Comp. Biochem. Physiol)Vol. 98B, No. 4, pp. ~ 1 4 , 1991 '0305-0491/91 $3.00 + 0.00 Printed in Great Britain © 1991 Pergamon Press pie

THE EFFECT OF ACCLIMATION TEMPERATURE ON ENZYME ACTIVITY IN DROSOPHILA MELANOGASTER

ANN M. BURNELL, CAROLANN REAPER and JOAN~ DOHERTY Biology Department, St Patrick's College, Maynooth, Co. Kildare, Ireland (Tel: 01 6285 222)

(Received 13 September 1990)

Abstract--1. The response to thermal acclimation of five key rate-limiting enzymes of intermediary metabolism and of six degradative enzymes was measured in tissue extracts of adult Drosophila melanogaster which had been acclimated for 4 days to 15, 25 or 30°C.

2. Three enzymes of intermediary metabolism (HK, a-GPDH and CO) showed positive thermal compensation, which is the type of response characteristic of the enzymes involved in energy metabolism in vertebrate ectotherms.

3. The data obtained for CS and G6PDH showed no evidence for increased activity of TCA cycle nor of the pentose phosphate pathway upon cold acclimation in D. melanogaster.

4. Two degradative enzymes, ADH and non-specific esterase, showed inverse thermal compensation which is the type of response characteristic of degradative enzymes in vertebrate ectotherms.

5. In contrast to the situation in vertebrate ectotherr/ts, catalase and the three lysosomal enzymes assayed (APH, acid DNase and acid RNase) displayed positive rather than inverse compensation.

6. The results presented here extend the data on the range of D. melanogaster enzymes which show compensation upon thermal acclimation and on the type of acclimation response which occurs.

INTRODUCTION

Temperature exerts a profound effect on the catalytic activity of enzymes. Nevertheless ectothermic organ- isms from different thermal environments and which have very different body temperatures succeed in maintaining relatively similar metabolic rates (reviewed by Hochachka and Somero, 1973, 1984; Hazel and Prosser, 1974; Sidell, 1983; Somero, 1983; Hoffmann, 1985; Alahiotis et al., 1987; Dunn, 1988). Thus while temperature shifts in vitro cause large effects on the catalytic activities of enzymes, when the catalytic activities are determined at the physiological temperature of the ectotherm, a strong conservation of catalytic function is noted. From studies of the amino acid sequences of lactate dehydrogenase (LDH) in thermophylic, mesophylic and psychro- philic bacteria, Zuber and colleagues have established that the temperature adaptation of an enzyme is the result of a long evolutionary process in which exten- sive amino acid substitutions take place (reviewed by Zuber, 1988).

In ~iddition to the long-term evolutionary changes which occur in the gene enzyme systems of an ectotherm in response to ambient temperature, many ectotherms also display short-term adaptations which occur within the life time of an individual. When these adaptive changes in phenotype result from experimental manipulations of environmental tem- perature in the laboratory, they are referred to as acclimations. As a result of the process of acclimation many ectothermic animals are able to retain relatively stable rates of metabolism in spite of a large change in environmental temperature.

The extent of metabolic compensation upon ac- climation varies depending on the organism and on the metabolic system being investigated. The range of

responses has been classified by Precht (1958) into five types: overcompensation; perfect compensation (the acclimated rates are the same at both tempera- tures); partial compensation; no compensation and inverse compensation (the acclimated rate in the cold is lower than the rate on direct transfer from warm to cold). The term "inverse compensation" has also been applied by Hochachka and Somero (1973) to metabolic reactions in which the acclimated rates are higher in the tissues of warm acclimated specimens.

Hazel and Prosser (1970, 1974) reviewed the litera- ture on enzyme acclimation and they noted that the enzymes which displayed positive thermal compen- sation were primarily associated with pathways of energy metabolism (e.g. glycolysis, the hexose mono- phosphate shunt, the citric acid cycle and electron transport enzymes), while those enzymes which dis- played inverse compensation upon acclimation were those associated with degradative processes such as the lysosomal and peroxisomal enzymes and those associated with nitrogen metabolism. Hazel and Prosser speculate that at high temperatures, when the metabolic rate of the organism is increased, there is a greater need for the removal of metabolic waste products and consequently the enzymes involved in these degradative processes display increased activity levels upon acclimation to high temperatures. Simi- larly, they argue that it would be advantageous for a cold acclimated animal to have increased activity of those enzymes involved in energy metabolism in order to compensate for reduced kinetic activity at low acclimation temperatures.

Hochachka and Somero (1973, 1984) hypothesize that the phenotypic adjustments which enable ecto- therms to hold their metabolic rates relatively constant following acclimation may result in an alteration in the amount of enzymes present in the

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6t0 A.M. BURNELL et al.

organism, "the quantitative strategy", or alterna- tively in the synthesis of isozyme or allozyme variants having adaptively different kinetic properties, "the qualitative strategy". However, they note (Hochachka and Somero, 1984) that "there are very few clear cut examples of allozymes possessing adap- tively different functional or structural properties that correlate with environmental differences". They also point out some limitations to the quantitive strategy--the solvent capacity of the cell is limited; a single enzyme may not function at the entire range of temperatures at which catalysis must be performed and protein synthesis is an energetically costly pro- cess. For those metabolic pathways in which a quan- titative strategy is employed on acclimation then a prime target for phenotypic adjustment might be the rate-limiting enzymes which control metabolite flux through metabolic pathways and whose maximal activities are usually considerably lower than those of the non-regulatory enzymes in the same pathway.

The majority of studies on acclimation to tempera- ture have relied on aquatic vertebrate ectotherms, usually fish and relatively few studies have employed insects. Yet, ectothermic insects are exposed to larger diurnal and seasonal changes in ambient temperature in temperate climates than are aquatic ectotherms. Drosophila melanogaster has a widespread distri- but ion in temperate regions and its gene enzyme systems have been subject to extensive investigations over three decades. Furthermore, the life span of D. melanogaster is short, 36.5 days at 25°C (Felix and Ramirez, 1967) and an acclimation response in D. melanogaster must occur over a relatively short time period if it is to be of adaptive value to the organism. In the study reported here the effect of acclimation on the activities of a range of key rate- limiting metabolic enzymes and of lysosomal and degradative enzymes was investigated. The objective of the investigation was to establish whether, follow- ing acclimation, the patterns of inverse compensation of degradative enzymes and of positive compensation of the enzymes of energy metabolism which are characteristic of vertebrate ectotherms could also be observed in D. melanogaster.

MATERIALS AND METHODS

Growth and acclimation of flies The Canton-S strain of D. melanogaster was used. The

flies were grown in the dark at 25°C on standard cornmeal medium (Lewis, 1960). Flies were acclimated by transferring adult flies, 0-12 hr post eclosion, without etherization to fresh food bottles which were then incubated for 4 days, in the dark, at one of three temperatures: 15, 25 or 30°C, In preliminary studies for this work we investigated the effect on enzyme activity of warm and cold acclimation for 1, 4 and 7 days and similar results were obtained whether the flies were acclimated for 4 or for 7 days.

Preparation of tissue extracts The acclimated flies were lightly etherized and were

homogenized at a concentration of 50 mg/rnl of extraction buffer, with the exception of the following assays, where I00 mg of flies per ml of extraction buffer was used: acid DNase, acid RNase, citrate synthase and cytochrome oxi- dase. The extraction buffers used were as follows: acid phosphatase, 45 mM Na3 citrate buffer, pH 4.9; catalase, 10raM K phosphate, pH 7.5; citrate synthase, 100raM

Tris-HC1, pH 8.0; cytochrome oxidase, I mM EDTA, 154 mM KCI, 5 mM MOPS, pH 7.4 and 0.1% (w/v) bovine serum albumin (O'Byrne-Ring et aL, 1981); for acid DNase and acid RNase, the flies were homogenized in distilled H20 and 20 mg/ml activated charcoal was added to decolorize the homogenates (Detwiler and MacIntyre, 1978). For the remaining enzymes, the extraction buffers used were those described in the assay references listed below.

Unless otherwise stated, the homogenates were centri- fuged at 28,000g at 4°C for 30rain and the resulting supernatants assayed immediately for enzyme activity. The mitochondrial fractions used for the cytochrome oxidase assays were obtained as follows: the homogenates were centrifuged at 830g for 15 rain at 4°C and the supernatants were recentrifuged at 13,000g for 15rain. The mito- chondrial pellets were resuspended in homogenizing buffer and centrifuged at 13,000g. The sedimented mitochondria were resuspended in homogenizing buffer and assayed for enzyme activity. The fractions used in the citrate synthase assays were obtained by centrifuging the homogenates at 1000g at 4°C for 10rain, the supernatants were recentri- fuged at 1000g for 10 rain and the resulting supernatants were assayed for enzyme activity.

The lysosomal extracts used for the acid DNase, acid RNase and acid phosphatase assays were obtained by centrifuging the homogenates at 3300g for 20rain at 4°C. The supernatants were mixed 1 : 1 (v/v) with 100 mM citrate phosphate buffer, pH 5.0, containing 0.2% Triton-X (acid DNase, acid RNase) or with 49 mM Na3 citrate buffer containing 0.2% Triton-X (acid phosphatase) and the ex- tracts were left at -20°C overnight before being assayed for enzyme activity.

Enzyme assays Each assay was replicated four times with the exception

of citrate synthase and catalase where each assay was replicated five times. Enzyme activity measurements were linearly correlated with incubation time (with the exception of catalase) and with the protein concentration of the tissue extract used in the enzyme assay. Since catalase activity levels decreased with incubation time, probably because of the toxic effect of substrate H202 to the enzyme, the initial reaction rate was used in calculating the spec. act. of catalase. The following enzymes were assayed spectrophoto- metrically against reagent blanks at 25°C as described in the accompanying references: hexokinase, by monitoring the formation of glucose-6-phosphate (Bergmeyer et aL, 1974); giucose-6-phosphate dehydrogenase (Bijlsma and van der Meulen-Brnijns, 1979); ~t-giycerophosphate dehydrogenase (O'Brien and Maclntyre, 1972; the concentration of sub- strates used was 100raM ~t-glyeerophosphate and 2 mM NAD); citrate synthase (Salvarry and Cazzulo, 1982); cyto- chrome oxidase was assayed by measuring the oxidation of reduced cytoehrome c at 550 nm (Smith, 1955). Reduced cytoehrome c was prepared as described by Wharton and Tsagoloff (1967). The concentration of reduced cytoehrome c used in the assay was 0.7 mM and the spec. act. was calculated using an Ess 0 value of 2.77 × 104M -~ em -t for reduced eytoehrome c. Alcohol dehydrogenase was assayed by monitoring the reduction of NAD at 340 nm. The reaction mixture contained 100raM ethanol and 2mM NAD in 100 mM Tris-HCl, pH 8.0. Catalase was assayed as described by Courtright (1967). The spec. act. of catalase was calculated using an ~ value for hydrogen peroxide of 39.4 M -I cm -l (Nelson and Kiesow, 1972).

The following enzymes were assayed using fixed time incubations as described in the accompanying references: acid DNase (Detwiler and MacIntyre, 1978); acid phos- phatase, using as substrate 7 mM disodiurn 4-nitrophenyl phosphate in 45 mM Na3 citrate buffer, pH 4.9 (Moss, 1984); esterase (Burnell and Wilkins, 1988). Acid RNase was assayed as follows: the reaction mixture contained 0.24 ml of 0.1 M citrate phosphate buffer, pH 5.0, 0.12ml RNA

Temperature acclimation in Drosophila 611

Table 1. The effect of temperature acclimation on the specific activities* (assayed at 25°C) of metabofic enzymes in Drosophila melanogaster. For each enzyme, mean values which do not differ significantly from each other are in italics

Acclimation temperature Relative activity

cold acclimated (15°C)

Enzyme n 15°C 25°C 30°C F warm accfimated (30°C)

Hexokinase 4 237.13 + 7.23 199.44 5:3.30 176.72 + 2.46 40.33t 1.34 Glucose-6-phosphate dehydrogenase 4 18.31 + 0.63 15.90 + 0.33 15.29 + 1.21 3.88 (N.S.) I. 19

-Glycerophosphate dehydrogenase 4 387.43 + 12.50 300.54 + 3.62 246.84 -4- 0.99 88.65t 1.56 Citrate synthase 5 182.69 5- 3.42 18Z52 + 3.85 194.15 5: 4. 71 2.10 (N.S.) 0.94 Cytochrome oxidase 4 287.52 +_ 16.81 235.44 + 13.83 189.95 5:6.55 14.12t 1.52

*nmol product formed/min/mg protein. tP < 0.005.

(yeast RNA, Boehringer, 1 mg/ml) and 0.02 ml tissue ex- tract. The mixture was incubated for 60 min at 25°C. The tubes were then placed in an ice bath and 0.038 ml of 3 M Na acetate pH 5.5 was added, followed by 1.05 ml ethanol. The residual RNA was precipitated overnight at -20°C, The tubes were centrifuged at 9000g for 30 min and the A s of the supernatant determined. Tissue blanks were obtained by withholding the tissue extracts until after the addition of the ethanol. The spec. act. of both acid DNase and acid RNase was calculated using an E260 of 1.0 x 104M -t cm -t for the soluble oligonucleotides (Dulaney and Touster, 1972).

Protein concentrations were measured using the pro- cedure of Lowry et al. (1951) using bovine serum albumin as standard.

Statistical analysis

One-way analysis of variance was used to determine if the differences in mean enzyme activity observed upon acclim- ation were statistically significant. The statistical significance of the differences observed between the individual treat- ments was determined using Duncan's (1955) new multiple range test. The level of significance for the latter test was set at P < 0.05.

RESULTS

The net effect of Precht type 1 and 2 compensat ion is that, when measured at an intermediate tempera- ture, the activity o f enzymes from cold acclimated organisms is greater than that of warm acclimated enzymes (Hazel and Prosser, 1970). Similarly, for those enzyme systems which display Precht type 5 compensat ion (inverse compensation) the activity of enzymes from warm acclimated organisms is greater than that of cold acclimated enzymes when measured at an intermediate temperature. Such an experimental approach was used in the study reported here and all enzyme assays were carried out at 25°C.

Intermediary metabol ism

The metabolic enzymes chosen for investigation were hexokinase (HK) which controls the entry of carbohydrates into the glycolytic pathway; glucose-6- phosphate dehydrogenase (G6PDH) which catalyses the first reaction of the pentose phosphate pathway, an alternative pathway to glycolysis for glucose utilization; ~t-glycerophosphate dehydrogenase (~tGPDH), the cytoplasmic enzyme of the glycerol 3-phosphate shuttle in insect flight muscles; citrate synthase (CS) which is the rate-limiting enzyme of the tricarboxylic acid (TCA) cycle and cytochrome c oxidase (CO). The CO complex is the terminal com- ponent of the mitochondrial electron transport chain and it transfers electrons to 02.

Tissue extracts from adult flies which had been acclimated for 4 days to 15, 25 or 30°C were assayed for enzyme activity at 25°C and the results obtained are presented in Table 1. Three of the enzymes assayed (HK, ~tGPDH and CO) showed an inverse relationship between acclimation temperature and enzyme activity. This is a Precht type 1-2 form of temperature compensat ion and thus the data ob- tained for these enzymes are in agreement with the hypothesis o f Hazel and Prosser (1970). However for the remaining two key metabolic enzymes studied, G 6 P D H and CS, no statistically significant difference in enzyme activity was observed upon cold or warm acclimation.

Degradatioe and lysosomal enzymes

Six degradative enzymes were assayed in tissue extracts f rom warm and cold acclimated flies and the results obtained are presented in Table 2. Three lysosomal enzymes were assayed: acid phosphatase (APH), acid DNase and acid RNase. In eukaryotes,

Table 2. The effect of temperature acclimation on the specific activities* (assayed at 25°C) of degradative enzymes in Drosophila melanogaJter. For each enzyme, mean values which do not differ significantly from each other are in italics (Duncan's multiple range test)

Acclimation temperature Relative activity

cold acclimated (15°C)

Enzyme n 15°C 25°C 30°C F warm acclimated (30°C)

Alcohol dehydrogenase 4 88.23 + 2.52 90.68 + 1.24 120.70 + 3.33 51.66~ 0.73 Esterase 4 24.73+_ 1 . 8 4 23.15+_ 0.72 61.59+_ 1.13 265.30~ 0.40 Catalaset 5 119.44 + 5.56 103.02 + 3.39 99.33 + 5.15 4.99:~ 1.20 Acid phosphatase 4 14.79 + 0.25 13.34 + 0.23 13.01 + 0.13 21.13~ 1.14 Acid DNase 4 3.15 + 0.17 3.29 + 0.08 1.24 + 0.04 110.28~ 2.54 Acid RNase 4 16.77+_0.73 15 .00+_0.30 11.38+_0.67 20.97§ 1.47

*nmol product formed/min/mg protein. t/zmol product formed/min/mg protein. :~P < 0.05, §P < 0.005.

612 A .M. BURNELL et al.

intracellular turnover of almost all types of macro- molecules occurs in lysosomes---discrete membrane- bounded structures which contain upwards of 40 different hydrolytic enzymes, most of which have acid pH optima. For both acid DNase and acid RNase no statistically significant difference in enzyme activity was observed on cold acclimation, however upon warm acclimation both enzymes displayed thermal compensation, i.e. the spec. act. was significantly lower in the warm acclimated extracts. In the case of APH, no significant difference was observed upon warm acclimation, however a slight but statistically significant increase in spec. act. was observed upon cold acclimation. A pattern of thermal compensation similar to that of APH was also observed for catalase, an enzyme which catalyses the detoxification of hydrogen peroxide. The levels of catalase detected were very high, being 3-4 orders of magnitude greater than the acti~,ity levels detected for the other degra- dative enzymes assayed in this study. The catalase values reported here are in agreement with those obtained by Nahmias and Bewley (1984) for D. melanogaster.

These four degradative enzymes then, showed a pattern of either increased activity upon cold acclimation or of decreased activity upon warm acclimation. This is a Precht type 1-2 form of com- pensation, which according to Hochachka and Somero is not characteristic of degradative enzymes. The data reported here for these four D. melanogaster degradative enzymes were confirmed in the repeated experiments.

The two remaining degradative enzymes investi- gated, alcohol dehydrogenase (ADH) and non- specific esterase, showed a very strong pattern of inverse compensation with activity levels being sig- nificantly higher in warm acclimated flies. This is a Precht type 5 form of temperature compensation which Hazel and Prosser (1970, 1974) consider to be the characteristic compensation response for degra- dative enzymes in ectotherms.

DISCUSSION

The results obtained in our study confirm that the pattern of positive thermal compensation which Hazel and Prosser (1970) found to be characteristic of the enzymes of energy metabolism in vertebrate ectotherms also occurs in D. melanogaster for the enzymes HK, GPDH and CO. For these three en- zymes, then, the increased spec. act. observed in cold acclimated flies should compensate for reduced kinetic energy at low temperatures and so maintain the flux of metabolites through the glycolytic pathway, the glycerol 3-phosphate shuttle and the mitochondrial electron transport chain at a level similar to that of the controls. We did not however observe an acclimation response for the metabolic enzymes G6PDH and CS. This observation suggests that acclimated Drosophila are not able to hold ~onstant the rate of metabolite flux through the pentose phosphate pathway and the TCA cycle following acclimation and thus the rate of flux through these pathways may depend on the ambient temperature.

A limited number of studies have been carried out in Drosophila on the effect of thermal acclimation upon enzyme activity. Positive thermal compensation has been observed in cold acclimated D. melanogaster for succinic dehydrogenase (Hunter and Cediel, 1970), for ADH (Sampsell and Baruette, 1985), and the data presented by Hall et al. (1980) indicate that inverse compensation of acetylcholine esterase activity occurs in D. melanogaster upon warm acclim- ation. Increased activity of MDH has also been observed in D. melanogaster following heat shock (40°C for 30 rain) by Goulielmos et al. (1986) and Goulielmos and Alahiotis (1989). Thus, the results presented here further extend the data on the range of D. melanogaster enzymes which show compen- sation upon thermal acclimation and on the type of acclimation response which occurs.

From his review of the rather limited number of studies on thermal acclimatization in ectothermic insects, Hoffmann (1985) concluded that (a) the magnitude of compensation for glycolytic enzymes is less than that characteristic of electron transport and TCA cycle enzymes and (b) adaptation to cold frequently shifts metabolism towards anabolism, accompanied by a marked activation of the pentose shunt enzymes. The data obtained in this study for CS and G6PDH, however, show no evidence for increased activity of the TCA cycle nor the pentose phosphate pathway upon cold acclimation in D. melanogaster. The large compensation response ob- served in ~GPDH upon cold acclimation is consistent with its key role in the glycerol 3-phosphate shuttle which is used to transfer reducing equivalents into the mitochondrion. The shuttle permits a high rate of re-oxidation of NADH and the complete oxidation of glucose to CO2 and H20 without the accumulation of lactate (Sacktor, 1975). The glycerol 3-phosphate shuttle carries reducing equivalents directly to the electron transport chain and this may be correlated with the increased cytochrome oxidase activity de- tected upon cold acclimation in this study. Thus, the positive thermal compensation displayed by ~ GPDH and CO may compensate for the lack of compen- sation displayed by CS, the key regulatory enzyme of the TCA cycle.

Of the six degradative enzymes assayed, only two, ADH and non-specific esterase, displayed inverse compensation. The increased activity observed in these two enzymes upon warm acclimation may form part of the adaptive response of Drosophila to increased environmental temperatures. ADH is an enzyme required for the detoxification of ethanol and this enzyme confers on Drosophila the ability to exploit alcohol-containing environments. ADH activity has been shown to be induced in D. melanogaster larvae as a result of an ethanol-stimu- lated increase in the amount of ADH mRNA (Geer et al., 1988; Kapoun et al., 1990). Since alcohol production in yeast is strongly temperature depen- dent (White and Munns, 1951), the level of alcohol production by the live yeast in standard Drosophila medium would be expected to increase at higher environmental temperatures. The non-specific ester- ases have a broad stibstrate specificity and among the physiological functions they have been postulated to serve in insects are digestion (Kapin and Ahmad,

Temperature acclimation in Drosophila 613

1980), juvenile hormone metabolism (DeKort and Granger, 1981), reproduction (Richmond et al., 1980) and insecticide degradation (Pasteur et ai., 1981; Devonshire et al., 1986; Mouches et al., 1986).

Catalase is an enzyme which usually displays inverse thermal compensation (Hazel and Prosser, 1974) but this response was not observed for D. melanogaster in our study. However the catalase activity levels in D. melanogaster were at least three orders of magnitude greater than the levels of activity of any other enzyme assayed; thus a further increase in catalase activity upon warm acclimation might not be required in D. melanogaster.

Relatively little attention has been paid to the thermal acclimation of lysosomal enzymes. However the trend observed, in general, is one of inverse compensation (e.g. Milanesi and Bird, 1972). By contrast the lysosomal enzymes investigated in the study reported here displayed positive thermal compensation which is suggestive of a decreased degradative role for these enzymes in D. melanogaster upon warm acclimation.

Data which demonstrate increased enzyme activity upon warm or cold acclimation do not necessarily imply an increased synthesis of the enzymes involved. Nevertheless, it seems likely that at least some of the acclimation responses in enzyme activity which we observed in this study (e.g. the 2.7-fold increase in esterase activity upon warm acclimation) are indicative of a very finely modulated control of enzyme synthesis during thermal acclimation in D. melanogaster. Experiments using protein synthesis inhibitors in vivo to test this possibility are currently in progress in our laboratory.

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