Detoxication capacity in the honey bee, Apis mellifera L

PESTICIDE

BIOCHEMISTRY

AND

Detoxication

PHYSIOLOGY

22, 360-368

(1984)

Capacity in the Honey Bee, Apis mellifera S. J. Yu, F. A. ROBINSON,

Department

of Entomology

& Nematology,

University

L.’

AND J. L. NATION of Florida,

Gainesville,

Florida

32611

Received November 10, 1983; accepted January 26, 1984 Various detoxifying enzymes, including microsomal oxidases, glutathione S-transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase, were assayed in adult worker bees (Apis mellifera L.) using midguts as the enzyme source. A cell-free system was used for all enzyme assays, except that microsomal oxidases required intact midgut because of the inhibitor encountered. Midgut microsomal preparations contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain the low microsomal oxidase activity in microsomes. All enzymes studied were active, suggesting that the high susceptibility of honey bees to insecticides is not due to low detoxication capacity. Sublethal exposure of honey bees to various insecticides had no effect on these enzyme activities, with the exception of permethrin which significantly stimulated the glutathione S-transferase, and malathion, which significantly inhibited the cxnaphthylacetate esterase and carboxylesterase. 0 1984 Academic Press. Inc. INTRODUCTION

This report concerns a study of various detoxication enzyme activities in worker honey bees. The effect of sublethal concentrations of various insecticides on these enzymes was also examined.

Severe losses of honey bee colonies have occurred in recent years because of the field applications of pesticides. According to Atkins (l), nearly 50% of the pesticides used are highly or moderately toxic to honey bees. The high susceptibility of honey bees to pesticides, especially carbamates, was suggested to be due to their lack of detoxication enzymes. This interpretation was based on the relatively low synergistic ratios in piperonyl butoxide/carbaryl combination studies (2). However, in their study of microsomal oxidation in honey bees, Gilbert and Wilkinson (3) found that microsomal oxidases, including epoxidase, hydroxylase, and O-demethylase, were active in larvae and adults. These microsomal enzymes are known to play a major role in the detoxication of insecticides (4). Other detoxifying enzymes such as glutathione S-transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase, most of which are known to be involved in insecticide resistance (5), have not been investigated in honey bees. t Florida Agricultural No.

Experiment

MATERIALS

Insects. Colonies of the honey bee, Apis melliferu L., were maintained at the University of Florida. Adult worker bees of mixed ages were collected from hives and used in this study. Chemicals. The chemicals (analytical grade) used in this study and their sources were aldrin, Shell Biosciences Laboratory (Sittingbourne, England); DDT, Chem Service, Inc. (West Chester, Pa.); permethrin, ICI Americas Inc., (Goldsboro, N.C.); malathion, American Cyanamid Company, (Princeton, N.J.); carbaryl, Union Carbide (Bound Brook, N.J.); methoxychlor, E.I. DuPont De Nemours & Company, (Wilmington, Del.); diflubenzuron, ThompsonHayward Chemical Company, (Kansas City, Kan.); glutathione, p-hydroxymercuribenzoate (PHMB), and phenylmethylsulfonyl fluoride (PMSF), Sigma Chemical Company (St. Louis, MO.); biphenyl, Ald-

Station Journal

5038. 360

0048-3575184

$3.00

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

DETOXICATION

CAPACITY

rich Chemical Company, Inc., (Milwaukee, Wise.); 4-hydroxybiphenyl, Eastman Kodak Company, (Rochester, N.Y.); and 18t4C]styrene oxide, Amersham Corporation, (Arlington Heights, Ill.). All other chemicals were of analytical quality and purchased from commercial suppliers. Enzyme assays. Adult worker honey bees were collected and placed in screen cages, and then put in the freezer compartment of a refrigerator for 5 min to immobilize the bees for dissection. When microsomal oxidase activities were measured, groups of 5-10 midguts were dissected from the bees in 1.15% KCl, and the intact midguts (contents included) were used as the enzyme source. Microsomal epoxidase activity was measured with aldrin as substrate. The 5-ml incubation mixture contained 5 midguts; 0.1 M sodium phosphate buffer, pH 7.5; an NADPH-generating system consisting of 1.8 kmol NADP, 18 bmol glucose 6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase; and 250 nmol aldrin in 0.1 ml methyl Cellosolve. Duplicate incubations were conducted in a water bath with shaking at 40°C (optimum temperature) in an atmosphere of air for 5 min. The reaction product, dieldrin, was extracted with 10 ml hexane and analyzed by gas chromatography as described previously (6). Microsomal N- and 0-demethylase activities were measured with p-chloro-N-methylaniline (PCMA) and p-nitroanisole as substrates, respectively, and 10 intact midguts as the enzyme source, as previously described (6). Microsomal hydroxylase activity was determined with biphenyl as substrate and 10 intact midguts as the enzyme source as described previously (7). To assay for epoxide hydrolase, groups of 50 midguts with gut contents removed were homogenized in 25 ml of ice-cold 0.1 M sodium phosphate buffer, pH 7.5, in a motor-driven tissue grinder for 30 set and filtered through cheesecloth. The homogenate was then centrifuged at 10,OOOg for 15 min in a Beckman L5-50E ultracentrifuge.

IN

HONEY

BEES

361

The pellet was discarded, and the supernatant, which had been filtered through glass wool, was recentrifuged at 105,OOOg for 60 min. The microsomal pellet was suspended in ice-cold 0.5 M Tris-HCl buffer, pH 9.0, to make a final concentration of 0.4 mg protein/ml, and was used immediately. The procedures above were conducted at 0 to 4°C. Epoxide hydrolase was assayed with styrene oxide as substrate using the method slightly modified from Oesch et al. (8). The incubation mixture consisted of 0.5 ml microsomal suspension and 7 p,l acetonitrile containing 8 kg cold styrene oxide and 0.6 p,g (100,000 dpm) [8-14C]styrene oxide. Duplicate incubations were carried out in an atmosphere of air with shaking at 37°C for 5 min. The reaction was stopped by addition of 10 ml petroleum ether, and the unreacted styrene oxide was extracted by the solvent. The petroleum ether was readily decanted by freezing the aqueous phase in a dry ice-acetone mixture. The same extraction was repeated again after the aqueous phase was thawed. The aqueous solution, which contained the polar product, [8-14C]styrene glycol. was then shaken with 2 ml ethyl acetate, and the product in the ethyl acetate was quantified by liquid scintillation counting. When glutathione S-transferases were assayed, groups of 50 cleaned midguts were homogenized in 0.1 M Tris-HCI buffer, pH 8.0 (for methyl iodide conjugation), pH 9.0 (for 3,4-dichloronitrobenzene (DCNB) conjugation), or pH 6.5 (for I-chloro-2,Cdinitrobenzene (CDNB) conjugation). The soluble fraction (105,OOOg supernatant) was prepared from the homogenate as described above and used as the enzyme source. Glutathione S-transferase activities were determined with DCNB, CDNB. and methyl iodide as substrates as described previously (9). Esterase and carboxylesterase activities were measured as follows: groups of 5 cleaned midguts were homogenized in 20 ml ice-cold 0.1 M sodium phosphate buffer. pH 7.0. The homogenate was filtered

362

YU, ROBINSON,

through cheesecloth, and the filtered homogenate was used as the enzyme source. Both esterase activities were assayed by the method of Van Asperen (10) using (Ynaphthylacetate (a-NA) as substrate. To measure carboxylesterase activity, eserine (lop4 M) and PHMB (1O-4 M) were included in the incubation mixture to eliminate cholinesterase and arylesterase, respectively. When acetylcholinesterase activity was measured, groups of 10 heads were removed from the bees and homogenized in 10 ml ice-cold 0.1 M sodium phosphate buffer, pH 8.0, in a motor-driven tissue grinder for 30 sec. The homogenate was filtered through cheesecloth, and the filtered homogenate was used as the enzyme source. Acetylcholinesterase activity was measured with acetylthiocholine as substrate as described by Ellman et al. (11). When DDT-dehydrochlorinase activity was measured, the enzyme source (soluble fraction) was prepared in 0.1 M sodium phosphate buffer, pH 7.4, as described earlier, and enzyme activity was determined as described by Yu and Terriere (12). Microsomal cytochrome P-450 content was determined by the method of Omura and Sato (13) using a Beckman Model 5260 uvlvis spectrophotometer equipped with a scattered transmission accessory. Protein was determined by the method of Bradford (14) using bovine serum albumin as standard. Determination of sublethal concentrations of insecticides. Metal screen cylin-

ders, 13.5 cm in height and 8.5 cm in diameter, covered with plastic Petri dishes at both ends, were used to confine the bees during the treatment. Each cage had two holes (2.5 cm in diameter) in the cover, one for water supply using a scintillation vial, and the other for food that was placed on a metal screen on the top of the hole. The food pellet was covered with aluminum foil. Food containing insecticides was prepared by first mixing the required amounts of insecticide in 20 ml acetone with 10 g pow-

AND NATION TABLE 1 Detoxifying Enzyme Activities in Adult Worker Honey Bees

Detoxifying enzymea Microsomal oxidasesb Epoxidation N-demethylation 0-demethylation Hydroxylation Glutathione S-transferases Aryltransferase (DCNB) Aryltransferase (CDNB) Alkyltransferase Esterases a-NA esterase Carboxylesterase AcetylcholinesteraseC Epoxide hydrolase DDT-dehydrochlorinase

Specific activity (nmol mini mg protein - L)d 34.96 217.95 57.50 101.83

f f -L k

2.63 49.98 9.03 5.53

5.64 ‘- 0.59 466.32 k 15.02 39.22 k 0.36 302.47 196.29 202.78 8.57 0.215

k 2 i r -+

30.60 25.43 15.17 1.67 0.023

’ Midguts were used as the enzyme source. b pmol mint midgut-‘; 0.736 mg protein/midgut. ’ Heads were used as the enzyme source. d Means * SE of two to three experiments, each with duplicate enzyme assays.

dered sugar and 8 g granular sugar. The acetone was evaporated under a hood and 2 ml water was added with thorough mixing. The food thus prepared was then used to determine the toxicity of various insecticides to honey bees. All treated bees were held at 26.7”C. Mortality was recorded after 3 days. Log dosage-probit lines were based on four to five points using three replicates of 20 worker honey bees each. Probit analysis was performed by the method of Finney (15). Sublethal concentration for each insecticide was estimated from the regression line, representing approximately one-third of the LC, value. The same procedures for preparation of insecticides in food and subsequent feeding were used to determine sublethal effects of insecticides on detoxication enzymes. RESULTS

Detoxication

Enzymes in Honey Bees

The results obtained

from assays of 12

DETOXICATION

CAPACITY

detoxifying enzymes in the midguts of worker honey bees are summarized in Table 1. Although acetylcholinesterase is not regarded as a detoxifying enzyme in the strict sense, it was included in this study because of its ability to hydrolyze acetylcholine, which is essential to the operation of nerves. With respect to the microsomal oxidases, we initially used microsomes prepared from midguts of worker bees in 0.1 M sodium phosphate buffer, pH 7.5, as the enzyme source, and found no significant activity of aldrin epoxidase. The results confirmed those of Gilbert and Wilkinson (3, 16), who found in the midguts of worker bees a potent inhibitor of microsomal oxidase that was released on tissue homogenization. Our studies also showed that the midgut microsomes contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain their low MFO activity. The addition of EDTA (1 mM), PMSF (1 mh4) or glycerol (10%) to the homogenization buffer did not improve the P-450 content. However, a significant amount of cytochrome P-450 (average of 0.381 nmol/mg protein) was obtained when the homogenization buffer contained 1.5% BSA, even though the epoxidase activity (4.54 pm01 mint mg protein-‘) was still low. Therefore, we decided to use the intact midguts without tissue homogenization as the enzyme source. It can be seen that honey bees contained various microsomal oxidases including epoxidase, N- and 0-demethylase, and hydroxylase, with N-demethylase being the most active and epoxidase the least. There was an eight-fold difference in activity between these two enzymes. Glutathione Stransferases were also active in the midguts with aryltransferase (against CDNB) being the highest. Esterases such as (Y-NA esterase, carboxylesterase, and acetylcholinesterase were all very active. The a-NA esterase from midgut homogenates was also characterized using eserine and PHMB as inhibitors of cholinesterase and arylesterase, respectively, as mentioned earlier.

IN HONEY

363

BEES

TABLE 2 Susceptibility of Adult Worker Homv Bees PI Various Insecticides Oral toxicity (ppm) Insecticide Methoxychlor Carbaryl Malathion Permethrin Diflubenzuron”

LC5,

65.67 1.44

4.61

95% Confidence limits of LC,,, 5.5.06-78.20 1.23- 1.66 2.40- 8.56

1.55- 3.33 __. ’ No mortality was observed up to 1000 ppm in the diet. 2.28

-

It was found that esterase activity consisted of 31% of arylesterase (portion of esterase activity inhibited by PHMB), 5% cholinesterase (portion of esterase activity inhibited by eserine), and 64% carboxylesterase (portion of esterase activity not inhibited by PHMB and eserine). The results also indicated that midgut microsomes from worker honey bees contained epoxide hydrolase and the soluble fraction contained active DDT-dehydrochlorinase. Sublethal Effect of Insecticides

The oral toxicity of various insecticides to worker honey bees is shown in Table 2. Each insecticide represents one of five major groups of insecticides currently in use. Based on LD,, values, the order of toxicity to honey bees was carbaryl > permethrin > malathion > mexthoxychlor > diflubenzuron. There was a 45.6-fold difference in LC,, value between carbaryl- and methoxychlor-treated bees. Permethrin appeared to be slightly less toxic than carbaryl, which was somewhat unexpected. Diflubenzuron did not show any toxicity to bees up to 1000 ppm in the diet; concentrations higher than this were not studied. The effect of a sublethal concentration of five insecticides on various detoxifying enzymes of honey bees is given in Table 3. It is seen that dietary insecticides at the sublethal concentrations did not significantly alter the activities of aldrin epoxidase, ace-

364

YU,

ROBINSON,

AND

NATION

tylcholinesterase, and epoxide hydrolase. In the case of glutathione S-transferase (against DCNB), however, permethrin was found to significantly induce the transferase as compared with the controls, but the others had no effect on the enzyme. On the other hand, both cw-NA esterase and carboxylesterase activities were significantly inhibited by dietary malathion whereas the other compounds were without effect. DISCUSSION

The results of the present study show that honey bees, as in the case of other insects, possess various detoxifying enzymes that might be expected to protect them from insecticide poisoning. For general purposes of comparison, the detoxifying enzyme activities of various insect pests are summarized in Table 4. In the case of microsomal oxidase, critical comparisons between honey bees and other species were difficult because of the inhibitor encountered in the midgut, which required our using intact midguts as the enzyme source. However, when oxidase activities were expressed on a protein basis using an average of 0.736 mg protein/midgut, it was found that the epoxidase activity (47.50 pmol min mg protein- ‘) in worker honey bees appeared to be twice as high as that reported in black blow flies (17), and was comparable to that in Madagascar cockroaches (18). It was about one-fourth to one-seventh that in house crickets (19), tobacco budworms (20), fall armyworms (21) and house flies (CSMA) (22), and about one-fifteenth that in southern armyworms (23) and southern mole crickets (Scupteriscus acletus) (24). Thus, epoxidase activity in adult honey bees seems to be generally lower than in other insect species. Honey bee larvae appeared to be different in this regard. Gilbert and Wilkinson (3) reported that the specific activity of the epoxidase in 7-day-old drone larvae, which apparently contained no inhibitor of microsomal oxidases, was comparable to that of other insect species and several mammals.

DETOXICATION

Detoxlfiing

Enzyme Aldrin epoxidation

CAPACITY

IN HONEY BEES

365

TABLE 4 Enzyme Activities in Various Insect Species

Species

Tissue source

Specific activity (nmol min-’ mg protein - ‘)

Reference

-.

Black blow fly Madagascar cockroach House cricket Tobacco budworm Fall armyworm House fly (CSMA) Southern armyworm Southern mole cricket

Headless fly Midgut Malpighian tubules Midgut Midgut Abdomen Midgut Midgut

0.023 0.088 0.183 0.296 0.214 0.356 0.659

Tobacco hornworm Fall armyworm Southern mole cricket Southern armyworm

Midgut Midgut Midgut Midgut

0.160 0.420 2.45 3.85

Gypsy moth Tobacco hornworm Fall armyworm Southern mole cricket House fly (sbo)

Alimentary canal Midgut Midgut Midgut Abdomen

0.067 0.103 0.056 0.230 0.343

Fall armyworm

Midgut

0.445

Tobacco budworm Fall armyworm Tobacco hornworm House fly (CSMA)

Midgut Midgut Midgut Whole fly

3.59 31.2 33.33 108.8

Glutathione Stransferase (CDNB)

Fall armyworm

Midgut

692.0

Glutathione Stransferase (CH,I)

Fall armyworm

Midgut

House fly (CSMA) Flesh fly Black blow fly Fall armyworm Gypsy moth

Hemolymph Hemolymph Hemolymph Midgut Midgut

76.33 205.0 240.3 1390 1004

30 31

Carboxylesterase

Fall armyworm Flesh fly House fly (CSMA) Black blow fly

Midgut Hemolymph Hemolymph Hemolymph

151.0 248 83.33 76.67

Yu, unpub. 31 30 31

Styrene epoxide hydrolase

Fall armyworm Flour beetle

Midgut Whole larva

25.9 15.9

Yu, unpub.

DDT-dehydrochlorinase

Tobacco homworm Fali armyworm

Midgut Midgut

PCMA N-demethylation

p-Nitroanisole O-demethylation

Biphenyl hydroxylation Glutathione Stransferase (DCNB)

a-NA esterase

0.750

18.0

0.673 0.288

17 18 19 20 21

22 23 24

25 21 24

26 27

25 21 24 28

7 20 9

25 29

9 9

31 32 33

34 25

Yu, unpub.

366

YU,

ROBINSON,

The N-demethylase activity (377.65 pmol min-’ mg protein-i) in these bees was twice as high as that in tobacco hornworms (25) and was comparable to that in fall armyworms (21), although about one-sixth to one tenth that in southern mole crickets (24) and southern armyworms (26). On the other hand, the O-demethylase activity (78.13 pmol min- 1 mg protein- ‘) was comparable to that in gypsy moths (27), tobacco hornworms (25) and fall armyworms (21), although it was about one-third to onefourth that in southern mole crickets (24) and house flies (sbo) (28). The hydroxylase activity (138.36 pmol min-’ mg protein-‘) was about one-third that in fall armyworms (7). As to glutathione S-transferases, the aryltransferase activity toward DCNB in worker bees was comparable to that in tobacco budworms (20), although about onefifth that in fall armyworms (9) and tobacco hornworms (25), and one-nineteenth that in house flies (CSMA) (29). The aryltransferase activity toward CDNB was comparable to that in fall armyworms (9). On the other hand, the alkyltransferase activity was about twice as high as that found in fall armyworms (9). Esterases appeared to be fairly active in worker honey bees as compared with other insect species. The a-NA esterase activity in these bees was higher than that in house flies (CSMA) (30), flesh flies, and black blow flies (31), although about one-third that in fall armyworms (32) and gypsy moths (33). In the case of the carboxylesterase, its activity in the bees was comparable to that in fall armyworms (Yu, unpublished results) and flesh flies (31), although about twice as high as that in house flies (CSMA) (30) and black blow flies (31). The epoxide hydrolase in bees was found to be low, about one-half that in flour beetles (34) and fall armyworms (Yu, unpublished results). DDT-dehydrochlorinase activity was one-third that in tobacco hornworms (25) and was comparable to that in fall armyworms (Yu, unpublished results).

AND

NATION

It is obvious, on the basis of these findings, that the high susceptibility of honey bees to certain insecticides (1) is not directly related to low detoxication capacity. The detoxication activity appears to be normal. In this regard, Gilbert and Wilkinson (3) suggested that, because of the lack of microsomal oxidase activity in the honey stomach, which functions as a reservoir for ingested nectar, the ingested insecticides are likely to penetrate through the honey stomach membrane to reach internal target sites without being detoxified by the midgut detoxifying enzymes. Although this explanation is plausible, we suggest an additional factor. Adult honey bees do not contain a large amount of fat body in the abdomen. Because the fat body is known to play an important role in detoxication as well as storage of insecticides, the extremely low fat body content in this insect may be, in part, responsible for their high susceptibility to insecticides. This is especially true when contact insecticides are considered. Insecticides have been shown to be inducers of various detoxifying enzymes including microsomal oxidases (35, 36), glutathione S-transferase (29), carboxylesterase (37), and DDT-dehydrochlorinase (12) in insects. This subject has recently been reviewed by Terriere (38) with respect to insecticide resistance. Our results (Table 3) show that, among those insecticides studied, only permethrin at the sublethal concentration significantly induced glutathione S-transferase in bees. Since this transferase is important in the detoxication of organophosphorus insecticides (5), the induction, if it occurs in the field, would increase the survival capacity of bees. On the other hand, the inhibition of esterase activities caused by sublethal exposure to malathion would make bees more susceptible to certain insecticides containing an ester linkage such as pyrethroids, organophosphates, and juvenile hormone analogs, which are known to be hydrolyzed by these enzymes.

DETOXICATION

CAPACITY

ACKNOWLEDGMENTS This work was supported in part by Environmental Protection Agency Cooperative Agreement CR808964010. We are grateful to Dr. D. L. Shankland and Dr. S. H. Kerr for reviewing the manuscript. The technical assistance of R. T. Ing and A. B. Bolten is appreciated. REFERENCES 1. E. L. Atkins, Injury to honey bees by poisoning, in “The Hive and the Honey Bee,” p. 663, Dadant & Sons, Hamilton, Illinois, 1975. 2. R. L. Metcalf, T. R. Fukuto, C. F. Wilkinson, M. H. Fahmay, S. Adb El-Aziz, and E. R. Metcalf, J. Agric. Food Chem. 14, 555 (1966). 3. M. D. Gilbert and C. E Wilkinson, Microsomal oxidases in the honey bee, Apis mellifera L., Pestic.

Biochem.

Physiol.

4, 56 (1974).

4. T. Nakatsugawa and M. A. Morelli, Microsomal oxidation and insecticide metabolism, in “Insecticide Biochemistry and Physiology” (C. E Wilkinson, Ed.), p. 61, Plenum, New York, 1976.

W. C. Dauterman and E. Hodgson, Detoxication mechanisms in insects, in “Biochemistry of Insects” (M. Rockstein, Ed.), p. 541, Academic Press, New York, 1978. 6. S. J. Yu, Induction of microsomal oxidases by host plants in the fall armyworm, Spodoptera frugiperda (J. E. Smith), Pestic. Biochem. Phy5.

siol.

17, 59 (1982).

7. S. J. Yu and R. T. Ing, Microsomal biphenyl hydroxylase of fall armyworm larvae and its induction by allelochemicals and host plants, Comp. Biochem. Physiol., In press. 8. E Oesch, D. M. Jerina, and J. Daly, A radiometric assay for hepatic epoxide hydrase activity with 7-3H styrene oxide, Biochim. Biophys. Acta 227,

(1972).

13. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes, J. Biol. Chem.

14. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding, Anal. Biochem. 72, 248 (1976). 15. D. J. Finney, “Probit Analysis,” 2nd ed., p. 236, Cambridge Univ. Press, London/New York, 1952.

16. M. D. Gilbert and C. F. Wilkinson, An inhibitor of microsomal oxidation from gut tissues of the honey bee (Apis mellifera), Comp. Biochem. Physiol.

239 (1964).

B 50, 613 (1975).

17. H. A. Rose and L. C. Terriere, Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and B-naphthoflavone, Pestic. Biochem. Physiol. 14, 275 (1980). 18. G. M. Benke, C. E Wilkinson, and J. N. Telford, Microsomal oxidases in a cockroach, Gromphadorhina

portentosa,

J. Econ.

Entomol.

65,

1221 (1972). 19. G. M. Benke and C. E Wilkinson, Microsomal oxidation in the house cricket, Acheta domesticus (L.), Pestic. Biochem. Physiol. 1, 19 (1971). 20. E Gould and E. Hodgson, Mixed function oxidase and glutathione transferase activity in last instar Heliothis virescens larvae, Pestic. Biochem. Physiol.

13, 34 (1980).

21. S. J. Yu, Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm, Pestic. Biochem. Physiol. 19, 330 (1983). 22. A. E Moldenke and L. C. Terriere, Cytochrome P-450 in insects. 3. Increase in substrate binding by microsomes from phenobarbital-induced house flies, Pestic. Biochem. Physiol. 16, 222 (1981).

23. R. I. Krieger, P. P. Feeny, and C. F. Wilkinson, Detoxication enzymes in the guts of caterpillars: An evolutionary answer to plant defenses’?

685(1971).

S. J. Yu. Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms, Pestic. Biochem. Physiol., In press. 10. K. Van Asperen, A study of housefly esterases by means of a sensitive calorimetric method, J. Insect Physiol. 8, 401 (1962). 11. G. L. Ellman, K. D. Courtney, V. Anders, Jr., and R. M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 881 (1961). 12. S. J. Yu and L. C. Terriere, Enzyme induction in the house fly: The specificity of the cyclodiene insecticides, Pestic. Biochem. Physiol. 2, 184 9.

367

IN HONEY BEES

Science

172, 579(1971).

24. S. J. Yu, Microsomal crickets, Scapteriscus bard and Scapteriscus Biochem.

Physiol.

oxidases

in the mole

acletus Rehn and Hevicinus Scudder, Pestir. 17, 170 (1982).

25. L. G. Tate, S. S. Nakat, and E. Hodgson, Comparison of detoxication activity in midgut and fat body during fifth instar development of the tobacco hornworm, Manduca sexta. Comp. Biochem.

Physiol.

C 72, 75 (1982).

L. B. Brattsten, C. F. Wilkinson, and M. M. Root, Microsomal hydroxylation of aniline in the southern armyworm, Spodoptera eridania. Insect Biochem. 6, 61.5 (1976). 27. A. J. Forgash and S. Ahmad, Hydroxylation and demethylation by gut microsomes of gypsy moth larvae, Znt. J. Biochem. 5, 11 (1974). 28. F. W. Plapp, Jr., L. G. Tate, and E. Hodgson, Biochemical genetics of oxidative resistance to dia-

26.

368

YU, ROBINSON, zinon in the house fly, Pestic. Biochem. Phy6, 175 (1976). T. Hayaoka and W. C. Dauterman, Induction of glutathione S-transferase by phenobarbital and pesticides in various house fly strains and its effect on toxicity, Pestic. Biochem. Physiol. 17, 113 (1982). W. C. J. Maa and L. C. Terriere, Age-dependent variation in enzymatic and electrophoretic properties of house fly (M. domestica) carboxylesterases. Comp. Biochem. Physiol. C 74, 461 (1983). W. C. J. Maa and L. C. Terriere, Age-dependent variation in enzymatic and electrophoretic properties of flesh fly (Sarcophaga bulluta) and blow fly (Phormia regina) carboxylesterases, Comp. Biochem. Physiol. C 74, 451 (1983). S. J. Yu, Age variation in insecticide susceptibility and detoxification capacity of fall armyworm (Lepidoptera: Noctuidae) larvae, J. &on. Entomol. 76, 219 (1983). M. A. Kapin and S. Ahmad, Esterases in larval tissues of gypsy moth, Lymantria dispar (L.):

AND NATION

siol.

29.

30.

31.

32.

33.

34.

35. 36. 37.

38.

Optimum assay conditions, quantification and characterization, Insecf Biochem. 10, 331 (1980). E. Cohen, Epoxide hydrase activity in the flour beetle, Tribolium castaneum (Coleoptera, Tenebrionidae), Comp. Biochem. Physiol. B 69, 29 (1971). C. R. Walker and L. C. Terriere, Induction of microsomal oxidases by dieldrin in Musca domestica, Entomof. Exp. Appl. 13, 260 (1970). F. W. Plapp, Jr. and J. Casida, Induction by DDT and dieldrin of insecticide metabolism by house fly enzymes, 1. &on. Entomo/. 4, 1091 (1970). A. Wongkobrat and D. L. Dahlman, Larval Manduca sexta hemolymph carboxylesterase activity during chronic exposure to insecticide containing diets, J. Econ. Entomol. 69, 237 (1976). L. C. Terriere, Enzyme induction, gene amplification and insect resistance to insecticides, in “Pest Resistance to Insecticides” (G. P. Georgiou and T. Saito, Eds.), p. 265, Plenum, New York, 1983.