Mode of action of etoxazole - ResearchGate

Pest Management Science Pest Manag Sci 62:379–382 (2006)
Rapid Report
Mode of action of etoxazole
Ralf Nauen 1∗ and Guy Smagghe 2
1 Bayer CropScience AG, Research Insecticides, Biology, Alfred Nobel Str. 50, D-40789 Monheim, Germany
2 Laboratory of Agrozoology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
Abstract: The mode of action of the 2,4-diphenyl-1,3-oxazoline acaricide/insecticide etoxazole has been argued to
be moulting inhibition, but experimental results supporting this hypothesis are lacking. This study investigated
the effect of etoxazole on chitin biosynthesis in the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera:
Noctuidae). Etoxazole induced moulting defects in fall armyworm larvae similar, if not identical, to those caused
by benzoylphenylureas, a well-known class of insecticidal chitin biosynthesis inhibitors. Furthermore, in contrast
to untreated larvae, the chitin content in the integuments of larvae several days after treatment did not differ from
that in freshly ecdysed individuals, thus suggesting strong chitin biosynthesis inhibition in vivo. Amoredetailed
investigation of the inhibitory potential by incubating cultured integument pieces from larvae of S. frugiperda with
[ 14 C]N-acetyl-D-glucosamine, a radiolabelled chitin precursor, revealed I 50 values of 2.95 and 0.071 µM for etoxazole
and triflumuron respectively. The incorporation of radiolabel into potassium hydroxide-resistant material was
inhibited by etoxazole in a dose-dependent manner. Based on these results, it is concluded that the acaricidal and
insecticidal mode of action of etoxazole is chitin biosynthesis inhibition.
© 2006 Society of Chemical Industry
Keywords: etoxazole; chitin biosynthesis; triflumuron; Spodoptera frugiperda; acaricide mode of action
1 INTRODUCTION
Etoxazole is the only commercialised compound
belonging to the chemical class of 2,4-diphenyl-1,3-
oxazolines. It was publicly introduced in 1994 and
launched in 1998 as an acaricide/insecticide with
a quite narrow spectrum of efficacy. 1,2 Main pests
targeted by etoxazole are tetranychid spider mites such
as Panonychus ssp. and Tetranychus ssp. Insecticidal
side effects on aphids, green rice leafhoppers and
diamondback moths have also been claimed but
are of lesser importance. Etoxazole is active against
eggs, larvae and nymphs of spider mites but lacks
any efficacy against male and female adults. 3 It
usually exhibits high efficacy and behaves like an
insect/mite growth regulator, considering its effects on
the different developmental stages mentioned above.
However, one of the major threats of new acaricidal
modes of action is the development of resistance, and,
owing to its high efficacy, resistance factors against
etoxazole could reach values of greater than 100 000
as described recently. 4 Other acaricides known to
affect spider mite growth include hexythiazox, but
so far no cross-resistance between hexythiazox and
etoxazole has been reported. 1 One of the major issues
in resistance management strategies is the rotation
of compounds with different modes of action in
order to prevent or delay the rapid development
of resistance, and it is therefore highly desirable to
elucidate the mode of action of newer compounds
in detail. However, the mode of action of etoxazole
has been reported to appear to be an inhibition
of the moulting process during mite development,
with a mechanism suggested to be similar to that
of benzoylphenylureas, a class of insecticides known
to interfere with chitin biosynthesis. 3 Etoxazole is
furthermore included in group 10 within the IRAC
(Insecticide Resistance Action Committee) mode of
action classification scheme, i.e. compounds with
unknown or non-specific mode of action as mite
growth regulators (www.irac-online.org).
Here we provide experimental evidence for the
very first time that etoxazole is physiologically
acting as a chitin biosynthesis inhibitor similar to
benzoylphenylurea insecticides.
2 MATERIAL AND METHODS
2.1 Insects
Larvae of the fall armyworm, Spodoptera frugiperda
(Smith) (Lepidoptera: Noctuidae), were reared in
10 cm × 10 cm × 20 cm plastic boxes on an artificial
diet based on bean meal under a 16/8 h light/dark
photoperiod at 27 ◦ C and 70% relative humidity.
Fourth-instar larvae were separated and kept individually
in petri dishes to prevent cannibalism. Pupae
and adults were maintained under the same conditions
in 20 cm × 20 cm × 10 cm plastic boxes. Male
and female adults were fed a saturated solution of
sucrose in water, and eggs deposited on filter paper
were transferred to the above-mentioned plastic boxes.
∗ Correspondence to: Ralf Nauen, Bayer CropScience AG, Research Insecticides, Biology, Alfred Nobel Str. 50, D-40789 Monheim, Germany
E-mail: ralf.nauen@bayercropscience.com
(Received 1 December 2005; accepted 5 December 2005)
Published online 22 March 2006; DOI: 10.1002/ps.1192
© 2006 Society of Chemical Industry. Pest Manag Sci 1526–498X/2006/$30.00

R Nauen, G Smagghe
2.2 Insecticides and chemicals
The etoxazole and triflumuron used were of technical
grade with purities of >98% and were obtained
in-house. N-Acetyl-D-[1- 14 C]glucosamine (GluNAc)
with a specific activity of 2.17 GBq mmol −1 was
purchased from Amersham BioSciences (Piscataway,
NJ, USA). All other chemicals and solvents were of
analytical grade and obtained from Sigma (Heidelberg,
Germany) or Merck (Darmstadt, Germany).
2.3 Gravimetric ex vivo determination of chitin
deposition in larvae of Spodoptera frugiperda
exposed to insecticide-treated leaves
Stock solutions of insecticides (1000 mg litre −1 )were
prepared in aqueous Triton X-100 solution (0.2 g
litre −1 ) and diluted accordingly. Circular leaf discs
(44 mm in diameter) cut from true leaves of 3-weekold
cabbage plants, Brassica olearacea L., were dipped
for 3 s in insecticidal solutions and transferred to petri
dishes containing a filter paper disc. Subsequently a
single freshly ecdysed sixth-instar larva of S. frugiperda
(165–190 mg) was transferred to each leaf disc. Four
freshly ecdysed sixth-instar larvae were directly frozen
at −80 ◦ C and served as 0 h controls. Larvae were
weighed and fed daily with fresh leaf discs (either
treated or untreated), and after 3 days at least three
groups of four larvae per test concentration (including
untreated controls) were assessed for symptoms of
poisoning and frozen at −80 ◦ C. After thawing, larvae
were dissected and integuments free of adhering tissue
(4–12 replicates per concentration) were dried for
2 h at 110 ◦ C. The integuments were weighed and
subsequently treated individually with boiling aqueous
potassium hydroxide (2.5 M,2ml)for2h.Afterwards,
integuments were washed with deionised water (2 ml),
hydrochloric acid (0.5 M, 2 ml) and ethanol (2 ml).
The remaining residue (chitin) was dried for 2 h at
100 ◦ C and weighed. The experiment was repeated at
least two times.
Examination for symptoms of poisoning was
conducted with fifth-instar larvae using the same
bioassay, except that larvae were not analysed for
integumental chitin content.
2.4 Inhibition of incorporation of
[ 14 C]N-acetyl-D-glucosamine into cultured
integuments of Spodoptera frugiperda
Early sixth-instar larvae of S. frugiperda were selected
and used for the preparation of integument fragments.
Procedures were basically the same as described
by Nakagawa et al. 5 After surface sterilisation of
larvae with 75% ethanol for 15 min, six integument
fragments of about 20 mm 2 were dissected aseptically
and incubated first at 27 ◦ C in 1 ml of sterile Grace’s
medium containing 2.2 µM 20-hydroxyecdysone (20E)
but not antibiotics or Bovine serum albumine (BSA).
After 24 h culture the fragments were transferred to
another sterile culture well containing 1 ml of Grace’s
medium with 0.2 nmol of 14 C-GluNAc but not
containing 20E. These conditions were chosen based
on earlier experiments. 5 To test the effects of etoxazole
and triflumuron, 1 µl aliquots of solutions of each
compound in dimethyl sulfoxide (DMSO) were added
to the culture wells to give a final concentration range
from 10 −10 to 10 −4 M. The integument fragments were
further incubated in the medium at 27 ◦ Cfor72h.
After incubation the culture medium was removed
from the well and the pieces of integument were
washed with deionised water (3 × 0.5ml).Tomeasure
the 14 C activity in the cultured tissues, integument
pieces were treated with an alkaline solution, washed
three times with distilled water and treated overnight
with tissue solubiliser in 20 ml glass liquid scintillation
vials. The amount of radiolabel was measured with
Ultima Gold cocktail (PerkinElmer LifeSciences,
Wellesley, MA) in a liquid scintillation counter as
described elsewhere. 6
Based on the dose–response curve, the activity
was expressed as pIC 50 , the negative decadic
logarithm of the mean concentration causing 50%
inhibition of the incorporation of 14 C-GluNAc into
the integument parts, using sigmoidal curve fitting
of GraphPad Prism software (Statcon, Witzenhausen,
Germany). Data are results of two repeat
measurements, and for each compound seven concentrations
were tested (n = 14). The goodness of
fit was expressed as R 2 . The 0% effect (highest
amount of incorporated radiolabel without inhibitor)
was 1622 dpm; the 100% effect (lowest amount of
incorporated radiolabel with 10 −4 M triflumuron) was
402 dpm.
3 RESULTS AND DISCUSSION
3.1 Effects of etoxazole on Spodoptera
frugiperda larvae
Potency-wise, etoxazole is a strong acaricide and
much less effective against fall armyworm larvae
than benzoylphenylureas such as triflumuron, which
has no acaricidal activity. However, physiological
and biochemical mode of action investigations with
juvenile spider mites are very difficult to conduct.
Therefore we have chosen S. frugiperda as an
appropriate model insect which is readily affected by
etoxazole, thus allowing us to study the mode of action
more closely.
Fifth-instar larvae of the fall armyworm feeding
on cabbage foliage treated with etoxazole responded
with obvious signs of moulting defects in a dosedependent
manner after 3 days, i.e. etoxazole was
acting slowly like an insect growth regulator. 7 Most
prominent symptoms were a double head capsule due
to the inability to shed the old one, and incomplete
ecdysis with subsequent loss of haemolymph
on the interface between the new head capsule and
thethoracicsegments.Thetreatmentoffallarmyworm
larvae with etoxazole resulted in symptoms of
poisoning similar, if not identical, to those of the benzoylphenylurea
insecticide triflumuron (Fig. 1). The
380 Pest Manag Sci 62:379–382 (2006)
DOI: 10.1002/ps

Etoxazole mode of action
Etoxazole
Chitin per larva (mg)
3.0
2.5
2.0
1.5
1.0
0.5
Etoxazole
Triflumuron
Triflumuron
0.0
1000
200
40
8
1.6
0.32
Untreated 3d
Concentration (mg litre −1 )
Untreated 0d
Figure 2. Inhibition of chitin formation in sixth-instar larvae of
Spodoptera frugiperda exposedtoetoxazoleandtriflumuronafter
foliar application. Three days after application, integuments were
dissected, dried and incubated in 2.5 M KOH and the remaining
residue was gravimetrically analysed. Control larvae (untreated) were
fed with untreated leaves.
Figure 1. Symptoms of poisoning by etoxazole and triflumuron in
fifth-instar larvae of Spodoptera frugiperda. Treated larvae (200 mg
litre −1 ) showed a double head capsule (arrows) and incomplete
ecdysis.
results obtained in this study confirmed hypothetical
considerations that the strong moulting inhibition
effects of 2,4-diphenyl-1,3-oxazolines such as etoxazole
lead to their larvicidal activity in a similar way to
the benzoylphenylureas. 3
The chitin content in integuments of sixth-instar
fall armyworm larvae which fed for 3 days on cabbage
leaves treated with concentrations of etoxazole
as low as 40 mg litre −1 did not increase compared
with 3-day-old untreated larvae. Interestingly, the
chitin content of etoxazole-treated larvae after 3 days
was not significantly different from that of freshly
moulted sixth-instar larvae (Fig. 2), thus indicating
its inhibitory potential on chitin biosynthesis. At a
concentration of 1.6 mg litre −1 , etoxazole no longer
inhibits chitin biosynthesis in vivo, whereas triflumuron
– originally designed to control lepidopteran
larvae – is still fully effective. The inhibition of chitin
biosynthesis with both compounds is clearly correlated
with larval mortality (data not shown).
3.2 Inhibition of incorporation of
[ 14 C]N-acetylglucosamine (GluNAc) into cultured
integuments of Spodoptera frugiperda
The dose–response relationships of etoxazole and triflumuron
to the inhibition of GluNAc incorporation
into pieces of integument of S. frugiperda are shown in
Fig. 3. Concentrations of ≤10 −7 M of etoxazole were
not inhibitory, but, at the highest concentration tested
Incorporation of GluNAc (dpm)
1750
1500
1250
1000
750
500
250
0
Etoxazole
Triflumuron
10 −11 10 −10 10 −9 10 −8 10 −7 10 −6 10 −5 10 −4 10 −3
Log concentration (M)
Figure 3. Dose–response curves for etoxazole and triflumuron on the
incorporation of GluNAc into pieces of the integument of Spodoptera
frugiperda. Curves are fitted based on two replicates per
concentration for each compound tested.
(10 −4 M), its incorporation inhibition was similar to
that of the reference compound triflumuron. This
confirms the potency of etoxazole to inhibit chitin
biosynthesis by interfering with the incorporation of
GluNAc into the integument, i.e. as described for
benzoylphenylureas. 5 However, based on pIC 50 values,
etoxazole (10 −5.53 M,2.95 µM) is about 40 times
less potent than triflumuron (10 −7.15 M, 70.8 nM) in
inhibiting 50% of KOH-resistant GluNAc incorporation
(Table 1). Although the inhibitory potential of
etoxazole is 40-fold less than that of triflumuron, it is
still rather specific in its effect, considering the pIC 50
value in the lower micromolar range. Furthermore,
etoxazole has been developed as an acaricide with only
insecticidal side effects such as described for aphids
and diamondback moth larvae. 3 The inhibitory potential
of triflumuron for the incorporation of GluNAc
into isolated pieces of fall armyworm integuments
Pest Manag Sci 62:379–382 (2006) 381
DOI: 10.1002/ps

R Nauen, G Smagghe
Table 1. Effect of etoxazole and triflumuron on the net amount of
KOH-resistant 14 C-GluNAc incorporated into the integument of
Spodoptera frugiperda. DataaregivenaspIC 50 (±standard error) and
the 95% fiducial limits after sigmoidal curve fitting of the
dose–response curves from Fig. 1. The goodness of fit is given as R 2
Compound pIC 50 (±SE) 95% fiducial limits R 2
Etoxazole 5.53 (±0.17) 5.15–5.90 0.90
Triflumuron 7.15 (±0.10) 6.93–7.36 0.98
is similar to that described for diflubenzuron on Chilo
suppressalis Walker (20 nM), thus indicating the validity
of the chosen assay format with S. frugiperda integuments.
Thus from our experimental results we conclude
that etoxazole is an acaricidal (and insecticidal) chitin
biosynthesis inhibitor with a mode of action similar
to that of benzoylphenylureas, i.e. inhibiting the
incorporation of the radiolabelled chitin precursor
[ 14 C]GluNAc into the integuments of pest invertebrates
affected by this compound.
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