Action of a novel nonsteroidal ecdysteroid mimic ... - Insects.ugent.be

Pestic. Sci. 1994, 42, 85-92
Action of a Novel Nonsteroidal Ecdysteroid
Mimic, Tebufenozide (RH-5992), on Insects
of Different Orders
Guy Smagghe & Danny Degheele"
Laboratory of Agrozoology, Faculty of Agricultural and Applied Biological Sciences, University of Gent,
Coupure Links 653, B-9000 Gent, Belgium
(Revised manuscript received 6 June 1994; accepted 15 June 1994)
Abstract: The nonsteroidal ecdysteroid agonist tebufenozide (RH-5992) was
tested on larval stages of a number of lepidopteran species by topical application
and by feeding on treated leaves. LC,, values in the range 0.03-0.10 mg litre-'
were obtained for third to sixth instars of Spodoptera exempta (Walker) when
insects were fed on leaves dipped in aqueous emulsions of the compound, while
first to fifth instars of Spodoptera exigua (Hiibner) were less susceptible (LC,,
values in the range 2.5-105 mg litre-'). When insects were topically treated,
susceptibility of last-instar larvae of Lepidoptera tested decreased in the order
Spodoptera exempta, Mamestra brassicae L., Spodoptera littoralis (Boisd.), S. exigua
and Galleria mellonella L. Tebufenozide induced a premature and lethal larval
moult in larval Lepidoptera within 24 h of treatment. Most larvae died in their
old larval cuticle. Other aberrations included inhibition of weight gain and feeding,
extrusion of the hindgut, loss of hemolymph and an abnormal and lethal pupation.
In contrast, tebufenozide at similar doses/concentrations had no activity on larval
instars of Leptinotarsa decemlineata (Say) and Diabrotica virgifera oirgifera
(LeConte), nymphs of Podisus sagitta (F.) and larvae of Locusta migratoria
migratorioides (R. & F.). It had a chemosterilizing activity in S. exigua, resulting
in a total inhibition of oviposition within two days of continuous treatment
at doses of > 10 mg litre-'. On the other hand, all deposited eggs were viable.
Similar effects on reproduction occurred in L. decernlineata, but at higher
concentrations.
1 INTRODUCTION several authors applying RH-5849 to larvae of different
Lepid~ptera.'-~*~-'~ T ebufenozide possesses a similar
Tebufenozide (RH-5992) is a novel synthetic nonsteroidal activity, though it is more potent and selective for
ecdysteroid agonist, which represents a new class of insect Lepid~ptera.~.~ As previously reported, RH-5849 apgrowth
regulators (IGRs). Its chemical structure, N-tert- peared to cause a variety of moulting and behavioural
butyl-N'-(4-ethylbenzoyl)-3,5-dimethylbenzohydrazide), effects in larval C~leoptera,~*~~~*'~~'~
and interfered in
is based on that of RH-5849, the prototype compound moulting and metamorphosis of some larval Crustacea."
of this group of insecticides. As previously reported for On the other hand, it was found that RH-5849 had no
RH-5849, this group of compounds induces a premature activity against Periplaneta americanu (L.),7 Podisus
and lethal larval moult by direct stimulation of the sagitta (F.) and Locusta migratoria migratorioides (R. &.
ecdysteroid receptors (EcR), especially in larval Lepidop- F.),' while in Oncopeltus fusciatus (Dallas) it caused the
tera.lP5 In this respect, these compounds mimic the formation of half-sized and sterile adult^.^
activity of the natural ecdysteroids, despite their different Ecdysteroids also have essential functions in controlling
molecular structure. This activity has been confirmed by processes involved in insect reproduction, e.g. stimulation
of vitellogenesis, release of the ovulation myotropin
* To whom correspondence should be addressed.
and cell stimulation for spermatocyte growth. 1 6 g 1
85
Pestic. Sci. 0031-613X/94/$09.00 0 1994 SCI. Printed in Great Britain

86 Guy Smagghe, Danny Degheele
Consequently, high concentrations of natural ecdysteroids
possess chemosterilizing properties in several
insect species.’8-20
The present study evaluates the toxicity and biological
activity of tebufenozide on insects of different orders,
families and genera. The compound was applied topically
or orally to various larval stages of several important
pest insects, namely; Lepidoptera: Spodoptera exigua
(Hiibner), Spodoptera exempta (Walker), Spodoptera
littoralis (Boisd.), Mamestra hrassicae L., and Galleria
mellonella L.: Coleoptera: Leptinotarsa decemlineata
(Say) and Diabrotica uirgijera uirgifera (LeConte); and
Orthoptera: L. migratoria migratorioides. Likewise,
tebufenozide was applied to nymphs of the predatory
Heteroptera, P. sagitta and Orius insidiosus (Say). This
research aimed to provide evidence for the novel mode
of action of tebufenozide, and its relatively high selectivity
for Lepidoptera compared to the prototype analogue
RH-5849. In addition, it evaluates the chemosterilizing
properties of tebufenozide in some species.
2.1 Chemicals
2 EXPERIMENTAL METHODS
Technical tebufenozide was obtained from Rohm and
Haas Company (Spring House, Pennsylvania, USA). All
solvents were of analytical grade.
2.2 Insects
S. exempta, the African armyworm, was reared on freshly
cut maize seedlings.’, Larvae of the beet armyworm, S.
exigua, and the Egyptian cotton leafworm, S. littoralis,
were fed on fresh castor-bean leaves, Ricinus communis
L.” The cabbage moth, M. brassicae, and the greater
wax moth, G. mellonella, were cultured as previously
de~cribed.’~ All stages of L. decemlineata, the Colorado
potato beetle, were fed on freshly cut potato foliage
(Solanum tuberosum L. cv ‘Bit~tJe’)~~ and D. uirgifera
uirgijera, the Western corn rootworm, on roots of live
maize seedings (Janssen, S., 1993, pers. comm.). Likewise,
the laboratory rearing of P. sagitta,2s 0. insidiosus26 and
L. migratoria rnigratorioide~~~ was carried out under
standard conditions.
2.3 Toxicity and insecticidal activity assay
For dipping experiments, different concentrations of
technical tebufenozide were prepared in water containing
02 ml litre-’ ‘Triton’ X-100. Freshly cut leaves were
dipped for 10 s, dried for 30-40 min at room temperature
in a fume hood and fed to the larvae ad libitum. For S.
exempta, D. uirgifera and L. migratoria larvae, freshly cut
maize seedlings were used; for S. exigua and S. littoralis,
fresh R. communis leaves and, for L. decemlineata, fresh
potato foliage. Leaves treated with water containing
0.2 ml litre-’ Triton X-100, were provided to the
controls.
For topical treatments, doses ranging from 001 to
40,000 ng per larva were applied by a dorsal administration
of 1 pl of methanolic solutions of technical
tebufenozide. Control specimens were treated with
methanol alone.
In another series of experiments, a high concentration
of 100 g litre-’ was prepared in dimethyl sulfoxide
(DMSO) and 1-2 p1 was topically applied on last-instars
of S. exempta and S. exigua. Control larvae were treated
with DMSO.
For P. sagitta nymphs, tebufenozide was topically
applied to the dorsal side, and, in a separate experiment,
to the ventral side because of the heavy dorsal sclerotization.
In another assay, P. sagitta nymphs were treated
orally by providing live last-instar S. exigua larvae as
prey. The latter were topically treated with 20 pg per
larva and allowed to take up the compound for 0.5 to 1 h.
In a preliminary experiment, the susceptibility of 0.
insidiosus nymphs to tebufenozide was also tested. As
food, fresh S. exigua eggs previously dipped for 10 min
in 1 g litre-’ (in methanol) and dried for 15 min, were
provided ad libitum to all nymphal stages (N,-N,) of
one generation. Controls were treated with methanol.
For both the leaf-dipping technique and topical
application, mortality percentages included both dead
and affected individuals. Mortality was monitored after
a time-period equal to the duration of the specific instar
plus 48 h for the last instar or 24 h for the other instars.
Mortality data were corrected for mortalities in the
controls and subjected to probit analysis using the
computer program POLO-PC.28 For each concentration,
20 to 40 insects were used and at least six different
concentrations were applied in order to calculate LC,,
and LD,, values.
Data on weight gain of treated specimens were also
determined and tested by one-way analysis of variance
(ANOVA) and mean values separated by a least significant
difference (LSD) multiple range test (P < 0.05).29
For internal abnormalities, control and treated specimens
were collected and fixed in 4% formaldehyde.
Subsequently, they were rinsed in distilled water and
dehydrated by passage through ethanol and xylol and
then embedded in paraplast. Sections were made with a
Historange microtome prior to examination with a light
microscope.
2.4 Effects on adult survival, fecundity and egg
viability of Spodoprera exigua
Different concentrations of tebufenozide were made in a
10% honeywater solution and supplied to the adults ad
libitum. Eight newly emerged S. exigua adults (sex ratio
1: 1) were placed together in a plastic box (1 1 x 1 1 x 16 cm)
the inside walls of which were covered with paper

~~
Tebufenozide in diflerent orders of insects 87
providing oviposition sites. Egg-laying and adult survival
were determined at 24-h intervals. Fecundity per female
was obtained by calculating the ratio of the number of
deposited eggs by the number of live females. Ovaries of
females which had stopped oviposition were examined.
For the determination of egg viability, eggs were placed
in a Petri dish (9 x 1.5 cm) containing a fresh castor-bean
leaf to prevent egg-eating cannibalism by newly emerged
larvae, and egg hatching was recorded. For each
concentration, three replicates of eight adults were set
up. Mean values of cumulative fecundity and egg viability
were tested by one-way ANOVA and separated by an
LSD-multiple range test (P < 005).29
2.5 Effects on adult survival, fecundity and egg
viability of Leptinotarsa decemlineata
Newly emerged adults (0-6 h old; sex ratio 1: 1) were
placed in a Plexiglas cylinder (1 3 x 20 cm) and freshly
cut potato leaves were provided ad libitum. After six days
of oviposition, leaves previously dipped in 0, 30 or
100 mg litre-' tebufenozide in water and then dried in a
fume hood for 30min were continuously provided as
food. Contact activity was also tested by treating adults
topically with 20 pg per adult, prepared in methanol, after
six days of oviposition. Controls were treated with
methanol alone. In both experiments, three replicates of
six adults were set up for each concentration. Data on
fecundity and egg viability were determined as described
for S. exigua. To prevent egg-eating cannibalism by newly
emerged larvae, freshly cut potato leaves were provided.
Ovaries of individuals which had stopped oviposition
were examined.
3 RESULTS
3.1 Toxicity and effects of tebufenozide on Lepidoptera
Table 1 shows the range of susceptibilities to tebufenozide
among the species tested. According to LC,, and LD,,
values, last-instar larvae of S. exempta were most
susceptible, followed by M. brassicae, and S. littoralis. To
kill 50% of treated S. exigua and G. mellonella larvae,
large amounts of tebufenozide were needed. Although
potencies differed markedly, similar insecticidal effects
were observed in all Lepidoptera. Treated last-instar
caterpillars (S. exempta: 2 3 ng larva-', 20.02 mg litre- ';
S. exigua: 2 20 ng larva- ', > 1 mg litre- '; S. littoralis:
2 10 ng larva-'; M. brassicae: 2 7 ng larva-'; G. mellonella:
2 300 ng larva- ') showed symptoms of a prematurely
induced and lethal larval moult. Within 24 h of
treatment, the head capsule had slipped down, revealing
double head capsules; moreover, underneath the old
capsule a fragile and (often) nonsclerotized new head
capsule was observed. Figure 1 exemplifies the formation
of a double cuticle in the thorax of a last-instar S. exigua
larva treated orally for 24 h with 3 mg litre- '. Similar
symptoms were observed in all treated Lepidoptera.
During this period, larval feeding and weight gain
(Fig. 2A,B) were significantly suppressed, and in some
cases loss of hemolymph and extrusion of the hindgut
were seen. Most treated larvae died in their old cuticle
shortly afterwards. In some cases, the old cuticle was
partially shed, with remnants remaining attached to
the new cuticle. Likewise, rupture of the imperfectly
formed new cuticle was sometimes observed. Adult
emergence did not occur at these concentrations. At lower
TABLE 1
Toxicity of Tebufenozide to Different Larval Stages of a Number of Lepidopteran Species"
Species Instar LD,, (ng larva-') LC,,, (mg litre-')
Relative potencyb
S. exempta
S. exigua
S. littoralis
M. brassicae
G. rnellonella
6th 6.75 (4.39-9.91; 2.53)
6th
5th
4th
3rd
5th 52.9 (35.5-74.5; 2.22)
5th
4th
3rd
2nd
1st
6th 11.02 (7.79-15.25; 1.84)
6th 8.53 (5.89-12.63; 1.40)
6th 571 (397-826; 1.89)
0.034 (0.028-0.042; 2.71)
0.085 (0.060-0.1 16; 0.47)
0.095 (0076-0.1 16; 0.60)
0'103 (0'083-0.127; 0.65)
2.5 (1.8-3.4; 3.00)
10.0 (5.9-14.2; 2.59)
8.5 (4.9-120; 2.57)
10.5 (5.1-16.3; 256)
9.7 (4.9- 15.2; 2.51)
74.1
52-9
87.1
80.0
73.8
75.6
44.0
19.4
23.5
18.7
21.2
59.0
186.4
13.1
Expressed as LD,, or LC,, values when insects were topically treated with or fed leaves dipped in aqueous emulsions of
tebufenozide, respectively. The 95% confidence interval, followed by the slope of the probit line are indicated in
parentheses.28
'Potency of tebufenozide relative to that of RH-5849, obtained by calculating the ratio of LD,, or LC,,
of RH-5849 (obtained under comparable conditions' 3, to that of tebufenozide.

88 Guy Smagghe, Danny Degheele
Fig. 1. Cross-section through the thorax of a last-instar S. exigua larva fed for 24 h on leaves previously dipped in an aqueous
solution of 3 mg litre- tebufenozide. Arrows indicate the formation of a double cuticle.
concentrations (S. exempta: 0.5-5 ng larva- ', 0.01-0.05
mg litre- '; S. exigua: 10-40 ng larva- ', 1-10 mg litre- ';
S. littoralis: 5-30 ng larva-'; M. brassicae: 3-30 ng
larva-'; G. mellonella: 100-750 ng larva- '), many lastinstar
larvae that did not appear to be affected underwent
an abnormal and lethal pupation. They were unable to
ecdyse successfully or to produce a normal pupal cuticle,
resulting frequently in larva-pupa intermediates. The
lowest concentrations (S. exempta: 60.01 ng larva- ',
60.001 mg litre-'; S. exigua: 60.1 ng larva-', d0.05
mg litre- '; S. littoralis: 60.3 ng larva- I; M. brassicae:
60.3 ng larva- '; G. mellonella: 6 10 ng larva-'), had no
effect on larval growth or development.
When tebufenozide, diluted in DMSO, was topically
applied at 100-200 pg per larva to last-instar larvae of
S. exernpta and S. exigua, nc signs of neurotoxicity were
noted. All larvae underwent premature head capsule
apolysis and died in their old cuticle within 24 h of
treatment.
It was also found that third- to fifth-instar larvae of
S. exempta and first- to fourth-instar larvae of S. exigua,
fed tebufenozide at 3 0.02-0.05 and 3 1-2 mg litre- ',
respectively, showed symptoms of a prematurely induced
and lethal moult within 24 h of treatment. Likewise, food
intake and weight gain ceased dramatically (P 6 0.05) as
compared to controls. Other symptoms of toxicity
included extrusion of the hindgut, loss of hemolymph,
and no, or an incomplete, shedding of the old cuticle.
Treated larvae died shortly afterwards. No visible effects
were observed at doses of 60.001 and d0.05 mg litre-'
for the various larval instars of S. exempta and S. exiyua,
respectively.
The effect on cumulative fecundity per female in
S. exigua is presented in Fig. 3. Tebufenozide rapidly
halted egg-laying in a dose-dependent manner. At
3 10 mg litre- ', oviposition was totally suppressed after
one or two days of continuous treatment. S. exigua
femaies treated with 1 mg litre- ' stopped egg-laying after
three or four days of treatment. Dissection of femaies
which had stopped oviposition suggested that the
formation of new oocytes was inhibited. Moreover, the
ovaries showed signs of degeneration and were therefore
frail. In the adults treated with 3 10 mg litre ~ ', death
appeared 4( k 1) days after treatment, vs. 9( f 1) days in
the control. In spite of a disastrous effect on fecundity,
all egs of treated S. exigua adults were equally viable
(P > 0.05). Mean percentages of egg hatching were
81 ( f 6), 75( f S), 79( f 8) and 76( k 8)% following application
of 0, 1, 10 and 100 mg litre-', respectively.
3.2 Toxicity and effects of tebufenozide in Coleoptera
Growth and ecdysis were not affected in L. decemlineata
larvae of all different instars (L,-L,) treated orally with
concentrations up to 50 mg litre-'. No significant differences
(P > 0.05) between weight gain of control and

Tebufenozide in different orders of insects 89
300
-m-
Control
250
0.005
=
h
f
''
200
c 0
- 150
m 0 15
2
I
c 100
I
50 4
----.__
0 24 48 12 96 120 144
Time (h into last-larval instar)
200
-c
Control
S 150 -A-
E
Y
E
0
1 I00
- E
m
c
I 50
250
200
B v
150
'E -
m
- ii 100
c
I
50
0
0 24 4a 12 96
Time (h into last-larval instar)
-0-
Contra\
..+.
50
0 24 4a 72 96 120 144
Time (h into last-larval instar)
Fig. 2. Effect of various concentrations of tebufenozide (mg
litre-') on weight gain of last-instar larvae of (A) S. exernpta,
(B) S. exigua and (C) L. decernlineata. Data represent the means
of three to four groups of 10 larvae. At various intervals, * and
O indicate a significant difference (P < 0.05) from the untreated
control.29
I
700
600
$500
c
c 2 400
P) >
- p 300
5 i
200
100
0
1 2 3 4 5 6 7
Time (days after first oviposition)
Fig. 3. Effect of various concentrations of tebufenozide (mg
litre-l) on the cumulative fecundity per female of S. exigua
adults by continuous feeding on treated honey-water. The
symbol * indicates that all adults were dead.
treated larvae (< 50 mg litre-I) were noted, as is shown
in Fig. 2C for the last-instar larvae. However, although no
toxicity was noted at concentrations up to 50 mg litre- ',
tremors and paralysis were induced at 100 mg litre-'
within 1 h of treatment in some cases.
Tebufenozide possessed no significant activity against
second- and last-instar larvae of D. virgifera virgifera,
since larval growth and development were unaffected
when freshly cut maize seedlings previously dipped in an
aqueous solution of 50 or lOOmglitre-' of the compound
were continuously supplied.
On the other hand, tebufenozide inhibited egg-laying
within a short time of uptake in L. decemlineata. In
six-day ovipositing females, oviposition was significantly
decreased (P < 0.05) within two days of continuous
feeding with potato leaves treated with 100 mg litre- '
(Fig. 4). Males and females also stopped feeding and
movement, further copulation was not observed and the
insects died shortly afterwards. On dissecting such
females, there was an absence of developing oocytes. At
30 mg litre- ', the cumulative number of eggs was only
significantly (P < 0.05) reduced relative to controls after
about 10 days of treatment. Up to this time, the average
fecundity of treated insects was lower than that of the
control insects, although this difference was not significant
at P = 0.05 level. At the end of the test, 243( f26) eggs
per female were deposited by these adults vs. 297( f 24)
in the control (Fig. 4). In adults topically treated with
20 pg per adult, oviposition was suppressed within two
or three days of treatment, resulting in 103(+9),
133( 1 1) and 154( _+ 13) eggs per female beetle after 2,4
and 15 days of treatment vs. 143(+12), 174(f13) and
297( f24) in the control (Fig. 4). On the other hand, egg
viability was similar (P < 0.05) in all treatments. Average
hatching percentages reached 73( f 8), 70( f 8) and
69( i 7)% for the groups fed continuously with leaves
dipped in 0, 30 and 100mglitre-', respectively, and

90 Guy Smagghe, Danny Degheele
P
‘3
350
-m-
5 150
5
0 100
04 I
0 5 10 15 20
Time (days after first oviposition)
Fig. 4. Effect of tebufenozide on the cumulative fecundity per
female of L. decemlineata adults, at 20 pg per adult by topical
treatment and at 30 or 100 mg litre-’ by continuous feeding
on treated potato leaves. The arrow indicates the moment when
topical treatment took place or oral application started. The
symbol * indicates that all adults were dead.
73( k 8) and 69( k 8)% for the groups treated topically
with 0 and 20 pg per adult, respectively.
3.3 Toxicity and effect of tebufenozide on Heteroptera
No effect on growth, weight gain or ecdysis was observed
on third- (N,) and last- (N5) instar nymphs of P. sagitta
at doses ranging from 1 to 40 pg per nymph, topically
applied either on the dorsal or on the ventral side.
Likewise, no toxicity and no effects on the development
of N, and N, bugs were noted when last-instar S. exigua
larvae previously treated with 20 pg per larva were
provided as prey.
In a preliminary assay with 0. insidiosus, no effects on
the development of any nymphal stage (N,-N,) or adult
formation were noted when fresh eggs of S. exigua
previously dipped in 1 g litre-’ tebufenozide were
supplied ad libitum.
3.4 Toxicity and effects of tebufenozide on Orthoptera
First- and second-instar L. migratoria migratorioides
larvae showed a pattern of growth and ecdysis into the
following instar after a continuous treatment with 30 or
100 mg litre-’ tebufenozide which was the same as that
of the control insects.
4 DISCUSSION
Tebufenozide acts primarily by inducing a premature and
lethal larval moult, especially in larval Lepidoptera. Our
results strengthen the concept that the compound
stimulates epidermal cells to undergo apolysis prematurely
and to synthesize a new larval cuticle by imitating the
activity of ecdysteroids. In addition, LC50 values of
tebufenozide against the different stages of S. exempta
and S. exigua illustrate the uniformity of response with
respect to the larval stage, which is consistent with the
pattern of RH-5849 against the different stages of several
Lepidoptera obtained under comparable conditions.2*’2
The minimum concentration of tebufenozide (by means
of a continuous oral application) needed to induce a
premature moult in the different larval instars was similar
for each species: in third- to sixth-instar larvae (L3-Ls)
of S. exempta at a concentration of 20.02 mg litre-’, and
in L1-L5 of S. exigua at 21 mg litre-’. LC50 values for
last instars reached lower levels, since abnormal and
lethal pupation are included in the mortality percentages
for last-intar larvae.
The inability of treated larvae to ecdyse out of the old
cuticle might be provoked by an interference with
eclosion hormone (EH) production/release, since it is
known that this hormone is released by a drop in
ecdysteroid titre prior to ecyd~is.~~ Residual amounts of
ecdysteroid agonist in the insect body may cause an
inhibition of EH-release. Similar results of death during
a prematurely promoted moult have been reported
previously, following application of natural ecdysteroids
to different insect species.31
The decrease in weight gain and feeding in larval
Lepidoptera treated with tebufenozide may result from
an ecdysonergic activity of the compound. It is well
known that cessation of feeding and weight gain occurs
prior to ecdysis as a result of ecdysteroid ~ecretion.~’ In
contrast, no such effects were observed in larvae of L.
decemlineata, D. virggera, P. sagitta and L. migratoria at
similar doses/concentrations of tebenufenozide.
The different toxicities of ecdysteroid agonists on
last-instar larvae of S. exempta, S. exigua and L.
decemlineata applied orally cannot be explained by
differences in retention, distribution and metabolic
detoxification of the compound in the insect body.”,33
Consequently, we consider that the wide range of
susceptibilities to the ecdysteroid-mimicking compounds
in the different insect species may be explained by
differences in the structure of the EcR and their binding
affinity for the ecdysteroid agonist ligand molecules. The
latter hypothesis is consistent with the concept that
structure and biochemical properties of EcR may differ
among insect species.34 In general, although the hypothesis
seems reasonable that the different toxicities of
ecdysteroid agonists are induced by differences in
EcR-binding affinity, further research is required.
In this study, it was noted that tebufenozide possessed
little or no insecticidal activity against larvae of two
coleopterans, L. decemlineata and D. virgqera virgqera,
against various nymphal stages of the predatory heteropterans,
P. sagitta and 0. insidiosus, or against larvae
of the orthopteran, L. migratoria migratorioides, as

Tebufenozide in diferent orders of insects 91
compared with Lepidoptera. These results agree with
Carlson, G. R. (1993, 1994, pers. comm.) who found
tebufenozide to be 200 to 3000 times less active than
RH-5849 against larvae of L. decemlineata, and confirmed
tebufenozide's target selectivity for Lepidoptera after
determining its pest-control spectrum using more than
150 different insect species. In this respect, the high
selectivity and potency of tebufenozide against larval
Lepidoptera makes this compound an excellent tool in
integrated pest management (IPM) programmes. Moreover,
its novel chemistry and mode of action suggest that
tebufenozide should control species resistant to other
classes of insecticides, including current IGRs.
Our data showed that tebufenozide affected the
ovarian growth in S. exigua and L. decemlineata, which
agreed with the results obtained for RH-5849 in several
Lepidoptera, Coleoptera and Diptera.z36*' 393s Likewise,
certain steroid ecdysone analogues inhibited ovarian
development of the house fly, Musca domestica L., the
flour beetle, Tribolium confusum Jacquelin du Val, and
the boll weevil, Anthonomus yrandis Boheman, and
resulted in a complete and permanent sterility.'8-20
However, tebufenozide's inhibiting activity in Lepidoptera
adults seems to be only a little stronger than that of
RH-5849.13 In contrast, tebufenozide was about 60 times
more potent than the prototype compound against the
different larval stages of S. exiyua.' In L. decemlineatn
adults, an increase in dose of tebufenozide was needed
to inhibit reproduction as compared with RH-5849.'
How exactly tebufenozide causes the observed effects
remains unknown. As with RH-5849,29631 3335 we presume
that tebufenozide inhibited egg-laying by causing a
(re)sorption of the ovarioles. However, it is unclear
whether the compound affected vitellogenesis directly or
indirectly. In general, many questions about the activity
of synthetic nonsteroidal ecdysteroid agonists in insect
reproduction still remain unanswered.
ACKNOWLEDGEMENTS
We are very grateful to G. R. Carlson (Rohm and Haas
Company, USA) for providing technical tebufenozide,
and also thank P. De Clercq, L. Tirry, L. Butaye, B. Roos
and M. Van de Veire (all from the authors' laboratory)
and S. Janssens (Plant Genetic Systems, Belgium) for
supplying specimens of some insect species. In addition,
P. De Clercq is thanked for critically reading the
manuscript. This work has been supported by grant No.
910071 of the IWONL (Institute for Encouragement of
Scientific Research in Industry and Agriculture, Belgium)
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