Enhanced pyrethroid hydrolysis in pyrethroid-resistant larvae of the tobacco budworm, Heliothis virescens (F.)

PESTICIDE

BIOCHEMISTRY

Enhanced

AND

Pyrethroid Tobacco

PHYSIOLOGY

28, 9-16 (1987)

Hydrolysis Budworm,

in Pyrethroid-Resistant Larvae Heliothis virescens (F.)’

of the

PATRICK E DOWD,~~YNTHIA C. GAGNE,* ANDTHOMAS C. SPARKS~ Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, and *American Cyanamid, Agricultural Research Division, P.O. Box 400, Princeton, New Jersey 08540 Received August 18, 1986; accepted November 26, 1986 Larvae of Heliothis virescens (F.) collected from the field in the Imperial Valley of California were reared in the laboratory and selected periodically with flucythrinate. At the end of the selection experiment, this Imperial Valley (IV) strain had LD,, values that were ca. 68, 53, 16, 23, and 2 times higher for flucythrinate, fenvalerate, DDT, carbaryl, and ethyl parathion, than a susceptible American Cyanamid laboratory strain (ACCO). This IV strain was then transferred to LSU, without further selection pressure, where it was found to be ca. 12.2-, 5.4-, and 25fold less susceptible to trans-permethrin. cis-permethrin, and fenvalerate, respectively, when compared with another laboratory strain (LSU lab). Piperonyl butoxide did not synergize trans-permethrin in the IV strain. Conversely, profenofos increased the toxicity of trans-permethrin to levels nearly identical to that for the LSU lab strain. The rates of hydrolysis were ca. 2- to 3-fold higher in the IV strain than in the LSU lab strain for all three pyrethroids. Isoelectric focusing also indicated higher levels of rrans-permethrin hydrolytic activity in the IV strain as opposed to the LSU lab strain as well as the presence of an additional peak of activity. These observations suggest that there is both a qualitative and a quantitative difference in the ability of the pyrethroid-resistant IV strain to hydrolyze pyrethroids. o 1987 Academic press, IX.

INTRODUCTION

there has been concern that it may also develop resistance to the pyrethroids. Recent problems with pyrethroids in the related Australian species, Hefiothis armigera (4, 5), and the development of pyrethroid-resistant strains of H. virescens in the laboratory (6, 7) has contributed to this concern. Studies on H. virescens from the Imperial Valley of southern California (8, 9) suggest that there has been a decline in susceptibility to the pyrethroids (but also see Ref. (10). Recent studies on H. virescens from the Imperial Valley demonstrated a 15 to 22-fold decrease in susceptibility to permethrin, relative to a susceptible strain, associated with a decrease in nerve sensitivity and an increase in rate of metabolism (11, 12). Since ester hydrolysis is a major metabolic pathway for many of the pyrethroids in insects (13), including H. virescens (14), the presence, nature, and potential importance of the enzyme(s) responsible for pyrethroid hydrolysis in H. virescens were investigated using a pyre-

Insecticide resistance has become one of the major concerns in insect pest management (1, 2). Without effective chemical control measures, many insect pest management programs would be in jeopardy. In the case of the tobacco budworm, He&this virescens (F.), the pyrethroids were introduced in the late 1970s at a time when this species had developed resistance to many of the available organophosphorus and carbamate insecticides (3). Today the pyrethroids are the primary insecticides used for the control of this and other insect pests. Since H. virescens has had a history of developing resistance to insecticides (3), * Approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript 86-17-0057. Z Present address: USDA-ARS, Northern Regional Research Center, 1815 North University Street, Peoria, 1161604. 3 To whom correspondence should be addressed. 9

0048-3575187 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

10

DOWD,

GAGNE,

throid-resistant strain from the Imperial Valley and a laboratory strain. MATERIALS

AND

METHODS

The LSU laboratory strain of H. was obtained from a colony collected from cotton in Louisiana in 1977 and annually supplemented with field-collected individuals. The pyrethroid-resistant strain was collected from the Imperial Valley (IV) of California in October of 198 1. Third instar larvae of the F, generation were found to be 7.3-fold less susceptible to flucythrinate than a susceptible laboratory colony maintained at American Cyanamid. Starting 4 months after colonization, the IV strain was selected with flucythrinate for the next 18 months. The resistance ratio eventually rose to ca. 68-fold for flucythrinate (Table l), after which insecticide selection was terminated and the colony was transferred to LSU. IV strain larvae used in the present study had been without selection pressure for ca. three to four generations prior to initiation of these studies. All larvae were reared on a pinto bean diet (15), at 27 + l”C., and 40 + 10% relative humidity, and on a 14L:lOD photoperiod. Insects used for enzyme assays were third or last (fifth) instar, feeding phase larvae. Only last instar larvae were used for isoelectric focusing and inhibition studies. Chemicals. 14C-Radiolabeled cis- and trans-permethrin (both at 57 mCi/mmol, labeled on the methylene carbon of the 3phenoxybenzyl alcohol), and unlabeled cisand trans-permethrin (99% pure) were a gift from FMC Corp. Radiolabeled [14C]fenvalerate (8.7 mCi/mmol, labeled on the chlorophenyl group of the acid moiety) and unlabeled fenvalerate were kindly provided by Shell Development Corp. The 2-(4-chlorophenyl)-3-methylbutryic acid was from Frinton, while the DEF (S,S,S-tri-n-butyl phosphorotrithiolate) and piperonyl butoxide (PB) were from Chem Service. Profenofos (0(4-bromo-2-chloroInsects. virescens

AND

SPARKS

phenyl) O-ethyl S-propyl phosphorothiolate) was from Ciba-Geigy. Mercuric chloride, l,lO-phenanthroline, and all other biochemicals were from Sigma. Bioassays. Larvae (35-45 mg) were treated topically on the dorsum of the thorax with the pyrethroid or pyrethroidsynergist combination (1: lo), which were applied in 1 ~1 of acetone. At least five doses were used per compound, and at least 30 insects were used per dose. The criterion for mortality (72 hr) was the inability of the larvae to translocate within 30 set of being prodded. Mortality was analyzed by probit analysis (16). Enzyme assays. Whole body and midgut homogenates of larvae were prepared as described previously (17). Midguts were dissected from chilled (on ice) larvae in pH 7.4, 0.1 M sodium phosphate buffer, and the contents were removed. The midguts or whole bodies were then homogenized in 1 ml of the buffer, centrifuged for 5 min at lOOOg, and the supernatant was filtered through a 0.45~pm HATF filter (Millipore Corp.). The filtrate was diluted in the above buffer containing 0.05% bovine serum albumin such that the rate of hydrolysis (at 35°C) for the three pyrethroids (all at 5 p&Y) was linear with time. Hydrolysis products were separated from the parent compounds either through solvent partitioning (cis- and trans-permethrin) (17) or C-18 reverse phase (Whatman) TLC using methanol as solvent (fenvalerate). Standards for the TLC separations included fenvalerate and 2-(4-chlorophenyl)-3-methylbutryic acid (acid moiety of fenvalerate). Following separation of the hydrolysis products, quantification of radioactivity was by liquid scintillation counting. Preliminary studies of the products resulting from the incubation of fenvalerate with the midgut or whole body homogenates indicated that only hydrolysis was occurring under the conditions described for the assay. The protein content of the homogenates was determined by the method of Bradford (18) as modified by Bio-Rad.

PYRETHROID

RESISTANCE

Inhibition studies. Inhibitors (in 1 ~1 of ethanol) were added to midgut homogenates (the most active tissue in H. virescens for hydrolyzing pyrethroids; Dowd and Sparks, unpublished) and preincubated at 35°C for 10 min. The trans-permethrin was then added, and the incubation continued for 20 min. All other aspects of these assays were the same as above. Inhibitory activity was based on concurrently run uninhibited enzyme samples. Isoelectric focusing. Isoelectric focusing was performed using an LKB Multiphor system as described previously (19). For the determination of initial areas of activity, LKB precast pH 3-10 polyacrylamide gels were used, with the more detailed studies being performed with narrow range gels (pH 4-6.5) cast using Pharmacia ampholytes. Samples (20 pi/track) of whole body homogenates (4/ml) were prepared as described above and applied to a prefocused gel (1 hr at 25 W) and then focused for 2 hr. The gels were sectioned (0.25 cm) and then assayed for trans-permethrin hydrolytic activity. RESULTS

When compared with a susceptible laboratory strain (ACCO), larvae collected from the Imperial Valley and reared through one generation were ca. 7.3-fold less susceptible to flucythrinate (Table 1). In the absence of selection pressure, this level of resistance dropped to levels that were equal to those for the susceptible strain. Once selection pressure was again started, resistance to flucythrinate quickly reappeared and eventually rose to rather high levels (68 x ). These flucythrinate-resistant insects were also highly resistant to fenvalerate and to a lesser degree also to DDT and carbaryl. There was essentially no cross-resistance to ethyl parathion (Table 1). The transfer of larvae to LSU, combined with a lack of selection pressure and the use of the LSU strain as a reference, all

IN Heliothis

11

contributed to a reduction in both the observed LD,, of the IV strain and the resistance ratios. Compared with the LSU laboratory strain, the IV strain of H. virescens was ca. 12.2x, 5.4x, and 2.5~ less susceptible to trans-permethrin, cis-permethrin, and fenvalerate, respectively (Table 2). PB did not significantly synergize the toxicity of trans-permethrin to the resistant strain or the LSU lab strain. While profenofos was able to synergize the toxicity of trans-permethrin by 4.7 x for the LSU lab strain, it was nearly 10 times more effective (46.8 x ) for the Imperial Valley strain (Table 2). With profenofos, the toxicity of the synergized mixture to the IV strain was nearly equal to that of the LSU lab strain (Table 2). Profenofos and PB alone were apparently nontoxic at the doses used (data not shown). The rates of hydrolysis for cis- and trans-permethrin and fenvalerate were approximately 2 x higher in third instar larvae of the IV strain than in larvae of the LSU lab strain (Table 3). Trans-permethrin was the most rapidly hydrolyzed pyrethroid, followed by fenvalerate and cis-permethrin. A similar trend in activity for the two strains was also noted for last instar larvae (Table 3). Three compounds, each representative of a class of pyrethroid hydrolysis inhibitor (organophosphates, sulfhydryl group reagents, and chelators, Ref. 19), were tested for their activity toward homogenates of both strains of H. virescens. DEF and mercuric chloride were highly effective inhibitors of truns-permethrin hydrolysis for both strains of H. virescens (Table 4), while 1, lo-phenanthroline was somewhat less active. Isoelectric focusing studies showed that truns-permethrin hydrolytic activity occurred over a fairly large region (pH 4.7-5.7) for both the IV and LSU lab strains of H. virescens (Fig. 1). Recovered activity was greater in the IV strain than in the LSU lab strain at two areas (pH 5.0-4.65, and pH 5.25-5.10). An addi-

12

DOWD, GAGNE,

AND SPARKS

TABLE Susceptibility

History

of Heliothis

virescens

1

Collectedfrom

the Imperial

Valley

L&o

Date

Comments

11-81 12-81 4-82 7-82 12-82 7-83 12-83 12-83 12-83 12-83 12-83 l-84

IV strain collected F, Selection starts

Compound

with Flucythrinate

RRb

bk)

ACCO-S

Flucythrinate Flucythrinate Flucythrinate Flucythrinate Flucythrinate Flucythrinate Fenvalerate DDT Carbaryl Ethyl parathion

and Selected

0.12 0.11 0.08 0.08 0.27 604.3 41.3 60.4

IV-R

IV/S

IV/482

0.88 0.09 0.34 1.90 4.70 5.43 14.3 9870.0 934.8 108.7

7.3 -

1.0 3.8 21.1 52.2 60.3 -

17.3 58.8 67.9 53.0 16.3 22.7 1.8

Moved to LSU

0 ACCO-S is a susceptible laboratory strain maintained at American Cyanamid; IV-R is the pyrethroid-resistant strain from the Imperial Valley. Av weight of larvae tested was 23 mg. b Resistance ratios (RR). LD,, of IV strain/LD,, of ACCO strain, or LD,, of IV strain/LD,, of IV strain on 4-82.

tional area of activity (pH 5.85-5.25) also seen in the IV strain.

was

DISCUSSION

Three groups of insecticides (organophosphorus, carbamate, and pyrethroid) are subject to metabolism via ester hydrolysis, but only for the organophosphorus insecticides has this metabolic pathway been of great importance as a resistance mechanism (13, 20, 21). For insects and arthropods that have developed resistance

to the pyrethroids, reduced penetration and especially active site insensitivity appear to be more important as resistance mechanisms (21, 22). For example, studies with pyrethroid-resistant strains of Musca domestica (22-25), Spodoptera exigua, and S. littoralis (26) found little or no role for ester hydrolysis. In Myzus persicae (27), an esterase (E4) that confers resistance to carbamates, organphosphates, and pyrethroids was able to hydrolyze the least active isomer (lS-trans) of permethrin. However,

TABLE Toxicity

of Pyrethroids

to LSU

2

Lab and Imperial

Valley

Strains

LSU lab strain Pyrethroid trans-Permethrin cis-Permethrin Fenvalerate trans-Permethrin + piperonyl butoxide trans-Permethrin + profenofos

of Heliothis

virescens

Imperial Valley strain

L&o b&T)

(CL)

SR”

LD,o bdg)

(C.I.)

3.70 0.725 0.880

(2.98-5.52) (0.572-0.882) (0.730-1.15)

-

45.4 3.90 2.18

8.52

(6.19-12.16)

0.4

52.9

0.780

(0.605-0.962)

4.7

0.970

SR

RRb

(36.1-55.1) (2.98-4.97) (1.80-3.28)

-

12.2 5.4 2.5

(32.0-65.4)

0.9

6.2

(0.720- 1.20)

46.8

1.3

a SR = synergist ratio; LD, for trans-permethrin/LD,, for trans-permethrin plus PB or profenofos. b RR = resistance ratio; LD,, for Imperial Valley strain/LD,, for lab strain. Weights of insects ranged from 35 to 45 mg. LD,,s were determined from at least five doses for each insecticide or insecticide combination, with 30 insects per dose; values were calculated by probit analysis (16). C.I. = 95% confidence intervals.

PYRETHROID

RESISTANCE

13

IN Heliothis

TABLE 3 Pyrethroid Hydrolysis in LSU Lab and Imperial Valley Strains of Heliothis virescens Hydrolysis (pmol/min/mg protein) Pyrethroid

LSU Lab strain

Imperial Valley strain

HRb

Third instar trans-Permethrin cis-Permethrin Fenvalerate

53.6 + 12.8 3.2 2 0.9 6.3 k 5.2

109.0 k 19.6 5.7 k 1.0 14.4 5 1.2

2.0 1.8 2.3

Last instar trans-Permethrin cis-Permethrin Fenvalerate

13.6 2 1.8 0.7 * 0.2 2.1 2 0.4

44.1 2 1.5 k 3.8 t

7.0 0.6 0.9

3.2 2.1 1.8

0 Values are means * SD for three assays of three replicates each for whole body homogenates. b HR = hydrolysis ratio, rate of hydrolysis for the IV strain/LSU lab strain.

the role of this e&erase in pyrethroid resistance may be that of sequestering pyrethroids rather than inactivating them (27). Although increased rates of hydrolysis of esterase substrates and synergism with the esterase inhibitor DEF suggest the involvement of hydrolytic enzymes in the resistance of S. littoralis to pyrethroids (28, 29), no actual studies using a pyrethroid as a substrate were performed. In the case of a pyrethroid resitant strain of Boophilus microplus, no change in the rate of permethrin hydrolysis was apparent (30). However, subsequent studies (31) found an enhancement in the rate of permethrin hydrolysis, as well as a higher level of synergism with coumaphos (an organophosphorus compound that would be expected to inhibit hydrolysis). Using isoelectric focusing to separate the enzymes responsible, further studies on strains of B. microp/us resistant and susceptible to pyrethroids demonstrated differences in numbers and total activity of esterases that would hydrolyze the pyrethroids (32). Similarly, permethrin-resistant strains of Amblyseius fallacis also possess higher levels of carboxylesterase activity capable of hydrolyzing permethrin (33). For H. virescens, pyrethroid resistance has been attributed to a combination of active site insensitivity, which may be related to a loss of specific binding sites (34), and

increased metabolism (11, 12). The pyrethroid-resistant strain of H. virescens used in the present study displayed cross-resistance to DDT and the other pyrethroids tested but not to the organophosphorus insecticide, ethyl parathion. These results are typical of those observed when active site insensitivity is present (22, 35). In the present study, the IV strain was also resistant to carbaryl in addition to DDT and the pyrethroids, suggsting the involvement of some form of metabolism. This observation was supported by the presence of higher rates of hydrolysis for three pyrethroids (in both larval instars tested) in the pyrethroid-resistant (IV) strain of H. virescens than in the LSU lab strain. Thus, the results of the present study are consistent with the presence of both active site insensitivity and enhanced metabolism in pyrethroid-resistant H. virescelzs. These results confirm the findings of the earlier studies (11, 12). Although Nicholson and Miller (11, 12) suggested that enhanced metabolism played a role in the pyrethroid resistance of H. virescens from the Imperial Valley, the nature of the enzyme systems involved was unclear. The present study clearly demonstrates that the enhanced metabolism is primarily a function of increased ester hydrolysis of the pyrethroids. While both strains are capable of hydrolyzing pyre-

14

DOWD,

GAGNE,

TABLE 4 Effects of Inhibitors on trans-Permethrin Hydrolysis by Homogenates of LSU Lab and Imperial Valley Strains of Heliothis virescens % Inhibition

at 10e4 M Imperial Valley strain

LSU Lab strain

Compound

&Cl, DEF 1, IO-Phenanthrohne (10-T M) Note. Values listed assays run in triplicate

100.0 97.6

* 0.0 k 4.1

99.6 98.8

-t- 0.7 k 2.7

68.9

2 6.7

63.1

r 7.1

are means 2 SD of at least on midgut homogenates.

two

throids, the isoelectric focusing studies indicate that the increased rate of pyrethroid ester hydrolysis in the resistant strain is a function of higher levels of enzyme activity and the presence of an additional enzyme not detected in the susceptible strain. Therefore, there are both quantitative and qualitative differences in the enzymes responsible for trans-permethrin hydrolysis in the IV strain of H. virescens. The importance of ester hydrolysis as a resistance mechanism is further supported by in vivo studies. Profenofos, a highly ef-

AND

SPARKS

fective inhibitor of pyrethroid hydrolysis and a pyrethroid synergist (36, 37), returned the LD,, values for trans-permethrin to nearly susceptible levels in the IV strain. These results suggest that organophosphate-pyrethroid combinations may potentially provide at least a short-term improvement in the control of pyrethroidresistant H. virescens. In contrast to profenofos, piperonyl butoxide, an inhibitor of oxidative metabolism, failed to synergize trans-permethrin in either the LSU lab or the resistant (IV) strain. Although not unequivocal, these results suggest that oxidative metabolism makes little or no contribution to the ability of the IV strain to resist the pyrethroids. The results of the present study indicate that pyrethroid-resistant H. virescens from the Imperial Valley of California exhibits an enhanced capacity to hydrolyze pyrethi-oids, which seems to contribute heavily to their ability to deal with these insecticides. Recent studies (19, 37) suggest that the enzymes responsible for the ester hydrolysis of pyrethroids in the soybean looper, Pseudoplusia includens, behave more like metaloproteases than carboxylesterases. Based on the effectiveness of mercuric chloride and the 1, lo-phenathroline as inhibitors of trans-permethrin hydrolysis in the present study, the same may be true for H. virescens. If H. virescens continues to decrease in its susceptibility toward the pyrethroids, the novel nature of the enzymes involved may make it possible to develop new synergists as counter-measures to pyrethroid resistance. ACKNOWLEDGMENTS

6.0

5.5

5.0

4.5

4.0

PH

1. Separation of tran-permethrin-hydrolyzing enzymes from Imperial Valley and lab strains of H. virescens by isoelectric focusing, representative example (points represent averages of two replicates). LSV lab strain (susceptible to trans-permethrin) (a) Imperial Valley strain (resistant to trans-permethrin) (D). Isoelectric focusing was performed on two separate occasions with two replicates per occasion. FIG.

We thank K. Albarez, M. Barrow, B. Bondy, and R. Rose for assistance in rearing insects, J. Graves and R. Rose for suggestions concerning the manuscript, and V. Daye for assistance in pressuring the IV strain. This research was supported in part, by the Chevron Chemical Co. REFERENCES 1. G. P. Georghiou and R. B. Mellon, Pesticide resistance in time and space, in “Pest Resistance to

PYRETHROID

RESISTANCE

Pesticides,” p. 1, (G. P. Georghiou and T. Saito, Eds.), Plenum, New York (1983). 2. G. P. Georghiou, The magnitude of the resistance problem. in “Pesticide Resistance: Strategies and Tactics for management” (Committee on strategies for the management of pesticide resistance pest populations), p. 14, National Academy Press, Washington, DC, 1986. 3. T. C. Sparks, Development of insecticide resistance in Heliothis zea and Heliothis virescens in North America, Bull. Entomol. Sot. Amer. 27, 186(1981). 4.

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15

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DOWD,

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27. A. L. Devonshire and G. D. Moores, A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate, and pyrethroid resistance in peach-potato aphids (Myzus persicae), Pestic. Biochem. Physiol. 18, 235 (1982). 28. M. R. Riskallah, Esterases and resistance to synthetic pyrethroids in the Egyptian cotton leafworm, Pestic. Biochem. Physiol. 19, 184 (1983). 29. M. R. Riskallah, S. F. Abd-Elaghfar, M. R. AboElghar, and M. E. Nassar, Development of resistance and cross resistance in fenvalerate and deltamethrin selected strains of Spodoptera littorahs (Boisd.), Pestic. Sci. 14, 508 (1983). 30. R. A. Nicholson, A. E. Chalmers, R. J. Hart, and R. G. Wilson, Pyrethroid action and degradation in the cattle tick (Boophilus microplus), in “Insecticide Neurobiology and Pesticide Action” (London: Society of Chemical Industry), p. 289, Whitstable Litho, Kent, England, 1980. 31. H. J. Schnitzerling, J. Nolan, and S. Hughes, Toxicology and metabolism of some synthetic pyrethroids in larvae of susceptible and resistant strains of the cattle tick, Boophilus microplus (Can.), Pestic. Sci.14, 64 (1983). 32. J. DeJersey, J. Nolen, P. A. Davey, and P. W. Riddles, Separation and characterization of the pyrethroid hydrolyzing esterases of the cattle

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tick, Boophilus microplus, Pestic. Biochem. Physiol. 23, 349 (1985). C. K. Chang and M. E. Whalon, Hydrolysis of permethrin by pyrethroid esterases from resistant and susceptible strains of Amblyseius fallacis (Acari: Phytoseiidae), Pestic. Biochem. Physiol. 25, 446 (1986). C. P Chang and F. W. Plapp, Jr., DDT and synthetic pyrethroids: Mode of action, selectivity, and mechanism of synergism in the tobacco budworm (Lepidoptera: Noctuidae) and a predator, Chrysopa carnea Stephens (Neuroptera: Chrysopidae), J. Econ. Entomol. 76, 1206 (1983). T. A. Miller and M. A. Adams, Mode of action of pyrethroids, in “Insecticide Mode of Action” (J. R. Coats, Ed.), p. 3, Academic Press, New York, 1982. L. C. Gaughan, J. L. Engel, and J. E. Casida, Pesticide interactions: Effects of organophosphate pesticides on the metabolism, toxicity, and persistence of selected pyrethroid insecticides, Pestic. Biochem. Physiol. 14, 81 (1980). P. E Dowd and T. C. Sparks, Inhibition of transpermethrin hydrolysis in Pseudoplusia includens (Walker) and use of inhibitors as pyrethroid synergists, Pestic. Biochem. Physiol. 27, 123 (1987).