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Activities of some microsomal enzymes of the yellow mealworm, Tenebrio molitor (Linné)
PESTICIDE
BIOCHEMISTRY
Activities
AND
PHYSIOLOGY
30, 40-45 (1988)
of Some Microsomal Tenebrio
II. Influence
Enzymes of the Yellow Mealworm, molitor (Linnb)
of Larval Weight on Basal Levels, Inducibility, with and without Aldrin Pretreatment
and Lindane
ELIANN EGAAS,* ELISABETHGRAMJENSEN,IANDJANNECHE
Toxicity
UTNE SKAARE?
*Norwegian Plant Protection Institute. Department of Entomology, P.O. Box 70. N-1432 and #The Norwegian College of Veterinary Medicine. Department of Pharmacology Dep. Oslo I I N-Oslo, Norway
Aas-NLH. NorwBay, and Toxicology,
Received January 26, 1987: accepted October I, 1987 The effect of larval body weight in the range 0.07 to 0.25 g on basal activity levels and inducibility were studied in microsomal enzymes from yellow mealworm carcass (soft tissue without gut). The lindane toxicity and the effect of aldrin pretreatment on lindane toxicity were studied in three different weight groups. 1. The basal levels of several mixed function oxidase activities decreased with increasing sample mean body weight. 2. The aldrin induction potential of cytochrome P-450 content and aldrin epoxidase activity was inversely related to the sample mean body weight. The larvae with a lower body weight contained a higher microsomal induction potential than larvae with a higher sample mean body weight. 3. Lindane toxicity was independent of the sample mean body weight. 4. Pretreatment with aldrin resulted in an increased lindane mortality in larvae with sample mean body weight of 0.09 g and a decreased lindane toxicity in larvae with sample mean body weight of 0.23 g to the corresponding controls. i. 1988 Academic Prw. Inc.
of the effect of aldrin pretreatment on the mortality of lindane-exposed larvae of different body weight.
INTRODUCTION
The importance of mixed-function oxidases (MFOs)’ in the metabolism of various insecticides in insects is well documented (I), and it appears likely that stages of changed activity of these enzymes may indicate periods of different insecticide susceptibility (2-6). The aim of the present study was to establish the basal activity levels and the induction potential of the MFO system in soft tissue without gut, here defined as carcass of the yellow mealworm, Tenebrio molitor, in relation to developmental stages. Both lindane and aldrin are known to induce the MFO system of many species (7), including the yellow mealworm (8), and as such may alter the metabolism and biological effect of different chemicals. Therefore to study whether enzyme induction would be reflected in an altered insecticide tolerance, we included an interaction study t Abbreviation dase.
used: MFO,
mixed-function
MATERIALSANDMETHODS
Insects. Larvae of the yellow mealworm, T. molitor, were initially bought in a local pet shop in Oslo, Norway, and reared at 25°C. The larvae and imago were maintained and treated as described previously (8). Eggs were collected from stock culture every two weeks and supplied with a fresh diet. Chemicals. Aldrin (99% purity) and lindane (99% purity) were purchased from Chem. Service Inc. (West Chester, PA). 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. Induction studies. The larvae were selected according to body weight and pooled within the weight ranges 0.07 to 0.11 g, 0.12 to0.14g,0.15to0.17g,0.18to0.20g,and
oxi-
40
0048-3575/88 $3.00 Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
Tenebrio
tnolitor
MICROSOMAL
0.21 to 0.25 g. Aldrin-fortified feed (62 ppm) was prepared by mixing ether solution of the pesticide into the meal and evaporating the ether. Corresponding amounts of ether only were added to the control feed. Groups of larvae were starved for 24 hr and exposed to the feed for 3 days. In a dose-response study, groups of larvae having body weight ranging from 0.20 to 0.22 g were given feed fortified with aldrin (6, 31, 62, 125 ppm). Interaction studies. From each of the resulting aldrin-exposed (62 ppm) or control larvae in each of the weight groups, 0.07 to 0.11 g, 0.15 to 0.17 g, and 0.21 to 0.25 g, 30 to 36 larvae were sampled and exposed to lindane-fortified feed (250 ppm). After 8 days, the larva1 mortality of all groups was registered. Enzyme prepurations and assays. Microsomal pellets of from 5 to 10 g of larvae were prepared per group. The intestine was removed through razorblade-made incisions at the oral and anal region. The remaining soft tissues was pressed out and added to 4 vol of preparation buffer (0.2 M potassium phosphate buffer, pH 7.8, 1 mM EDTA, and 2 mM reduced glutathione) and homogenized by hand. All further preparations and assays were described earlier (8). Cytochrome h-5 was measured and calculated as described by Omura and Sato (9) using an extinction coefficient of 185 mM-’ * cd M424-410). Statistics. Statistical analysis was carried out by simple linear regression and by Fisher-Irwing test for two by two tables (10). using a significance level of 0.05. RESULTS
Basal activity levels of the MFO system of larval carcass in relation to larval mean body weight. When the mean larval body weight increased by 110% from 0.09 to 0.19 g, the carcass microsomal content of cytochrome P-450 from larvae given control feed decreased linearily by 55% (y =
ENZYME
is s x g l" ZE EE 25 4
z
ACTIVITY,
0.8
_
0.6
_
Es .E E . 0.4
_
0.2
_
41
II
B
1 0 09 0.07* 11
d--,hh 0.13 0 12-3.14
MEAN
AND
RANGE
0.16 c).15*.17
0.19
0.23 3.21-0.25
O.l8+X?O
OF BODY
WEIGHT
(g)
FIG. I. Cytochxm~~ P-450 contrnf (A) und aldrin epoxida.ce ucti\,ity (B) in c’urcuss microsomes prepured ,frotn yellorc, meol~r~ortn o,f differenf hod> ti,eights (0.07 [o 0.25 g) feeding on (Cl) hu,snl or (ml uldrin-f~ortified.f~ed (62 ppm)for 3 drays. A// r~tlues in (A) und control wlrres in (B) ure arithmetic tneun.s C SD of tow IO three different experiments, each ussuy in duplicute. Aldrin-treated ~v~ltcrs in (B) urc urithmetic meuns k SD of duplicate a.s.\uys ,frotn one to tkw expcvimenrs.
-2.8~ + 0.76, r2 = 0.96) (Fig. 1A). There was no significant difference between the content of cytochrome P-450 in larvae with mean body weights 0.19 and 0.23 g. Similarily, carcass microsomes from larvae with mean body weight 0.19 g contained only 50% of the aldrin epoxidase activity of the larvae with mean body weight 0.09 g. Between 0.09 and 0.19 g of body weight, the activity fell linearily (y = -0.5x + 0.15. r2 = 0.98). and between and 0.19 and 0.23 g there was no significant reduction in the aldrin epoxidase activity (Fig. 1B). Furthermore, the reductase activity and the cytochrome b-5 content in carcass micro-
42
EGAAS,
JENSEN,
somes linearily decreased with increasing body weight (y = -483x + 135, r2 = 0.96, and y = -0.86x + 0.21, r2 = 0.98, respectively) (Fig. 2). When the mean body weight was increased from 0.09 to 0.21 g, the reductase activity and the cytochrome b-5 content were reduced from 100% to, respectively, 36 and 29%. Induced activity levels of the MFO system of larval carcass in relation to larval mean body weight. Aldrin treatment
of larvae with mean body weight 0.09 g resulted in a 20% increase in the cytochrome P-450 content as compared to the control larvae (Fig. 1A). The content of cytochrome P-450 in the aldrin-treated groups also decreased linearily with increasing mean body weight (y = -4x + 0.98, r2 = 0.96). The effect of aldrin treatment on the microsomal cytochrome P-450 content was highest in the groups with the lower mean body weights, and was not significant in the larvae groups with mean body weight equal to or higher than 0.19 g. This finding was confirmed in a separate experiment, where groups of larvae with mean body weights ranging from 0.20 to 0.22 g were given al-
AND
SKAARE
drin-fortified feed (6, 31, 62, and 125 ppm). After 3 days of exposure, there was no significant difference in the carcass microsomal cytochrome P-450 prepared from aldrin-treated and control larvae. Like the cytochrome P-450, the effect of aldrin treatment on the aldrin epoxidase was greatest in the smaller larvae and decreased, although not linearly, with increasing body weight (Fig. 1B). Aldrintreated larvae with mean body weight 0.09 and 0.13 g contained, respectively, 9 and 3 times higher epoxidase activity than the corresponding control. Aldrin-treated larvae with body weights ranging from 0.15 to 0.25 g contained approximately twice the epoxidase activity of the corresponding controls. Aldrin treatment did not increase the reductase or cytochrome b-5 activities in any of the weight groups studied. Aldrin-lindane interaction study. Mortality was not significantly different in the three weight groups treated with lindane (Fig. 3). The 3 days of aldrin pretreatment did not result in any visible signs of intoxi100
80 3 > I-
60
i 2
40
5 I 20
-
i 0.09 0.07-0.11
MEAN
0.09 MEAN
0.13
BODY
0.17
WEIGHT
0.21
(9)
FIG. 2. Redactase activity (A) and b-5 content (B) in carcass microsomes prepared from yellow mealworm of different body weights (0.07 to 0.25 g).
BODY
0.16 0.15-017
AND
0.23 0.21-0.25
RANGE
WEIGHT
OF (g)
FIG. 3. Mortality in yellow mealworm after 8 days exposure to lindane-fortified feed (250 ppm). Each weight group consists of(m) 30 to 36 aldrin-pretreated /arvae (62 ppm) and (0) 30 to 36 control larvae pretreated with control feed.
Tenebrio
mditor
MICROSOMAL
cation of the larvae, and there was no reduction in mean body weight relative to the controls. However, following aldrin pretreatment, lindane mortality differed significantly between the weight groups. Thus, aldrin pretreatment significantly raised the lindane mortality of the larvae group with mean body weight 0.09 g to 94% as compared to 75% in the control group. Conversely, aldrin pretreatment significantly reduced the lindane mortality of the larvae group with mean body weight 0.23 g to 43%. as compared to the corresponding 77% of the control group. In the group with mean body weight 0.16 g, aldrin pretreatment did not significantly affect the lindane mortality. DISCUSSION
The larvae of the yellow mealworm may pass 9 to 20 instars before pupation (11). Thus, isolation of instars related to specific developmental stages may be difficult. Furthermore, the head capsule width is not a suitable parameter of developmental stages (12). Insect larval weight can be altered by age, genetic variation, and nutrition. Pupae has been observed from larvae ranging from 0.09 to 0.25 g of body weight. However, in our laboratory culture we have noticed that the highest frequency of pupating larvae is well above 0.20 g of body weight. The selection of yellow mealworm based on sample body weight revealed that 96 to 98% of the variation in all the MFO parameters studied, can be atttributed to the variation in weight between 0.09 and 0.19 g. Thus, in the present study we have chosen to classify the developmental stages of yellow mealworm by means of body weight. Like microsomes prepared from the midgut of several other larval species (13), all yellow mealworm carcass MFO activities declined toward the end of the last instar, which is characterized by slow immobilization. However, in the weight range 0.09 to 0.19 g, the increase in yellow meal-
ENZYME
ACTIVITY,
II
43
worm body weight may consist of tissue constituents devoid of MFO activity, which could then be partly responsible for the reduction of the basal content of cytochrome P-450. The observed relationship between carcass MFO activities and the mean body weight of yellow mealworm samples corresponds well with the results published by Wells et al. (6). They compared microsomal content of cytochrome P-450 from decapitated, whole homogenized third and fifth instar of the tufted apple budmoth. Platynota ideusulis, and showed that the fifth instar had a lower content of cytochrome P-450. In microsomes prepared from aldrintreated yellow mealworm larvae, the body weight variance explained 96% of the variance in the cytochrome P-450 content. However, unlike the control larvae. body weight was not a suitable parameter in predicting the effect of aldrin treatment on the microsomal aldrin epoxidase activity. Obviously, the measurements done reflecting the concentration of cytochrome P-450 in the microsomal pool do not pick up the increased amount of the particular cytochrome P-450 species that may be involved in the aldrin epoxidation. Thus, the high induction potential of aldrin epoxidase of the lower weight larvae (0.07 to 0.14 g) as compared to the heavier larvae cannot be explained by this model of protein dilution on an individual basis. The fat-body is an amorphous mass of tissue in close contact with both the integument and the gut. Thus, Wilkinson and Brattsten (14) suggested that the fat-body MFO system might provide protection from integumental entry of toxicants. Several factors, including a smaller insecticide storage potential, higher penetration of insecticides through the cuticle (15), a higher ratio of surface area to volume, and a smaller midgut MFO activity and induction potential in the younger larvae (5. 16-18) suggest that a higher fat-body MFO activity
44
EGAAS,
JENSEN,
and inducibility would be more beneficial to earlier compared to later instars. Whereas the yellow mealworm mortality after lindane treatment was independent of the body weight between 0.07 and 0.25 g, pretreatment with aldrin introduced a body weight related change in the mortality after lindane treatment. A mechanism of the aldrin mediated change in lindane toxicity in yellow mealworm may be related to the observed induction of the MFO system. However, a possible protective influence of an induced MFO system in other tissues, like the midgut, against lindane mortality of larvae in the highest weight groups, cannot be disregarded. Unfortunately, the fragility of the larvae gut made it impossible to prepare and study the MFO activity of the midgut microsomes. Furthermore, the larvae with mean body weight 0.09 g seemed to be susceptible to an enhancing effect of the aldrin treatment on the lindane toxicity. This may be related to a more efticient conversion of aldrin to its more toxic metabolite dieldrin in the larvae of 0.09 g compared to the heavier larvae. Thus, Wells et al. (6) found that the microsomal fraction of the third instar of the tufted apple budmoth was superior to the fifth instar microsomal preparation in producing both water-soluble azinphosmethyl metabelites and the toxic oxygen analog of azinphosmethyl. Correspondingly, they found decreasing toxicity and lower content of cytochrome P-450 with increasing instars of the tufted apple budmoth. However, further elucidation of the relationship between the observed MFO induction and lindane tolerance requires investigations on the different cytochrome P-450 species that may be involved, as well as the toxicokinetics and dynamics of lindane, aldrin, and dieldrin in different weight groups of the yellow mealworm. ACKNOWLEDGMENTS This project was funded by The Agricultural Research Council of Norway. The technical assistance of
AND SKAARE Inger Halvorsen and Maalfrid Tofteberg Bjerke in preparing the manuscript is gratefully appreciated. REFERENCES 1. A. H. Conney and J. J. Burns, Metabolic interaction among environmental chemicals and drugs, Science 178, 576 (1972). 2. R. T. Gast, The relationship of weight of lepidopterous larvae to effectiveness of topically applied insecticides, .I. Econ. Entomol. 52, 1115 (1959). 3. C. R. Harris and F. Gore, Toxicological studies on cutworms. VIII. Toxicity of three insecticides to the various stages in development of the Darksided cutworm, J. Econ. Entomol. 64, 1049 (1971). 4. S. Ahmad and A. J. Forgash, Toxicity of carbaryl and diazinon to gypsy moth larvae, changes in relation to larval growth, J. Econ. Entomol. 68, 803 (1975). 5. S. J. Yu, Age variation in insecticide susceptibility and detoxification capacity of fall armyworm (Lepidoptera: Noctuidae) larvae, J. Econ. Entomol. 76, 219 (1983). 6. D. S. Wells, G. C. Rock, and W. C. Dauterman, Studies on the mechanisms responsible for variable toxicity of azinphosmethyl to various larval instars of the tufted apple budmoth, Platynotu idaeusalis. Pesf. Biochem. Physiol. 20, 238 (1983). 7. Wayland J. Hayes, Jr., “Pesticides Studied in Man,” pp. 234, 237, Williams & Wilkins, Baltimore/London, 1982. 8. E. Egaas, E. Gram Jensen, and J. U. Skaare, Activities of some microsomal enzymes of the yellow mealworm, Tenebrio molitor (Linne). I. Basal levels and inducibility, Pest. Biochem. Physiol. 30, 35 (1988). 9. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 2370 (1964). 10. J. L. Hodges, Jr., and E. L. Lehmann, “Basic Concepts of Probability and Statistics,” 2nd ed., Holden-Day, San Francisco, 1970. 11. R. Mehl, personal communication, National Institute of Public Health, Department of Entomology, Geitemyrsvn. 75, 0462 N-Oslo 4. Norway. 12. S. Kobro, personal communication, Norwegian Plant Protection Institute, Department of Entomology, Box 70, N-1432 Aas-NLH, Norway. 13. R. I. Krieger, C. E Wilkinson, L. J. Hicks, and E. F. Taschenberg, Aldrin epoxidation, dihydroisodrin hydroxylation and p-chloro-l\i-methylaniline demethylation in six species of saturniid larvae, J. Econ. Entomol. 69, 1 (1976).
Tenebrio
m&or
MICROSOMAL
14. C. F. Wilkinson and L. B. Brattsten, Microsomal drug metabolizing enzymes in insects, Drug Metab. Rev. 1, 153 (1972). 15. R. T. Gast, Factors involved in different susceptibility of corn earworm larvae to DDT, J. Econ. Enromol. 54, 1203 (1961). 16. R. 1. Krieger and C. F. Wilkinson, Microsomdl mixed-function oxidases in insects. I. Localization and properties of an enzyme system affecting aldrin epoxidation in larvae of the
ENZYME
ACTIVITY,
southern armyworm
45
II (Prodenia
eridanitr).
Bio-
18, 1403 (1969). 17. L. B. Brattsten, Biochemical defense mechanisms in herbivores against plant allelochemicals. in “Herbivores: Their Interaction and Secondary Plant Metabolites” (G. A. Rosenthal and D. H. Janzen, Eds.), p. 199, Academic Press. Neh York, 1979. 18. S. J. Yu. Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm. Prsfic, Biochem. Physiol. 19. 330 ( 1983). them.
Phurmucol.
BIOCHEMISTRY
Activities
AND
PHYSIOLOGY
30, 40-45 (1988)
of Some Microsomal Tenebrio
II. Influence
Enzymes of the Yellow Mealworm, molitor (Linnb)
of Larval Weight on Basal Levels, Inducibility, with and without Aldrin Pretreatment
and Lindane
ELIANN EGAAS,* ELISABETHGRAMJENSEN,IANDJANNECHE
Toxicity
UTNE SKAARE?
*Norwegian Plant Protection Institute. Department of Entomology, P.O. Box 70. N-1432 and #The Norwegian College of Veterinary Medicine. Department of Pharmacology Dep. Oslo I I N-Oslo, Norway
Aas-NLH. NorwBay, and Toxicology,
Received January 26, 1987: accepted October I, 1987 The effect of larval body weight in the range 0.07 to 0.25 g on basal activity levels and inducibility were studied in microsomal enzymes from yellow mealworm carcass (soft tissue without gut). The lindane toxicity and the effect of aldrin pretreatment on lindane toxicity were studied in three different weight groups. 1. The basal levels of several mixed function oxidase activities decreased with increasing sample mean body weight. 2. The aldrin induction potential of cytochrome P-450 content and aldrin epoxidase activity was inversely related to the sample mean body weight. The larvae with a lower body weight contained a higher microsomal induction potential than larvae with a higher sample mean body weight. 3. Lindane toxicity was independent of the sample mean body weight. 4. Pretreatment with aldrin resulted in an increased lindane mortality in larvae with sample mean body weight of 0.09 g and a decreased lindane toxicity in larvae with sample mean body weight of 0.23 g to the corresponding controls. i. 1988 Academic Prw. Inc.
of the effect of aldrin pretreatment on the mortality of lindane-exposed larvae of different body weight.
INTRODUCTION
The importance of mixed-function oxidases (MFOs)’ in the metabolism of various insecticides in insects is well documented (I), and it appears likely that stages of changed activity of these enzymes may indicate periods of different insecticide susceptibility (2-6). The aim of the present study was to establish the basal activity levels and the induction potential of the MFO system in soft tissue without gut, here defined as carcass of the yellow mealworm, Tenebrio molitor, in relation to developmental stages. Both lindane and aldrin are known to induce the MFO system of many species (7), including the yellow mealworm (8), and as such may alter the metabolism and biological effect of different chemicals. Therefore to study whether enzyme induction would be reflected in an altered insecticide tolerance, we included an interaction study t Abbreviation dase.
used: MFO,
mixed-function
MATERIALSANDMETHODS
Insects. Larvae of the yellow mealworm, T. molitor, were initially bought in a local pet shop in Oslo, Norway, and reared at 25°C. The larvae and imago were maintained and treated as described previously (8). Eggs were collected from stock culture every two weeks and supplied with a fresh diet. Chemicals. Aldrin (99% purity) and lindane (99% purity) were purchased from Chem. Service Inc. (West Chester, PA). 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. Induction studies. The larvae were selected according to body weight and pooled within the weight ranges 0.07 to 0.11 g, 0.12 to0.14g,0.15to0.17g,0.18to0.20g,and
oxi-
40
0048-3575/88 $3.00 Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
Tenebrio
tnolitor
MICROSOMAL
0.21 to 0.25 g. Aldrin-fortified feed (62 ppm) was prepared by mixing ether solution of the pesticide into the meal and evaporating the ether. Corresponding amounts of ether only were added to the control feed. Groups of larvae were starved for 24 hr and exposed to the feed for 3 days. In a dose-response study, groups of larvae having body weight ranging from 0.20 to 0.22 g were given feed fortified with aldrin (6, 31, 62, 125 ppm). Interaction studies. From each of the resulting aldrin-exposed (62 ppm) or control larvae in each of the weight groups, 0.07 to 0.11 g, 0.15 to 0.17 g, and 0.21 to 0.25 g, 30 to 36 larvae were sampled and exposed to lindane-fortified feed (250 ppm). After 8 days, the larva1 mortality of all groups was registered. Enzyme prepurations and assays. Microsomal pellets of from 5 to 10 g of larvae were prepared per group. The intestine was removed through razorblade-made incisions at the oral and anal region. The remaining soft tissues was pressed out and added to 4 vol of preparation buffer (0.2 M potassium phosphate buffer, pH 7.8, 1 mM EDTA, and 2 mM reduced glutathione) and homogenized by hand. All further preparations and assays were described earlier (8). Cytochrome h-5 was measured and calculated as described by Omura and Sato (9) using an extinction coefficient of 185 mM-’ * cd M424-410). Statistics. Statistical analysis was carried out by simple linear regression and by Fisher-Irwing test for two by two tables (10). using a significance level of 0.05. RESULTS
Basal activity levels of the MFO system of larval carcass in relation to larval mean body weight. When the mean larval body weight increased by 110% from 0.09 to 0.19 g, the carcass microsomal content of cytochrome P-450 from larvae given control feed decreased linearily by 55% (y =
ENZYME
is s x g l" ZE EE 25 4
z
ACTIVITY,
0.8
_
0.6
_
Es .E E . 0.4
_
0.2
_
41
II
B
1 0 09 0.07* 11
d--,hh 0.13 0 12-3.14
MEAN
AND
RANGE
0.16 c).15*.17
0.19
0.23 3.21-0.25
O.l8+X?O
OF BODY
WEIGHT
(g)
FIG. I. Cytochxm~~ P-450 contrnf (A) und aldrin epoxida.ce ucti\,ity (B) in c’urcuss microsomes prepured ,frotn yellorc, meol~r~ortn o,f differenf hod> ti,eights (0.07 [o 0.25 g) feeding on (Cl) hu,snl or (ml uldrin-f~ortified.f~ed (62 ppm)for 3 drays. A// r~tlues in (A) und control wlrres in (B) ure arithmetic tneun.s C SD of tow IO three different experiments, each ussuy in duplicute. Aldrin-treated ~v~ltcrs in (B) urc urithmetic meuns k SD of duplicate a.s.\uys ,frotn one to tkw expcvimenrs.
-2.8~ + 0.76, r2 = 0.96) (Fig. 1A). There was no significant difference between the content of cytochrome P-450 in larvae with mean body weights 0.19 and 0.23 g. Similarily, carcass microsomes from larvae with mean body weight 0.19 g contained only 50% of the aldrin epoxidase activity of the larvae with mean body weight 0.09 g. Between 0.09 and 0.19 g of body weight, the activity fell linearily (y = -0.5x + 0.15. r2 = 0.98). and between and 0.19 and 0.23 g there was no significant reduction in the aldrin epoxidase activity (Fig. 1B). Furthermore, the reductase activity and the cytochrome b-5 content in carcass micro-
42
EGAAS,
JENSEN,
somes linearily decreased with increasing body weight (y = -483x + 135, r2 = 0.96, and y = -0.86x + 0.21, r2 = 0.98, respectively) (Fig. 2). When the mean body weight was increased from 0.09 to 0.21 g, the reductase activity and the cytochrome b-5 content were reduced from 100% to, respectively, 36 and 29%. Induced activity levels of the MFO system of larval carcass in relation to larval mean body weight. Aldrin treatment
of larvae with mean body weight 0.09 g resulted in a 20% increase in the cytochrome P-450 content as compared to the control larvae (Fig. 1A). The content of cytochrome P-450 in the aldrin-treated groups also decreased linearily with increasing mean body weight (y = -4x + 0.98, r2 = 0.96). The effect of aldrin treatment on the microsomal cytochrome P-450 content was highest in the groups with the lower mean body weights, and was not significant in the larvae groups with mean body weight equal to or higher than 0.19 g. This finding was confirmed in a separate experiment, where groups of larvae with mean body weights ranging from 0.20 to 0.22 g were given al-
AND
SKAARE
drin-fortified feed (6, 31, 62, and 125 ppm). After 3 days of exposure, there was no significant difference in the carcass microsomal cytochrome P-450 prepared from aldrin-treated and control larvae. Like the cytochrome P-450, the effect of aldrin treatment on the aldrin epoxidase was greatest in the smaller larvae and decreased, although not linearly, with increasing body weight (Fig. 1B). Aldrintreated larvae with mean body weight 0.09 and 0.13 g contained, respectively, 9 and 3 times higher epoxidase activity than the corresponding control. Aldrin-treated larvae with body weights ranging from 0.15 to 0.25 g contained approximately twice the epoxidase activity of the corresponding controls. Aldrin treatment did not increase the reductase or cytochrome b-5 activities in any of the weight groups studied. Aldrin-lindane interaction study. Mortality was not significantly different in the three weight groups treated with lindane (Fig. 3). The 3 days of aldrin pretreatment did not result in any visible signs of intoxi100
80 3 > I-
60
i 2
40
5 I 20
-
i 0.09 0.07-0.11
MEAN
0.09 MEAN
0.13
BODY
0.17
WEIGHT
0.21
(9)
FIG. 2. Redactase activity (A) and b-5 content (B) in carcass microsomes prepared from yellow mealworm of different body weights (0.07 to 0.25 g).
BODY
0.16 0.15-017
AND
0.23 0.21-0.25
RANGE
WEIGHT
OF (g)
FIG. 3. Mortality in yellow mealworm after 8 days exposure to lindane-fortified feed (250 ppm). Each weight group consists of(m) 30 to 36 aldrin-pretreated /arvae (62 ppm) and (0) 30 to 36 control larvae pretreated with control feed.
Tenebrio
mditor
MICROSOMAL
cation of the larvae, and there was no reduction in mean body weight relative to the controls. However, following aldrin pretreatment, lindane mortality differed significantly between the weight groups. Thus, aldrin pretreatment significantly raised the lindane mortality of the larvae group with mean body weight 0.09 g to 94% as compared to 75% in the control group. Conversely, aldrin pretreatment significantly reduced the lindane mortality of the larvae group with mean body weight 0.23 g to 43%. as compared to the corresponding 77% of the control group. In the group with mean body weight 0.16 g, aldrin pretreatment did not significantly affect the lindane mortality. DISCUSSION
The larvae of the yellow mealworm may pass 9 to 20 instars before pupation (11). Thus, isolation of instars related to specific developmental stages may be difficult. Furthermore, the head capsule width is not a suitable parameter of developmental stages (12). Insect larval weight can be altered by age, genetic variation, and nutrition. Pupae has been observed from larvae ranging from 0.09 to 0.25 g of body weight. However, in our laboratory culture we have noticed that the highest frequency of pupating larvae is well above 0.20 g of body weight. The selection of yellow mealworm based on sample body weight revealed that 96 to 98% of the variation in all the MFO parameters studied, can be atttributed to the variation in weight between 0.09 and 0.19 g. Thus, in the present study we have chosen to classify the developmental stages of yellow mealworm by means of body weight. Like microsomes prepared from the midgut of several other larval species (13), all yellow mealworm carcass MFO activities declined toward the end of the last instar, which is characterized by slow immobilization. However, in the weight range 0.09 to 0.19 g, the increase in yellow meal-
ENZYME
ACTIVITY,
II
43
worm body weight may consist of tissue constituents devoid of MFO activity, which could then be partly responsible for the reduction of the basal content of cytochrome P-450. The observed relationship between carcass MFO activities and the mean body weight of yellow mealworm samples corresponds well with the results published by Wells et al. (6). They compared microsomal content of cytochrome P-450 from decapitated, whole homogenized third and fifth instar of the tufted apple budmoth. Platynota ideusulis, and showed that the fifth instar had a lower content of cytochrome P-450. In microsomes prepared from aldrintreated yellow mealworm larvae, the body weight variance explained 96% of the variance in the cytochrome P-450 content. However, unlike the control larvae. body weight was not a suitable parameter in predicting the effect of aldrin treatment on the microsomal aldrin epoxidase activity. Obviously, the measurements done reflecting the concentration of cytochrome P-450 in the microsomal pool do not pick up the increased amount of the particular cytochrome P-450 species that may be involved in the aldrin epoxidation. Thus, the high induction potential of aldrin epoxidase of the lower weight larvae (0.07 to 0.14 g) as compared to the heavier larvae cannot be explained by this model of protein dilution on an individual basis. The fat-body is an amorphous mass of tissue in close contact with both the integument and the gut. Thus, Wilkinson and Brattsten (14) suggested that the fat-body MFO system might provide protection from integumental entry of toxicants. Several factors, including a smaller insecticide storage potential, higher penetration of insecticides through the cuticle (15), a higher ratio of surface area to volume, and a smaller midgut MFO activity and induction potential in the younger larvae (5. 16-18) suggest that a higher fat-body MFO activity
44
EGAAS,
JENSEN,
and inducibility would be more beneficial to earlier compared to later instars. Whereas the yellow mealworm mortality after lindane treatment was independent of the body weight between 0.07 and 0.25 g, pretreatment with aldrin introduced a body weight related change in the mortality after lindane treatment. A mechanism of the aldrin mediated change in lindane toxicity in yellow mealworm may be related to the observed induction of the MFO system. However, a possible protective influence of an induced MFO system in other tissues, like the midgut, against lindane mortality of larvae in the highest weight groups, cannot be disregarded. Unfortunately, the fragility of the larvae gut made it impossible to prepare and study the MFO activity of the midgut microsomes. Furthermore, the larvae with mean body weight 0.09 g seemed to be susceptible to an enhancing effect of the aldrin treatment on the lindane toxicity. This may be related to a more efticient conversion of aldrin to its more toxic metabolite dieldrin in the larvae of 0.09 g compared to the heavier larvae. Thus, Wells et al. (6) found that the microsomal fraction of the third instar of the tufted apple budmoth was superior to the fifth instar microsomal preparation in producing both water-soluble azinphosmethyl metabelites and the toxic oxygen analog of azinphosmethyl. Correspondingly, they found decreasing toxicity and lower content of cytochrome P-450 with increasing instars of the tufted apple budmoth. However, further elucidation of the relationship between the observed MFO induction and lindane tolerance requires investigations on the different cytochrome P-450 species that may be involved, as well as the toxicokinetics and dynamics of lindane, aldrin, and dieldrin in different weight groups of the yellow mealworm. ACKNOWLEDGMENTS This project was funded by The Agricultural Research Council of Norway. The technical assistance of
AND SKAARE Inger Halvorsen and Maalfrid Tofteberg Bjerke in preparing the manuscript is gratefully appreciated. REFERENCES 1. A. H. Conney and J. J. Burns, Metabolic interaction among environmental chemicals and drugs, Science 178, 576 (1972). 2. R. T. Gast, The relationship of weight of lepidopterous larvae to effectiveness of topically applied insecticides, .I. Econ. Entomol. 52, 1115 (1959). 3. C. R. Harris and F. Gore, Toxicological studies on cutworms. VIII. Toxicity of three insecticides to the various stages in development of the Darksided cutworm, J. Econ. Entomol. 64, 1049 (1971). 4. S. Ahmad and A. J. Forgash, Toxicity of carbaryl and diazinon to gypsy moth larvae, changes in relation to larval growth, J. Econ. Entomol. 68, 803 (1975). 5. S. J. Yu, Age variation in insecticide susceptibility and detoxification capacity of fall armyworm (Lepidoptera: Noctuidae) larvae, J. Econ. Entomol. 76, 219 (1983). 6. D. S. Wells, G. C. Rock, and W. C. Dauterman, Studies on the mechanisms responsible for variable toxicity of azinphosmethyl to various larval instars of the tufted apple budmoth, Platynotu idaeusalis. Pesf. Biochem. Physiol. 20, 238 (1983). 7. Wayland J. Hayes, Jr., “Pesticides Studied in Man,” pp. 234, 237, Williams & Wilkins, Baltimore/London, 1982. 8. E. Egaas, E. Gram Jensen, and J. U. Skaare, Activities of some microsomal enzymes of the yellow mealworm, Tenebrio molitor (Linne). I. Basal levels and inducibility, Pest. Biochem. Physiol. 30, 35 (1988). 9. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 2370 (1964). 10. J. L. Hodges, Jr., and E. L. Lehmann, “Basic Concepts of Probability and Statistics,” 2nd ed., Holden-Day, San Francisco, 1970. 11. R. Mehl, personal communication, National Institute of Public Health, Department of Entomology, Geitemyrsvn. 75, 0462 N-Oslo 4. Norway. 12. S. Kobro, personal communication, Norwegian Plant Protection Institute, Department of Entomology, Box 70, N-1432 Aas-NLH, Norway. 13. R. I. Krieger, C. E Wilkinson, L. J. Hicks, and E. F. Taschenberg, Aldrin epoxidation, dihydroisodrin hydroxylation and p-chloro-l\i-methylaniline demethylation in six species of saturniid larvae, J. Econ. Entomol. 69, 1 (1976).
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Bio-
18, 1403 (1969). 17. L. B. Brattsten, Biochemical defense mechanisms in herbivores against plant allelochemicals. in “Herbivores: Their Interaction and Secondary Plant Metabolites” (G. A. Rosenthal and D. H. Janzen, Eds.), p. 199, Academic Press. Neh York, 1979. 18. S. J. Yu. Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm. Prsfic, Biochem. Physiol. 19. 330 ( 1983). them.
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