Development of pyrethroid substrates for esterases associated with pyrethroid resistance in the tobacco budworm, Heliothis virescens (F.)

Article abstract of DOI:10.1021/jf0493472

Assays to detect esterases associated with resistance to organophosphorus and pyrethroid insecticides in larvae of H. virescens were developed and evaluated. Cross-resistance to a variety of insecticides was measured in strains resulting from selection with either profenofos (OP-R) or cypermethrin (PYR-R), and resistance in both strains appeared to have a metabolic component. Esters were synthesized that coupled 3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropanecarboxylate, the acid moiety of some pyrethroid insecticides, with groups (e.g., p-nitrophenyl-) that could be detected spectrophotometrically following hydrolysis of the resulting esters. Activities toward these pyrethroid esters were significantly higher in both resistant strains than those in a susceptible reference strain. In addition, all pyrethroid esters significantly increased the toxicity of cypermethrin in bioassays with larvae from both PYR-R and OP-R strains. The biological and biochemical activities of these compounds are compared with those with more conventional esterase substrates and insecticide synergists, and the utility of pyrethroid esters as components of rapid assays for detecting esterases associated with insecticide resistance is discussed.

Full text of DOI:10.1021/jf0493472

J. Agric. Food Chem. 2004, 52, 6539−6545 6539
Development of Pyrethroid Substrates for Esterases Associated
with Pyrethroid Resistance in the Tobacco Budworm, Heliothis
virescens (F.)
HUAZHANG HUANG AND JAMES A. OTTEA*
Department of Entomology, Louisiana State University Agricultural Center,
Baton Rouge, Louisiana 70803
Assays to detect esterases associated with resistance to organophosphorus and pyrethroid insecticides
in larvae of H. virescens were developed and evaluated. Cross-resistance to a variety of insecticides
was measured in strains resulting from selection with either profenofos (OP-R) or cypermethrin (PYR-
R), and resistance in both strains appeared to have a metabolic component. Esters were synthesized
that coupled 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, the acid moiety of some
pyrethroid insecticides, with groups (e.g., p-nitrophenyl-) that could be detected spectrophotometrically
following hydrolysis of the resulting esters. Activities toward these pyrethroid esters were significantly
higher in both resistant strains than those in a susceptible reference strain. In addition, all pyrethroid
esters significantly increased the toxicity of cypermethrin in bioassays with larvae from both PYR-R
and OP-R strains. The biological and biochemical activities of these compounds are compared with
those with more conventional esterase substrates and insecticide synergists, and the utility of pyrethroid
esters as components of rapid assays for detecting esterases associated with insecticide resistance
is discussed.
KEYWORDS: Tobacco budworm; pyrethroid esters; insecticide synergists; cross-resistance; esterases
INTRODUCTION
The disadvantages of these assays are similar to those of assays
with insecticide synergists: Model (i.e., noninsecticide) sub-
strates may not be selectively metabolized by enzymes respon-
sible for resistance. For example, 1-naphthyl acetate (1-NA),
which has been used extensively as a model substrate in
resistance studies, is a poor indicator of resistance-associated
esterases from pyrethroid resistant H. Virescens (12).
Developing more specific synergists and substrates for
resistance-associated enzymes would enhance the accuracy of
assays for detecting resistance. Our working hypothesis is that
the specificity with which resistance-associated esterases are
detected may be enhanced by using reagents that are structurally
similar to the insecticide that is resisted. The approach used
was to modify the structure of a commonly used pyrethroid
insecticide such that the product of hydrolysis is detectable by
spectrophotometry. A similar approach has been used to study
esterases associated with pyrethroid hydrolysis in the cattle tick
Boophilus microplus (13) and in human serum (14). The major
objective of this study was to synthesize pyrethroid esters and
test them both as synergists of pyrethroid toxicity and as
substrates for esterases in insecticide susceptible and resistant
strains of H. Virescens.
Insecticide resistance is a major impediment to the effective
management of field populations of the tobacco budworm,
Heliothis Virescens (1-3). Resistance may result from enhanced
enzymatic detoxication or reduced penetration of the insecticide
and/or reduced sensitivity at the site of action of an insecticide
(4, 5). Expression of all three resistance mechanisms has been
reported in H. Virescens (2, 3, 6).
The ability to discriminate among these three biochemical
mechanisms is essential for the development and optimization
of insecticide resistance management strategies. At present, there
are a number of techniques available to identify insecticide
resistance mechanisms (7); however, most methodologies are
designed for use in the laboratory and are difficult to utilize in
field situations without modification. Insecticide bioassays with
synergists are fast, easy to use, and have a potential field utility,
but the specificity of these techniques is often questionable
because multiple forms of detoxifying enzymes may be ex-
pressed concurrently in resistant insects (8), and the specificity
of these enzymes toward a single inhibitor is variable (9, 10).
Therefore, the lack of synergism of insecticide toxicity might
indicate that either a detoxifying enzyme is not responsible for
resistance or a resistance-associated enzyme is present but not
inhibited by the synergist used (11).
MATERIALS AND METHODS
In addition to synergist bioassays, colorimetric measurements
of enzyme activities have been used to detect changes in
detoxifying enzymes expressed in insecticide resistant insects.
Chemicals. Permethrin (94.6%), bifenthrin (96%), tefluthrin (97%),
cypermethrin (96%), and acephate (97.6%) were kindly provided by
FMC Corporation (Princeton, NJ). Technical grade profenofos (89%)
10.1021/jf0493472 CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/28/2004

6540 J. Agric. Food Chem., Vol. 52, No. 21, 2004
Huang and Ottea
Table 1. Structures and Physical Characteristics of Pyrethroid Esters
a Purity was calculated based on relative peak area. b Rt
) ) relative intensity, percent.
retention time. c RI
was a gift from Novartis (Greensboro, NC). Indoxacarb (100%) and
spinosyn A (100%) were gifts from DuPont Agricultural Products
(Newark, DE) and DowElanco Corporation (Indianapolis, IN), respec-
tively. trans-Fenfluthrin and 2,3,6-trichloro-3-(2-propynyloxy)benzene
(TCPB; >95% purity) were synthesized as described previously (15)
and recrystallized before use. S,S,S-Tributylphosphorotrithioate (DEF;
98.7% purity) was provided by Bayer Corporation (Kansas City, MO),
and piperonyl butoxide (PBO; 90-95% purity) was purchased from
Fluka Chemical Corporation (Milwaukee, WI). Fast Blue B salt (90%
purity), 1-NA (98% purity), benzenethiol (97% purity), S-methyl
thiobutanoate (SMTB, 98% purity), 1-naphthol (99% purity), 2-naphthol
(99% purity), acetyl chloride (97% purity), thionyl chloride (99%
purity), phosphorus pentoxide (98% purity), 5,5′-dithio bis(2-nitroben-
zoic acid) (DTNB, 99% purity), 4-nitrophenol (99% purity), and benzoyl
chloride (99% purity) were obtained from Aldrich Chemical Co.
(Milwaukee, WI). Silica gel (28-200 mesh, average pore diameter 22
Å) and 2-naphthyl acetate were purchased from Sigma Chemical Co.,
Inc. (St. Louis, MO). The methyl ester of 3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropane carboxylate (PA; cis/trans ) 40/60) was pur-
chased from Fisher Scientific (Pittsburgh, PA). All other chemicals were
of analytical quality and were purchased from commercial suppliers.
a solid product, which was recrystallized from hexane/ether (1:1, v/v);
yield, 18.0 g (86%).
The trans and cis isomers of PA were separated using the method
of Foggassy et al. (16) with modifications. A mixture of PA (20.9 g,
0.1 mol) and benzene (100 mL) was stirred at 27 °C for 5 h. The
suspension was filtered, and the filter residue was recrystallized from
benzene to give 5.0 g of pure cis-PA (60% recovery). After benzene
was removed from the filtrate, the solid product was stirred for 5 h at
27 °C in 100 mL of petroleum ether and then filtered to give 2.5 g of
a solid product, which was recrystallized from hexane to give 1.8 g of
pure trans-PA (14% recovery).
Two methods were used to prepare esters from PA. First, thionyl
chloride (0.1 mol, 8.7 mL) was added dropwise into a mixture of PA
(0.025 mol, 5.25 g) and 100 mL of benzene (60% in hexane) and then
refluxed for 3 h at 50-60 °C. The solvent and excess thionyl chloride
were removed from the reaction mixture, and an oily product (PA-
chloride) was obtained (13). Freshly prepared PA-Cl (0.025 mol) was
added to a mixture containing 50 mL of benzene (60% in hexane) and
p-nitrophenol (3.5 g, 0.025 mol) and refluxed for 4 h at 50-60 °C.
The reaction mixture was washed with saturated NaHCO3 (20 mL ×
3) and 50% ethanol-water (50 mL × 2), and then purified by column
chromatography (silica gel; 28-200 mesh, average pore diameter
22 Å) with hexane:ethyl acetate (70:30) as the developing phase to
obtain 5.2 g of p-NPPA (62.5% yield). A similar method was used to
prepare thiophenyl acetate (TPA) and the 1- or 2-naphthyl esters of
PA (1-NPA or 2-NPA). After they were washed with NaHCO3 (20
mL × 3) and 50% ethanol-water (50 mL × 2), the final reaction
mixtures were purified by column chromatography using hexane:ethyl
acetate (80:20 for TPA or 100:10 for NPA esters). The yields of TPA,
1-NPA, and 2-NPA were 72, 79.7, and 80.5%, respectively.
Synthesis of Pyrethroid Esters. Pyrethroid esters that were
synthesized and tested include the following: thiophenyl, 3-(2,2-
dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (TPPA), p-nitro-
phenyl, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate
(p-NPPA), and 1- or 2-naphthyl, 3-(2,2-dichlorovinyl)-2,2-dimeth-
ylcyclopropane carboxylate (1- or 2-NPA; Table 1). The preparation
of PA from its methyl ester followed the procedure of Shan et al. (15).
A mixture of the methyl ester of PA (22.3 g, 0.1 mol), sodium hydroxide
(12 g, 0.3 mol), and ethanol-water (1:1, 250 mL) was refluxed for 12
h, concentrated under reduced pressure, diluted with 150 mL of water,
and extracted with ethyl ether (3 × 30 mL). The aqueous phase was
acidified with HCl (37.4%, 30 mL), and the oily precipitate was
extracted into ether (3 × 30 mL), washed with water (2 × 25 mL),
and then dried over anhydrous Na2SO4 overnight. The solution was
filtered, and ethyl ether was evaporated under reduced pressure to obtain
A second method (direct esterification between an acid and an
alcohol under dehydrating conditions) was used to prepare TPPA (17,
18). A reaction mixture containing cis- or trans-PA (0.01 mol),
thiophenol (0.011 mol), polyphosphate ester (20 mL), chloroform (80
mL), and pyridine (10 drops) was stirred for 12 h at room temperature.
The final mixture was washed with saturated NaHCO3 (2 × 50 mL)

Evaluation of Novel Pyrethroid Substrates
J. Agric. Food Chem., Vol. 52, No. 21, 2004 6541
and then extracted with chloroform (2 × 40 mL). The combined organic
phase was dried over anhydrous Na2SO4 overnight. After the solvent
was removed, the solid product was purified by column chromatography
(silica gel; 28-200 mesh, average pore diameter 22 Å) with a mixture
of hexane:ethyl acetate (70:30) as the developing phase. The yields of
cis-and trans-TPPA were 7.30 (96.5%) and 6.95 g (93.0%), respectively.
corrected for nonenzymatic hydrolysis using reactions without protein
as the control.
Activities of esterases toward 1-NA, 1-NPA, and 2-NPA were
measured by modifying the method of Gomori (22). Reaction mixtures
contained (final concentration in 250 µL): homogenate (60 µg of
protein), phosphate buffer (0.1 mM; pH 7.0), and a solution containing
Fast Blue B (1.2 mM) and substrate (2.15, 0.67, or 0.67 mM for 1-NA,
1-NPA, or 2-NPA, respectively). Changes in optical density at 450 nm
were measured during the initial 10 min of the reactions, and rates
were converted to nmol min-1 using the extinction coefficient 9.25
mM-1 250 µL-1 for 1-naphthol (23). Esterase activities toward SMTB,
TPA, and cis- or trans-TPPA were measured by the method of Ellman
et al. (24), as modified by van Asperen (25). Reaction mixtures
contained (final concentrations in 300 µL): homogenate (30, 30, or
120 µg of protein for SMTB, TPA, or cis-/trans-TPPA, respectively),
DTNB (0.05 mM), phosphate buffer (0.1 mM; pH 7.6, 7.6 or 8.0 for
SMTB, cis-/trans-TPPA, or TPA, respectively), and substrate (2.0, 1.33,
or 1.33 for SMTB, cis-/trans-TPPA, or TPA, respectively). Changes
in optical density at 405 nm were measured during the initial 10 min
(or 1 min for TPA) of the reactions, and rates were converted to nmol
min-1 using an experimentally derived “extinction coefficient” of 8.34
mM-1 300 µL-1. Finally, the esterase activity toward p-NPPA was
measured using the method of Kinoshita et al. (26) as modified by
Hansen and Hodgson (27). The reaction mixtures contained (final
concentrations in 300 µL): homogenate (360 µg of protein), phosphate
buffer (0.1 mM; pH 7.6), and substrate (1.33 mM). Changes in optical
density at 405 nm were measured during the first 10 min of the
reactions, and rates were converted to nmol min-1 using an experi-
mentally derived extinction coefficient of 11.4 mM-1 300 µL-1 for
p-nitrophenol. Homogenates from at least 30 larvae were used in assays
with each substrate. Protein concentrations were measured from diluted
homogenates by the method of Bradford (28) using bovine serum
albumin as the standard. Results were analyzed and compared using
analysis of variance (ANOVA)-Tukey’s HSD test (P e 0.01).
Identification of Products. The products were identified using gas
chromatography-mass spectrometry (GC-MS) on a Finnigan GCQ
(Trace GC 2000 coupled with Polaris mass spectral detector) (Table
1). A silica capillary GC column (DB-5MS; 30 m × 0.25 mm × 0.25
µm; J &W Scientific, Folsom, CA) was operated at 60 °C for 1 min,
increased to 150 °C at 5 °C/min increments, held for 15 min at this
temperature, and then increased to 300 °C at a rate of 5 °C/min, where
it was held for the final 10 min. The injector port was operated in the
splitless mode at 200 °C, and helium was used as the carrier gas at 1.2
mL/min. The mass spectral detector was set on full scan mode (m/z
41-400). The purity was calculated by relative peak area.
Insects. A susceptible strain of H. Virescens (LSU-S) was established
in 1977 (19) and has been reared in the laboratory since that time
without exposure to insecticides. Larvae from the LSU-S strain were
selected for five consecutive generations with topical applications of
profenofos at doses corresponding to the LD60 for each generation (3.5,
4.6, 8.5, 13.6, and 23.8 µg/individual). After 1 year without selection,
individual larvae were treated with 25 µg of profenofos (an approximate
LD20) and the survivors (OP-R strain) were used for biological assays.
Similarly, larvae from the LSU-S strain were selected for four
consecutive generations with topical applications of cypermethrin at
doses corresponding to the LD60 (0.22, 0.4, 0.5, and 0.6 µg/individual).
After one generation without selection, larvae were treated with 1 µg
of cypermethrin (an approximate LD90), and the next generation (PYR-R
strain) was used for biochemical and biological assays.
Toxicological Assays. Fifth instars (day 1), weighing 180 ( 20 mg,
were treated on the midthoracic dorsum with 1 µL of insecticide (in
acetone) or with acetone alone (control). Dose-mortality relationships
for each insecticide were assessed from assays with at least five doses
and 30 insects per dose. To evaluate the effects of conventional
synergists (i.e., PBO, DEF, and TCPB) and pyrethroid esters on the
toxicity of cypermethrin or profenofos, compounds were applied to
the midabdominal dorsum 30 min prior to application of the insecticide.
In preliminary assays, doses (0-60 µg/larva) of the five pyrethroid
esters (1- or 2-NPA, cis- or trans-TPPA, and p-NPPA) were topically
applied to larvae from the LSU-S strain, followed by a dose of
cypermethrin corresponding to the LD20 (0.15 µg/larva). Doses of the
pyrethroid esters causing maximum mortality in these tests were used
in assays with resistant strains. All pyrethroid esters were nontoxic to
LSU-S larvae at 70 µg/larva. In tests with PBO, DEF, and TCPB, a
dose of 50 µg/larva was used (15). For all bioassays, larvae that were
treated with either 1 µL of acetone or the synergist alone served as
controls. After treatment, larvae were maintained at 27 °C, and mortality
was recorded after 72 h. The criterion for mortality was the lack of
coordinated movement within 30 s after being prodded with a sharpened
pencil. The results were corrected for control mortality with Abbott’s
formula (20) and then analyzed by Finney’s method (21). The
differences between LD50 values were considered significant if the 95%
fiducial limits (FLs) did not overlap. Resistance ratios (RR) were
calculated as LD50 of resistant strain/LD50 of susceptible strain. The
synergism ratio (SR) was calculated as: LD50 of insecticide alone/
LD50 of insecticide plus synergist.
RESULTS
Characterization of Resistant Strains. The selection of
LSU-S larvae with either profenofos (OP-R strain) or cyper-
methrin (PYR-R) resulted in resistance to all insecticides tested
except acephate in the PYR-R strain (Table 2). Resistance was
highest to the insecticide used as the selecting agent (i.e., 18.1-
fold resistance to profenofos in the OP-R strain and 19.6-fold
resistance to cypermethrin in the PYR-R strain). In tests with
OP-R larvae, resistance was also high to indoxacarb (15.8-fold)
and to pyrethroids containing 3-phenoxybenzyl- groups (i.e.,
12.9-fold to cypermethrin and 7.2-fold to permethrin). Similarly,
in cypermethrin-selected (PYR-R) larvae, resistance was also
high to permethrin (12.4-fold) but lower for pyrethroids with
nonphenoxybenzyl alcohols such as bifenthrin (3-phenyl 2-meth-
ylbenzyl alcohol; 5.1-fold), fenfluthrin (pentafluorobenzyl al-
cohol; 2.91-fold), or tefluthrin (4-methyl tetrafluorobenzyl
alcohol; 2.18-fold). Finally, as compared with OP-R larvae,
cross-resistance to profenofos and indoxacarb was lower (4.53-
and 5.57-fold, respectively) in larvae from the PYR-R strain.
Synergism of Insecticide Toxicity. All of the pyrethroid
esters tested synergized the toxicity of cypermethrin to LSU
larvae (Figure 1). The NPA esters were slightly more potent
than the TPPA compounds, and differences in potencies of the
geometric (for TPPA) and positional isomers (for NPA) were
minimal. Maximal levels of mortality (50-63%) were similar
among compounds within the range of doses tested, but optimal
doses for synergism differed and ranged from 20 (for 1- and
2-NPA) to 50 µg/larva (for p-NPPA).
Biochemical Assays. Homogenates from individual fifth instars were
used as the enzyme source for measuring esterase activities toward all
substrates. Individual larvae were dissected, and the digestive system
was evacuated. The remaining carcass was homogenized using an all-
glass homogenizer with 500 µL of ice-cold, 0.1 M sodium phosphate
buffer (pH 6-8). Homogenates were centrifuged at 12600g for 15 min
at 4 °C, and the resulting supernatants were held in ice and used in
enzyme assays within 20 min of preparation. For all substrates, assay
conditions (i.e., pH, temperature, substrate concentration, and protein
dependence) were optimized in preliminary experiments (data not
shown). Activities were measured in 96 well microplates using a
Thermomax Plate Reader (Molecular Devices, Palo Alto, CA) and were
Effects of nonpyrethroid compounds (i.e., DEF, PBO, and
TCPB) on insecticide toxicity varied depending on the insect
strain and insecticide used for assays (Table 3). In tests with
the LSU-S strain, PBO synergized cypermethrin toxicity but
was an antagonist of profenofos toxicity. In contrast, PBO had

6542 J. Agric. Food Chem., Vol. 52, No. 21, 2004
Huang and Ottea
Table 3. Effects of Compounds on Profenofos or Cypermethrin
Toxicity in Fifth Stadium Larvae from Susceptible (LSU-S) and
Resistant (OP-R or PYR-R) Strains of H. virescens
Table 2. Insecticide Susceptibility of Larval H. virescens from
Insecticide Susceptible (LSU-S) and Resistant (OP-R and PYR-R)
Strains
straina
insecticide
LD50 (95% FLb)
slope (SDc)
ø
-square
RRd
straina
treatmentb
(µ
g)
LD50 (95% FL)c
slope (SD) SRd RRe
LSU-S profenofos
cypermethrin
permethrin
2.90 (2.40
0.19 (0.16
0.14 (0.11
0.51 (0.42
1.64 (1.22
0.05 (0.04
0.27 (0.22
−
−
−
−
−
−
−
3.56)
0.24)
0.19)
0.69)
2.12)
0.06)
0.33)
2.63 (0.40)
2.81 (0.39)
1.91 (0.25)
2.65 (0.52)
2.99 (0.40)
3.12 (0.42)
4.46 (0.64)
4.26 (0.61)
1.66 (0.29)
3.88 (0.57)
1.38
2.90
0.93
0.05
3.73
3.08
2.05
1.65
0.49
1.76
1.06
3.99
2.38
0.16
2.03
1.11
4.76
0.64
2.17
1.56
0.57
6.75
3.18
8.40
0.44
9.48
0.19
1
1
1
1
1
1
1
1
1
18.1
12.9
LSU-S cyper alone
0.19 (0.16
0.09 (0.08
0.22 (0.16
0.12 (0.08
2.90 (2.40
7.24 (5.71
8.02 (6.52
1.63 (1.23
3.75 (3.16
3.28 (2.55
4.81 (3.84
1.74 (1.43
1.84 (1.27
2.08 (1.80
1.75 (1.32
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.24) 2.81 (0.39)
cyper
cyper
cyper
+
+
+
PBO (50)
DEF (50)
TCPB (50)
0.10)e 4.02 (0.38) 2.05
0.38) 1.75 (0.34) 0.85
0.18) 2.61 (0.36) 1.66
3.60) 2.63 (0.40)
tefluthrin
trans-fenfluthrin
bifenthrin
indoxacarb
acephate
spinosyn A
prof alone
prof
prof
prof
+
+
+
PBO (50)
DEF (50)
TCPB (50)
10.4)e 2.47 (0.53) 0.40
11.2)e 2.06 (0.48) 0.36
1.97)e 2.93 (0.51) 1.78
4.33) 3.34 (0.48)
25.8 (22.3
1.84 (1.35
52.3 (46.0
−
−
−
29.4)
2.66)
59.0)e
PYR-R cyper alone
19.7
OP-R
profenofos
cypermethrin
permethrin
tefluthrin
cyper
cyper
cyper
cyper
cyper
cyper
cyper
cyper
+
+
+
+
+
+
+
+
PBO (50)
DEF (50)
4.20) 1.83 (0.24) 1.14 17.3
6.07) 2.14 (0.32) 0.78 25.3
2.46 (1.86
1.03 (0.54
1.68 (1.38
4.30 (3.70
0.17 (0.13
4.29 (3.18
−
−
−
−
−
−
3.43)e 6.49 (0.88)
1.75)e 2.05 (0.31)
2.01)e 3.00 (0.42)
4.88)e 4.94 (0.64)
0.24)e 1.77 (0.34)
5.87)e 1.62 (0.23)
7.20
TCPB (50)
1-NPA(20)
2-NPA (20)
cis-TPPA (30)
trans-TPPA (40) 1.42 (1.17
p-NPPA (50) 1.11 (0.91
2.13)e 2.69 (0.35) 2.16
3.00)e 2.99 (0.48) 2.04
9.16
9.68
3.30
2.59
3.47
trans-fenfluthrin
bifenthrin
indoxacarb
acephate
2.48)e 3.31 (0.54) 1.80 10.9
2.69)e 3.54 (0.60) 2.14
1.76)e 2.49 (0.38) 2.64
1.36)e 2.41 (0.35) 3.34
9.21
7.47
5.84
15.8
69.1 (55.0
−
86.8)e
5.49)e 1.52 (0.22)
16.2)e
2.49 (0.39)
3.64 (0.53)
2.76
2.19
4.53
19.6
12.4
2.18
2.91
5.10
5.57
1.53
2.99
spinosyn A
4.03 (2.85
−
OP-R
prof alone
52.3 (46.0
67.7 (58.8
59.5 (42.5
−
−
−
59.2)
79.8)
91.2)
3.88 (0.57)
2.89 (0.41) 0.77 23.3
3.72 (0.56) 0.88 20.5
18.0
PYR-R profenofos
cypermethrin
permethrin
13.2 (10.4
3.75 (3.16
5.59 (4.27
1.11 (0.96
4.83 (3.91
0.26 (0.18
1.51 (1.02
−
prof
prof
prof
+
+
+
PBO (50)
DEF (50)
TCPB (50)
−
−
−
−
−
−
4.33)e 3.34 (0.48)
7.91)e 1.77 (0.28)
1.31)e 1.77 (0.28)
6.25)e 4.49 (0.65)
0.41)e 2.97 (0.38)
2.06)e 1.68 (0.29)
8.35 (6.29
2.46 (1.86
0.84 (0.55
0.68 (0.56
0.67 (0.56
−
−
−
−
−
−
−
10.3)e 2.84 (0.43) 6.26
2.88
12.9
4.42
3.58
3.53
3.68
4.16
tefluthrin
trans-fenfluthrin
bifenthrin
indoxacarb
acephate
spinosyn A
cyper alone
3.43) 6.49 (0.88)
cyper
cyper
cyper
cyper
cyper
+
+
+
+
+
1-NPA (20)
2-NPA (20)
cis-TPPA (30)
trans-TPPA (40) 0.70 (0.42
p-NPPA (50) 0.79 (0.53
1.26)e 2.84 (0.41) 2.93
0.81)e 3.14 (0.43) 3.62
0.78)e 3.12 (0.47) 3.67
1.05)e 3.17 (0.42) 3.51
1.14)e 2.59 (0.39) 3.11
39.4 (24.8
−
62.5)
5.09 (0.61)
5.49 (3.79
−
7.93)e 1.30 (0.21)
a Strains: LSU-S, insecticide susceptible; OP-R, profenofos selected; PYR-R,
cypermethrin selected. b FL 95% fiducial limits. c SD standard deviation.d RR
is defined as LD50 of resistant strain/LD50 of LSU-S strain. e Denotes a value that
a Strains: LSU-S, insecticide susceptible; OP-R, profenofos selected; PYR-R,
cypermethrin selected. b Treatments: cyper, cypermethrin; prof, profenofos.
)
)
c FD
)
95% fiducial limits. d SR defined as LD50 of insecticide alone/LD50 of
is significantly different (P
strain.
e
0.05) from the corresponding value for the LSU-S
insecticide plus synergist. e Value is significantly different from that of the insecticide
alone treatment.
Table 4. Esterase Activities toward Pyrethroid and Nonpyrethroid
Estersa
strainb
substrate
LSU-S
OP-R
R/S
PYR-R
R/S
1-NA
TPA
SMTB
1-NPA
2-NPA
cis-TPPA
trans-TPPA
p-NPPA
73.5 (9.51)B
345 (79.5)B
98.5 (15.5)C
16.6 (2.19)C
12.6 (3.24)B
0.82 (1.62)C
1.43 (1.64)C
0.42 (0.36)B
91.5 (12.9)A
582 (126)A
214 (33.9)A
39.6 (5.55)B
35.2 (7.09)A
2.02 (0.69)B
6.57 (1.10)B
1.21 (0.65)A
1.25
1.68
2.17
2.39
2.12
2.46
4.59
2.88
85.1 (15.9)A
621 (158)A
188 (40.8)B
50.9 (3.47)A
38.6 (3.28)A
6.88 (0.87)A
10.6 (1.37)A
0.96 (0.72)A
1.16
1.80
1.91
3.07
3.06
8.39
7.41
2.28
-
1
-1
a Values represent mean activities (nmol min mg prot
;
±
SD) based on
triplicate assays with 30 larvae from each strain. For comparing activities toward
each substrate, values with the same letters are not significantly different (ANOVA
Tukey’s HSD test, P
0.01). b Strains: LSU-S, reference susceptible strain; OP-
R, profenofos selected; PYR-R, cypermethrin selected.
−
e
Figure 1. Mortality measured after sequential topical application of various
doses of synergists followed by 0.15 g of cypermethrin onto fifth stadium
(day 1) larvae of H. virescens from the LSU-S strain.
strain, SRs were similar among the five pyrethroid esters and
ranged from 2.93 (for 1-NPA) to 3.67 (for cis-TPPA). Similarly,
in tests with PYR-R larvae, SRs for the pyrethroid esters ranged
from 1.80 (for 2-NPA) to 3.34 (for p-NPPA).
µ
no significant effect on the toxicity of cypermethrin or pro-
fenofos in either the OP-R or the PYR-R strains. Similarly, DEF
antagonized profenofos toxicity in the LSU-S strain but had no
significant effect on profenofos toxicity in the OP-R strain or
on cypermethrin toxicity in the LSU-S or PYR-R strain. In
contrast, TCPB was the most potent synergist of profenofos
toxicity in the LSU-S and OP-R strains and synergized
cypermethrin toxicity in PYR-R larvae but had no significant
effect on cypermethrin toxicity in the LSU-S strain.
Esterase Activities toward Pyrethroid and Nonpyrethroid
Substrates. Regardless of the substrate used, activities were
significantly greater (Tukey’s HSD; P e 0.01) in larvae from
the resistant rather than the susceptible strains (Table 4). In
addition, esterase activities toward the nonpyrethroid substrates
(1-NA, TPA, and SMTB) were significantly greater (Tukey’s
HSD; P e 0.01) than those measured with the pyrethroid esters
(Table 4). For all three strains, activities were greatest in assays
with TPA and lowest toward p-NPPA. However, differences
in activities toward nonpyrethroid esters between the susceptible
All pyrethroids esters significantly increased cypermethrin
toxicity in both OP-R and PYR-R strains (Table 3). In the OP-R

Evaluation of Novel Pyrethroid Substrates
J. Agric. Food Chem., Vol. 52, No. 21, 2004 6543
and the resistant strains were not dramatic and ranged from 1.16-
(for 1-NA) to 2.17-fold (for SMTB) in the PYR-R and OP-R
strains, respectively. Also, activities toward nonpyrethroid
substrates were generally similar between the insecticide
resistant strains, although the activity with SMTB was statisti-
cally greater in the OP-R than the PYR-R strain. Differences
in activities toward pyrethroid esters between resistant and
susceptible strains were greater than those measured with
nonpyrethroid esters and ranged from 3.1- and 2.8-fold (for
2-NPA) to 8.4- and 2.7-fold (for cis-TPPA) in the PYR-R and
OP-R strains, respectively. In addition, in tests with the LSU-S
strain, no activities were detectable toward cis-TPPA, trans-
TPPA, and p-NPPA in five, four, and one of the tested
individuals, respectively. Finally, esterase activities toward three
of the pyrethroid substrates (1-NPA, cis-TPPA, and trans-TPPA)
were significantly greater in PYR-R larvae than those from the
OP-R strain, and differences ranged from 1.3-fold for 1-NPA
to 3.4-fold for cis-TPPA.
metabolism by insecticide resistant H. Virescens (37, 38). Further
support for involvement of P450 monooxygenases (P450 MOs)
in resistance in the OP-R and PYR-R strains was measured in
synergist assays, where coapplication of TCPB, a P450 MO
inhibitor, significantly increased the toxicity of cypermethrin.
In contrast, PBO, which has been widely used to ascertain the
involvement of P450 MOs in resistance, had a lesser, nonsig-
nificant effect on cypermethrin toxicity in the resistant strains.
This result is similar to those from previous reports (10, 15)
and underscores the need to use multiple synergists in “diag-
nostic” bioassays (3).
Results from the current study also support previous findings
that enhanced ester hydrolysis contributes to resistance in H.
Virescens (3, 39-41). In a previous study, products of hydrolysis
were the predominant metabolites following topical application
of cypermethrin to pyrethroid resistant larvae from field-
collected and laboratory-selected strains of this insect (41). In
current tests, pyrethroid esters were among the most active
synergists of cypermethrin toxicity in the PYR-R strain.
Synergism was greatest with p-NPPA, which increased cyper-
methrin toxicity by 3.34-fold and reduced the RR in these insects
from 20- to 5.8-fold. The residual resistance measured may
reflect the action of additional mechanisms (e.g., P450 MO or
reduced target site sensitivity). The expression of oxidase-based
resistance is suggested based on the activity of TCPB as a
synergist; however, the effect of this compound on esterases
has not been examined directly. Alternatively, the doses of
synergists used for these tests, which were chosen based on
tests using larvae from the LSU-S strain, may have been
suboptimal (i.e., too low) for larvae from the resistant strains.
Thus, results measured may reflect incomplete inhibition of
esterases.
DISCUSSION
Currently available bioassays provide little insight into the
biochemical mechanism underlying resistance. Knowledge of
the mechanism expressed in resistant pests would allow
countermeasures to be tailored toward the mechanism being
expressed in resistant members of pest populations. Synergist
bioassays are commonly used to test for the involvement of
metabolic resistance mechanisms; however, the specificity of
synergists for detoxifying enzymes associated with resistance
is often questionable (3, 29, 30). In addition, synergists have
been shown to affect insecticide toxicity via mechanisms that
do not involve metabolism (e.g., decreasing or increasing
cuticular penetration; 30-32). In the present study, we tested
the hypothesis that compounds with structural similarity to
pyrethroid insecticides will be more specific synergists of
pyrethroid toxicity than compounds that are structurally unre-
lated.
Selection of larval H. Virescens with either cypermethrin or
profenofos resulted in cross-resistance to an array of insecticides,
some of which act at differing target sites. Both OP-R and
PYR-R larvae expressed resistance to profenofos, an organo-
phosphorus insecticide that inhibits the enzyme, acetylcholin-
esterase, and also to pyrethroid insecticides, which alter function
of voltage sensitive sodium channels. This aspect of the
resistance profile suggests that a metabolic mechanism is at least
partially associated with resistance in these strains. In tests with
the OP-R strain, a high level of resistance (over 15-fold) was
also measured to indoxacarb, which is a blocker of insect sodium
channels (33) and alters the functioning of nicotinic acetylcho-
line receptors in mammals (34). This finding was unexpected
because these insects had not been exposed to this compound.
However, very high levels of resistance to indoxacarb have been
reported in obliquebanded leafroller, Choristoneura rosaceana,
prior to significant exposure to this insecticide (35, 36).
Varying degrees of cross-resistance among the pyrethroids
tested were measured in larvae from both resistant strains. In
cypermethrin-selected, PYR-R larvae, resistance to pyrethroids
with 3-phenoxybenzyl alcohols (i.e., cypermethrin and per-
methrin) was greater than to pyrethroids lacking this group
(bifenthrin, trans-fenfluthrin, and tefluthrin). Similar results were
observed in tests with OP-R larvae, suggesting that the phe-
noxybenzyl group of cypermethrin and permethrin contains
targets for metabolism in these strains. This finding is consistent
with those from previous studies showing that NADPH-
dependent ring hydroxylation at the 2′- and 4′-positions of the
phenoxybenzyl alcohol is a predominant route of pyrethroid
A role for esterases in resistance in these strains is also
supported by results from biochemical assays. Esterase activities
toward a structurally diverse array of substrates were higher in
both PYR-R and OP-R strains as compared with those in LSU-S
larvae. Whereas activities in all strains were higher toward
nonpyrethroid esters (i.e., 1- NA, TPA, and SMTB) than
pyrethroid esters, differences in activities between susceptible
and resistant strains were greater in assays with the pyrethroid
esters. Furthermore, in comparisons between resistant strains,
esterase activities toward 1-NPA and cis- and trans-TPPA were
higher in PYR-R than OP-R larvae. These data suggest that
substrate selectivities of esterases differ among resistant and
susceptible larvae and that esterase activities associated with
resistance to pyrethroid and organophosphorus insecticides may
be measured with substrates that are structurally similar to the
insecticide that is resisted. Because insecticide metabolism was
not measured in the current study, our conclusions are neces-
sarily tenuous and further studies are required. However, these
results provide a foundation for subsequent efforts for use of
such compounds for development of rapid assays to detect
esterase-based resistance in field-collected H. Virescens.
ACKNOWLEDGMENT
We thank Lane Foil and Mike Stout for critically reviewing
this manuscript. This article has been approved for publication
by the Director of the Louisiana Agricultural Experiment Station
as Manuscript Number 04-26-0175.
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Received for review April 22, 2004. Revised manuscript received July
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JF0493472