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Activation of phosphorylase in response to cold and heat stress in the flesh fly, Sarcophaga crassipalpis
J. Insecl Physrol. Vol. 36, No. 8. pp. 549-553, Printed in Great Britain. All rights reserved
1990 Copyright
0022-1910/90 $3.00 + 0.00 gc’ 1990 Pergamon Press plc
ACTIVATION OF PHOSPHORYLASE IN RESPONSE TO COLD AND HEAT STRESS IN THE FLESH FLY, SARCOPHAGA CHENG-PING Department of Entomology,
CRASSIPALPIS
CHENand
DAVID L. DENLINGER*
Ohio State University, 1735 Neil Avenue, Columbus, OH 43210, U.S.A.
(Received 9 January 1990; revised 6 March 1990) Abstract-Glycogen phosphorylase can be activated in diapausing pupae and nondiapausing pharate adults of the flesh fly, Sarcophaga crussipalpis in response to both cold and heat stress. The greatest activation was observed at 0 and above 30°C. Activation was rapid: following exposure to 0°C activated phosphorylase increased 36-S% within 30min. The proportion of activated phosphorylase was consistently lower in diapausing pupae than in nondiapausing pharate adults. Among the nondiapausing stages, only pharate adults and adults responded to cold stress (2 h at 0°C) and heat stress (2 h at 40°C) by activating phosphorylase. No such response was observed in larvae or young pupae. During diapause, the proportion of activated phosphorylase was highest at the onset of diapause, declined and remained fairly constant throughout the remainder of diapause. Diapausing pupae held continuously at WC maintained a higher proportion of activated phosphorylase throughout the diapause than pupae held at 20°C. Cold stress consistently activated phosphorylase in diapausing pupae held at 2O”C, but heat stress elicited this response only during the last half of diapause. The activation of phosphorylase by brief periods of chilling and the activation observed in diapausing pupae held continuously at low temperature is consistent with our earlier work showing an elevation of glycerol under these conditions.
Ke_v Word Index:
Glycogen phosphorylase;
cold shock; heat shock; pupa1 diapause; Sarcophugu
crassipalpis
INTRODUCTION
a source of energy in all tissues, serves both as a metabolic reserve and a synthetic “building block” for macromolecules. The insect fat body is the site of the major glycogen reserves that are utilized for metabolism of lipids, carbohydrates and proteins (Wyatt, 1957; Steele, 1982). Glycogen phosphorylase, present in the fat body and flight muscle of many insects, exists in two interconvertible forms: an active phosphorylated a form and an inactive dephosphorylated b form (Stevenson and Wyatt, 1964; Van Marrewijk et al., 1988). Phosphorylase can be activated by starvation (Goldsworthy, 1970; Gade, 1981), flight activity (Van Marrewijk et al., 1980; Ziegler and Schulz, 1986), exposure to cold (Ziegler and Wyatt, 1975; Ziegler et al., 1979; Bahjou et al., 1988), and by a factor extracted from the corpora cardiaca (Steele, 1963; Wiens and Gilbert, 1967; Goldsworthy, 1969; Gade, 1981). In diapause pupae of the silkmoth, Hyalophora cecropiu, cold exposure (4°C) increases glycogen phosphorylase activity of the fat body about 50% and glycerol accumulates in the haemolymph (Ziegler ef al., 1979). Thus, this enzyme acts as one of the key enzymes regulating synthesis of cryoprotectants, i.e. glycerol, trehalose or other low molecular weight substances synthesized from glycogen by overwintering diapause insects. The activation of phosphorylase by low temperature exposure, however, is not restricted to diapausing insects. Phosphorylase activity can also be elevated rapidly by low Glycogen,
*To whom all correspondence
should be addressed.
temperature in fat body cultures from Tenebriu molitar, a nondiapausing species (Bahjou et al., 1988). The fact that fat body phosphorylase kinase continues to function at low temperature, while phosphorylase phosphatase does not, favours the activity of phosphorylase (Hayakawa and Chino. 1983; Hayakawa, 1985). In Surcophuga brief exposure to moderately low or high temperature generates protection against injury caused by more extreme low or high temperature. Elevation of glycerol is at least partially responsible for the protection against cold-shock injury that is generated by a brief period of chilling, but we have no evidence that glycerol protects against heat-shock injury (Chen et al., 1987, 1990). The rapid elevation of glycerol at low temperature suggests an important role for phosphorylase in protecting the fly from cold-shock injury. In these experiments with S. crassipalpis we test the possibility that activation of phosphorylase may be stimulated by cold and heat stress. In addition, we examine this response in different developmental stages and in diapausing pupae. MATERIALS AND METHODS
Animals
A colony of the flesh fly, Sarcophaga crassipalpis Macquart, was reared in the laboratory as described by Denlinger (1972). Parental adults were reared at 25°C with either a diapause-inducing photoperiod (12 h light-12 h dark) or a nondiapause photoperiod (15 h light-9 h dark). Larvae and pupae were 549
550
CHENG-PING CHEN
and
maintained either at 20 or 25-C under the maternal photophase. Under the short-day conditions at 20°C a high incidence ( > 95%) of pupal diapause is produced. The developmental status of each pupa was determined by removing the anterior portion of the puparium and looking for signs of antenna1 formation and the eye-pigmentation characteristic of pharate adult development (Fraenkel and Hsiao, 1968). Chemicals
NADP, cyclic AMP, oyster glycogen, dithiothreitol (DTT), EDTA, glucose-l ,6-diphosphate, glucose-6phosphate dehydrogenase and phosphoglucomutase were purchased from Sigma (St Louis, MO.). All other chemicals were reagent grade. The glycogen was treated with charcoal (Merck, activated) before use, to remove traces of AMP (Stevenson and Wyatt, 1964). Preparation of phosphorylase
Abdomens of nondiapausing pharate adults or whole bodies of other stages were immediately homogenized with a plastic disposable pestle (Kontes, Vineland, N.J.) in 1 ml homogenizing buffer (20 mM triethanolamine acetate, 5 mM EDTA, 20 mM NaF. pH 7.0) in a microfuge tube and centrifuged at 8000 g at 4’C for IOmin. The supernatant, between the pellet and a fatty surface layer, was withdrawn and used for the assay. The extracts were stored at - 4°C and analysed within 1 week. Most assays were done within 2 days.
DAVID L. DENLINGER
ing A340min a spectrophotometer (Varian DMS 100) for at least 15 min. A comparison of phosphorylase activity obtained in the presence and absence of 5’-AMP permitted calculation of the amount of enzyme which was present in the active form. Data expression and analysis
Activity of phosphorylase was expressed as % phosphorylase a of total phosphorylase. The increase of phosphorylase activity was expressed by comparing % phosphorylase a of the treatment with the value for the controls. Statistical significance of differences were estimated using SAS programs (SAS Institute Inc., 1985). RESULTS
Effect of temperature on phosphorylase
Diapausing pupae (13 days in diapause) reared at 20°C and exposed for 2 h to temperatures ranging from 0 to 45°C significantly increased the proportion of phosphorylase a at temperatures below and above the rearing temperature: 3472% increase at temperatures 15a)“C and 41-76% increase at 30-45”C [Fig. 1 (A)]. Nondiapausing pharate adults (red-eye stage), which were reared at 25°C and received a l-h exposure to each of the experimental temperatures (0-SOC) showed a significant increase of % phosphorylase a at 0°C (60%) and at high temperatures in the range of 30-50°C (31-71% increase) [Fig. I (B)]. Phosphorylase activation was thus evident at
Phosphorylase assa)
Phosphorylase was assayed in the direction of glycogen breakdown with a coupled enzyme system slightly modified from Childress and Sacktor (1970) and Ziegler et al. (1979). All assays were conducted with 1 ml reaction mixture containing a final concentration of 38 mM triethanolamine acetate, 4.8 mM imidazole, 4.8 mM magnesium acetate, 1.9 mM EDTA, 1.3 mM DTT, 76 mM potassium phosphate, 10.5 mg/ml glycogen, 0.57 mM NADP. 1.2pM glucose- 1,6_diphosphate, 0.89 units glucose-6-phosphate dehydrogenase and 0.28 units phosphoglucomutase (pH 7.0). The enzymes were diluted with buffer slightly modified from Childress and Sacktor (1970): glucose-6-phosphate dehydrogenase was prepared with a diluting buffer containing 20 mM triethanolamine acetate, 5 mM imidazole, 1 mM magnesium acetate and 0.002% bovine serum albumin (pH 7.0); phosphoglucomutase was diluted with a buffer containing 10 mM triethanolamine acetate, 0.1 mM EDTA. 1 mM DTT and 0.10% bovine serum albumin (pH 7.0). The non-enzymatic components were prepared as several stock solutions, each adjusted to pH 7.0. The reaction mixture was prepared freshly and used within 24 h. Two cuvettes containing I ml reaction mixture each were equilibrated at 30°C in a shaking waterbath. To measure activity of phosphorylase a, 50 ~1 supernatant was added to the mixture in a cuvette. and a second 50 ~1 aliquot of supernatant and 50 ~1 40 mM 5’-AMP (pH 7.0) were added to the reaction mixture in the other cuvette to assay total phosphorylase. Reduction of NADP was measured by record-
6o
(A)
r
’
60
-s 40
0
0
5
10 15 20 25 30 35 40 45 Temperature
50
(“C)
Fig. I. Effects of different temperatures on % phosphorylase a in diapausing (A) and non diapausing (B) flesh flies. Diapausing pupae (13 days in diapause) were reared at 20 C and treated 2 h at each temperature. Nondiapausing pharate adults (red-eye stage) were reared at 25°C and exposed for I h to each temperature. R + SE, % phosphorylase a of 5 replicates. For the diapausing pupae all increases were significantly higher than the response at the rearing temperature (20°C). except 25°C [T (LSD) test, P < 0.051. In the nondiapausing group. the response at the rearing temperature (25°C) differed significantly (P < 0.05) from the response at 0 and 3&5O”C by T (LSD) test, GLM Procedure, SAS System.
Temperature effect on phosphorylase 0°C and above 30°C for both diapausing and nondiapausing flies. Under these experimental conditions, values for diapausing pupae were consistently lower than values obtained for nondiapausing individuals.
60
60
551
r
Feeding
larvae
40
E#ect of cold exposure time on phosphorylase
Nondiapausing pharate adults and 13-day diapausing pupae were analysed for phosphorylase activity following various durations of exposure to 0°C in viuo (Fig. 2). 0°C Was chosen as the test temperature because brief exposure to 0°C is known to induce a rapid cold-hardening response that protects against cold-shock injury and to elevate glycerol concentrations (Chen et al., 1987). For diapausing pupae % phosphorylase a was again relatively lower than in nondiapausing flies. In the nondiapausing pharate adults % phosphorylase a increased from 36.1% at 25°C to 52.8% within 30 min of exposure to 0°C and increased to 57.9% within 1 h, i.e. 60% increase overall. The proportion of phosphorylase a remained elevated for exposure times up to 24 h. For diapausing pupae phosphorylase activity increased from 18.4% at 20°C to its highest value (38.9%) within 1 h of exposure to 0°C. Though the % phosphorylase a in diapausing pupae remained consistently lower than in nondiapausing individuals the % increase was higher in diapausing pupae (100% increase for 1 h exposure to 0°C vs 67% maximal increase observed in nondiapausing flies after a 2-h exposure to 0°C).
Y-
Wondenng
Phorate
adults
Phosphorylase activation at d@erent stages of nondiapause development
To determine the effect of cold stress (2 h at 0°C) and heat stress (2 h at 40°C) on elevation of active phosphorylase at different stages of nondiapause development five stages of S. crassipalpis reared at 25°C were exposed to 0 or 40°C for 2 h and then enzyme activity was compared to that of flies held continuously at 25C (Fig. 3). From the overall data in Fig 3 significant differences were found for the temperatures (F = 25.0, P < 0.001) and developmental stages (F = 10.7, P < 0.001) tested. The overall means of % phosphorylase a pooled from the data
1
1
2
3
4
’
(
24
Time at 0°C (hl
Fig 2. Increase of phosphorylase a in response to increased exposure to O‘C. 13-Day diapausing pupae (D) reared at 20 C and nondiapausing pharate adults (red-eye stage) (ND) reared at 25°C were exposed to 0°C in tko. X & SE, % phosphorylase a of 5 replicates. The % phosphorylase a at each interval was significantly higher (P < 0.05) than the value at time 0. except for the 24 h exposure of diapausing pupae analysed by T (LSD) test, GLM Procedure, SAS System.
larvae
(red-eye
stage
1
Adults 60
”
0 Temperature
40
25
(“C)
Fig. 3. % Phosphorylase a in different developmental stages of nondiapausing flies in response to cold stress (2 h at 0°C) and heat stress (2 h at 4OC). R + SE, % phosphorylase a of 5 replicates. Treatments indicated by different letters are significantly different (P < 0.05) T(LSD) test analysed by GLM Procedure, SAS System.
for all the stages tested were 0°C (57.4%) > 40°C (51.5%) > 25°C (41.3%) and differences between any two temperatures were significant (LSD test, P < 0.05). Phosphorylase a varied from 36 to 49% at 25°C in different stages of nondiapausing flies. Significant differences (LSD test, P < 0.05) were observed between larval (feeding and wandering larva), pupal-pharate adult and adult groups. However, significant differences of phosphorylase induced by cold and heat stress were only detected in pharate adults and adult stages. Phosphorylase activation during diapause
Diapausing pupae were held at either 0 or 20 C from the inception of diapause and phosphorylase activity was determined at IO-day intervals (Fig. 4). At all ages the proportion of active enzyme was greater at 0°C. At 20°C % phosphorylase a was
552
CHENG-PING
5Or
CHEN
and
DAVID
t.
DENL~NGER
adults a l-h exposure to OC produced a 60% increase of phosphorylase a in our experiments, and in diapausing pupae (13 days in diapause) a 2-h exposure caused a 72% increase. A similar cold activation of phosphorylase has been noted in other species. When diapausing pupae of H. cecropia were transferred from 2.5 to O’C fat body phosphorylase a increased about 7-fold in 1 h (Ziegler and Wyatt, 1975). A 2-h exposure of fat body from T. molitor larvae to 4°C increased phosphorylase a 65% (Bahjou et al., 1988). High temperature also elevated the proportion of 40 50 60 70 phosphorylase a in the flesh fly. Diapausing pupae Days an diapouse and non-diapausing pharate adults heat stressed for l-2 h at 3&4O’C increased their proportion of Fig. 4. Effect of temperature stress (2 h at 0 C or 2 h at 4O’C) and long time acclimation at 0 and 2O’C on % phosphorylase a 31-71% and 41-76% respectively. phosphorylase a in diapausing pupae of S. crussipalpis. Lethal high temperatures (45-5O’C kill nondiapaus.f’ k SE, % phosphorylase a of 3-5 replicates. In case where ing pharate adults) caused the greatest increase in no error bars are shown, the SE was smaller than the symbol phosphorylase a (7671% increase) for nondiapausshown on the graph. % Phosphorylase a is affected signifiing pharate adults. This high temperature increase cantly by temperature and age during diapause. analysed by may be part of the organism’s heat shock response. 2-way ANOVA (GLM procedure. SAS system): Model Little information is available on the effect of heat y =A (Age) T (Temperature), d.f. =9. F = 33.71. shock on activity of glycogen phosphorylase. But, P < 0.0001; For Age, d.f. = 6, F = 26.27, P < 0.0001; For phosphorylase is known to be activated by physical Temperature, d.f. = 3. F = 48.59. P -c 0.0001. All temperature treatments differ significantly (P < 0.05) as analysed by injury in silkmoth pupae (Stevenson and Wyatt, T (LSD) test, GLM procedure. SAS system. Curve is the 1964) and by flight activity, which normally is accombest-fit generated by a nonlinear regression program. panied by an increase in body temperature (Van Marrewijk et al., 1980; Goldsworthy, 1983; Ziegler highest at the onset of diapause and then declined to and Schulz, 1986). a relatively stable value for the remainder of the Two temperatures, 0 and 4O’C, were previously diapause period. Likewise, at O’C. the highest value identified as the optimal temperatures to generate biological protection against lethal cold- and heatwas observed at the beginning of diapause and subsequent values were lower. shock injury respectively in pharate adults of S. crassipalpis (Chen et al.. 1987, 1990) and we have Response to cold stress (2 h at O’C) or heat stress (2 h at 40°C) was also monitored in diapausing pupae now demonstrated a concomitant elevation of phosphorylase a at these same temperatures. As suggested reared at 2CrC (Fig. 4). Throughout diapause cold stress consistently elevated the proportion of by previous work (Steele, 1982) activation of phosphorylase may vary with developmental stage. Our phosphorylase a. But, heat stress was ineffective in results indicate that different developmental stages of elevating phosphorylase a during the first half of nondiapausing flies respond differently to cold and diapause. Only late in diapause (after 40 days) was heat stress capable of elevating the proportion of heat shock. Among nondiapausing stages activation of phosphorylase was only observed in pharate adults active phosphorylase. and adult stages. Diapausing pupae can respond to either short-term DISCUSSION or long-term exposure to cold by activating phosphorylase. A higher % phosphorylase a was observed in Phosphorylase activity can be measured in either response to long-term cold acclimation than by a the direction of glycogen synthesis or breakdown. The degradation (spectrophotometric) assay embrief cold stress. Both short- and long-term exposure to 0 C previously was known to markedly increase ployed in this experiment is generally considered to be glycerol concentration (Chen et al., 1987; Lee et al., comparable to the synthesis (phosphate) assay but may overestimate phosphorylase a due to reduction 1987). In diapausing pupae H. cecropiu exposure to of NADP by other enzymes (Ziegler et al.. 1979). In 4 C also caused the active form of fat body phosphoour work with whole body extractions of flesh flies we rylase to increase, followed by a rise of haemolymph glycerol concentration (Ziegler et al., 1979). The found 20-35% phosphorylase a in untreated diapausing pupa and nondiapausing pharate adult. These biochemical consequences of high temperature elevation of phosphorylase is not clear. We have no values are higher than results obtained with the degradation assay in other insects: 810% in fat body evidence that glycerol is elevated by heat shock (Chen of diapausing pupae of Hyalophora cecropia, 17% in et al., 1990). In diapausing pupae a 2-h exposure to 40 C activates phosphorylase significantly only durfat body of Locusta migratoria adults (Ziegler et al.. 1979) and 7% in larvae and adult males of Manduca ing the last half of diapause. Interestingly, this change sexta (Siegert and Ziegler, 1983; Ziegler and Schulz, in response coincides with several other events that occur at this same time: pupae switch from utilization 1986). Activation of phosphorylase was evident at temof fat to nonfat reserves (Adedokun and Denlinger, 1984), they become highly sensitive to stimulation by peratures above and below the rearing temperature. The optimal low temperature for activating phospho20-hydroxyecdysone (Denlinger et al., 1988) and rylase was 0 C for both diapausing pupae and nondithey become less tolerant of low ( - 17-C) temperature (Lee et al.. 1987). While the implications of a apausing pharate adults. In nondiapausing pharate
4iii’
Temperature effect on phosphorylase switch in the high temperature response remain obscure the changes underscore the dynamic nature of diapause development. Acknowledgements-We thank Dr Richard E. Lee Jr for his review of the manuscript. This research was supported by USDA-CRGO Grant No. 88-37153-3473 and NSF Grant No. DCB-8811317. REFERENCES
Enr. 9, 361-364.
Bahjou A., Gourdoux L., Moreau R. and Dutrieu J. (1988) In vitro regulation of glycogen phosphorylase of the larval fat body of Tenebrio molitor. Camp. Biochem. Physiol. 89B, 233-237.
Chen C-P. Denlinger D. L. and Lee R. E. Jr (1987) Cold-shock injury and rapid cold hardening in the flesh &ssipal&.
Gen. Camp. Endocr.
14,
78-85.
Goldsworthy G. J. (1983) The endocrine control of flight metabolism in locusts. Ado. Insecf Phvsiol. 17. 149-204. Hayakawa Y. (1985) Activation mechanism of’insect fat body phosphorylase by cold: phosphorylase kinase, phosphatase and ATP level. Insect Biochem. 15, 123-128. Hayakawa Y. and Chino H. (1983) Insect fat body phosphorylase kinase is Ca’+-independent and acts even at 0°C. Biochem. Biophys. Acta 746, 14-17. Lee R. E. Jr, Chen C-P., Meacham M. H. and Denlinger
Adedokun T. A. and Denlinger D. L. (1984) Cold hardiness: a component of the diapause syndrome in pupae of the flesh flies, Sarcophaga crassipalpis and S. bullata. Physiol.
By, Sarcophagi
on glucogen phosphorylase.
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Chen C-P.. Lee R. E. Jr and Denlinaer D. L. (1990) Cold \ shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures. Physiol. Ent. In Press. Childress C. C. and Sacktor 9. (1970) Regulation of glycogen metabolism in insect flight muscle. J. biol. Chem. 245, I
2927-2936.
Denlinger D. L. (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biol. Bull. 142, 1l-24. Denlinger D. L., Giebultowicz J. and Adedokin T. (1988) Insect diapause: dynamics of hormone sensitivity and vulnerability to environmental stress. In Endocrinological Frontiers ii Physiological Insect Ecology. (Edited by Sehnal F.. Zabza A. and Denlinaer D. L.). DO. 309324. Wroclaw Technical University Piess, W&&w. Fraenkel G. and Hsiao C. (1968) Morphological and endocrinological aspects of pupal diapause in a flesh fly, Sarcophaga argyrostoma (Robineau-Desvoidy). J. Insect Physiol. 14, 707-718.
Gade G. (198 1) Activation of fat body glycogen phosphorylase in Locusta migratoria by corpus cardiacum extract and synthetic adipokinetic hormone. J. Insect Physiol. 27, 155-161.
Goldsworthy G. J. (1969) Hyperglycaemic factors from the corpus cardiacum of Locusta migraroria. J. Insect Physiol. 15, 2131-2140. Goldsworthy G. J. (1970) The action of hyperglycaemic factors from the corpus cardiacum of Locusta migratoria
D. L. (1987) Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis. J. Insect Physiol. 8, 587-592.
SAS Institute Inc. (1985) SAS User’s Guide: Stafistics, Version 5 Edition, Cary N.C. Siegert K. and Ziegler R. (1983) A hormone from the corpora cardiaca controls fat body glycogen phosphorylase during starvation in tobacco homworm larvae. Nature 301, 526527. Steele J. E. (1963) The site of action of insect hyperglycaemic hormone. Gen. Camp. Endocr. 3, 4652. Steele J. E. (1982) Glycogen phosphorylase in insects. Insect Biochem. 12, 131-147.
Stevenson E. and Wyatt G. R. (1964) Glycogen phosphorylase and its activation in silkmoth fat body. Archs Biochem. Biophys. 108, 42&429. Van Marrewijk W. J. A., Van den Broek A. Th. M. and Beenakkers A.M.Th. (1980) Regulation of glycogenolysis in the locust fat body during flight. Insect Biochem. 10, 675-679. Van Marrewijk W. J. A., Van den Broek A. Th. M., De Graan P. N. E. and Beenakkers A. M. Th. (1988) Formation of partially phosphorylated glycogen phosphorylase in the fat body of the migratory locust. Insect Biochem. 18, 821-827. Wiens A. W. and Gilbert L. I. (1967) The phosphorylase system of the silkmoth Hyalophora cecropia. Camp. Biochem. Physiol. 21, 145-160.
Wyatt G. R. (1975) Regulation of protein and carbohydrate metabolism in insect fat body. Verh. Dtsch. Zool. Ges. 1974, 209-226 Ziegler R. and Schulz M. (1986) Regulation of carbohydrate metabolism during flight in Manduca sexta J. Insect Physiol. 32, 997-1001.
Ziegler R. and Wyatt G. R. (1975) Phosphorylase and glycerol production activated by cold in diapausing silkmoth pupae. Nature 254, 622623. Ziegler R., Ashida M., Fallon A. M., Wimer L. T., Wyatt S. S. and Wyatt G. R. (1979) Regulation of glycogen phosphorylase in fat body of Cecropia silkmoth pupae. J. camp. Physiol. 131, 321-332.
1990 Copyright
0022-1910/90 $3.00 + 0.00 gc’ 1990 Pergamon Press plc
ACTIVATION OF PHOSPHORYLASE IN RESPONSE TO COLD AND HEAT STRESS IN THE FLESH FLY, SARCOPHAGA CHENG-PING Department of Entomology,
CRASSIPALPIS
CHENand
DAVID L. DENLINGER*
Ohio State University, 1735 Neil Avenue, Columbus, OH 43210, U.S.A.
(Received 9 January 1990; revised 6 March 1990) Abstract-Glycogen phosphorylase can be activated in diapausing pupae and nondiapausing pharate adults of the flesh fly, Sarcophaga crussipalpis in response to both cold and heat stress. The greatest activation was observed at 0 and above 30°C. Activation was rapid: following exposure to 0°C activated phosphorylase increased 36-S% within 30min. The proportion of activated phosphorylase was consistently lower in diapausing pupae than in nondiapausing pharate adults. Among the nondiapausing stages, only pharate adults and adults responded to cold stress (2 h at 0°C) and heat stress (2 h at 40°C) by activating phosphorylase. No such response was observed in larvae or young pupae. During diapause, the proportion of activated phosphorylase was highest at the onset of diapause, declined and remained fairly constant throughout the remainder of diapause. Diapausing pupae held continuously at WC maintained a higher proportion of activated phosphorylase throughout the diapause than pupae held at 20°C. Cold stress consistently activated phosphorylase in diapausing pupae held at 2O”C, but heat stress elicited this response only during the last half of diapause. The activation of phosphorylase by brief periods of chilling and the activation observed in diapausing pupae held continuously at low temperature is consistent with our earlier work showing an elevation of glycerol under these conditions.
Ke_v Word Index:
Glycogen phosphorylase;
cold shock; heat shock; pupa1 diapause; Sarcophugu
crassipalpis
INTRODUCTION
a source of energy in all tissues, serves both as a metabolic reserve and a synthetic “building block” for macromolecules. The insect fat body is the site of the major glycogen reserves that are utilized for metabolism of lipids, carbohydrates and proteins (Wyatt, 1957; Steele, 1982). Glycogen phosphorylase, present in the fat body and flight muscle of many insects, exists in two interconvertible forms: an active phosphorylated a form and an inactive dephosphorylated b form (Stevenson and Wyatt, 1964; Van Marrewijk et al., 1988). Phosphorylase can be activated by starvation (Goldsworthy, 1970; Gade, 1981), flight activity (Van Marrewijk et al., 1980; Ziegler and Schulz, 1986), exposure to cold (Ziegler and Wyatt, 1975; Ziegler et al., 1979; Bahjou et al., 1988), and by a factor extracted from the corpora cardiaca (Steele, 1963; Wiens and Gilbert, 1967; Goldsworthy, 1969; Gade, 1981). In diapause pupae of the silkmoth, Hyalophora cecropiu, cold exposure (4°C) increases glycogen phosphorylase activity of the fat body about 50% and glycerol accumulates in the haemolymph (Ziegler ef al., 1979). Thus, this enzyme acts as one of the key enzymes regulating synthesis of cryoprotectants, i.e. glycerol, trehalose or other low molecular weight substances synthesized from glycogen by overwintering diapause insects. The activation of phosphorylase by low temperature exposure, however, is not restricted to diapausing insects. Phosphorylase activity can also be elevated rapidly by low Glycogen,
*To whom all correspondence
should be addressed.
temperature in fat body cultures from Tenebriu molitar, a nondiapausing species (Bahjou et al., 1988). The fact that fat body phosphorylase kinase continues to function at low temperature, while phosphorylase phosphatase does not, favours the activity of phosphorylase (Hayakawa and Chino. 1983; Hayakawa, 1985). In Surcophuga brief exposure to moderately low or high temperature generates protection against injury caused by more extreme low or high temperature. Elevation of glycerol is at least partially responsible for the protection against cold-shock injury that is generated by a brief period of chilling, but we have no evidence that glycerol protects against heat-shock injury (Chen et al., 1987, 1990). The rapid elevation of glycerol at low temperature suggests an important role for phosphorylase in protecting the fly from cold-shock injury. In these experiments with S. crassipalpis we test the possibility that activation of phosphorylase may be stimulated by cold and heat stress. In addition, we examine this response in different developmental stages and in diapausing pupae. MATERIALS AND METHODS
Animals
A colony of the flesh fly, Sarcophaga crassipalpis Macquart, was reared in the laboratory as described by Denlinger (1972). Parental adults were reared at 25°C with either a diapause-inducing photoperiod (12 h light-12 h dark) or a nondiapause photoperiod (15 h light-9 h dark). Larvae and pupae were 549
550
CHENG-PING CHEN
and
maintained either at 20 or 25-C under the maternal photophase. Under the short-day conditions at 20°C a high incidence ( > 95%) of pupal diapause is produced. The developmental status of each pupa was determined by removing the anterior portion of the puparium and looking for signs of antenna1 formation and the eye-pigmentation characteristic of pharate adult development (Fraenkel and Hsiao, 1968). Chemicals
NADP, cyclic AMP, oyster glycogen, dithiothreitol (DTT), EDTA, glucose-l ,6-diphosphate, glucose-6phosphate dehydrogenase and phosphoglucomutase were purchased from Sigma (St Louis, MO.). All other chemicals were reagent grade. The glycogen was treated with charcoal (Merck, activated) before use, to remove traces of AMP (Stevenson and Wyatt, 1964). Preparation of phosphorylase
Abdomens of nondiapausing pharate adults or whole bodies of other stages were immediately homogenized with a plastic disposable pestle (Kontes, Vineland, N.J.) in 1 ml homogenizing buffer (20 mM triethanolamine acetate, 5 mM EDTA, 20 mM NaF. pH 7.0) in a microfuge tube and centrifuged at 8000 g at 4’C for IOmin. The supernatant, between the pellet and a fatty surface layer, was withdrawn and used for the assay. The extracts were stored at - 4°C and analysed within 1 week. Most assays were done within 2 days.
DAVID L. DENLINGER
ing A340min a spectrophotometer (Varian DMS 100) for at least 15 min. A comparison of phosphorylase activity obtained in the presence and absence of 5’-AMP permitted calculation of the amount of enzyme which was present in the active form. Data expression and analysis
Activity of phosphorylase was expressed as % phosphorylase a of total phosphorylase. The increase of phosphorylase activity was expressed by comparing % phosphorylase a of the treatment with the value for the controls. Statistical significance of differences were estimated using SAS programs (SAS Institute Inc., 1985). RESULTS
Effect of temperature on phosphorylase
Diapausing pupae (13 days in diapause) reared at 20°C and exposed for 2 h to temperatures ranging from 0 to 45°C significantly increased the proportion of phosphorylase a at temperatures below and above the rearing temperature: 3472% increase at temperatures 15a)“C and 41-76% increase at 30-45”C [Fig. 1 (A)]. Nondiapausing pharate adults (red-eye stage), which were reared at 25°C and received a l-h exposure to each of the experimental temperatures (0-SOC) showed a significant increase of % phosphorylase a at 0°C (60%) and at high temperatures in the range of 30-50°C (31-71% increase) [Fig. I (B)]. Phosphorylase activation was thus evident at
Phosphorylase assa)
Phosphorylase was assayed in the direction of glycogen breakdown with a coupled enzyme system slightly modified from Childress and Sacktor (1970) and Ziegler et al. (1979). All assays were conducted with 1 ml reaction mixture containing a final concentration of 38 mM triethanolamine acetate, 4.8 mM imidazole, 4.8 mM magnesium acetate, 1.9 mM EDTA, 1.3 mM DTT, 76 mM potassium phosphate, 10.5 mg/ml glycogen, 0.57 mM NADP. 1.2pM glucose- 1,6_diphosphate, 0.89 units glucose-6-phosphate dehydrogenase and 0.28 units phosphoglucomutase (pH 7.0). The enzymes were diluted with buffer slightly modified from Childress and Sacktor (1970): glucose-6-phosphate dehydrogenase was prepared with a diluting buffer containing 20 mM triethanolamine acetate, 5 mM imidazole, 1 mM magnesium acetate and 0.002% bovine serum albumin (pH 7.0); phosphoglucomutase was diluted with a buffer containing 10 mM triethanolamine acetate, 0.1 mM EDTA. 1 mM DTT and 0.10% bovine serum albumin (pH 7.0). The non-enzymatic components were prepared as several stock solutions, each adjusted to pH 7.0. The reaction mixture was prepared freshly and used within 24 h. Two cuvettes containing I ml reaction mixture each were equilibrated at 30°C in a shaking waterbath. To measure activity of phosphorylase a, 50 ~1 supernatant was added to the mixture in a cuvette. and a second 50 ~1 aliquot of supernatant and 50 ~1 40 mM 5’-AMP (pH 7.0) were added to the reaction mixture in the other cuvette to assay total phosphorylase. Reduction of NADP was measured by record-
6o
(A)
r
’
60
-s 40
0
0
5
10 15 20 25 30 35 40 45 Temperature
50
(“C)
Fig. I. Effects of different temperatures on % phosphorylase a in diapausing (A) and non diapausing (B) flesh flies. Diapausing pupae (13 days in diapause) were reared at 20 C and treated 2 h at each temperature. Nondiapausing pharate adults (red-eye stage) were reared at 25°C and exposed for I h to each temperature. R + SE, % phosphorylase a of 5 replicates. For the diapausing pupae all increases were significantly higher than the response at the rearing temperature (20°C). except 25°C [T (LSD) test, P < 0.051. In the nondiapausing group. the response at the rearing temperature (25°C) differed significantly (P < 0.05) from the response at 0 and 3&5O”C by T (LSD) test, GLM Procedure, SAS System.
Temperature effect on phosphorylase 0°C and above 30°C for both diapausing and nondiapausing flies. Under these experimental conditions, values for diapausing pupae were consistently lower than values obtained for nondiapausing individuals.
60
60
551
r
Feeding
larvae
40
E#ect of cold exposure time on phosphorylase
Nondiapausing pharate adults and 13-day diapausing pupae were analysed for phosphorylase activity following various durations of exposure to 0°C in viuo (Fig. 2). 0°C Was chosen as the test temperature because brief exposure to 0°C is known to induce a rapid cold-hardening response that protects against cold-shock injury and to elevate glycerol concentrations (Chen et al., 1987). For diapausing pupae % phosphorylase a was again relatively lower than in nondiapausing flies. In the nondiapausing pharate adults % phosphorylase a increased from 36.1% at 25°C to 52.8% within 30 min of exposure to 0°C and increased to 57.9% within 1 h, i.e. 60% increase overall. The proportion of phosphorylase a remained elevated for exposure times up to 24 h. For diapausing pupae phosphorylase activity increased from 18.4% at 20°C to its highest value (38.9%) within 1 h of exposure to 0°C. Though the % phosphorylase a in diapausing pupae remained consistently lower than in nondiapausing individuals the % increase was higher in diapausing pupae (100% increase for 1 h exposure to 0°C vs 67% maximal increase observed in nondiapausing flies after a 2-h exposure to 0°C).
Y-
Wondenng
Phorate
adults
Phosphorylase activation at d@erent stages of nondiapause development
To determine the effect of cold stress (2 h at 0°C) and heat stress (2 h at 40°C) on elevation of active phosphorylase at different stages of nondiapause development five stages of S. crassipalpis reared at 25°C were exposed to 0 or 40°C for 2 h and then enzyme activity was compared to that of flies held continuously at 25C (Fig. 3). From the overall data in Fig 3 significant differences were found for the temperatures (F = 25.0, P < 0.001) and developmental stages (F = 10.7, P < 0.001) tested. The overall means of % phosphorylase a pooled from the data
1
1
2
3
4
’
(
24
Time at 0°C (hl
Fig 2. Increase of phosphorylase a in response to increased exposure to O‘C. 13-Day diapausing pupae (D) reared at 20 C and nondiapausing pharate adults (red-eye stage) (ND) reared at 25°C were exposed to 0°C in tko. X & SE, % phosphorylase a of 5 replicates. The % phosphorylase a at each interval was significantly higher (P < 0.05) than the value at time 0. except for the 24 h exposure of diapausing pupae analysed by T (LSD) test, GLM Procedure, SAS System.
larvae
(red-eye
stage
1
Adults 60
”
0 Temperature
40
25
(“C)
Fig. 3. % Phosphorylase a in different developmental stages of nondiapausing flies in response to cold stress (2 h at 0°C) and heat stress (2 h at 4OC). R + SE, % phosphorylase a of 5 replicates. Treatments indicated by different letters are significantly different (P < 0.05) T(LSD) test analysed by GLM Procedure, SAS System.
for all the stages tested were 0°C (57.4%) > 40°C (51.5%) > 25°C (41.3%) and differences between any two temperatures were significant (LSD test, P < 0.05). Phosphorylase a varied from 36 to 49% at 25°C in different stages of nondiapausing flies. Significant differences (LSD test, P < 0.05) were observed between larval (feeding and wandering larva), pupal-pharate adult and adult groups. However, significant differences of phosphorylase induced by cold and heat stress were only detected in pharate adults and adult stages. Phosphorylase activation during diapause
Diapausing pupae were held at either 0 or 20 C from the inception of diapause and phosphorylase activity was determined at IO-day intervals (Fig. 4). At all ages the proportion of active enzyme was greater at 0°C. At 20°C % phosphorylase a was
552
CHENG-PING
5Or
CHEN
and
DAVID
t.
DENL~NGER
adults a l-h exposure to OC produced a 60% increase of phosphorylase a in our experiments, and in diapausing pupae (13 days in diapause) a 2-h exposure caused a 72% increase. A similar cold activation of phosphorylase has been noted in other species. When diapausing pupae of H. cecropia were transferred from 2.5 to O’C fat body phosphorylase a increased about 7-fold in 1 h (Ziegler and Wyatt, 1975). A 2-h exposure of fat body from T. molitor larvae to 4°C increased phosphorylase a 65% (Bahjou et al., 1988). High temperature also elevated the proportion of 40 50 60 70 phosphorylase a in the flesh fly. Diapausing pupae Days an diapouse and non-diapausing pharate adults heat stressed for l-2 h at 3&4O’C increased their proportion of Fig. 4. Effect of temperature stress (2 h at 0 C or 2 h at 4O’C) and long time acclimation at 0 and 2O’C on % phosphorylase a 31-71% and 41-76% respectively. phosphorylase a in diapausing pupae of S. crussipalpis. Lethal high temperatures (45-5O’C kill nondiapaus.f’ k SE, % phosphorylase a of 3-5 replicates. In case where ing pharate adults) caused the greatest increase in no error bars are shown, the SE was smaller than the symbol phosphorylase a (7671% increase) for nondiapausshown on the graph. % Phosphorylase a is affected signifiing pharate adults. This high temperature increase cantly by temperature and age during diapause. analysed by may be part of the organism’s heat shock response. 2-way ANOVA (GLM procedure. SAS system): Model Little information is available on the effect of heat y =A (Age) T (Temperature), d.f. =9. F = 33.71. shock on activity of glycogen phosphorylase. But, P < 0.0001; For Age, d.f. = 6, F = 26.27, P < 0.0001; For phosphorylase is known to be activated by physical Temperature, d.f. = 3. F = 48.59. P -c 0.0001. All temperature treatments differ significantly (P < 0.05) as analysed by injury in silkmoth pupae (Stevenson and Wyatt, T (LSD) test, GLM procedure. SAS system. Curve is the 1964) and by flight activity, which normally is accombest-fit generated by a nonlinear regression program. panied by an increase in body temperature (Van Marrewijk et al., 1980; Goldsworthy, 1983; Ziegler highest at the onset of diapause and then declined to and Schulz, 1986). a relatively stable value for the remainder of the Two temperatures, 0 and 4O’C, were previously diapause period. Likewise, at O’C. the highest value identified as the optimal temperatures to generate biological protection against lethal cold- and heatwas observed at the beginning of diapause and subsequent values were lower. shock injury respectively in pharate adults of S. crassipalpis (Chen et al.. 1987, 1990) and we have Response to cold stress (2 h at O’C) or heat stress (2 h at 40°C) was also monitored in diapausing pupae now demonstrated a concomitant elevation of phosphorylase a at these same temperatures. As suggested reared at 2CrC (Fig. 4). Throughout diapause cold stress consistently elevated the proportion of by previous work (Steele, 1982) activation of phosphorylase may vary with developmental stage. Our phosphorylase a. But, heat stress was ineffective in results indicate that different developmental stages of elevating phosphorylase a during the first half of nondiapausing flies respond differently to cold and diapause. Only late in diapause (after 40 days) was heat stress capable of elevating the proportion of heat shock. Among nondiapausing stages activation of phosphorylase was only observed in pharate adults active phosphorylase. and adult stages. Diapausing pupae can respond to either short-term DISCUSSION or long-term exposure to cold by activating phosphorylase. A higher % phosphorylase a was observed in Phosphorylase activity can be measured in either response to long-term cold acclimation than by a the direction of glycogen synthesis or breakdown. The degradation (spectrophotometric) assay embrief cold stress. Both short- and long-term exposure to 0 C previously was known to markedly increase ployed in this experiment is generally considered to be glycerol concentration (Chen et al., 1987; Lee et al., comparable to the synthesis (phosphate) assay but may overestimate phosphorylase a due to reduction 1987). In diapausing pupae H. cecropiu exposure to of NADP by other enzymes (Ziegler et al.. 1979). In 4 C also caused the active form of fat body phosphoour work with whole body extractions of flesh flies we rylase to increase, followed by a rise of haemolymph glycerol concentration (Ziegler et al., 1979). The found 20-35% phosphorylase a in untreated diapausing pupa and nondiapausing pharate adult. These biochemical consequences of high temperature elevation of phosphorylase is not clear. We have no values are higher than results obtained with the degradation assay in other insects: 810% in fat body evidence that glycerol is elevated by heat shock (Chen of diapausing pupae of Hyalophora cecropia, 17% in et al., 1990). In diapausing pupae a 2-h exposure to 40 C activates phosphorylase significantly only durfat body of Locusta migratoria adults (Ziegler et al.. 1979) and 7% in larvae and adult males of Manduca ing the last half of diapause. Interestingly, this change sexta (Siegert and Ziegler, 1983; Ziegler and Schulz, in response coincides with several other events that occur at this same time: pupae switch from utilization 1986). Activation of phosphorylase was evident at temof fat to nonfat reserves (Adedokun and Denlinger, 1984), they become highly sensitive to stimulation by peratures above and below the rearing temperature. The optimal low temperature for activating phospho20-hydroxyecdysone (Denlinger et al., 1988) and rylase was 0 C for both diapausing pupae and nondithey become less tolerant of low ( - 17-C) temperature (Lee et al.. 1987). While the implications of a apausing pharate adults. In nondiapausing pharate
4iii’
Temperature effect on phosphorylase switch in the high temperature response remain obscure the changes underscore the dynamic nature of diapause development. Acknowledgements-We thank Dr Richard E. Lee Jr for his review of the manuscript. This research was supported by USDA-CRGO Grant No. 88-37153-3473 and NSF Grant No. DCB-8811317. REFERENCES
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