Toxicity and Neurophysiological Effects of Fipronil and Its Oxidative Sulfone Metabolite on European Corn Borer Larvae (Lepidoptera: Crambidae)

Pesticide Biochemistry and Physiology 71, 97–106 (2001) doi:10.1006/pest.2001.2564, available online at http://www.idealibrary.com on

Toxicity and Neurophysiological Effects of Fipronil and Its Oxidative Sulfone Metabolite on European Corn Borer Larvae (Lepidoptera: Crambidae) Eric W. Durham, Michael E. Scharf,1 and Blair D. Siegfried2 Department of Entomology, University of Nebraska, 202 Plant Industry Building, Lincoln, Nebraska 68583-0816 Received February 28, 2001; accepted June 21, 2001 The phenylpyrazole insecticide fipronil is the first compound of its class to be registered for commercial use. The mode of action of this class of insecticides involves antagonism of the inhibitory neurotransmitter, ␥ -aminobutyric acid (GABA). The sulfone metabolite of fipronil has been reported to be similar in toxicity to the parent compound. In this study, the toxicity and neurophysiological effects of fipronil and the sulfone metabolite were determined for European corn borer larvae. Fipronil was very toxic to neonate European corn borer larvae in feeding bioassays (LC50 ⫽ 3.34 ng a.i./cm2 of treated diet) and to fifth instars in topical bioassays (LD50 ⫽ 18.78 ng/insect). The sulfone metabolite was slightly more toxic to neonate larvae (LC50 ⫽ 1.44 ng a.i./cm2) and equally toxic to fifth instar larvae (LD50 ⫽ 19.54 ng/insect) compared with the parent compound. Neonate larvae preexposed to piperonyl butoxide (PBO) residues coated on the inside of glass scintillation vials for 6 h at 100 ␮g/vial resulted in significant antagonism of fipronil toxicity (LC50 ⫽ 4.39 ng a.i./cm2), whereas preexposure to PBO at 1 ␮g/vial had no effect (LC50 ⫽ 2.91 ng a.i./cm2). Fifth instars topically treated with 10 ␮g of PBO caused significant antagonism of fipronil toxicity (LD50 ⫽ 34.41 ng/insect). Electrophysiological recordings of spontaneous electrical activity were conducted on isolated ventral nerve cords from fifth instar larvae. Results from these experiments indicate that fipronil and its sulfone metabolite both reverse the inhibitory effect of GABA on spontaneous electrical activity. Fipronil, however, caused an increase in spontaneous electrical activity relative to that of the sulfone metabolite. Key Words: phenylpyrazole; fipronil; fipronil–sulfone; electrophysiology; European corn borer.

INTRODUCTION

Le Conte, in many Midwest states including Nebraska. Fipronil’s mode of action involves disruption of chloride ion flow by interacting at the ␥ -aminobutyric acid (GABA)-gated chloride channel of the central nervous system, similar to the cyclodiene class of insecticides (1, 10–14). Fipronil metabolism has been studied both in vivo and in vitro in both insect and mammalian systems. Importantly, the compound possesses a thioether functional group that is metabolized to the corresponding sulfone via cytochrome P450dependent microsomal monooxygenases. This sulfone metabolite appears to possess properties similar to those of the parent molecule in terms of toxicity and neurophysiological effects (15–17). Competitive binding assays with 1-[(4-ethynyl)phenyl]-4-n-propyl-2,6,7-trioxabicyclo-[2.2.2]octane (EBOB), a ligand specific for the GABA

Fipronil is the first phenylpyrazole insecticide licensed for commercial use (1) and is toxic to both piercing–sucking and chewing insects. Fipronil formulations have been developed for soil, foliar, bait, and seed treatment applications (1) and has shown activity across a broad spectrum of insect orders, including Blattaria (2–4), Diptera (2, 5, 6), Hymenoptera (7), Orthoptera (8), and Lepidoptera (9). A fipronil formulation (Regent) was recently introduced for control of corn insects such as the European corn borer (ECB), Ostrinia nubilalis Hu¨bner, and western corn rootworm, Diabrotica virgifera virgifera 1 Present Address: Department of Entomology, Purdue University, West Laffayette, IN 47507-1158. 2 To whom reprint requests should be addressed. Fax: (402) 472-4687. E-mail: [email protected].

97 0048-3575/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

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receptor, suggest that the binding site for fipronil and fipronil–sulfone are similar if not the same (17). Recordings of spontaneous electrical discharge from isolated nerve cords of western corn rootworms (Diabrotica virgifera virgifera L) (15) also indicate a similar mode of action for fipronil and the sulfone metabolite. Studies using the cytochrome P450 inhibitor piperonyl butoxide (PBO), which should inhibit sulfone formation, have provided variable results. In German cockroaches (Blattella germanica L.), PBO is antagonistic to fipronil toxicity (4) or has no effect (2), and in house flies (Musca domestica L.), PBO is synergistic (2, 10). In western corn rootworms, fipronil toxicity was unaffected by pretreatment with PBO (15). Although fipronil has been targeted for O. nubilalis control in maize, there is little information on the toxicity and physiological interaction between this insecticide and the crop pest. Based on the potential for metabolic activation of the parent molecule, the primary objectives of the following series of experiments were to (1) compare the toxicity of fipronil and its sulfone metabolite, (2) determine whether the cytochrome P450 inhibitor piperonyl butoxide affects fipronil toxicity, and (3) use neurophysiological techniques to investigate neural responses to fipronil, its sulfone metabolite, and other known GABA antagonists. MATERIALS AND METHODS

Insects An O. nubilalis insecticide-susceptible laboratory strain was used in all experiments. This lab colony was initiated from field-collected adults obtained from field corn in Saunders County, Nebraska during June 1994 and June 1995 (18) and was reared according to procedures developed by the USDA-ARS Corn Insects Research Unit, Ames, Iowa (19). Larvae were maintained on wheat germ-based diet at 27 ⫾ 0.7⬚C with a photoperiod of 24:0 (light:dark) h and 80% relative humidity (RH) (20). At pupation, insects were transferred to mating cages where emergent adults were maintained with 8-h scotophase at 18 ⫾ 0.7⬚C and

16-h photophase at 27 ⫾ 0.7⬚C with 80% relative humidity. The mating cages where misted twice a day with water and supplied with adult diet for maximum egg production (21). The egg masses were collected and incubated within polystyrene petri dishes on moistened filter paper until hatching. Chemicals Technical-grade fipronil (5-amino-1-(2,6dichloro-4-trifluoromethylphenyl)-3-cyano-4trifluoromethanesulfinylprazole; 97.1% AI) and the sulfone analog (5-amino-1-(2,6dichloro-4-trifluoromethylphenyl)-3-cyano-4trifluoromethylsulfonylpyrazole; 99.9% AI) were provided by Rhoˆne-Poulenc Ag Co. (Research Triangle Park, NC). Dieldrin and PBO were purchased from Crescent Chemical Co. (Hauppage, NY). Picrotoxinin, dimethyl sulfoxide (DMSO), and GABA were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of reagent grade or better. Bioassays Stock solutions of technical-grade fipronil, its sulfone metabolite, and PBO were prepared in acetone. A dilution of PBO (1 or 100 ␮g/0.5 ml) or acetone was pipetted into 20-ml borosilicateglass scintillation vials. The vials were rolled until dry in a fume hood, providing uniform distribution of PBO on the inside of the glass vials. Twenty-five to 30 O. nubilalis egg masses were added to the vials which were placed in a growth chamber (16.3⬚C in 24 h light with 80% RH) to synchronize larval emergence. The synchronized egg masses were allowed to hatch at room temperature (25⬚C), and all larvae in the treated vials were exposed to the PBO residue for 6 h after hatching. For feeding bioassays, stock solutions of fipronil and fipronil–sulfone were prepared in a 0.1% Triton X-100 solution. Serial dilutions of the insecticide stocks were used to treat artificial diet in 128-well bioassay trays (each well 16 mm diameter ⫻ 16 mm height; CD International, Pitman, NJ) (18). Neonate European corn borer larvae, either with or without PBO pretreatment,

FIPRONIL TOXICITY TO EUROPEAN CORN BORER

were placed on the insecticide-treated diet. Diet treated with 0.1% Triton X-100 solution was used for controls. Six (12 for fipronil) replicates of 16 larvae per insecticide concentration and controls were conducted for each insecticide bioassay. Larval mortality was assessed after 48 h as the inability to move or respond to probing. For topical bioassays, newly hatched ECB larvae were transferred onto 1 ml rearing diet in 128-well bioassay trays which were placed in a growth chamber at 27⬚C in 24 h light and 80% RH for 8 days after which individual larvae (5th instar) were randomly selected and treated with insecticides. Larval treatments consisted of fipronil alone, fipronil with piperonyl butoxide pretreatment (10 ␮g/insect applied in 0.5 ␮l of acetone 1 h prior to treatment), and fipronil– sulfone. Three replicates of 16 insects per dose per insecticide treatment were used. Insects were anesthetized by refrigeration, and insecticides were applied with a repeating dispenser in 0.5 ␮l of acetone to the dorsal abdomen. Groups of 16 treated larvae were divided into two groups of 8 per dose and placed in loosely capped 20-ml glass scintillation vials. The vials were held at 24⬚C with 16-h photophase and 8-h scotophase in 80% RH until mortality was assessed at 48 h. Probit analysis (22) of larval mortality was performed with POLO as adapted for PC use (23). A likelihood ratio test was conducted to test the hypothesis that all LCX or LDX values (lethal concentration or lethal dose at which a percentage mortality X was attained) were equal. Neurophysiology Equipment A custom-made suction recording electrode (with reference) was used for all neurophysiological observations. The recording electrode was fitted with 1.0-mm borosilicate capillary tubing (World Precision Instruments, Sarasota, FL) pulled to a fine point with a micropipette puller (MI Industrial Science Associates Inc., Ridgewood, NY). Recording and reference electrodes were designed from 0.68-mm-diameter silver wire and attached to a differential amplifier (DP-301; Warner Instruments, Hamden, CT), which was interfaced with computerized

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hardware (MacLab/2e; ADInstruments, Milford, MA) and software designed to function as an eight-channel chart recorder (Chart Version 3.5.7; ADInstruments). Recording chambers were prepared by the pouring of melted dissection wax into 5 ⫻ 0.9-cm polystyrene petri plates (Gelman Science; Ann Arbor, MI) and then a 3.5 ⫻ 0.75-cm depression in the wax was formed to hold nerve preparations and solutions. Recording chambers were changed periodically to minimize contamination. Neurophysiological Studies One- and 2-day-old fifth instars were used for all electrophysiology experiments. While submerged in physiological saline (185 mM sodium chloride, 10 mM potassium chloride, 5 mM calcium chloride, 5 mM magnesium chloride, 5 mM HEPES sodium salt, and 20 mM glucose, pH 7.1), the larvae were dissected longitudinally on the dorsal surface. The gut and fat bodies were removed from the dissected larvae, exposing the ventral nerve cord. The ventral nerve cord was severed anterior to the third thoracic ganglion and posterior to the second abdominal ganglion. By use of a micromanipulator (No. 1203; Narishige Inc., Japan), the suction electrode was positioned posterior to the first abdominal ganglion. A 10-cc syringe provided suction to attach the electrode to the ventral nerve cord. Spontaneous electrical activity from ventral nerve cord preparations was monitored and recorded by standard electrophysiological techniques and general methods derived from Bloomquist et al. (24) and Scharf and Siegfried (15). Baseline recordings of spontaneous electrical activity were achieved by the setting of a threshold with the software’s “counter” function to obtain 100–500 threshold-surpassing electrical bursts per min. The baseline recording continued for 5 min and was then stopped momentarily to allow physiological saline containing 5 mM GABA to be perfused onto the preparation as the preexisting saline was removed. Electrical activity was observed for 5 min to monitor the GABA effect. The recording

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was stopped, and the preparation received an application randomly chosen from a serial dilution of fipronil or sulfone ranging from 20 to 0.625 ␮M in 5 mM GABA saline. Each preparation received only one treatment with the chosen toxin concentration, and the highest concentration of DMSO (insecticide carrier, 0.4% v/v) was used as the experimental control. The electrical activity of the insecticide-treated preparation was recorded for at least 20 min. Three or more replicates were used per insecticide concentration, resulting in a minimum total sample size of 21 larvae per neurophysiological experiment. GABA-gated chloride channel antagonists dieldrin and picrotoxinin (10 ␮M) were also used in neurophysiological experiments to evaluate the effect of known GABA antagonists on spontaneous electrical activity. The effect of each compound on spontaneous electrical activity was quantified by comparison of the change in counts per min in relation to the average GABA effect or average baseline counts during the initial 5-min recording. Results from the concentration-range assays were quantified in comparison to the average GABA-quieting activity following three baseline recordings. The results were combined into four groups of 5-min intervals (1–5, 6–10, 11– 15, and 16–20 min) for each compound following exposure.

RESULTS

Fipronil and Sulfone Toxicity The sulfone metabolite of fipronil was more toxic to neonate O. nubilalis larvae than the parent compound (LC50 ⫽ 1.44 and 3.34 ng a.i./cm2, respectively) (Table 1). Pretreatment of larvae with 100 ␮g PBO resulted in significant antagonism of fipronil toxicity (LC50 ⫽ 4.39 ng a.i/cm2); however, pretreatment of larvae with 1 ␮g PBO did not significantly affect fipronil toxicity (LC50 ⫽ 2.91 ng a.i/ cm2) as determined by overlapping fiducial limits. In contrast to results form bioassay with neonate larvae, topical bioassays with fifth instars indicated that fipronil and the sulfone metabolite were equally toxic (LD50 ⫽ 18.8 and 19.5 ng/insect, respectively) (Table 2). Additionally, pretreatment of larvae with PBO (10 ␮g/insect) caused significant antagonism of fipronil toxicity based on nonoverlapping fiducial limits at both the LD50 and the LD90. Neurophysiological Recording A representative neurophysiologcal recording of spontaneous electrical activity from isolated ventral nerve cords from fifth instar O. nubilalis larvae is presented in Fig. 1. Preparations perfused with 5 mM GABA in physiological saline showed a reduction in spontaneous electrical activity in relation to the baseline, supporting

TABLE 1 Toxicity of Fipronil, Fipronil ⫹ PBO, and the Sulfone Metabolite to Neonate European Corn Borer Larvae as Assessed by Contact/Feeding Bioassays Treatment

na

Fipronil Fipronil ⫹ PBOe Fipronil ⫹ PBO f Sulfone

1536 768 768 768

a b c d e f

␹2

b

2.56 2.58 4.24 4.68

Slope (⫾SE)

LC50 (95% FL)c

TR50d

LC90 (95% FL)c

TR90d

5.68 5.59 7.29 4.16

3.34 2.91 4.39 1.44

— 0.87 1.31 0.43

5.62 4.93 6.58 2.94

— 0.88 1.17 0.52

(⫾0.45) (⫾0.66) (⫾0.84) (⫾0.49)

(3.13–3.55) (2.62–3.19) (4.07–4.72) (1.24–1.62)

(5.18–6.23) (4.40–5.79) (5.98–7.54) (2.56–3.54)

The number of insects on which each probit analysis is based. Pearson ␹ 2 value indicating goodness-of-fit of the crude mortality data (all P values are acceptable; P ⬍ 0.05). Lethal concentrations (ng a.i./cm2 diet surface); FL, 95% fiducial limits. Toxicity ratios ⫽ fipronil ⫹ PBO or sulfone LC50 and LC90/fipronil LC50 and LC90. Neonate larvae preexposed to 1 ␮g of PBO/20 ml scintillation vial 6 h before insecticide exposure. Neonate larvae preexposed to 100 ␮g of PBO/20 ml scintillation vial 6 h before insecticide exposure.

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TABLE 2 Toxicity of Fipronil, Fipronil ⫹ PBO, and the Sulfone Metabolite to Fifth Instar European Corn Borer Larvae as Assessed by Topical Bioassays Treatment

na

Fipronil Fipronil ⫹ PBOe Sulfone

384 384 384

a b c d e

␹2

b

0.01 2.43 0.43

Slope (SE)

LD50 (95% FL)c

TR50d

LD90 (95% FL)c

TR90d

6.76 (⫾1.20) 3.40 (⫾0.47) 4.98 (⫾0.84)

18.78 (16.16–21.27) 34.41 (27.24–41.41) 19.54 (16.40–22.42)

— 1.83 1.04

29.06 (25.20–36.83) 82.03 (66.65–110.71) 35.34 (29.96–46.54)

— 2.82 1.22

The number of insects on which each probit analysis is based. Pearson ␹ 2 value indicating goodness-of-fit of the crude mortality data (all P values are acceptable; P ⬍ 0.05). Lethal dose (ng/insect); FL, 95% fiducial limits. Average larval weight ⫾ SD ⫽ 42.4 ⫾ 7.6 mg. Toxicity ratios ⫽ fipronil ⫹ PBO or sulfone LD50 and LD90/fipronil LD50 and LD90. Fifth instar larvae were treated with PBO (10 ␮g) 1 h before insecticide treatment.

the inhibitory effect of this neurotransmitter at synaptic junctions of the central nervous system (Fig. 1). A return of spontaneous electrical activity occurred when the GABA solution was removed and replaced with physiological saline (results not shown). The inhibitory effect of GABA on spontaneous electrical activity was consistently reversed by perfusion with 10 ␮M fipronil (Fig. 1), resulting in increased bursting activity. A moderate degree of variation in baseline activity in nerve preparation was encountered, and threshold values were adjusted to provide baseline counts of bursting activity that ranged from 100 to 500 counts per min in the presence of physiological saline. Neural preparations remained viable for at least 1 h after dissection in physiological saline and exhibited relatively small changes in electrical activity during 30-min recordings (Fig. 2A).

Neural preparations perfused with 5 mM GABA in saline after a 5-min baseline recording indicated a suppression of electrical activity relative to its baseline. Perfusion with saline containing GABA ⫹ DMSO (control, 0.4% v/v) did not increase spontaneous bursting activity relative to the baseline obtained after perfusion with GABA–saline. Fipronil (10 ␮M) elicited pronounced increases in electrical activity beyond the threshold in the presence of 5 mM GABA (Fig. 2B). The oxidative sulfone metabolite (10 ␮M), like its parent compound fipronil, increased spontaneous activity exceeding the threshold relative to both its average baseline and a representative GABA ⫹ DMSO control (Fig. 2C). However, the increase in spontaneous electrical activity was less pronounced than that to similar fipronil

FIG. 1. Spontaneous electrical activity from a representative O. nubilalis neural preparation from fifth instar larvae. Baseline activity in physiological saline (A), GABA (5 mM) in physiological saline (B), and fipronil (10 ␮M) ⫹ GABA (5 mM) in physiological saline (C). The threshold (dotted line) is representative of a threshold resulting in 100–500 baseline counts per min, as was used throughout the study.

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concentrations. Two other known GABA antagonists, dieldrin (10 ␮M) and picrotoxinin (10 ␮M), also reversed the GABA quieting effect and increased spontaneous electrical activity compared with the pretreated baseline (Fig. 2D). To further evaluate the effects of fipronil and the sulfone analog on spontaneous electrical activity from O. nubilalis neural preparations, a range of concentrations was compared for each compound. The average spontaneous electrical bursting for the controls (n ⫽ 3) treated with DMSO (0.4% v/v) did not increase in frequency compared to activity from preparations treated with GABA alone during the 20-min recordings. During the first 5 min, fipronil did not increase

bursting activity for concentrations below 5 ␮M (Fig. 3A). The average bursting activity for the preparations treated with 5, 10, and 20 ␮M fipronil concentrations exhibited a concentration-dependent increase in activity (Fig. 3A). The sulfone metabolite produced more sporadic results, although increased activity was noted at lower concentrations than those with fipronil during the 1- to 5-min recording interval (Fig. 3A). Higher concentrations of sulfone produced greater bursting activity relative to controls, although the magnitude of increase was considerably smaller compared to that of fipronil. During the 16- to 20-min interval, the average bursting activity for 1.25 and 5 ␮M fipronil increased, whereas that of 10 ␮M

FIG. 2. Representative time course plots of spontaneous electrical activity for O. nubilalis larvae, quantified by determination of the change of threshold-exceeding bursting frequency in relation to a 5-min average pretreatment baseline. Normal spontaneous activity in physiological saline and controls with 5 mM GABA alone or 5 mM GABA ⫹ DMSO (A); 10 ␮M fipronil and control (B); 10 ␮M sulfone metabolite and control (C); 10 ␮M dieldrin, picrotoxinin, and control (D). Solvent controls and insecticide perfusions occurred in the presence of 5 mM GABA. Arrows at 5 (a) and 10 (b) min indicate initiation of GABA and DMSO or insecticide perfusion, respectively.

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FIG. 2—Continued

decreased slightly and that of 20 ␮M decreased sharply relative to the average 1- to 5-min activity (Fig. 3B). Average spontaneous activity from preparations treated with 5, 10, and 20 ␮M sulfone decreased but remained elevated compared to control activity (Fig. 3B). DISCUSSION

The role of cytochrome P450 in fipronil metabolism and sulfone formation by European corn borer larvae is uncertain, although it is clear that the sulfone metabolite is as toxic or more toxic than the parent compound. The fipronil– sulfone metabolite was significantly more toxic than the parent compound fipronil in neonate bioassays, but for fifth instar larvae fipronil and fipronil–sulfone were equally toxic. Fipronil alone or in combination with PBO was highly toxic to both neonate and late instar European corn borer larvae. Piperonyl butoxide, a common

synergist for insecticides that are metabolized by microsomal oxidases, significantly antagonized fipronil toxicity both in neonate and in fifth instar larvae. The slight antagonism of fipronil toxicity noted for O. nubilalis larvae indicates that oxidative formation of the sulfone metabolite may be an important determinant in the overall toxicity of this compound. It is difficult to determine the cause of differences between neonate and fifth instar larvae in relative toxicity of fipronil and the sulfone metabolite. The discrepancies are likely to be the result of toxicokinetic (uptake, accumulation at target sites, and rate of metabolism) differences between the two compounds. It is important to note that both fipronil and fipronil– sulfone are very toxic. As a consequence, the metabolism of fipronil to the sulfone metabolite by monooxygenases should not affect fipronil toxicity, and pest species that have evolved

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FIG. 3. Concentration-dependent effects of fipronil and its sulfone metabolite on the bursting activity of O. nubilalis neural preparations from fifth instar larvae, at (A) 1–5 min and (B) 16–20 min following perfusion. Shown are results from at least three perfused nerve cords (n ⫽ 3 to 5). Results are the 5-min averages ⫾ SE of threshold-exceeding bursting activity calculated in relation to GABA-quieting for the same preparations. Separate preparations were used for each concentration and insecticide. All control and insecticide perfusions were in the presence of 5 mM GABA, and the control contained DMSO at a quantity identical to that of 20 ␮M insecticide concentration (0.4% v/v).

enhanced oxidative metabolism as a resistanceconferring mechanism may not exhibit crossresistance to fipronil. The electrophysiological techniques used to measure changes in spontaneous electrical activity in larval central neurons were adequate for the detection of neurophysiological affects of GABA antagonists, including fipronil and its sulfone metabolite. It is clear that all GABA antagonists tested had excitatory effects on the central nervous system of the borer larvae. Consistently, both the fipronil and the sulfone metabolite caused increased bursting activity relative to that of control preparations. However, the

parent molecule, fipronil, consistently caused increased bursting activity relative to the sulfone metabolite. These results are somewhat contradictory to results from the bioassays, which indicated that the sulfone was equal to or greater than fipronil in toxicity to O. nubilalis larvae. This discrepancy may be related to differences in the rates at which the molecules enter the organism, accumulate, or interact with the target site. The sulfone metabolite may also possess greater affinity for the target site. Radioligand binding studies showed that fipronil–sulfone (IC50: house fly, 1.5; rat, 315) displaced the chloride channel ligand, EBOB, more readily in

FIPRONIL TOXICITY TO EUROPEAN CORN BORER

house fly and rat brain than the parent compound fipronil (IC50: house fly, 8; rat, 772) (1). The results from the present studies indicate that both fipronil and fipronil–sulfone act on the O. nubilalis central nervous system and cause increased spontaneous electrical activity in isolated nerve cords that is likely to occur due to an interaction with the GABA-gated chloride ionophore complex. The differences noted between toxicity and neurophysiological effects suggest differing toxicokinetics in fipronil and its sulfone metabolite. Most importantly, these results indicate that both fipronil and fipronil– sulfone are equally toxic and both result in increased spontaneous electrical activity of the central nervous system.

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ACKNOWLEDGMENTS L. Meinke and S. Parimi provided critical review of an earlier draft of the manuscript. Terrence Spencer assisted in laboratory rearing of O. nubilalis used in this investigation. This study was supported in part by Rhoˆne-Poulenc Ag Co. (Research Triangle Park, NC). This is paper No. 13281 of the Journal Series of the University of Nebraska Agricultural Research Division and Contribution No. 1093 of the Department of Entomology, University of Nebraska-Lincoln.

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