 t h e a c t i v i t y o f t h i s e n z y m e does n o t v a r y a d a p t i v e l y w i t h a c c l i m a t i o n t e m p e r a t u r e in t h e t w o s t e n o t h e r m a l s p e c i e s s t u d i e d . TABLE 1--GLUTAMATE-ASPARTATETRANSAMINASEACTIVITY IN EURYTHERMALDrosophila
Stock
Temperature (°C)
Activity (mg/hr) *
/zg Protein nitrogen/mg*
Activity t (rag protein nitrogen/hr)
Sex
N
15 15 15 15 25 25
M F M F M F
10 10 10 10 10 10
0"53 0'49 0.50 0.48 0"56 0"43
(0'06) (0'05) (0.07) (0.06) (0"11) (0"05)
9'4 9'4 9.9 9.7 10'0 10'1
(0"8) (0"8) (1.5) (1.3) (1-7) (1'6)
56 52 51 49 56 43
15 15 25 25
M F M F
7 7 8 8
0'40 0"38 0"32 0'31
(0"01) (0"05) (0"02) (0"03)
8'9 8'1 7"5 7'5
(1"9) (1"0) (0"5) (0"7)
45 47 43 41
D. melanogaster 10 pairs 10 pairs 120 120 5 pairs 5 pairs
D. immigrans 8 pairs 8 pairs 3 pairs 3 pairs
* Figures in parentheses are standard deviations. t Activity is/xmoles oxaloacetate produced/rag protein nitrogen per hour at 20°C. TABLE 2--GLUTAMATE-ASPARTATETRANSAMINASEACTIVITYIN STENOTHERMALDrosophila
Stock
Temperature (°C) Sex
N
Activity (mg/hr) *
/zg Protein nitrogen/mg*
Activity t (rag protein nitrogen/hr)
D. pseudoobscura 10 pairs 10 pairs 5 pairs 5 pairs
15 15 25 25
M F M F
10 10 10 10
0"38 0"36 0"41 0.41
(0-04) (0"05) (0"04) (0.06)
8-4 (1'3) 8"9 (0'8) 9"3 (1'0) 10-3 (2"3)
45 40 44 40
15 15 25 25
M F M F
10 10 10 10
0"48 0"42 0-47 0"41
(0"06) (0'06) (0"14) (0"08)
10'2 (1 '1) 9"9 (0-8) 9'3 (1'7) 9-4 (1'7)
47 42 51 44
D. willistoni 10 pairs 10 pairs 5 pairs 5 pairs
* Figures in parentheses are standard deviations. Activity is /zmoles oxaloacetate produced/mg protein nitrogen per hour at 20°C.
254
MARY
Jo BURR AND ALICE S. HUNTER
Although it is not the purpose of this study, it is of interest to compare the transaminase activity in the two sexes. Male D. melanogaster grown at 25°C contain higher glutamate-aspartate transaminase levels than do females of the same temperature stock (P = 0.01). A similar sex difference is seen in young adults of D. willistoni maintained at 15°C or at 25°C (P = 0.10). In D. melanogaster grown at 15°C, D. immigrans and D. pseudoobscura there are no significant differences in transaminase activity of the two sexes. When glutamate-aspartate transaminase activity is expressed on a protein nitrogen rather than fresh weight basis, the same comparisons can be made of relative activities between flies grown at 15°C and at 25°C and between the sexes as can be seen in Tables 1 and 2. DISCUSSION This is part of an investigation testing the theory that eurythermal species of Drosophila are more capable of temperature adaptation than stenothermal species. Physiological temperature adaptation (which may be environmentally induced or genetic) is interpreted as a shift toward a lower metabolic rate after acclimation to a higher than "normal" temperature and vice versa. The D. melanogaster, D. immigrans and D. pseudoobscura used here were originally collected from the relatively "cool" environment (average temperature 15°C) of Bogotfi. Stocks of each species were maintained at the "normal" temperature of 15°C and others were acclimated at 25°C. The first two species are considered eurythermal and the third is stenothermal. The D. will#toni were collected from a warm environment (temperature 25-30°C) and a stock was maintained at the normal temperature of 25°C and another stock was grown at 15°C. T h e stenothermal species, D. pseudoobscura and D. willistoni, show no indication of temperature adaptation with respect to transaminase activity. With D. melanogaster, the females have significantly lower transaminase activity after growth at the higher temperature of 25°C compared with the stocks maintained at the more "normal" temperature of 15°C. Two different stocks were grown at 15°C because temperature affects inversely the size of flies and those grown under optimal conditions such as the 10-pair stocks are considerably larger than the 25°C grown flies. Therefore crowding (120 flies) was used in order to obtain a fly comparable in size to that grown at 25°C. A comparison of the 120-fly 15°C stock with the 5-pair 25°C stock controls any possible variation in transaminase activity with respect to size of fly. The male D. melanogaster does not give evidence of temperature adaptation with respect to transaminase activity. This point is of interest because similar results were found previously with oxygen consumption; that is, females adapt and males do not (Hunter, 1964). The glutamate-aspartate transaminase activity of D. immigrans acclimated at a higher temperature of 25°C is significantly lower in both sexes than that of the flies maintained at 15°C. This is the same as was found previously with respect to oxygen consumption (Hunter, 1968) and upholds the theory being tested. The present data support a previous suggestion that female and not male D.
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melanogaster undergo the process of temperature adaptation (Hunter, 1964). This does not seem to apply to all eurythermal species of Drosophila since in D. immigrans both sexes show adaptation in transaminase activity and in both D. immigrans and D. hydei both sexes show adaptation in oxygen consumption. Whether it is characteristic of D. melanogaster as a species or only of the Bogot~i stock used in this study is not known. Hunter (1964) suggested that adaptation restricted to organs specific to the female could explain the occurrence of temperature adaptation in the one sex only. Insect transaminases are localized in a variety of organs and on a wet weight basis are particularly active in Malpighian tubules and the fat body (Kilby & Neville, 1957; McAllan & Chefurka, 1961; Chen & BachmannDiem, 1964). A fivefold increase in glutamate-aspartate transaminase activity in whole houseflies during pupal-adult transformation is correlated with elevated levels of the enzyme in thoracic muscle alone (McAllan & Chefurka, 1961). The fact that other tissues examined did not exhibit a concomitant rise in activity indicates that organ specific responses of the transaminase system may occur in insects. Specific changes in metabolism of different tissues with temperature acclimation have been reported for other poikilotherms (Rieck et al., 1960). Cold acclimation results in an elevation of liver and muscle transaminase activities (Harmon, 1963 ; Klain & Vaughan, 1963). Klain & Vaughan (1963) have shown that the increase of transaminase activity in cold-acclimated rats is related to greater food consumption and is substrate induced. This observation suggests the possibility that the basis for higher transaminase levels in cold-grown eurythermal species of Drosophila might also be subtrate induction. Anders et al. (1964) have shown marked increases in the size of the amino acid pool in D. melanogaster larvae and pupae grown at 15°C but the higher concentration of most amino acids is accompanied by a decrease in the glutamic acid content. Perhaps this is related to increased transamination of the glutamic acid which makes availablesubstrates for the Krebs cycle. High transaminase activity may be associated with increased rates of protein synthesis or with an active energy metabolism. In several species of Drosophila the protein content/mg of cold-gro~n flies does not differ significantly from that of flies raised at 25°C (Burr & Hunter, 1969). In the present case a relationship between rates of respiration and transamination seems more plausible. Activities of glutamate-aspartate transaminase in D. pseudoobscura and D. willistoni do not differ significantly with acclimation temperature; no physiological adaptation is observed in these species (Hunter, 1965, 1966). In D. melanogaster and D. immigrans, comparable changes in oxygen consumption and transaminase activity are observed after acclimation to higher temperature (Hunter, 1964, 1968). In general, little evidence was obtained for adaptive changes in respiratory enzymes (Hunter & Cediel, 1970). These facts taken together suggest that respiratory control in Drosophila may be related to variable rates of amino acid conversions and that temperature-dependent modifications in the rate of respiration result from changes in the level of substrates feeding into the Krebs cycle rather than changes in activity of respiratory enzymes.
256
MARY Jo BURR AND ALICE S. HUNTER
REFERENCES ANDERS VON E., DRAWERTF., ANDERSA. & REUTHERK. H. (1964) Ober kausale Zusammenhange zwischen der Zuchttemperatur, dem Aminos~iuren-Pool u n d einigen quantitativen morphologischen Phanen bei Drosophila melanogaster. Z. Naturf. 196, 495-499. BURR M. J. & HUNTER A. S. (1969) Effects of temperature on Drosophila--V. Weight and water, protein and RNA content. Comp. Biochem. Physiol. 29, 647-652. CHEN P. S. & BACHMANN-DIEM C. (1964) Studies on the transamination reactions in the larval fat body of Drosophila melanogaster. J. Insect Physiol. 10, 819-829. COHEN P. P. (1955) Estimation of animal transaminases. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. II, pp. 178-184. Academic Press, New York. EKBERG D. R. (1962) Anaerobic and aerobic metabolism in gills of the crucian carp adapted to high and low temperatures. Comp. Biochem. Physiol. 5, 123-128. FREED J. (1965) Changes in activity of cytochrome oxidase during adaptation of goldfish to different temperatures. Comp. Biochem. Physiol. 14, 651-659. HANNON J. P. (1963) Current status of carbohydrate metabolism in the cold-acclimatized mammal. Fedn Proc. 22, 856-861. HOCHACHKA P. W. & HAYS F. R. (1963) Effect of temperature acclimation on pathways of glucose metabolism in trout. Canad.ff. Zool. 40, 261-270. HUNTER A. S. (1964) Effects of temperature on Drosophila--I. Respiration of D. melanogaster grown at different temperatures. Comp. Biochem. Physiol. 11, 411-417. HUNTER A. S. (1965) Effects of temperature on Drosophila--II. Respiration of D. pseudoobscura and D. viracochi grown at different temperatures. Comp. Biochem, Physiol. 16, 411417. HUNTER A. S. (1966) Effects of temperature on DrosophiIa--III. Respiration of D. willistoni and D. hydei grown at different temperatures. Comp. Biochem. Physiol. 19, 171-177. HUNTER A. S. (1968) Effects of temperature on Drosophila--IV. Adaptation of D. immigrans. Comp. Biochem. Physiol. 24, 327-333. HUNTER A. S. & CEDIEL N. (1970) Effects of temperature on Drosophila--VI. Respiratory enzymes. Comp. Biochem. Physiol. Submitted for publication. KANUNGO M. S. & PROSSER C. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures--II. Oxygen consumption of liver homogenate; oxygen consumption and oxidative phosphorylation of liver mitochondria, ft. cell. comp. Physiol. 54, 265-274. KILBY B. A. ~ NEVILLE E. (1957) Amino acid metabolism in locust tissues. J. Biol. 34, 276289. KLAIN G. J. & VAUGHAN D. A. (1963) Alterations of protein metabolism during coldacclimation. Fedn Proc. 22, 862-867. LAYNE E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. In JVlethods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. III, pp. 4 4 7 4 5 4 . Academic Press, New York. ~IcALLAN J. W. & CHEFURKAW. (1961) Some physiological aspects of glutamate-aspartate transamination in insects. Comp. Biochem. Physiol. 2, 290-299. RIECK A. F., BEOLI J. A. • BLASKOVICSM. E. (1960) Oxygen consumption of whole animal and tissues in temperature acclimated amphibians, Proc. Soc. exp. Biol. Med. 103, 436439.
Key Word Index--Insect biochemistry; Drosophila ; glutamate-aspartate transaminase; temperature adaptation.
E F F E C T S OF T E M P E R A T U R E ON D R O S O P H I L A - - V I I . GLUTAMATE-ASPARTATE TRANSAMINASE ACTIVITY MARY JO BURR and ALICE S. HUNTER Physiology Laboratory, Civil Aeromedical Research Institute, Oklahoma City, Oklahoma (Received 4 May 1970) A b s t r a c t - - 1 . Glutamate-aspartate transaminase activity in homogenates of young adults is reported for stocks of Drosophila melanogaster, D. immigrans, D. pseudoobscura and D. willistoni grown at 15 and 25°C. 2. Female D. melanogaster acclimated at 25°C, have lower glutamateaspartate transaminase activity, measured at 20°C, than do those grown at the "normal" temperature of 15 °C. 3. Glutamate-aspartate transaminase activity of young adult males and females of D. immigrans acclimated at 25°C is lower than that of flies grown at 15 °C when measured at 20°C. 4. Transaminase activity does not vary with acclimation temperature in young adults of the two stenothermal species, D. pseudoobscura and D. willistoni. 5. The data reported support the theory that eurythermal species of Drosophila have greater capacity for physiological adaptation than do stenothermal species. It is theorized that respiratory control in Drosophila may be related to rates of amino acid conversions and the level of substrates entering the Krebs cycle.
INTRODUCTION IT HAS been suggested that eurythermal species of Drosophila (those distributed widely with respect to temperature) have a superior capacity for metabolic adaptation to different temperatures than do stenothermal species (Hunter, 1964). Females of D. melanogaster and both sexes of D. hydei and D. immigrans have a lower rate of respiration when acclimated to a temperature higher than normal (Hunter, 1964, 1965, 1968). These species are widely distributed in regions of both high and low temperature and are considered eurythermal. On the other hand, the stenothermal species studied, D. pseudoobscura, D. viracochi and D. willistoni, do not show adaptive changes in oxygen consumption after acclimation to different temperature (Hunter, 1965, 1966). Attempts to explain the presence or lack of respiratory adaptation on the basis of differences in enzyme activity were unsuccessful. In other poikilotherms examples of changes in enzymes of carbohydrate metabolism with adaptation to different temperatures exist (see, for example, Kanungo & Prosser, 1959; Ekberg, 1962; Hochachka, 1963; Freed, 1965). Of the Krebs cycle reactions studied in Drosophila, however, the variations of oxidative enzyme activities with temperature did not parallel the respiratory changes (Hunter & Cediel, 1970). 251
252
MARY Jo BURRAND ALICE S. HUNTER
Anders et al. (1964) have demonstrated the level of free amino acids to be inversely related to the environmental temperature in D. melanogaster. T h e concentration of glutamic acid, however, decreases with decreasing temperatures. It was therefore felt that,a study of enzymes in amino acid metabolism might yield information on temperature adaptation on the enzyme level in Drosophila and might also help clarify the basis of respiratory adaptation in eurythermal species. T h e present paper reports the activity of glutamate-aspartate transaminase in crude homogenate fractions of two eurythermal species, D. melanogaster and D. immigrans, and of two stenothermal species, D. pseudoobscura and D. willistoni. MATERIALS AND METHODS Descriptions and maintenance of the Drosophila stocks have been reported (Hunter, 1964, 1965, 1968). Young adult flies of the same age as those used in respiration studies were chosen for the enzyme experiments. The flies grown at 25°C were collected at 29-3I days after the parents had been set up, while those at 15°C were used at 50-60 days. Each temperature stock had been growing for several generations since the original collection and no attempt was made to distinguish environmentally induced short-term adaptations from genetic adaptations. The transaminase system employed (Cohen, I955) contained 0'045 mg pyridoxal phosphate, 60/zM DL-aspartic acid, 30/zM ketoglutarate and 1"5 ml of a supernatant fraction of homogenate made up to a total volume of 4"8 ml 0'05 M phosphate buffer, pH 7"6. The appearance of oxaloacetate was determined by the increase in optical density at 280 m/z. The reaction was carried out at 20°C in a Beckman DU Spectrophotometer and was followed for 30 min with readings taken at 5 min intervals. The homogenate was prepared from approximately 24 mg fresh weight of flies in 6 ml of the buffer and was centrifuged 1"5 min to a maximum of 400 g. The resultant supernatant fraction used for the enzyme assay contained 40-50 per cent of the protein of the original homogenate and approximately 70 per cent of the glutamate-aspartate transaminase activity present in the whole homogenate. Similar supernatant fractions were prepared for protein analyses from more concentrated homogenates of sibling flies (70 mg fresh wt./6 ml buffer). The protein fraction obtained after precipitation with perchloric acid was solubilized in hot 1 N NaOH, and the concentration of protein was determined by the Biuret method (Layne, 1957). RESULTS T h e glutamate-aspartate transaminase activity in /xmoles of oxaloacetate p r o d u c e d / m g fresh fly wt. per hour is reported in Table 1 for homogenates of D. melanogaster and D. immigrans and in Table 2 for D. pseucloobscura and D. willistoni. I n general, activity of the enzyme is higher in D. melanogaster than in D. pseudoobscura and D. immigrans. D. willistoni possesses an intermediate level of the transaminase. In the eurythermal species, D. melanogaster, there is no statistically significant difference in the glutamate-aspartate transaminase activity expressed/mg wet wt. of males grown at the different temperatures. Females acclimated at 25°C have a lower rate of transamination than do those grown at 15°C when measured at 20°C (P = 0.02, P = 0-10). Within the two D. melanogaster stocks growing at 15°C there are no significant differences in transaminase activity between the 10pair and the 120-fly stocks.
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I n b o t h sexes o f D. immigrans y o u n g a d u l t s o f t h e 25°C stock have l o w e r activities o f g l u t a m a t e - a s p a r t a t e t r a n s a m i n a s e t h a n d o t h e c o r r e s p o n d i n g 15°C flies ( P = 0.01). A s seen in T a b l e 2 t h e a c t i v i t y o f t h i s e n z y m e does n o t v a r y a d a p t i v e l y w i t h a c c l i m a t i o n t e m p e r a t u r e in t h e t w o s t e n o t h e r m a l s p e c i e s s t u d i e d . TABLE 1--GLUTAMATE-ASPARTATETRANSAMINASEACTIVITY IN EURYTHERMALDrosophila
Stock
Temperature (°C)
Activity (mg/hr) *
/zg Protein nitrogen/mg*
Activity t (rag protein nitrogen/hr)
Sex
N
15 15 15 15 25 25
M F M F M F
10 10 10 10 10 10
0"53 0'49 0.50 0.48 0"56 0"43
(0'06) (0'05) (0.07) (0.06) (0"11) (0"05)
9'4 9'4 9.9 9.7 10'0 10'1
(0"8) (0"8) (1.5) (1.3) (1-7) (1'6)
56 52 51 49 56 43
15 15 25 25
M F M F
7 7 8 8
0'40 0"38 0"32 0'31
(0"01) (0"05) (0"02) (0"03)
8'9 8'1 7"5 7'5
(1"9) (1"0) (0"5) (0"7)
45 47 43 41
D. melanogaster 10 pairs 10 pairs 120 120 5 pairs 5 pairs
D. immigrans 8 pairs 8 pairs 3 pairs 3 pairs
* Figures in parentheses are standard deviations. t Activity is/xmoles oxaloacetate produced/rag protein nitrogen per hour at 20°C. TABLE 2--GLUTAMATE-ASPARTATETRANSAMINASEACTIVITYIN STENOTHERMALDrosophila
Stock
Temperature (°C) Sex
N
Activity (mg/hr) *
/zg Protein nitrogen/mg*
Activity t (rag protein nitrogen/hr)
D. pseudoobscura 10 pairs 10 pairs 5 pairs 5 pairs
15 15 25 25
M F M F
10 10 10 10
0"38 0"36 0"41 0.41
(0-04) (0"05) (0"04) (0.06)
8-4 (1'3) 8"9 (0'8) 9"3 (1'0) 10-3 (2"3)
45 40 44 40
15 15 25 25
M F M F
10 10 10 10
0"48 0"42 0-47 0"41
(0"06) (0'06) (0"14) (0"08)
10'2 (1 '1) 9"9 (0-8) 9'3 (1'7) 9-4 (1'7)
47 42 51 44
D. willistoni 10 pairs 10 pairs 5 pairs 5 pairs
* Figures in parentheses are standard deviations. Activity is /zmoles oxaloacetate produced/mg protein nitrogen per hour at 20°C.
254
MARY
Jo BURR AND ALICE S. HUNTER
Although it is not the purpose of this study, it is of interest to compare the transaminase activity in the two sexes. Male D. melanogaster grown at 25°C contain higher glutamate-aspartate transaminase levels than do females of the same temperature stock (P = 0.01). A similar sex difference is seen in young adults of D. willistoni maintained at 15°C or at 25°C (P = 0.10). In D. melanogaster grown at 15°C, D. immigrans and D. pseudoobscura there are no significant differences in transaminase activity of the two sexes. When glutamate-aspartate transaminase activity is expressed on a protein nitrogen rather than fresh weight basis, the same comparisons can be made of relative activities between flies grown at 15°C and at 25°C and between the sexes as can be seen in Tables 1 and 2. DISCUSSION This is part of an investigation testing the theory that eurythermal species of Drosophila are more capable of temperature adaptation than stenothermal species. Physiological temperature adaptation (which may be environmentally induced or genetic) is interpreted as a shift toward a lower metabolic rate after acclimation to a higher than "normal" temperature and vice versa. The D. melanogaster, D. immigrans and D. pseudoobscura used here were originally collected from the relatively "cool" environment (average temperature 15°C) of Bogotfi. Stocks of each species were maintained at the "normal" temperature of 15°C and others were acclimated at 25°C. The first two species are considered eurythermal and the third is stenothermal. The D. will#toni were collected from a warm environment (temperature 25-30°C) and a stock was maintained at the normal temperature of 25°C and another stock was grown at 15°C. T h e stenothermal species, D. pseudoobscura and D. willistoni, show no indication of temperature adaptation with respect to transaminase activity. With D. melanogaster, the females have significantly lower transaminase activity after growth at the higher temperature of 25°C compared with the stocks maintained at the more "normal" temperature of 15°C. Two different stocks were grown at 15°C because temperature affects inversely the size of flies and those grown under optimal conditions such as the 10-pair stocks are considerably larger than the 25°C grown flies. Therefore crowding (120 flies) was used in order to obtain a fly comparable in size to that grown at 25°C. A comparison of the 120-fly 15°C stock with the 5-pair 25°C stock controls any possible variation in transaminase activity with respect to size of fly. The male D. melanogaster does not give evidence of temperature adaptation with respect to transaminase activity. This point is of interest because similar results were found previously with oxygen consumption; that is, females adapt and males do not (Hunter, 1964). The glutamate-aspartate transaminase activity of D. immigrans acclimated at a higher temperature of 25°C is significantly lower in both sexes than that of the flies maintained at 15°C. This is the same as was found previously with respect to oxygen consumption (Hunter, 1968) and upholds the theory being tested. The present data support a previous suggestion that female and not male D.
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melanogaster undergo the process of temperature adaptation (Hunter, 1964). This does not seem to apply to all eurythermal species of Drosophila since in D. immigrans both sexes show adaptation in transaminase activity and in both D. immigrans and D. hydei both sexes show adaptation in oxygen consumption. Whether it is characteristic of D. melanogaster as a species or only of the Bogot~i stock used in this study is not known. Hunter (1964) suggested that adaptation restricted to organs specific to the female could explain the occurrence of temperature adaptation in the one sex only. Insect transaminases are localized in a variety of organs and on a wet weight basis are particularly active in Malpighian tubules and the fat body (Kilby & Neville, 1957; McAllan & Chefurka, 1961; Chen & BachmannDiem, 1964). A fivefold increase in glutamate-aspartate transaminase activity in whole houseflies during pupal-adult transformation is correlated with elevated levels of the enzyme in thoracic muscle alone (McAllan & Chefurka, 1961). The fact that other tissues examined did not exhibit a concomitant rise in activity indicates that organ specific responses of the transaminase system may occur in insects. Specific changes in metabolism of different tissues with temperature acclimation have been reported for other poikilotherms (Rieck et al., 1960). Cold acclimation results in an elevation of liver and muscle transaminase activities (Harmon, 1963 ; Klain & Vaughan, 1963). Klain & Vaughan (1963) have shown that the increase of transaminase activity in cold-acclimated rats is related to greater food consumption and is substrate induced. This observation suggests the possibility that the basis for higher transaminase levels in cold-grown eurythermal species of Drosophila might also be subtrate induction. Anders et al. (1964) have shown marked increases in the size of the amino acid pool in D. melanogaster larvae and pupae grown at 15°C but the higher concentration of most amino acids is accompanied by a decrease in the glutamic acid content. Perhaps this is related to increased transamination of the glutamic acid which makes availablesubstrates for the Krebs cycle. High transaminase activity may be associated with increased rates of protein synthesis or with an active energy metabolism. In several species of Drosophila the protein content/mg of cold-gro~n flies does not differ significantly from that of flies raised at 25°C (Burr & Hunter, 1969). In the present case a relationship between rates of respiration and transamination seems more plausible. Activities of glutamate-aspartate transaminase in D. pseudoobscura and D. willistoni do not differ significantly with acclimation temperature; no physiological adaptation is observed in these species (Hunter, 1965, 1966). In D. melanogaster and D. immigrans, comparable changes in oxygen consumption and transaminase activity are observed after acclimation to higher temperature (Hunter, 1964, 1968). In general, little evidence was obtained for adaptive changes in respiratory enzymes (Hunter & Cediel, 1970). These facts taken together suggest that respiratory control in Drosophila may be related to variable rates of amino acid conversions and that temperature-dependent modifications in the rate of respiration result from changes in the level of substrates feeding into the Krebs cycle rather than changes in activity of respiratory enzymes.
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MARY Jo BURR AND ALICE S. HUNTER
REFERENCES ANDERS VON E., DRAWERTF., ANDERSA. & REUTHERK. H. (1964) Ober kausale Zusammenhange zwischen der Zuchttemperatur, dem Aminos~iuren-Pool u n d einigen quantitativen morphologischen Phanen bei Drosophila melanogaster. Z. Naturf. 196, 495-499. BURR M. J. & HUNTER A. S. (1969) Effects of temperature on Drosophila--V. Weight and water, protein and RNA content. Comp. Biochem. Physiol. 29, 647-652. CHEN P. S. & BACHMANN-DIEM C. (1964) Studies on the transamination reactions in the larval fat body of Drosophila melanogaster. J. Insect Physiol. 10, 819-829. COHEN P. P. (1955) Estimation of animal transaminases. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. II, pp. 178-184. Academic Press, New York. EKBERG D. R. (1962) Anaerobic and aerobic metabolism in gills of the crucian carp adapted to high and low temperatures. Comp. Biochem. Physiol. 5, 123-128. FREED J. (1965) Changes in activity of cytochrome oxidase during adaptation of goldfish to different temperatures. Comp. Biochem. Physiol. 14, 651-659. HANNON J. P. (1963) Current status of carbohydrate metabolism in the cold-acclimatized mammal. Fedn Proc. 22, 856-861. HOCHACHKA P. W. & HAYS F. R. (1963) Effect of temperature acclimation on pathways of glucose metabolism in trout. Canad.ff. Zool. 40, 261-270. HUNTER A. S. (1964) Effects of temperature on Drosophila--I. Respiration of D. melanogaster grown at different temperatures. Comp. Biochem. Physiol. 11, 411-417. HUNTER A. S. (1965) Effects of temperature on Drosophila--II. Respiration of D. pseudoobscura and D. viracochi grown at different temperatures. Comp. Biochem, Physiol. 16, 411417. HUNTER A. S. (1966) Effects of temperature on DrosophiIa--III. Respiration of D. willistoni and D. hydei grown at different temperatures. Comp. Biochem. Physiol. 19, 171-177. HUNTER A. S. (1968) Effects of temperature on Drosophila--IV. Adaptation of D. immigrans. Comp. Biochem. Physiol. 24, 327-333. HUNTER A. S. & CEDIEL N. (1970) Effects of temperature on Drosophila--VI. Respiratory enzymes. Comp. Biochem. Physiol. Submitted for publication. KANUNGO M. S. & PROSSER C. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures--II. Oxygen consumption of liver homogenate; oxygen consumption and oxidative phosphorylation of liver mitochondria, ft. cell. comp. Physiol. 54, 265-274. KILBY B. A. ~ NEVILLE E. (1957) Amino acid metabolism in locust tissues. J. Biol. 34, 276289. KLAIN G. J. & VAUGHAN D. A. (1963) Alterations of protein metabolism during coldacclimation. Fedn Proc. 22, 862-867. LAYNE E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. In JVlethods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. III, pp. 4 4 7 4 5 4 . Academic Press, New York. ~IcALLAN J. W. & CHEFURKAW. (1961) Some physiological aspects of glutamate-aspartate transamination in insects. Comp. Biochem. Physiol. 2, 290-299. RIECK A. F., BEOLI J. A. • BLASKOVICSM. E. (1960) Oxygen consumption of whole animal and tissues in temperature acclimated amphibians, Proc. Soc. exp. Biol. Med. 103, 436439.
Key Word Index--Insect biochemistry; Drosophila ; glutamate-aspartate transaminase; temperature adaptation.