Mode of action of etoxazole - ResearchGate

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