Cypermethrin synergism by pyrethroid esterase inhibitors in adults of the whitefly Bemisia tabaci

PESTICIDE

BIOCHEMISTRY

Cypermethrin

PHYSIOLOGY

28, 155-162

(1987)

Synergism by Pyrethroid Esterase Inhibitors of the Whitefly Bemisia tabaci

ISAAC ISHAAYA,” *Department Isruel,

AND

ZMIRA MENDELSON,*

K. R. SIMON AXHER,?

of Entomology und fDepurtnrent qf To.ricology~ and $Pesticide Chemistry and Toxicology Laboratory. University of Culijbrniu, Berkeley. Received

August

7, 1985: accepted

in Adults

AND JOHN E. CASIDA~

ARO, The Volcmi Center, Bet Dugrrn 50-250, Departrrzer2t qf Entomolo~iccd Sciences. Culjfornirr 94720 April

14, 1986

Pyrethroid esterases are a major factor in the tolerance of adults of the whitefly Bemesia tahaci to cypermethrin and related pyrethroids. This conclusion is based on the substrate specificity of these esterases and their inhibition by organophosphorus compounds acting as synergists. Whitefly esterases hydrolyze trans-permethrin faster than its cis-isomer or its ol-cyano analogs truns- and ck-cypermethrin and deltamethrin. With trutrs-permethrin as the substrate, these pyrethroid esterases are sensitive to in vitro inhibition by monocrotophos and methamidophos (the active metabolite of acephate) with 50% inhibition at 9 x IO-’ and 10m5 M. respectively. The potency of cypermethrin under glasshouse conditions is synergized 5- to 50-fold by monocrotophos, acephate or methidathion and it is also greatly increased by profenofos, with synergist:pyrethroid ratios ranging from I:8 to 8: I. Cypermethrin toxicity under field conditions in cotton is strongly synergized and the effective period for whitefly control is prolonged by adding an equal weight of monocrotophos or acephate or 8 parts of methidathion to I part pyrethroid. Mouse liver pyrethroid esterases hydrolyzing cis-cypermethrin are inhibited by low intraperitoneal doses of profenofos and acephate but not monocrotophos and methidathion. The high magnitude of cypermethrin synergism in whiteflies is not repeated in mice. perhaps due to differences in the toxicological importance and inhibitor specificities of esterases involved in detoxification. Is 19X7 Academic Presc. Inc

INTRODUCTION

Pyrethroids are detoxified in insects by the action of both esterases and oxidases (l-8). In larvae of the Egyptian cotton leafworm (Spodoptera littoralis) and the cabbage looper (Trichoplusia nil, the rate of pyrethroid hydrolysis is higher with the trans- than with the cis-isomers, corresponding to their relative insecticidal activities (1, 6). Under laboratory conditions the toxicity of cis-cypermethrin is synergized by profenofos about 20-fold against T. ni (I) and by profenofos and monocrotophos about threefold against S. littoralis (6).

’ Abbreviations used: BSA, bovine serum albumin; ip, intraperitoneal: LC,,, lethal concentration for 50% of the organisms: LD,,, lethal dose for 507~ of the organisms; MTG, methoxytriglycol; RLT,,, 50% residual lethal time at which potency drops to one-half of the original value.

Phenyl saligenin cyclic phosphonate synergizes the toxicity of trans-permethrin over 60-fold against Chrysopa cnrnea larvae (4). On the other hand, in Triholium cnstcrneum larvae, oxidases seem to be more important than esterases for pyrethroid detoxification, since oxidase (but not esterase) inhibitors synergize the toxicity of several pyrethroids (7). Apparently the predominant pathway of pyrethroid detoxification in insects, whether hydrolytic or oxidative, depends largely on the insect species and to some extent on the individual pyrethroid involved. The present report and a preliminary abstract (9) consider biochemical and toxicological aspects of cypermethrin synergism by organophosphorus insecticides in adults of the whitefly Bemisia tabuci, an important pest of cotton and vegetable crops and a target for control by cypermethrin and other pyrethroids.

155 0048-3575187 Copyright All rights

$3.00

‘d 1987 by Academic Press. Inc. of repruduction in any form rexwed.

156

ISHAAYA

MATERIALS

AND

METHODS

The labeled pyrethroids were obtained and purified as described previously (1, 4, 6, 10). The r4C-labeled acid preparation of cis- and trans-permethrin was lRS, that of cis- and Irans-cypermethrin was lRS,(rS, and that of [14C]deltamethrin was (IR,c&)-cis. The unlabeled pyrethroids used were (IR,aS)-cis-cypermethrin (Roussel-Uclaf, Paris, France) of >95% stereochemical purity and emulsifiable concentrates of 10% Cymbush (cypermethrin with a 60:40 cis:trans ratio, Makhteshim, Be’er Sheva, Israel), 20% Fenom (cypermethrin with an 80:20 cis:trans ratio, Ciba-Geigy, Basel, Switzerland), and 50% Fenom-S (a mixture of 5.4% Fenom and 44.6% methidathion, Ciba-Geigy). Organophosphorus insecticides tested as candidate pyrethroid synergists were emulsifiable concentrates of 50% profenofos (Ciba-Geigy), 40% monocrotophos (Makhteshim), and 40% methidathion (CibaGeigy) and a soluble concentrate of 75% acephate (Chevron, Richmond, CA). Higher-purity samples were used for enzyme inhibition and mouse toxicity assays, i.e., monocrotophos (97%, Shell Co., London, U.K.), profenofos (91%, CibaGeigy), methidathion (99.6%, Ciba-Geigy), and recrystallized acephate and methamidophos (Chevron). Whitefly pyrethroid esterase assays. The standard enzyme solution was prepared at 0-3°C by homogenizing 30 mg of whitefly adults (-1000 adults) in 6 ml water. The postmitochondrial 12,OOOg supernatant (microsome + soluble) fraction obtained after 15 min centrifugation was assayed for pyrethroid esterase activity. The enzyme assay based on a procedure described previously (I, 4, 6) was optimized in 0.8 ml reaction mixture consisting of 0.2 ml enzyme solution (equivalent to I mg adult weight and approximating 35 adults and 100 kg protein), 0.4 ml 0.1 M glycine-NaOH buffer at pH 9.3, and 14C-labeled pyrethroid (up to 2.5 nmol, -2000 cpm) in 0.2 ml 0.2% bovine serum albumin (BSA)’ soChemicals.

ET

AL.

lution with incubation for 60 min at 37°C. The substrate was solubilized by adding 100 t~.l ethanolic solution of 14C-labeled pyrethroid to 10 ml 0.2% BSA solution. Enzyme activity was terminated by adding 5 ml scintillation mixture and determined as described previously ( 1). The inhibitors were dissolved in ethanol which was then diluted lOO-fold with water to give the inhibitor solutions (I, 6). The final inhibitor concentration is expressed as that present in 0.2 ml of preincubation medium consisting of 0.1 ml enzyme solution and 0. I ml inhibitor solution. The indicated amount of ethanol had no appreciable effect on enzyme activity. The enzyme reaction in 0.8 ml final volume at 37°C was initiated after 15 min preincubation by adding the buffer and substrate, and enzyme activity was determined as described above. Whitefly

synergism

assays.

B. tahaci

were reared on cotton seedlings in a glasshouse at 26 2 2°C. For glasshouse assays, cotton seedlings were sprayed until runoff with the pyrethroid and/or synergist using an electric sprayer. Six replicates of 12- 15 whitefly adults confined in leaf cages were exposed to treated plants at various intervals after application, and kept under glasshouse conditions at 26 ? 2°C for 48-hr mortality determination. For field assays, cotton plants (60-80 cm high) were sprayed until runoff with the pyrethroid and/or synergist using a knapsack sprayer. Three replicates of a 3-m row were used for each treatment, from which 15 branch samples were cut at various intervals and maintained in water for residual toxicity determination. Twelve to 15 adults were placed on each branch sample as above and kept under glasshouse conditions for mortality determination. With this procedure 24 hr exposure was used, since leaf wilting was observed after 48 hr. Both glasshouse and field assays were carried out in the summer seasons of 1983 and 1984 at Bet Dagan . Mouse synergism and pyrethroid terase assays. Male Swiss albino

es-

mice

SYNERGIZED

PYRETHROIDS

FOR

WHITEFLY

157

CONTROL

60

6

9

II

IO

0

30

60

TIME

PH

(mm

so )

ENZYME

PROTEIN,

pg

FIG. I. Effect of pH. incubation time. und enzyme protein lervl on hydrolysis of trcrtzs-pert?lethritl by Bemisiu tabnci esteruse( Enzyme protein refirs to the postmitochorzdrinl supernatant fraction. The g/wine-sodium hydroxide buffer QWS used. Each orrot\’ designcries the stundard crssay condition.

(18-22 g) were treated ip with the test compounds using MTG as the carrier vehicle (1 ~1 g-i body wt) or as controls with MTG alone. In toxicity studies, the candidate synergist was administered at 3 mg kg-’ (monocrotophos), 16 mg kg-i (profenofos, methamidophos, and acephate), or 150 mg kg-l (piperonyl butoxide) 1 hr be-

go‘1 60 -

60 -

01

I

20

1

I

30

I

I

I

40

I 50

1

I

fore (lR,aS)-cis-cypermethrin for mortality determinations 24 hr later. LD,, values are estimates based on studies with 74 mice (pyrethroid only) or 21-34 mice (each pyrethroid-synergist combination). For assays of liver pyrethroid esterases, the mice were treated with the organophosphorus compound at 0.25 to 16 mg kg-l or with MTG alone and sacrificed after 1 hr by cervical dislocation. The preparation of liver pyrethroid esterase and assay of its activity were modified from earlier studies (2, 11, 12). The liver was homogenized at 25% (w/v) in 0.1 M sodium phosphate buffer, pH 7.4, and centrifuged at 15,OOOg for 15 min. The supernatant was diluted 50fold with water and used as the enzyme solution. The standard enzyme assay consisted of 0.8 ml 0.1 M sodium phosphate buffer, pH 7.4, 0.8 ml enzyme solution (representing 4 mg liver or 100 pg protein), and [14C]cis-cypermethrin (-2.5 nmol and -2000 cpm) in 0.4 ml 0.2% BSA. The mixture was incubated for 15 min at 37°C. Enzyme activity was terminated by adding 10 ml liquid scintillation mixture and determined as described previously (2).

I

60

RESULTS TEMPERATURE,

FIG. 2. IZffct ?f trans-permethrin

of reaction

temperature by Bemisia rubuci

%

on hydrolysis esteruse(

Whitej7y pyrethroid

methrin

hydrolysis

esterase. trans-Perby the postmitochon-

158

ISHAAYA

Hydrolysis

of Permethrin

and Cypermethrin ,frorn

ET AL.

TABLE Isomers Whitej7y Hydrolysis

and Deltamethrirt Adults

trQnS

CiS

Permethrin Cypermethrin Deltamethrin

45.7 + O.@ 9.6 -c I.Oh

36.4 -+ 1.2< 14.0 t 0.2’ 20.7 + I.Od

standard letters

conditions. Data are averages of eight replicates are significantly different at the 1% level.

drial supernatant fraction is optimal at pH 9.2-9.4 in the glycine-NaOH buffer with incubation for up to 60 min using 100 pg protein, the standard conditions selected (Fig. 1). Although the highest esterase activity is obtained at 49°C (Fig. 2), 37°C was adopted since it approximates the environmental temperature of the whitefly in the summer season. With these assay conditions, trans-permethrin is hydrolyzed faster than its cis isomer or its a-cyano analogs trnnsand cis-cypermethrin and delta-

60 -

CONCENTRATION,

by Pyrethroid

M

3. Effect of organophosphorus insecticides in vitro on hydrolysis of trans-permethrin by Bemisia tabaci esterase(s): (0) monocrotophos: (0) methidathion; (A) acephate; (A) methamidophos. The concentrations of the inhibitors are those present itI the 0.2 ml enzyme-inhibitor preincubation medium (see Materials and Methods). Data are averages o,f four replicates with their SE values.

Esterase(s)

(‘Z)”

Pyrethroid

a Hydrolysis in 60 min under Means with different superscript

FIG.

I

Ratio translcis 1.45 0.69 with

their

SE values.

methrin (Table 1). trans-Permethrin was therefore used as the standard substrate. Monocrotophos is the most potent of the candidate pyrethroid esterase inhibitors, i.e., 50% inhibition at 9 x 10U7 M monocrotophos, 10m5 M methamidophos, and 6 x IO-’ M methidathion, and the enzyme is totally inhibited at lop4 M of each compound (Fig. 3). Acephate is not an inhibitor of pyrethroid esterase activity in vitro. Whitefly synergism. Monocrotophos, profenofos, and acephate are efficient synergists for formulated cypermethrin in glasshouse assays against B. tnbuci adults (Table 2 and Fig. 4). Addition of monocrotophos to a cypermethrin formulation (Cymbush) at a ratio of 1: I a.i., and of profenofos at a ratio of 1:4 a.i., strongly synergizes the toxicity of cypermethrin and prolongs its activity. The RLT,, value of 40 ppm cypermethrin is -3 days vs -16 days with the mixtures (Fig. 4). According to log dose-probit mortality plots, monocrotophos synergizes the toxicity of cypermethrin about 50- and 38-fold at 3 and 10 days after treatment, respectively, while synergism by acephate under similar experimental conditions is 5- and lo-fold at 1 and 6 days after treatment, respectively (Table 2). On cotton under field conditions, the RLT,, value is moderately prolonged by an equal amount of either acephate or monocrotophos, i.e., the RLT,, of 200 ppm cypermethrin is al day vs -5 days with the mixture (Table 3). Cypermethrin synergism obtained under these conditions is much

SYNERGIZED

PYRETHROIDS

FOR WHITEFLY

TABLE Effect

of Monocrotophos,

159

CONTROL

2

Acephate, and Methidathion, Toxicity of Cypermethrin against

under Glasshouse Conditions, Betnisia tabaci Adults

on the Residual

LC,, (ppm) [95% confidence limitsIb

SynergistO and days after application

Cypermethrin + organophosphorus compound

Cypermethrin

Monocrotophos 3 10 Acephate 1 6 Methidathion 7

Synergistic ratio

151 [96-3021 750’

3 20

[2-41 [13-391

50 38

151 [136-1671 316 [267-4011

32’ 33 115-471

5 10

39 [29-481

1.9 [0.2-4.11

20

Q Cypermethrin of normal cis:trans ratio (Cymbush) was used in the monocrotophos and the acephate assays, and that of high cis:trans ratio (Fenom) in the methidathion assay. Synergist concentrations were 40 ppm monocrotophos and 20 ppm acephate. In the methidathion assay, Fenom was compared with the commercial formulation Fenom-S (8: 1, methidathion:Fenom mixture). b LC, values and 95% confidence limits were calculated according to the probit procedure of the SAS statistical package (“SAS User’s Guide Statistics,” 1982 ed., Cary, NC). c The values on the probit curve were not sufficient to obtain 95% confidence limits.

less pronounced conditions. Methidathion

than that under glasshouse is an effective I

-2

FIG. 4. Synergistic

cyper-

I

L 6 16 32 DAYS AFTER TREATMENT

effect of monocrotophos and profenofos, under glasshouse conditions on cotton seedlings, on the toxicity and residual effectiveness of cypermethrin to Bemisia tabaci adults: (O-O) 40 ppm cypermethrin: (W-0) 10 ppm profenofos; (A- -A) 40 ppm monocrotophos; (0-O) 40 ppm cypermethrin plus 10 ppm profenofos; (A-A) 40 ppm cypermethrin plus 40 ppm monocrotophos. A logarithmic scale is used for time and a probit scale for percentage mortality.

methrin synergist under both glasshouse and field conditions when used in a commercial mixture of I part cypermethrin to 8 parts methidathion (Fenom-S). The amount of methidathion present in Fenom-S in these assays, when tested alone, has no appreciable effect on B. tabaci adults. At an equivalent pyrethroid concentration, Fenom-S is much more toxic than Fenom (Fig. 5), with 20-fold synergism at the LC,, level (Table 2). Fenom is more potent than Cymbush under glasshouse conditions (Table 2), as expected from its higher content of the &-isomer of cypermethrin. In a comparison under field conditions of three commercial formulations, each with 200 ppm cypermethrin, the synergized cypermethrin (Fenom-S) is considerably more effective than the nonsynergized cypermethrin (Cymbush or Fenom; Fig. 6). Mouse synergism and pyrethroid esterase. A preliminary study establishes that

the ip LD,, of (lR,aS)-cis-cypermethrin (7.6 mg kg-‘) is reduced only slightly (4.9-5.4 mg kg-r) in mice treated ip 1 hr earlier with profenofos, methidathion, or acephate at 16 mg kg-l. A higher degree of synergism occurs with either monocrotophos (3 mg kg-‘) or piperonyl butoxide

160

ISHAAYA

Effect

of Acephafe

and Monocrotophos.

ET AL.

TABLE 3 under Field Conditions, on the Residual against Bemisia tabaci Adalts Mortality

Synergists and days after application Acephate” 1 4 6 Monocrotophos” I 3 6

12

Toxicity

of Cypermethrin

t% -t SE) Cypermethrin

(200 ppm)

Cypermethrin (200 ppm)

Organophosphate (200 ppm)

32 2 3 25 k 5 17 t 4

23 +- 2 822 I?1

85 -t 4” 74 2 5” 36 t 4*

50 2 4 42 k 4 16 k 3 0

28 27

k t II 2

89 i 3* 84 ” 3* 35 5 6** 321

0

4 3 3

+ organophosphate

(200 ppm)

* Significantly different at the 5% level from the sum of mortality obtained with cypermethrin and organophosphate separately. ** Significantly different at the 5% level from the mortality obtained with cypermethrin alone. a The acephate and monocrotophos treatments were carried out in different sets of experiments and times in the summer season of 1983. The variability in the residual toxicity of cypermethrin may result from different climatic conditions

(150 mg kg-‘), which lowers the cypermethrin LD,, to 3.2-3.5 mg kg-‘. Profenofos and acephate, administered ip, in-

L-L-d I 2 DAYS AFTER

iREAT&NT

hibit the liver esterases hydrolyzing cis-cypermethrin by 50% at 2-3 mg kg-’ (Fig. 7). By contrast, methidathion and monocro-

16

FIG. 5. Synergistic effect of methidathion, under glasshouse conditions on cotton seedlings. on the to-vicity and residual effeectiveness of cypermethrin to Bemisia tabaci adults: (0) 30 ppm cypermethrin (Fenom); (0) 30 ppm cypermethrin plas 250 ppm methidathiorz (Fenom-S). Methidathion alone at 250 ppm gave no mortality. Scales for plotting are as in Fig. 4.

FIG. 6. Toxicity and residual effectiveness of three commercial formulations qf cypermethrin, under field conditions on cotton plants, to Bemisia tabaci adults: (0) 200 ppm cypermethrin (Cymbush); (0) 200 ppm cypermethrin (Fenom); (A) 200 ppm cypermethrin plus 1600 ppm methidathion (Fenom-S). Scales for plotting are as in Fig. 4.

SYNERGIZED

“,L, 0.25

PYRETHROIDS

,

,

I

4

CONCENTRATION,

,I 16

mg

kg-’

FIG. 7. Effect oforganophosphorus insecticides in \*ivo on hydrolysis of cis-cypermethrin by mouse live1 esterase(s): (m) profenofos; (A) acephate; (0) methidathion; (0) monocrotophos. Enzyme crssuys were carried out I hr after injecting the mice +\Gth ,sarious r~oncentrations of ench test compound. Treutments involved three to five repkates of tvt’o mice euch. Burs represent SE values of the mecrru.

tophos have no appreciable 16 mg kg-‘, respectively.

effect at 4 and

DISCUSSION

Both hydrolytic and oxidative processes are involved in the metabolism of permethrin, cypermethrin, and deltamethrin, so that the choice of an optimal synergist depends on the predominant pathway of pyrethroid detoxification in the insect of interest (5, 8, 13-17). The effectiveness of esterase inhibitors as synergists with B. tabaci enables us to add it to the list of species for which esterases are the limiting factor in pyrethroid toxicity, i.e., T. ni (I), S. littoralis (6), and C. carnea (4), in contrast to T. castaneum and Musca domestica vicina, for which oxidase inhibitors are the most effective synergists (7, 13). Organophosphorus insecticides inhibiting pyrethroid esterase activity in B. tabaci synergize, to varying extents, the toxicity of cypermethrin under both glasshouse and field conditions. Among the compounds tested, monocrotophos is the

FOR

WHITEFLY

CONTROL

161

most potent inhibitor of the whitefly pyrethroid esterase activity and most effectively synergizes the toxicity of cypermethrin under glasshouse conditions. Methidathion and acephate probably undergo in Go activation as cypermethrin synergists, by oxidation of methidathion to its oxygen analog and hydrolysis of acephate to methamidophos. Thus. acephate does not inhibit pyrethroid esterase activity whereas methamidophos shows a pronounced inhibitory effect. Cypermethrin synergism under glasshouse conditions is over 20-fold by monocrotophos or methidathion and up to lofold by acephate. Pyrethroid synergism is less pronounced in the field than under glasshouse conditions. This may be due in part to more extensive photodegradation of the synergist and/or the pyrethroid under field conditions. Optimization of a synergist for pyrethroids must consider its possible effects on mammalian toxicity. The compounds examined here are more effective synergists in whiteflies than in mice. The selectivity of monocrotophos and methidathion may be due in part to their poor activity as inhibitors of the mouse liver pyrethroid esterase(s). Mice also appear to depend on oxidases as well as esterases in cypermethrin detoxification. This limited comparison of whiteflies and mice indicates the possibility of selective synergism in insects compared with in mammals. ACKNOWLEDGMENTS This paper constitutes contribution No. 1324-E, 1985 series, from the Agricultural Research Organization. the Volcani Center. Bet Dagan. Israel. The research was supported in part by Grant I-249-81 from the United States-Israel Binational Agricultural Research and Development (BARD) Fund. and by Grant PO1 ES00049 from the U.S. National Institute of Environmental Health Sciences. The field assays were supported in part by the Cotton Council of Israel and CTS Chemical Co.. Tel Aviv. The authors thank CibaGeigy (Basel, Switzerland), Chevron (Richmond, CA), and Makhteshim (Be’er Sheva, Israel) for supplying most of the test chemicals.

162

ISHAAYA REFERENCES

1. I. lshaaya and J. E. Casida, Properties and toxicological significance of esterases hydrolyzing permethrin and cypermethrin in Trichoplusia ni larval gut and integument, Pesric. Biochem. Physiol.

and Toxicology” (D. H. Hutson and T. R. Roberts, Eds.), Vol. 3, pp. 401-435 (1983). 9. I. Ishaaya, Z. Mendelson. K. R. S. Ascher, and J. E. Casida. Mixtures of synthetic pyrethroids and organophosphorus compounds for controlling the whitefly. Bemisia tabaci. Phytoparusi-

14, 178 (1980).

tica 13, 76 (1985).

2. L C. Gaughan, J. L. Engel, and J. E. Casida. Pesticide interactions: Effects of organophosphorus pesticides on the metabolism. toxicity and persistence of selected pyrethroid insecticides, Pestic. Biochem. Physiol. 14, 81 .-(1980). ~~_.. 3. Y. A. 1. Abdel-Aal and D. M. Soderlund, Pyrethroid-hydrolyzing esterases in southern armyworm larvae: Tissue distribution, kinetic properties, and selective inhibition, Pestic. Biothem. Physiol. 14, 282 (1980). 4. I. Ishaaya and J. E. Casida, Pyrethroid esterase(s) may contribute to natural pyrethroid tolerance of larvae of the common green lacewing, Enl,iron. Entomol. 10, 681 (1981). 5. I. Ishaaya and J. E. Casida, Pyrethroid detoxification and synergism in insects. in “Pesticide Chemistry: Human Welfare and the Environment,” (J. Miyamoto and P. C. Kearney, Eds.), Voi. 3, pp. 307-310, Pergamon, New York (1983). 6. I. Ishaaya, K. R. S. Ascher, and J. E. Casida. Pyrethroid synergism by esterase inhibition in Spodoptera

ET AL.

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Protect.

2, 335

(1983). 7. I. Ishaaya, A. Elsner, K. R. S. Ascher, and J. E. Casida. Synthetic pyrethroids: Toxicity and synergism on dietary exposure of Tribolium castaneum (Herbst) larvae. Pestic. Sci. 14, 367 (1983). 8. D. M. Soderlund, J. R. Sanborn, and P. W. Lee. Metabolism of pyrethrins and pyrethroids in insects. in “Progress in Pesticide Biochemistry

10.

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T. Shono. K. Ohsawa, and J. E. Casida. Metabolism of truns- and cis-permethrin. ~rans- and cis-cypermethrin. and decamethrin by microsomal enzymes, J. Agric. Food Chem. 27, 316 (1979). L. T. Jao and J. E. Casida. Insect pyrethroid-hydrolyzing esterase, Pestic. Biochem. Physiol. 4, 465 (1974). T. Suzuki and J. Miyamoto. Purification and properties of pyrethroid carboxyesterase in rat liver microsome. Pestic. Biochem. Physiol. 8, 186 (1978). I. Ishaaya, S. Yablonski, K. R. S. Ascher, and J. E. Casida, Pyrethroid synergism and prevention of emergence in Tribolium castaneum and Mrrsca domestica b*icina by the insect growth regulator RO 13-5223, Phytoparasitica 12, 99 (1984). D. M. Soderlund and J. E. Casida, Substrate specificity of mouse-liver microsomal enzymes in pyrethroid metabolism, Amer. Chem. Sot. Symp. Ser. 42, 162 (1977). J. E. Casida and L. 0. Ruzo, Metabolic chemistry of pyrethroid insecticides, Pestic. Sci. 11, 257 (1980). W. S. Bigley and F. W. Plapp, Jr., Metabolism of cis- and trans-[14C]permethrin by the tobacco budworm and the bollworm, J. Agric. Food Chem.

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26, 1128 (1978).

J. S. Holden. Absorption and metabolism of permethrin and cypermethrin in the cockroach and the cotton leafworm larvae, Pestic. Sci. 10, 295 (1979).