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Activities of some microsomal enzymes of the yellow mealworm, Tenebrio molitor (Linné)
PESTICIDE
BIOCHEMISTRY
Activities
AND
PHYSIOLOGY
30, 35-39 (1988)
of Some Microsomal Enzymes of the Yellow Mealworm, Tenebrio molitor (Lint-k) I. Basal Levels and lnducibility
ELIANN EGAAS,* ELISABETHGRAMJENSEN,?
ANDJANNECHE UTNE SKAARE-~
*Norwegian Plant Protection Institute, Department of Entomology. P.O. Box 70. N-1432 and fThe Norwegian College of Veterinary Medicine, Department of Pharmuco1og.v Dep. Oslo 1, N-Oslo. Nor\+,a)
Aas-NLH. Notxltry. clnd To.vkolo,qv.
Received January 26. 1987: accepted September I. 1987 The basal levels and the induction potential of some microsomal enzymes of the yellow mealworm soft tissue without gut was established. The insecticides aldrin, lindane. and endosulfan were administered in the feed for 3 days of exposure. Cytochrome P-420. NADPH-cytochrome (’ reductase, aldrin epoxidase, and heptachlor epoxidase were chosen as mixed-function oxidase activity parameters. The maximal induction value of cytochrome P-450 was reached at SO00 ppm of endosulfan and was 334% of the control value. The maximal induction values of the aldrin and heptachlor epoxidase and the NADPH cytochrome c reductase activities were reached at 2500 ppm of endosulfan and were 5622, 1390, and 324% of the respective control values. c IYXX AGI~WW Press. Inc.
INTRODUCTION
worm, Tenebrio molitor, and to investigate the adaptive potential of the activities by exposure to different levels of the halogenated hydrocarbon insecticides aldrin, lindane, and endosulfan.
The microsomal enxymes of the mixedfunction oxidase (MFO)’ system are a group of enzymes dependent on cytochrome P-450. They are widely distributed through the phylogenesis and are known to play an important role in the primary degradation of drugs, pesticides, and other xenobiotics (I-3). In insect larvae, the MFO systems of lepidopterous larvae have been extensively studied (4, 5). Fewer data concerning MFO activity are available on coleopteran larva1 tissues (6). Halogenated hydrocarbon insecticides are known MFO inducers in several species (7). In the target organisms, the ability of the halogenated hydrocarbon insecticides to stimulate their own metabolism and that of other pesticides has been linked to the phenomenons of resistance and cross-resistance (8). The aim of the present study was to establish the basal levels of MFO activities in a coleopteran larvae, the yellow mealt Abbreviation dase.
used: MFO,
mixed-function
MATERIALSANDMETHODS
Insects. The yellow mealworm, T. molitor, were initially bought in a local pet animal shop in Oslo, Norway, and reared for 4 to 8 weeks in our laboratory. The larvae were maintained in plastic buckets (35 x 25 x 13 cm) on a bed of sand and a basal mixture of whole-meal flour and wheat bran (3:l). To provide moisture, slices of apples were administered. At the start of an experiment, the larvae were selected by body weight. During the experiments, each group of 60 to 70 larvae was kept in glass vials (10 x 8 x 6 cm) on a l-cm bed of sand and 40 g basal flour mixture. The larvae were kept at 25°C and 60% humidity in a Mode1 Psycrotherm R27 incubator. Chemicals. Aldrin (99% purity) and lindane (99% purity) were purchased from Chem. Service Inc. (West Chester, PA).
oxi.
35 0048-3575188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
36
EGAAS,
JENSEN,
Heptachlor (100% purity) was purchased from Unilab Research Corp. (Berkeley, CA). Endosulfan (98.8% purity) was a gift from Dr. H. 0. Friestad, Chemical Division, Agricultural University of Norway. NADH, NADPH, cytochrome c, and reduced glutathione were all obtained from Sigma (St. Louis, MO). All other chemicals were of the highest purity commercially available. Preparation of microsomal enzymes. Microsomal pellet was obtained by pressing out the soft tissue through razorblade-made incisions at the oral and anal regions. After removing the gut, the remaining tissue, which in the following is named “carcass,” was added to 4 vol of buffer A (0.2 M potassium phosphate buffer, pH 7.8, 1 mM EDTA, and 2 mM reduced glutathione). The mixture was homogenized in a PotterElvehjem homogenizer by three manual strokes, rotating the pistil continually. The homogenate was centrifuged at 10,OOOg for 15 min (0 to 4°C) in a Sorvall centrifuge, Model RC-2. The postmitochondrial supernatant was filtered through glass-wool and recentrifuged at 210,OOOg for 25 min (34°C) in a Centrikon ultracentrifuge, Model T-2070 (9). The microsomal pellet was rinsed once with buffer B (0.3 M potassium phosphate buffer, pH 7.8, 1 mM EDTA) and suspended in the same buffer to make a final concentration of 2 to 4 mg protein/ml. An aliquot of the microsomal suspension was used immediately for enzyme assays, while the remaining suspension was added glycerol to a final concentration of 40% before measuring the content of cytochrome P-450. All tissue preparations were kept at 0 to 4°C until analyzed. Enzyme assays. Aldrin and heptachlor epoxidase assays were analyzed according to Moldenke and Terriere (10) with the exception that buffer A was used as buffer during the incubations and NADPH was added instead of a NADPH-generating system. The relationship between the aldrin epoxidase activity and the protein concentration was studied with microsomal
AND SKAARE
protein obtained from control and aldrintreated larvae (62 ppm in the feed for 3 days). Using protein from control larvae, this relationship was linear over the range 0.5 to 3.8 mg protein per incubation. The epoxidase activity prepared from aldrintreated larvae was linear with the protein concentration up to about 2.0 mg per incubation. Dieldrin and heptachlor epoxide were extracted with cyclohexane and analyzed on a Varian gas chromatograph, Model 3700, equipped with an electron capture detector. The column used was 2.5 m x 3 mm i.d. and packed with 1.5% SP-2250 and 1.95% SP-2401 on IOO- 120 mesh Supelcoport. The temperature program was 190°C for 1 min, 3”C/min, 205°C for 6 min. Recovery of dieldrin and heptachlor epoxide was 106- 120%. The reproducibility was & 2.0%. No dieldrin from an in vivo epoxidation of aldrin was found in microsomes from aldrin-treated larvae. NADPH-cytochrome c’ reductase (shortened “reductase”) activity was determined in 5.5 ml incubation mixture containing 4.5 ml buffer B, 0.5 ml tissue homogenate (containing 0.5 to 1.5 mg of protein), 0.5 ml cytochrome c (1.5 mM in buffer B), and 20 ~1 NADPH (10 mM in buffer B). The reaction was registered for 2 to 4 min at 25°C. The extinction coefficient used was 21 mM-t * cm-’ (A,&. Cytochrome P-450 concentration was measured and calculated by the method of Johannesen and DePierre (11). Both sample and reference microsomes were treated with 20 t.~l of NADH (10 m&Z) before adding sodium dithionite to sample cuvette. Thus, we obtained a 50% reduction of the apparent cytochrome P-420 content of the corresponding suspension without NADH. The content and peak position at 448 nm of cytochrome P-450 was not changed by the addition of NADH. The content of cytochrome P-450 always surpassed the content of cytochrome P-420 more than seven times. All spectra were recorded on a double beam Beckman spectrophotometer, Model
Tenrhrio
molitor
MICROSOMAL
35. or with a double beam Kontron spectrophotometer, Model Unikon 860. Protein determinations were done by the Bio-Rad method of Bradford (12), using lyophilized bovine serum albumin (1.21 mg/ml) as standard. Induction studies. The larvae were initially starved for 24 hr. The compounds tested as inducers were dissolved in ether and mixed with the feed (aldrin 31 ppm, lindane from 6 to 250 ppm, and endosulfan from 10 to 10,000 ppm). A corresponding amount of ether only was added to the feed of the control groups. After evaporation of the ether, each group was exposed to their respective diets for 3 days. Larval weight was registered at the end of each experiment. Statistics. Statistical analysis was carried out with the Mann-Whitney test on the difference between two population means (13), using a significance level of 0.05. RESULTS
AND
DISCUSSION
The problems arising from the occurrence of endogenous inhibitors in insect tissue have usually been solved by choosing test insects from which a specific tissue, active in microsomal oxidation, can be isolated (4, 14-16). However, Farnsworth er al. (17) obtained aldrin epoxidase active microsomes from the larval soft tissue without gut, named carcass, of the alfalfa (Autograph californica) and the cabbage looper larvae (Trichoplusia ni). The correspondingly prepared yellow mealworm carcass consisted mainly of fatbody. Other tissues, like gonads or Malpigian tubules, could not be distinguished. Unlike fat-body from cockroach, Periplaneta americana (14), the carcass of the yellow mealworm seemed to be without any endogenous inhibitor. Thus, the postmicrosomal supernatant obtained from yellow mealworm carcass was without reductase activity (18-20). The corresponding microsomal cytochrome P-450 content was almost four times higher than in fat-body microsomes from uninduced
ENZYME
ACTIVITY,
I
37
last (sixth) instars of southern armyworm (Spodoptera eridania) (16) and carcass microsomes from alfalfa and cabbage looper larvae ( 17). The aldrin epoxidase was, respectively, 30, 50, and 150% of the corresponding value in the southern armyworm and the alfalfa and the cabbage looper larvae. Furthermore, the reductase activity in microsomes from the yellow mealworm carcass was half the corresponding value in the southern armyworm. Despite a rather heterogenous, nonsyncronous population of larvae ranging from 0.14 to 0.19 g of body weight, microsomes from aldrin-treated larvae contained a significant increase in both the cytochrome P-450. aldrin, and heptachlor epoxidase values relative to the corresponding controls (Table 1). The 14% reduction in mean body weight in the larvae groups exposed to the high lindane dose (Table 1) is comparable to an I 1% reduction found in the body weight of larvae which had been starved for 4 days (unpublished result). Furthermore, lindanetreated larvae showed signs of heavy intoxication. Therefore, the microsomes prepared from the lindane-treated groups might contain more inert protein from dying larvae relative to the control. This may explain why the epoxidation assays revealed an inducing effect of the lindane treatment, whereas the cytochrome P-450 assay did not (Table 1). In a separate doseresponse experiment, no increase in any of the MFO activity parameters was found in larvae that had been exposed to different doses of lindane (6, 31. 125 ppm). However, a 13% reduction in the mean body weight relative to the controls, and visible signs of intoxication, were observed in the groups that received 125 ppm of lindane. Exposure to endosulfan in the feed (10 ppm) significantly raised only the heptachlor epoxidase and the reductase activities relative to the corresponding activities of the controls (Table 1). However, endosulfan was the only insecticide tested that induced the reductase activity relative to
EGAAS,
38
JENSEN,
AND SKAARE
TABLE
1
Microsomal Epoxidase Activities, Reductase Activity. and Content of Cytochrome Yellow Mealworm Exposed to Aldrin (31 ppm), Lindane (250 ppm). or Endosulfnn for 3 Days
Insecticide Name Control Aldrin Lindane Endosulfan
Dose (wm) 0 31 250
10
Loss of body weight” (%) 3.8 7.3 14.4 7.0
? ” + k
2.0b 1.7 3.4* 2.4
Epoxidases (pmol/min. mgp) Aldrin 75.7 298.0 122.0 107.0
2 k z? 2
Reductase (nmolimin, mgp)
Heptachlor 21.8 57.0* lJ.O* 49.0
19.7 i 9.5 58.8 -+ 22.8* 36.5 ? 13.5* 35.8 z 9.9*
35.1 37.1 37.9 48.8
u A body weight range of 0.14 to 0.19 g was selected. b All numbers are arithmetic means 5 SD of five separate experiments experiment includes two parallels. * Significantly different from the corresponding control (p < 0.05).
the control values. In a separate experiment on a group of larvae with a more homogeneous body weight range of 0.16 to 0.17 g, doses from 50 ppm of endosulfan were highly effective in inducing both the cytochrome P-450 content, aldrin and heptachlor epoxidase, and the reductase activities (Table 2). During the 3 days of the experiment, larvae given more than 1250 ppm gradually stopped feeding and stretched out motionless on top of the flour. Thus, the absorption of endosulfan through the integument might have continued after the larvae stopped feeding. However, not even
2 11.0 f 8.4 2 9.1
?. 13.1*
Cytochrome (nmol/mgp) P-450 0.28 i 0.32 f 0.33 ” 0.35 2
0.01 0.04* 0.05 0.08
performed during 3 weeks, each
a dose of 10,000 ppm increased the mortality of the larvae. This is in contrast to lindane, which reached a lethal tissue concentration when the inducing effect was barely significant. Thus, it seems resonable to suggest that the remarkable inducing effect of endosulfan may be connected to the great tolerance to this insecticide by the yellow mealworm. ACKNOWLEDGMENTS Thanks are due to Dr. Jorgen Stenersen for initiating this project, funded by The Agricultural Research Council of Norway. The technical assistance of
TABLE Microsomal
P-450 in Carcass from (10 ppm) in the Feed
2
Epoxidase Activities, Reductase Activity, and Content of Cytochrome P-450 in Carcass Yellow Mealworm Exposed to Different Doses of Endosulfan in the Feed for 3 Daysapb
from
% of control activity Endosulfan dose @pm)
Epoxidase Aldrin
Reductase
0’ 50 250
100
100
424 1947
249
124
631
132
1250
3513
860
2500 5000
5622 5365 2228
203 324 275 76
10000
100
Cytochrome Heptachlor
1390 1305 610
P-450
loo 121 168 215 285 334 234
a All numbers are either arithmetic means of two assays (cytochrome P-450) or single assay. b A body weight range of 0.16 to 0.17 g was selected. c Control values: aldrin epoxidase, 34.2 pmol/min, mgp; heptachlor epoxidase, 23.6 pmol/min, mgp; reductase, 59.6 nmol/min, mgp; and cytochrome P-450, 0.24 2 0.01 nmol/mgp.
Tenebrio
molitor
MICROSOMAL
Inger Halvorsen and Maalfrid Tofteberg Bjerke in preparing this manuscript is gratefully appreciated. REFERENCES
1. L. B. Brattsten and C. F. Wilkinson, Herbivoreplant interactions: Mixed-function oxidases and secondary plant substances, Science 196, 1349 (1977). 2. L. B. Brattsten, Ecological significance of mixedfunction oxidations, Drug Metab. Rev. 10, 35 (1979). 3.
M. Agosin and A. S. Perry, in “The Physiology of Insecta” (M. Rochstein, Ed.), Vol. V. 2nd ed.. p. 537. Academic Press, New prk/London, 1974.
I,. B. Brattsten. Biochemical defense mechanisms in herbivores against plant allelochemicals, in “Herbivores: Their Interaction with Secondary Plant Metabolites” (G. A. Rosenthal and D. H. Janzen, Eds.). p. 199, Academic Press, New York, 1979. 5. S. Ahmad, Roles of mixed-function oxidases in insect herbivory. Proc. 5rh Int. Symp. lnsect-
4.
Plant
R&u.
(1982).
6. E. Cohen, Studies on several microsomal enzymes in two strains of Tribdium custutzeutn (Tenebrionidae. Coleoptera), Camp. B&hem. Physiol.
C 71, 123 (1982).
W. J. Hayes. Jr.. “Pesticides Studied in Man,” Williams & Wilkins. Baltimore/London, 1982. 8. A. H. Conney and J. J. Burns. Metabolic interaction among environmental chemicals and drugs, Science 178, 576 (1972). 9. L. B. Brattsten and C. A. Gunderson, Isolation of insect microsomal oxidases by rapid centrifugation. Pestic. Biochem. Physiol. 16, 187 (1981). 10. A. E Moldenke and L. C. Terriere, Cytochrome P-450 in insects. 3. Increase in substrate binding by microsomes from phenobarbital-induced house flies, Pesfic. Biochem. Physiol. 7.
16, 222 (1981).
1I. K. A. Johannesen and J. W. DePierre, Measure-
ENZYME
ACTIVITY,
39
I
ment of cytochrome P-450 in the presence of large amount of contaminating hemoglobin and methemoglobin, Anal. Biochem. 86, 725 (1978). 12. M. M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248 (1976). 13. M. Ben-Horim and H. Levy, “Statistics Decision and Applications in Business and Economics.” Random House Business Division. New York. 1984. 14. T. Nakatsugawa and P. A. Dahm. Parathion activation enzymes in the fat body microsomes of the American cockroach. J. Econ. Entrmzd. 58. 500 (196%.
15. R. J. Kuhr and C. W. Hessney, Toxicity and metabolism of methomyl in the European corn borer, Pestic. Biocbem. Physiol. 7, 301 (1977). 16. L. B. Brattsten. S. L. Price. and C. A. Gunderson. Microsomal oxidases in midgut and fatbody tissues of a broadly herbivorous insect larva, Spodopteru eridaniu Cramer Noctuidae. Camp. Biochem. Physiol. C 66, 231 t 1980). 17. D. E. Farnsworth. R. E. Berry. S. J. Yu. and L. C. Terriere. Aldrin epoxidase activity and cytochrome P-450 content of microsomes prepared from alfalfa and cabbage looper larvae fed various plant diets. Pest. Biwhem. Phy.rio/. 15, 158 (1981).
18. R. I. Krieger and C. F. Wilkinson. An endogenous inhibitor of microsomal mixed-function oxidases in homogenates of the southern armyworm (Prodenitr eridunru), Biochem. J. 116. 781 (1970).
19. L. B. Brattsten and C. F. Wilkinson. A microsomal enzyme inhibitor in the gut contents of the house cricket (Achetu domesticfrs). Camp. B&hem.
Physiol.
B 45, 59 (1973).
20. S. Orrenius, M. Berggren, P. Moldeus. and R. I. Krieger. Mechanism of inhibition of microsomal mixed function oxidation by the gut-contents inhibitor of the southern armyworm (Prodenier rridnneu).
Biochem.
J. 124, 427 t 1971 I.
BIOCHEMISTRY
Activities
AND
PHYSIOLOGY
30, 35-39 (1988)
of Some Microsomal Enzymes of the Yellow Mealworm, Tenebrio molitor (Lint-k) I. Basal Levels and lnducibility
ELIANN EGAAS,* ELISABETHGRAMJENSEN,?
ANDJANNECHE UTNE SKAARE-~
*Norwegian Plant Protection Institute, Department of Entomology. P.O. Box 70. N-1432 and fThe Norwegian College of Veterinary Medicine, Department of Pharmuco1og.v Dep. Oslo 1, N-Oslo. Nor\+,a)
Aas-NLH. Notxltry. clnd To.vkolo,qv.
Received January 26. 1987: accepted September I. 1987 The basal levels and the induction potential of some microsomal enzymes of the yellow mealworm soft tissue without gut was established. The insecticides aldrin, lindane. and endosulfan were administered in the feed for 3 days of exposure. Cytochrome P-420. NADPH-cytochrome (’ reductase, aldrin epoxidase, and heptachlor epoxidase were chosen as mixed-function oxidase activity parameters. The maximal induction value of cytochrome P-450 was reached at SO00 ppm of endosulfan and was 334% of the control value. The maximal induction values of the aldrin and heptachlor epoxidase and the NADPH cytochrome c reductase activities were reached at 2500 ppm of endosulfan and were 5622, 1390, and 324% of the respective control values. c IYXX AGI~WW Press. Inc.
INTRODUCTION
worm, Tenebrio molitor, and to investigate the adaptive potential of the activities by exposure to different levels of the halogenated hydrocarbon insecticides aldrin, lindane, and endosulfan.
The microsomal enxymes of the mixedfunction oxidase (MFO)’ system are a group of enzymes dependent on cytochrome P-450. They are widely distributed through the phylogenesis and are known to play an important role in the primary degradation of drugs, pesticides, and other xenobiotics (I-3). In insect larvae, the MFO systems of lepidopterous larvae have been extensively studied (4, 5). Fewer data concerning MFO activity are available on coleopteran larva1 tissues (6). Halogenated hydrocarbon insecticides are known MFO inducers in several species (7). In the target organisms, the ability of the halogenated hydrocarbon insecticides to stimulate their own metabolism and that of other pesticides has been linked to the phenomenons of resistance and cross-resistance (8). The aim of the present study was to establish the basal levels of MFO activities in a coleopteran larvae, the yellow mealt Abbreviation dase.
used: MFO,
mixed-function
MATERIALSANDMETHODS
Insects. The yellow mealworm, T. molitor, were initially bought in a local pet animal shop in Oslo, Norway, and reared for 4 to 8 weeks in our laboratory. The larvae were maintained in plastic buckets (35 x 25 x 13 cm) on a bed of sand and a basal mixture of whole-meal flour and wheat bran (3:l). To provide moisture, slices of apples were administered. At the start of an experiment, the larvae were selected by body weight. During the experiments, each group of 60 to 70 larvae was kept in glass vials (10 x 8 x 6 cm) on a l-cm bed of sand and 40 g basal flour mixture. The larvae were kept at 25°C and 60% humidity in a Mode1 Psycrotherm R27 incubator. Chemicals. Aldrin (99% purity) and lindane (99% purity) were purchased from Chem. Service Inc. (West Chester, PA).
oxi.
35 0048-3575188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
36
EGAAS,
JENSEN,
Heptachlor (100% purity) was purchased from Unilab Research Corp. (Berkeley, CA). Endosulfan (98.8% purity) was a gift from Dr. H. 0. Friestad, Chemical Division, Agricultural University of Norway. NADH, NADPH, cytochrome c, and reduced glutathione were all obtained from Sigma (St. Louis, MO). All other chemicals were of the highest purity commercially available. Preparation of microsomal enzymes. Microsomal pellet was obtained by pressing out the soft tissue through razorblade-made incisions at the oral and anal regions. After removing the gut, the remaining tissue, which in the following is named “carcass,” was added to 4 vol of buffer A (0.2 M potassium phosphate buffer, pH 7.8, 1 mM EDTA, and 2 mM reduced glutathione). The mixture was homogenized in a PotterElvehjem homogenizer by three manual strokes, rotating the pistil continually. The homogenate was centrifuged at 10,OOOg for 15 min (0 to 4°C) in a Sorvall centrifuge, Model RC-2. The postmitochondrial supernatant was filtered through glass-wool and recentrifuged at 210,OOOg for 25 min (34°C) in a Centrikon ultracentrifuge, Model T-2070 (9). The microsomal pellet was rinsed once with buffer B (0.3 M potassium phosphate buffer, pH 7.8, 1 mM EDTA) and suspended in the same buffer to make a final concentration of 2 to 4 mg protein/ml. An aliquot of the microsomal suspension was used immediately for enzyme assays, while the remaining suspension was added glycerol to a final concentration of 40% before measuring the content of cytochrome P-450. All tissue preparations were kept at 0 to 4°C until analyzed. Enzyme assays. Aldrin and heptachlor epoxidase assays were analyzed according to Moldenke and Terriere (10) with the exception that buffer A was used as buffer during the incubations and NADPH was added instead of a NADPH-generating system. The relationship between the aldrin epoxidase activity and the protein concentration was studied with microsomal
AND SKAARE
protein obtained from control and aldrintreated larvae (62 ppm in the feed for 3 days). Using protein from control larvae, this relationship was linear over the range 0.5 to 3.8 mg protein per incubation. The epoxidase activity prepared from aldrintreated larvae was linear with the protein concentration up to about 2.0 mg per incubation. Dieldrin and heptachlor epoxide were extracted with cyclohexane and analyzed on a Varian gas chromatograph, Model 3700, equipped with an electron capture detector. The column used was 2.5 m x 3 mm i.d. and packed with 1.5% SP-2250 and 1.95% SP-2401 on IOO- 120 mesh Supelcoport. The temperature program was 190°C for 1 min, 3”C/min, 205°C for 6 min. Recovery of dieldrin and heptachlor epoxide was 106- 120%. The reproducibility was & 2.0%. No dieldrin from an in vivo epoxidation of aldrin was found in microsomes from aldrin-treated larvae. NADPH-cytochrome c’ reductase (shortened “reductase”) activity was determined in 5.5 ml incubation mixture containing 4.5 ml buffer B, 0.5 ml tissue homogenate (containing 0.5 to 1.5 mg of protein), 0.5 ml cytochrome c (1.5 mM in buffer B), and 20 ~1 NADPH (10 mM in buffer B). The reaction was registered for 2 to 4 min at 25°C. The extinction coefficient used was 21 mM-t * cm-’ (A,&. Cytochrome P-450 concentration was measured and calculated by the method of Johannesen and DePierre (11). Both sample and reference microsomes were treated with 20 t.~l of NADH (10 m&Z) before adding sodium dithionite to sample cuvette. Thus, we obtained a 50% reduction of the apparent cytochrome P-420 content of the corresponding suspension without NADH. The content and peak position at 448 nm of cytochrome P-450 was not changed by the addition of NADH. The content of cytochrome P-450 always surpassed the content of cytochrome P-420 more than seven times. All spectra were recorded on a double beam Beckman spectrophotometer, Model
Tenrhrio
molitor
MICROSOMAL
35. or with a double beam Kontron spectrophotometer, Model Unikon 860. Protein determinations were done by the Bio-Rad method of Bradford (12), using lyophilized bovine serum albumin (1.21 mg/ml) as standard. Induction studies. The larvae were initially starved for 24 hr. The compounds tested as inducers were dissolved in ether and mixed with the feed (aldrin 31 ppm, lindane from 6 to 250 ppm, and endosulfan from 10 to 10,000 ppm). A corresponding amount of ether only was added to the feed of the control groups. After evaporation of the ether, each group was exposed to their respective diets for 3 days. Larval weight was registered at the end of each experiment. Statistics. Statistical analysis was carried out with the Mann-Whitney test on the difference between two population means (13), using a significance level of 0.05. RESULTS
AND
DISCUSSION
The problems arising from the occurrence of endogenous inhibitors in insect tissue have usually been solved by choosing test insects from which a specific tissue, active in microsomal oxidation, can be isolated (4, 14-16). However, Farnsworth er al. (17) obtained aldrin epoxidase active microsomes from the larval soft tissue without gut, named carcass, of the alfalfa (Autograph californica) and the cabbage looper larvae (Trichoplusia ni). The correspondingly prepared yellow mealworm carcass consisted mainly of fatbody. Other tissues, like gonads or Malpigian tubules, could not be distinguished. Unlike fat-body from cockroach, Periplaneta americana (14), the carcass of the yellow mealworm seemed to be without any endogenous inhibitor. Thus, the postmicrosomal supernatant obtained from yellow mealworm carcass was without reductase activity (18-20). The corresponding microsomal cytochrome P-450 content was almost four times higher than in fat-body microsomes from uninduced
ENZYME
ACTIVITY,
I
37
last (sixth) instars of southern armyworm (Spodoptera eridania) (16) and carcass microsomes from alfalfa and cabbage looper larvae ( 17). The aldrin epoxidase was, respectively, 30, 50, and 150% of the corresponding value in the southern armyworm and the alfalfa and the cabbage looper larvae. Furthermore, the reductase activity in microsomes from the yellow mealworm carcass was half the corresponding value in the southern armyworm. Despite a rather heterogenous, nonsyncronous population of larvae ranging from 0.14 to 0.19 g of body weight, microsomes from aldrin-treated larvae contained a significant increase in both the cytochrome P-450. aldrin, and heptachlor epoxidase values relative to the corresponding controls (Table 1). The 14% reduction in mean body weight in the larvae groups exposed to the high lindane dose (Table 1) is comparable to an I 1% reduction found in the body weight of larvae which had been starved for 4 days (unpublished result). Furthermore, lindanetreated larvae showed signs of heavy intoxication. Therefore, the microsomes prepared from the lindane-treated groups might contain more inert protein from dying larvae relative to the control. This may explain why the epoxidation assays revealed an inducing effect of the lindane treatment, whereas the cytochrome P-450 assay did not (Table 1). In a separate doseresponse experiment, no increase in any of the MFO activity parameters was found in larvae that had been exposed to different doses of lindane (6, 31. 125 ppm). However, a 13% reduction in the mean body weight relative to the controls, and visible signs of intoxication, were observed in the groups that received 125 ppm of lindane. Exposure to endosulfan in the feed (10 ppm) significantly raised only the heptachlor epoxidase and the reductase activities relative to the corresponding activities of the controls (Table 1). However, endosulfan was the only insecticide tested that induced the reductase activity relative to
EGAAS,
38
JENSEN,
AND SKAARE
TABLE
1
Microsomal Epoxidase Activities, Reductase Activity. and Content of Cytochrome Yellow Mealworm Exposed to Aldrin (31 ppm), Lindane (250 ppm). or Endosulfnn for 3 Days
Insecticide Name Control Aldrin Lindane Endosulfan
Dose (wm) 0 31 250
10
Loss of body weight” (%) 3.8 7.3 14.4 7.0
? ” + k
2.0b 1.7 3.4* 2.4
Epoxidases (pmol/min. mgp) Aldrin 75.7 298.0 122.0 107.0
2 k z? 2
Reductase (nmolimin, mgp)
Heptachlor 21.8 57.0* lJ.O* 49.0
19.7 i 9.5 58.8 -+ 22.8* 36.5 ? 13.5* 35.8 z 9.9*
35.1 37.1 37.9 48.8
u A body weight range of 0.14 to 0.19 g was selected. b All numbers are arithmetic means 5 SD of five separate experiments experiment includes two parallels. * Significantly different from the corresponding control (p < 0.05).
the control values. In a separate experiment on a group of larvae with a more homogeneous body weight range of 0.16 to 0.17 g, doses from 50 ppm of endosulfan were highly effective in inducing both the cytochrome P-450 content, aldrin and heptachlor epoxidase, and the reductase activities (Table 2). During the 3 days of the experiment, larvae given more than 1250 ppm gradually stopped feeding and stretched out motionless on top of the flour. Thus, the absorption of endosulfan through the integument might have continued after the larvae stopped feeding. However, not even
2 11.0 f 8.4 2 9.1
?. 13.1*
Cytochrome (nmol/mgp) P-450 0.28 i 0.32 f 0.33 ” 0.35 2
0.01 0.04* 0.05 0.08
performed during 3 weeks, each
a dose of 10,000 ppm increased the mortality of the larvae. This is in contrast to lindane, which reached a lethal tissue concentration when the inducing effect was barely significant. Thus, it seems resonable to suggest that the remarkable inducing effect of endosulfan may be connected to the great tolerance to this insecticide by the yellow mealworm. ACKNOWLEDGMENTS Thanks are due to Dr. Jorgen Stenersen for initiating this project, funded by The Agricultural Research Council of Norway. The technical assistance of
TABLE Microsomal
P-450 in Carcass from (10 ppm) in the Feed
2
Epoxidase Activities, Reductase Activity, and Content of Cytochrome P-450 in Carcass Yellow Mealworm Exposed to Different Doses of Endosulfan in the Feed for 3 Daysapb
from
% of control activity Endosulfan dose @pm)
Epoxidase Aldrin
Reductase
0’ 50 250
100
100
424 1947
249
124
631
132
1250
3513
860
2500 5000
5622 5365 2228
203 324 275 76
10000
100
Cytochrome Heptachlor
1390 1305 610
P-450
loo 121 168 215 285 334 234
a All numbers are either arithmetic means of two assays (cytochrome P-450) or single assay. b A body weight range of 0.16 to 0.17 g was selected. c Control values: aldrin epoxidase, 34.2 pmol/min, mgp; heptachlor epoxidase, 23.6 pmol/min, mgp; reductase, 59.6 nmol/min, mgp; and cytochrome P-450, 0.24 2 0.01 nmol/mgp.
Tenebrio
molitor
MICROSOMAL
Inger Halvorsen and Maalfrid Tofteberg Bjerke in preparing this manuscript is gratefully appreciated. REFERENCES
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