892 M. Kostyukovsky et al. Insect Biochemistry and Molecular Biology 30 2000 891–897
tha dominica
Fabricius and
Sitophilus oryzae
Linnaeus are all economically important pests of stored products, which feed on a wide range of commodities
Arbogast, 1991. Repeated pesticide treatments have led to the increasing incidence of resistance and consequent
serious control problems of these species in certain cases Subramanyam and Hagstrum, 1996. The objective of
this study was to compare the efficacy of the juvenile hormone agonist methoprene which is in use as control
agent against insect pests of stored products, to the juv- enile hormone agonist pyriproxyfen, and two ecdysteroid
agonists, RH-5849 and tebufenozide.
2. Materials and methods
2.1. Insects The test insect strains were reared in the laboratory
over 10 years without any contact with insecticides. Sito- philus oryzae and Rhyzopertha dominica were reared on
whole soft wheat, and Tribolium castaneum was reared on wheat flour. The actellic-resistant strain of T. cas-
taneum was obtained by raising the laboratory strain on a culture medium with gradually increasing concen-
trations up to 4.5 ppm of the organophosphorus insecti- cide, actellic 50 e.c. pirimiphos-methyl, for around
20 generations. The insects in the last five generations were raised on 4.5 ppm of actellic without increasing the
concentration. All these insects were maintained in 1 l glass jars with paper covers and bred at 27
± 0.5
° C,
70 ±
5 r.h. and light:dark cycle of 8:16 h. 2.2. Insecticides
Four compounds were used in our experiments: Methoprene,
[1,isopropyl2E,4E-11-methoxy-3,7,11- trimethyldodeca-2,4-dienoate], supplied by Zoecon Cor-
poration, California, USA. Pyriproxyfen, 2-[1-methyl-2- 4-phenoxy-phenoxyethoxy]pyridine, supplied by Sum-
itomo Chemical Corporation, Ltd., Japan. RH-5849: dibenzoyl
hydrazines, 1,2-dibenzoy,1-tert-butyl
hydrazine, and Tebufenozide, 3,5-dimethylbenzoic acid 1-1,1-dimethylethyl-2-4-ethylbenzoylhydrazide, both
supplied by Rohm and Haas Corporation, Spring House, PA, USA.
2.3. Treatments Stock solutions of the tested IGRs in acetone were
prepared at a concentration of 5 mgml. Appropriate amounts of the compounds to be tested were dissolved
in 10 ml acetone100 g medium and were thoroughly mixed with the food of each of the target insects. Food
treated with acetone alone was used as a control. Care was taken that all acetone had evaporated from the
treated and control samples, by mixing and ventilating the culture medium for 24 h, before insects were added
to all treated food. For each treatment 20 adults aged 14–18 days after emergence from the pupae, to allow
mating, when used. The insects were placed in a 100 ml jar with 20 g of wheat flour T. castaneum or whole
wheat R. dominica and S. oryzae. All insects were kept at 25
± 0.5
° C and 70
± 5 r.h. at a 8:16 h light:dark cycle.
After a period of 15 days during which the insects were allowed to oviposit eggs, the parental adults were
removed and their mortality was recorded. The emerged F
1
adults were counted twice a week and removed until emergence was complete. The developmental course and
external morphology of larvae, pupae and adults of T. castaneum were observed. In the cases of R. dominica
and S. oryzae, only live adults found externally were recorded, since the larvae and pupae develop inside the
grain kernels. The standard error of the life span means and numbers of F
1
adults were calculated. Significance of differences was analyzed using ANOVA test
P,0.05. The LC
50
and LC
90
were calculated according to POLO-PC probit analysis LeOra Software, 1987.
3. Results
3.1. Tribolium castaneum The external morphology, mortality in the various
developmental stages, life span, numbers and percentage reduction of F
1
adults of susceptible S and actellic- resistant R strains of T. castaneum are given in Table
1 and Table 2. The maximum recorded mortality of par- ental adults among all treatments and the control was
5 not shown. The results showed that methoprene at concentrations of 0.1 ppm and higher wholly prevented
the development of F
1
S-strain adults: most larvae were abnormal and died in the larval stage except for a small
number which died during pupation. As the concen- tration increased, the life span lengthened from 105 days
at 0.1 ppm to 139 days at 3 ppm, while that of control was only 67 days. These data were obtained from obser-
vation extending from the introduction of F
adults to the appearance of all F
1
adults or 95 mortality of larvae and pupae. The R-strain was less sensitive to methop-
rene: only at a concentration of 3 ppm was a 100 reduction of F
1
adults obtained. Insect mortality occurred in the pupal stage and during adult emergence but the
life span was lengthened only to 82 days compared with that of the S-strain. With pyriproxyfen, all larvae of both
S-strain and R-strain appeared abnormal in their external morphology, and died in this stage at all tested concen-
trations, but their life span was very greatly extended, to 158 and 300 days in S-strain and R-strain, respect-
ively. In addition, both methoprene and pyriproxyfen,
893 M. Kostyukovsky et al. Insect Biochemistry and Molecular Biology 30 2000 891–897
Table 1 The biological activity of JH agonists Methoprene and Pyriproxifen on development of F
1
adults of susceptible S and actellic-resistant R strains of Tribolium castaneum
a
External Stage of abnormal
Life span No of adults
reduction of IGRs
Strain Conc. ppm
morphology death
days ±
S.E.M.
b
F
1
± S.E.M.
c
F
1 d
Methoprene S
3 L
larva 1394.3
l
100 1
L larva
1175.4
h
100 larva, pupation
0.5 lL
1053.6
g
100 few
larva, pupation 0.1
lL 1055.4
g
100 few
R 3
lpP pupa, emergence
822.9
f
100 1
lpaA emergence, adult
823.2
f
13.00.4
l
81.9 0.5
lpaA adult few
822.5
f
56.71.3
h
21.2 0.1
lpaA none
823.3
f
1096.8
f
251.4 Pyriproxyfen
S 2
L larva
1586.4
g
100 1
L larva
1587.5
g
100 0.5
L larva
1585.7
g
100 0.1
L larva
1586.2
g
100 R
2 L
larva 3007.9
k
100 1
L larva
30010.2
k
100 0.5
L larva
3009.8
k
100 0.1
L larva
30012.3
k
100 Control
S lpa
none 672.3
e
69.30.8
e
R lpa
none 673.5
e
72.00.8
e a
The data are the average of three experiments, each with three replicates. l,L =
larvae; p,P =
pupae; a,A =
adults; l,p and a =
morphology completely normal; L,P and A
= abnormal.
b
From the introduction of F adults to the occurrence of all F
1
adults or death of 95 larvae or pupae.
c
Number of normal adults obtained at the end of F
1
generation period.
d
In comparison with control.
e–p
Different letters indicate statistically significant difference of averages in the same column with 95 confidence.
Table 2 The biological activity of ecdysone agonists RH-5849 and Tebufenozide on development of F
1
adults of susceptible S and actellic-resistant R strains of Tribolium castaneum
a
External Stage of abnormal
Life span No. of adults
reduction of IGRs
Strain Conc. ppm
morphology death
days ±
S.E.M.
b
F1 ±
S.E.M.
c
F
1 d
RH-5849 S
10 lpPa
pupa 785.9
f
5.30.4
n
92.4 7.5
lpPa pupa
786.4
f
9.30.4
m
86.6 5
lpa pupation
634.9
e
37.30.9
j
46.2 2.5
lpa pupation
633.7
e
47.31.5
I
31.7 R
10 lpa
pupation 786.7
f
2.00.8
o
97.2 pupation, adult
7.5 lpa
786.1
f
6.30.9
n
91.2 few
Tebufenozide S
20 lpPa
pupa 787.8
f
14.00.8
l
79.8 15
lpPa pupa
787.4
f
21.71.2
k
68.7 10
lpa pupation
635.4
e
56.71.3
h
18.2 R
20 lpaA
pupation, adult 788.4
f
0.70.4
p
99.0 15
lpaA pupation, adult
786.7
f
6.00.4
n
91.7 Control
S lpa
none 672.3
e
69.30.8
e
R lpa
none 673.5
e
72.00.8
e a
The data are the average of three experiments, each with three replicates. l,L =
larvae; p,P =
pupae; a,A =
adults; 1,p and a =
morphology completely normal; L,P and A
= abnormal.
b
From the introduction of F adults to the occurrence of all F
1
adults or death of 95 larvae or pupae.
c
Number of normal adults obtained at the end of F
1
generation period.
d
In comparison with control.
e–p
Different letters indicate statistically significant difference of averages in the same column with 95 confidence.
produced giant larvae with a darker color than those of the control, possibly, as a result of their longer life span.
The ecdysone agonists, RH-5849 and tebufenozide, showed relatively lower toxicity to T. castaneum Table
2 compared with JH agonists. RH-5849 at concen- trations of 2.5, 5, 7.5 and 10 ppm reduced the F
1
S-strain adults to 32, 46, 87 and 92, respectively, with minor
increase in the life span of the treated insects compared with control. Insect mortality occurred during pupation
and in the pupal stage.
894 M. Kostyukovsky et al. Insect Biochemistry and Molecular Biology 30 2000 891–897
In contrast to their responses to the JH agonist metho- prene, both R and S-strains of Tribolium were suscep-
tible to the ecdysone agonist RH-5849: concentrations of 7.5 and 10 ppm of RH-5849 achieved 91 and 97
reduction of F
1
adults, respectively Table 2. The toxicity of tebufenozide was far lower than that
of RH-5849. Exposure of the S-strain to 5, 10, 15 and 20 ppm of tebufenozide caused reduction in F
1
adults of only 12, 18, 69 and 80, respectively. The R-strain was
relatively more susceptible than the S-strain, and 92 and 99 control of F
1
was obtained at 15 and 20 ppm, respectively.
3.2. Rhyzopertha dominica Table 3 shows the numbers and the control percent-
ages of F
1
adults of a susceptible laboratory strain of R. dominica, and the LC
50
and LC
90
of the four IGRs. The maximum mortality of parental adults among all treat-
ments and the control was 7. The life span of the F
1
generation for all treatments including control was about 62 days, which suggests that the four IGRs had no effect
on the life span of this species. Methoprene and pyrip- roxyfen showed high toxicity to R. dominica: at a con-
centration of 0.1 ppm, a reduction of 99 of F
1
adults was obtained. The concentration of either juvenoids,
methoprene or pyriproxyfen, required for 50 or 90 F
1
adult reduction compared with control was less than 0.1 ppm. RH-5849 was less effective: 10 ppm caused 97
reduction of F
1
adults, and LC
50
and LC
90
were 2 and 5 ppm, respectively. Tebufenozide was found to be least
Table 3 The biological activity of various IGRs on development of F1 adults of susceptible laboratory strain of Rhyzopertha dominica
a
IGRs Concentration
No. of adults Reduction of F
1
adults ppm
F
1
± S.E.M.
b
LC
50
ppm
c
LC
90
ppm
c
Slope ±
S.E.M. Methoprene
3
o
100 ,0.10
,0.10 0.54 0.49
1 0.70.5
m
99.7 0.5
1.00.8
m
99.5 0.1
1.30.8
m
99.3 Pyriproxyfen
2 0.30.3
n
99.9 ,0.10
,0.10 1
0.50.4
m
99.8 0.5
0.70.6
m
99.7 0.1
0.70.6
m
99.7 RH-5849
10 4.71.0
l
97.6 2.12
5.26 3.25 0.29
7.5 8.70.7
k
95.6 1.80–2.41
4.79–5.89 5
18.32.2
i
90.7 2.5
83.74.8
g
57.6 Tebufenozide
20 7.71.0
k
96.1 7.65
13.79 5.00 0.29
15 10.31.3
j
94.8 5.97–9.19
11.29–19.49 10
53.33.5
h
73.0 5
164.77.8
f
16.5 Control
197.36.7
e a
The data are the average of four experiments, each with three replicates. POLO-PC LeOra Software, 1987 was used for probit analysis. Number of normal adults obtained at the end of F
1
generation period.
b
reduction of F
1
adults emerging compared with control.
c
LC
50
, LC
90
— concentration levels required for 50 or 90 reduction of F
1
adults compared with control. 95 confident limits are in parentheses.
e–o
Different letters indicate statistically significant difference of averages in the same column with 95 confidence.
active on this pest: control of F
1
adults was 96 at 20 ppm, and LC
50
and LC
90
were 7 and 14 ppm, respect- ively.
3.3. Sitophilus oryzae The biological activity of the four IGRs on F
1
devel- opment of the susceptible laboratory strain of S. oryzae
is shown in Table 4. The maximum mortality recorded for parental adults, including the control was 7. The
life span of the F
1
generation in all treatments including control was about 42 days, which indicates that, as with
R. dominica, the four IGRs also had no effect on the life span of S. oryzae. Pyriproxyfen showed very high
insecticidal effect: it reduced a number of F
1
adults by 97 at 2.5 ppm, and its LC
50
and LC
90
were only 0.3 and 1.2 ppm, respectively. However, methoprene, RH-
5849 and tebufenozide were much less effective for the control of this species: methoprene at 10 ppm, and RH-
5849 and tebufenozide at 20 ppm caused reduction of F
1
adults by only 29, 44 and 40, respectively. The LC
50
and LC
90
were 13 and 23 ppm for methoprene, 21 and 46 ppm for RH-5849, and for tebufenozide 22 and 32
ppm, respectively. In summary, comparison among the LC
50
and LC
90
of the four IGRs against the three insects on which they
were tested showed that pyriproxifen was the most potent: its LC
90
for T. castaneum and R. dominica was lower than 0.1 ppm and that for S. oryzae 1.2 ppm.
Methoprene showed lower activity, followed by RH- 5849 and tebufenozide Table 5.
895 M. Kostyukovsky et al. Insect Biochemistry and Molecular Biology 30 2000 891–897
Table 4 The biological activity of various IGRs on development of F
1
adults of susceptible laboratory strain of Sitophilus oryzae
a
IGRs Concentration
No. of adults Reduction of F
1
adults ppm
F
1
± S.E.M.
b
LC
50
ppm
c
LC
90
ppm
c
Slope ±
S.E.M. Methoprene
10 163.34.9
i
29.3 12.89
23.32 4.98 0.65
7.5 203.76.4
h
11.8 11.64–15.16
18.81–33.32 5
226.011.4
e
2.2 2.5
250.710.8
g
28.5 Pyriproxyfen
2.5 6.90.8
o
97.0 0.28
1.18 2.06 0.22
2 9.00.9
n
96.1 0.20–0.35
1.03–1.38 1
27.01.6
m
88.3 0.5
72.13.0
l
68.8 RH-5849
20 128.97.9
k
44.2 21.00
46.48 3.72 0.45
15 152.98.1
i
33.8 10
207.910.7
h
10.0 5
267.313.8
f
215.7 Tebufenozide
20 138.712.3
j
40.0 21.57
31.71 7.66 0.83
15 204.39.3
h
11.6 20.51–23.14
28.42–37.42 10
230.115.8
e
0.4 Control
231.013.2
e a
The data are the average of four experiments, each with three replicates. POLO-PC LeOra Software, 1987 was used for probit analysis. Number of normal adults obtained at the end of F
1
generation period.
b
reduction of F
1
adults emerging compared with control.
c
LC
50
, LC
90
— concentration levels required for 50 or 90 reduction of F
1
adults compared with control. 95 confident limits are in parentheses.
e–o
Different letters indicate statistically significant difference of averages in the same column with 95 confidence.
Table 5 LC
50
and LC
90
ppm of the four IGRs tested on T. castaneum, R. dominica and S. oryzae
IGRs T. castaneum
R. dominica S. oryzae R
S S
S LC
50
LC
90
LC
50
LC
90
LC
50
LC
90
LC
50
LC
90
Pyriproxyfen ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 0.3
1.2 Methoprene
0.8 1.9
,0.1 ,0.1 ,0.1 ,0.1 13 23
RH-5849 5
7 5
9 2
5 21
46 Tebufenozide
11 15
14 22
8 14
22 32
4. Discussion and conclusions
