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Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
Efficacy of Insect Growth Regulators against Red
Tef Worm, Mentaxya ignicollis (Walker)
(Lepdoptera: Noctuidae)
1Tariku Tesfaye, Mulugeta Negeri2 and Mohammed Dawd3
1Ethiopian Institute of Agricultural Research, Jimma Agricultural Research Center
2Department of Plant Science and Horticulture, Ambo University
3Ethiopian Institute of Agricultural Research, Ambo Plant Protection Research Center
Abstract
Tef (Eragrostis tef (Zucc.), Trotter: Poaceae) is a staple food crop of Ethiopia where it is
originated and diversified. Red tef worm (Mentaxya ignicollis) is a serious pest of tef
grown on clay soils. Hence the present study emphasized on the evaluation of insect
growth regulators to control Red Tef Worm. Laboratory study were carried out in
completely randomized design with two insect growth regulators (lufenuron at doses
20, 40 and 60g a.i/ha and teflubenzuron at doses of 75, 112.5 and 150g a.i./ha)
against 3rd instar larvae of RTW. The green house study was carried out in
randomized complete block design with the two Chitin synthesize Inhibitors (CSIs)
(lufenuron at dose of 40g a.i./ha and teflubenzuron at dose of 112.5g a.i./ha).
Efficacy of CSIs in affecting the hatchablity of the eggs was also studied. From the
laboratory and greenhouse experiments the IGRs, lufenuron and teflubenzuron,
caused mortality after affecting the developmental stage of RTW larvae and also
inhibited egg hatchability. In general, lefenuron (40g a.i./ha) was found to be
effective and and showed high potency against 3rd instar larvae of RTW under
laboratory and greenhouse conditions. Since the CSIs are safe to the environment and
other beneficial organisms, it is recommended to be verified for usage under open
and large field conditions for the control of RTW.
Key words: Lufenuron, Red tef worm , Teflubenzuron
Tariku Tesfaye et al. [14]
Journal of Science and Sustainable Development (JSSD), 2013, 1(2), 13-24
Introduction
Tef (Eragrostis tef (Zucc.), Trotter:
Poaceae) is a staple food crop of
Ethiopia, where it is originated and
diversified. Over 2.8 million hectares
of land is covered with tef every year
with a predicted 1228 kgha-1 mean
productivity at national level (CSA,
2011).
Red tef worm (RTW) (Mentaxya.
ignicollis) is a serious pest of tef grown
on black or heavy, deeply cracking
clay soils. The status of red tef worm,
M. ignicollis as a major pest of tef was
reported from Shewa, Kefa, Gojam, in
some places in Tigray and Wollega
regional states of the country
(Tadesse, 1987). It can cause up to 30%
loss in yield (IAR, 1986).
Control measures of RTW, including
cultural, chemical and microbial
methods have been attempted to some
extent (Tadesse, 1987a, 1987b).
However, they were not adequate to
minimize the density of RTW and
thereby alleviate the yield loss caused
by the pest. On the other hand, use of
synthetic insecticides causes
environmental pollution, pest
resistance and toxicity to other non-
target organisms.
Previously, no research has been done
with insect growth regulators to
control RTW. Chitin synthesis
inhibitors, lufenuron and
teflubenzuron, are extensively
available nowadays and are being
tested both in the laboratory and field
condition (Arnold et al., 2009; Kai et
al., 2009; Tassou and Schulz, 2009;
Mansur et al., 2010).
Hence in this research, insect growth
regulators, lufenuron and
teflubenzuron were used in both
laboratory and greenhouse studies on
RTW to provide information, assist
the development of an integrated pest
management program and provide
management options for the farmer.
Therefore, the present study was
carried out under laboratory and
greenhouse conditions to measure
efficacy of insect growth regulators
(lufenuron and teflubenzuron) and
determine effective dose against the
larvae of M. ignicollis under laboratory
and greenhouse conditions.
Materials and Methods
Growing of tef plants on
pots
The tef variety (Kuncho) was sown on
pots at the recommended rate of
25kgha-1.The sizes of the pots were
18x30cm. The pots were filled with
clay, compost and sandy soil in the
ratio of 1:2:1 respectively. The
experiment was carried out at Ambo
Plant Protection Research Center
(APPRC).
Rearing of red tef worm
(RTW)
The larvae of red tef worm were
collected from infested tef fields in
South West Shoa Zone, Becho and
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
Saden Sodo woredas early in the
morning on tef plants at grain filling
stage. The collected larvae were
transferred to plastic bowls which
were quarter filled with mixture of
fine sand and black soil and were
provided with fresh tef seedlings
every 24 hours and kept under
temperature of 26+2oC.
The larvae pupated in the plastic
bowls at the depth of 3-9cm. The soil
in the plastic bowls with pupae were
wetted and kept undisturbed. On an
average, 15 days after pupation adults
started to emerge. To culture the
adults, tef seedlings were grown on
small pots and kept in the cage (1.5m
x1.5m). Then emerged adults were
carefully transferred to the cage with
3:1, female to male ratio and provided
with 10% sugar solution (Tadesse and
Matthews, 1986) by sprinkling on the
tef seedlings, placing cotton wool
soaked in sugar solution in small cups
in the cage as well as suspending
cotton wool which was wetted with
the solution. Every day, the sugar
solution was sprinkled and the cotton
in the cups was changed. As an
alternative zigzag shaped paper were
suspended on the corner to facilitate
oviposition. Three days after
emergence, adults started oviposition.
The eggs were laid on the underside
of tef leaves, on the suspended paper,
and on the surface of cage (nylon
cage). Ten to fourteen days after
oviposition, eggs hatched and the
larvae fed on the leaves of the
seedlings.
Efficacy of Lufenuron and
Teflubenzuron in the
laboratory
Two insect growth regulators
(lufenuron 50% EC and teflubenzuron
15% SC) and endosulfan were
obtained from Abel Agrisher Ethiopia
PLC. And were evaluated at three
rates each by using sterile distilled
water: teflubenzuron 15% SC (150,
112.5 and 75 g a.i.ha-1) and lufenuron
50% EC (60, 40 and 20 g a.i.ha-1).
Endosulfan 35%EC at 700g a.i./ha and
unteareted checks were used. The
doses were chosen from a preliminary
trial carried out on related and other
insect species (N.S.Butter et al., 2003
and Bakr et. al., 2008).
Bio-assay on larvae
Total of 240 3rd instar larvae were used
for this experiment. Fresh chopped tef
leaves were kept in each Petri dish
(12.5cm diameter). Ten third instars
larvae were transferred to each
Petridish and the treatments were
sprayed using hand sprayer on the
leaves and on the surface of the
larvae. Larvae were allowed to feed
the treated leaves for 24 hours (Bakr,
et.al, 2008). The control insects were
allowed to feed on untreated leaves.
All the treatments were kept under
the same laboratory condition. The
experiment was carried out in a
completely randomized design with
eight treatments in three replications.
Fresh chopped leaves of tef were
replaced every day. Larval mortality
was recorded every 24 hours for ten
consecutive days.
Tariku Tesfaye et al. [16]
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
Bio-assay on egg
Three hundred twenty eggs were used
for the experiment and were obtained
from laboratory reared M. ignicollis.
Doses of 112.5 g a.i.ha-1 and 40 g a.i.ha-
1 were prepared for teflubenzuron and
lufenuron, respectively. These doses
were selected from the the preceding
laboratory based on their effectiveness
against the larvae. Sixteen Petri dishes
of 12.5cm diameter lined with filter
paper were prepared. Twenty black
headed eggs which develop to larvae
were transferred into each Petri dish
carefully using camel brush.
Individual treatments were applied
topically to eggs. Control eggs were
treated with sterilized distilled water
and the standard check, endosulfan
35%EC was applied at 700 g a.i ha-1.
The treated eggs were kept at the
temperature of 27+1oC, 65-85% RH
and 12L: 12D photoperiods until
larval hatch. Hatchability percentage
was recorded every 24 hours for five
consecutive days after application.
The embryocidal effect of the
treatments on developing embryo was
calculated as the percentage of
embryos that died in the eggs.
Verification of IGRs in
greenhouse
The experiment was conducted at
APPRC, entomology greenhouse in a
randomized completely block design
with three replications.
The treatments were: Teflubenzuron@
112.5g a.i/ha, Lufenuron@40g a.i/ha,
Endosulfan@ 700g a.i/ha and
Untreated check.
Ten third instar larvae of RTW were
placed on each tef plants pot at grain
filling stage. The treatments were
applied using hand sprayer, early in
the morning. Larval mortality was
assessed every 24 hrs for 10
consecutive days after treatment
application.
Data analysis
Larvae and egg mortality under each
treatment in both laboratory and
greenhouse conditions was corrected
using Abbott (1925) formula and the
corrected mortality data of the IGRs
in laboratory and greenhouse
conditions were analyzed using one
way analysis SAS program (SAS,
2005). LD50 and LT50 were also
calculated using SAS probit analysis.
CM (%) = (T-C)/(100-C)*100
Where, CM is Corrected mortality
T is Percent mortality in treated larvae of RTW
C is Percent mortality in untreated larvae of RTW
Results and Discussion
Efficacy of Lufenuron and Teflu-
benzuron against 3rd Instar Larvae of
RTW under Laboratory and
Greenhouse Conditions.
The results of laboratory study
showed that there were significant
differences in larval mortality between
untreated check and the other
treatments (Table 1). Lufenuron at the
dose of 40 and 60 g a.i./ha caused
significant mortality of 96.29 and
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
100%, respectively when compared
with the other treatments; however,
no significant differences was
observed with standard check
(endosulfan(100%)). On the other
hand, there was no significant
differences between teflubenzuron at
112.5g a.i./ha and 150g a.i/ha and the
lowest dose of lufenuron, 20g a.i/ha
(84..26%). This indicated that
lufenuron was more effective than
teflubenzuron in causing mortality of
RTW.
The data on lufenuron and
teflubenzuron potency against the 3rd
instar larvae of RTW in greenhouse
are presented in Table 2. Both IGRs
caused significant mortality of the
larvae ten days after treatment
application, compared to the
untreated control. Lufenuron (94.45%)
was not significantly different from
the standard check (97%), but
teflubenzuron (80.56%) was inferior.
Significant differences (p<0.001)
between lufenuron (94.45%) and
teflubenzuron (80.56%) with respect to
the larval mortality were also
observed from the results.
Most of the dead larvae treated with
lufenuron and teflubenzuron were
dark and shriveled and the old
exoskeletons were still attached to the
lower part of the abdomen. Prior to
death, the treated larvae remained
motionless and were unable to feed on
the provided tef seedlings.
Ratnakaran et al., (1985) justified that
the inability of larvae treated with
chitin synthesis inhibitors insect
growth regulators to feed on the
leaves could have been caused by the
displacement of the mandible and
labrum or the blockage of the gut.
Fogal(1977) also reported that the
incomplete clearance of the larval gut
at moult as well as the reduced
amount of chitin in the newly moulted
mouth parts could prevent the larvae
of Diprion similis from feeding after
ecdysis. The symptoms exhibited by
the treated RTW larvae were
consistent with symptoms reported
for some other species of insects such
as Lucilia cuprina, Manduca sexta and
Lymantria dispar treated with chitin
synthesis inhibitors (Abdel-Monem et
al. 1980; Kotze, 1992; Root and
Dauterman 1996)
Nagesh and Varma (1997), who
reported that the application of
lufenuron on diamond back moth
caused high percentage of mortality in
larvae compared with teflubenzuron.
Kim et al., (2000) have shown also that
lufenuron was highly effective (>80%
efficacy) against diamondback moth
larvae. Ivan et al., (2011) reported that
lufenuron showed high toxicity
against larvae of S. littoralis in
comparison with tebufenozide.
Lufenuron caused 100% mortality in
larvae that were fed with food
containing a high concentration of the
compound (0.01 ppm) (Ivan et al.,
2011). Within a 24 hour period from
the beginning of precocious molting,
the larvae developed elongated heads,
and stopped feeding. In this “sleeping
stage” the larvae died after 2–3 days
(Ivan et al., 2011). Lufenuron exhibited
more efficiency on both 2nd and 4th
larval instars of H. armigera in
Tariku Tesfaye et al. [18]
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
laboratory bio-assays in terms of
toxicity and speed of kill compared
with flufenoxuron and triflumuron
(Arnold et al., 2009). This study also
agreed with that of Abdel Rahman et
al., (2007) when they tested the direct
and latent effects of lufenuron and a
lufenuron mixture on the
development of S. littoralis larvae and
reported that lufenuron has toxic
effects on tested larval instars.
Based on this study, the comparative
effects of lufenuron and teflubenzuron
on the 3rd larval instar of RTW
indicated that lufenuron has the
potential to kill the larvae more
effectively than teflubenzuron.
Table 1. Cumulative Percent Mortality of 3rd Instar
Larvae of RTW when Treated with Lufenuron
and Teflubenzuron under Laboratory Condition
Treatments
Means(
+
SE)
Teflubenzuron @ 75g a.i.ha
-1
76.75
+
0.93d
Teflubenzuron @ 112.5 g a.i.ha
-1
81.02
+
3.24cd
Teflubenzuron @ 150 g a. i.ha
-1
88.43
+
0.46bc
Lufenuron @ 20 g a. i. ha
-1
84.26
+
4.63cd
Lufenuron @ 40 g a.i.ha
-1
96.27
+
3.7ab
Lufenuron @ 60
g a. i.ha
-1
100
+
0.00a
Endosulfan@700g a.i.ha
-1
(
standard check)
100
+
0.00a
Untreated check
13.33
+
3.33e
CV=5.82%
Means followed by the same letters are not significantly
different by Student Newman Keuls (SNK) test (P<0.001)
Table 2. Percent Mortality of 3rd Instar Larvae of RTW
Treated with Lufenuron and Teflubenzuron in
Greenhouse
Treatments
Means(
+
SE)
Teflubenzuron (112.5g a.i./ha)
36.25
+
1.25b
Lufenuron (40g a.i./ha)
91.25
+
1.25a
Endosulfan(700g a.i./ha)
92.5
+
1.45a
Untreated control
17.5
+
1.45c
CV=7.87% Means followed by the same letters are not
significantly different by Student Newman Keuls (SNK)
test (P<0.001)
Lethal dose determination
The results showed that lufenuron
was more effective than
teflubenzuron, as it had lower LD50
(9.88) and LD90 (24.79) values (Table
3). The relative potency values
indicated that lufenuron was more
effective than teflubenzuron with 1.21
and 12.15 times great potency against
3rd instar larvae of RTW at the LD50
and LD90 level respectively than
teflubenzuron.
El-sayed et al. 2011, reported that the
relative potency values indicated that
lufenuron was more effective than
flufenoxuron and triflumuron with 2.5
and 9.5 times great potency at the
LD50 level, respectively, and 3 and 5.8
times higher potency than
flufenoxuron and triflumuron at the
LD90 level, respectively.
In agreement with this study, the
comparative effects of lufenuron,
flufenoxuron and triflumuron on the
2nd and 4th larval instar of Spodoptera
littoralis indicate that lufenuron has
the potential to kill Spodoptera littoralis
larvae more efficiently than
flufenoxuron and triflumuron. And it
is also likely to be more efficient in the
field compared with the other tested
insecticides (El-Sayed et al.2011). The
efficiency of lufenuron, teflubenzuron
and flufenoxuron against third and
fifth instars of Spodoptera littoralis,
were also investigated by Bayoumi et
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
al. (1998) under laboratory conditions.
They showed that third instars are
more sensitive to lufenuron.
The present study indicated that the
lufenuron was more toxic than
teflubenzuron to 3rd instar of RTW
larvae. Therefore, it is recommended
to use the lower dose of lufenuron
than teflubenzuron to bring more
larval mortality of RTW.
Table 3. LD50 and LD90 of Teflubenzuron and Lufenuron against Larvae of RTW
Treatments
LD50 (95% CI) a
LD90(95% CI)
Relative potency
b
LD
50
LD
90
Teflubenzuron
11.95
301.19
1.21
12.15
Lufenuron
9.88(4.08
-
13.84)
24.79(20.14
-
30.09)
1
1
a LD50 or LD90 and 95% fiducial limits (CLs) are given in g of a.i.
b Relative potency is calculated as LD50 or LD90 of the tested IGRs/LD50 or LD90 of the most effective IGR
Lethal time determination
The median lethal time (LT50) values
of lufenuron, teflubenzuron and
endosulfan tested on the 3rd larval
instar of RTW are shown in Table 4.
The time required for 50% mortality
decreased with increasing dose in
both tested CSIs, however, there is no
dramatic changes from lufenuron 40
to 60g a.i./ha on 3rd larval instar
which were 3.55 and 3.38 days
respectively. Similarly, at high doses
of lufenuron (60g a.i/ha) and
teflubenzuron (150g a.i/ha)
approximate days (3.38 and 3.82
respectively) to kill 50% of the larvae
was observed; however, at their lower
doses, 20 and 75g a.i./ha respectively,
lufenuron caused 50% larval death
within 4.91 days where as
teflubenzuron caused within 5.37
days, which means that lufenuron is
more toxic when both are used at their
lower dose. Endosulfan caused 50%
death within not more than one day.
This indicated that it is more toxic to
3rd instar larvae of RTW.
The result indicated that lufenuron
exhibited more efficiency in killing
50% of 3rd instar larvae of RTW faster
than teflubenzuron at lower dose, but
slower than endosulfan at any doses.
At their individual high doses, they
showed almost similar toxicity to the
larvae within the days not more than
four. On the other hand the result of
medial lethal time indicated that
lufenuron is more toxic to 3rd instar
larvae of RTW, since it caused 50%
mortality at the dose less than half
(60g a.i./ha) of teflubenzuron (150g
a.i./ha) in the same days interval. This
data show that there is no need for
using very high concentrations of
lufenuron to get the pest controlled.
Generally, lufenuron is preferred than
teflubenzuron economically, because
it cause immediate 50% mortality at
lower dose.
Tariku Tesfaye et al. [20]
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
Table 4. LT50 and LT90 of Teflubenzuron and Lufenuron to 3rd Instar Larvae of
RTW under Laboratory Condition
Treatments
LT
50
(95%CL)
a
Teflubenzuron@75g a.i.ha
-1
5.37( 4.99
-
5.7
6)
Teflubenzuron@112.5g a.i.ha
-1
4.35 (3.95
-
4.76)
Teflubenzuron@150g a.i.ha
-1
3.82 (3.43
-
4.20)
Lufenuron @ 20 g a. i. ha
-1
4.91 (4.38
-
5.48)
Lufenuron @ 40 g a.i.ha
-1
3.55 (3.20
-
3.89)
Lufenuron @ 60 g a. i.ha
-1
3.38
(3.07
-
3.68)
Endosulfan@700g a.i.ha
-1
0.983374
a LT50 and 95% fiducial limits (CLs) are given in days
Potency of Teflubenzuron
and Lufenuron against egg
hatchablity of RTW
Effect of teflubenzuron and lufenuron
was examined (Table 5). Eggs of RTW
were observed unhatched when
treated with the CSIs and the standard
check. Shrinkage, death of 1st instar
larvae in the egg and partial hatch
(part of larvae were attached with the
body of the egg) were the symptoms
observed during the experiment
(Fig.1). On the contrary, normal 1st
instars larvae were hatched in the
untreated eggs. Sallam (1999) reported
that the developed embryos were
unabled to perforate the surrounding
vitelline membrane, it could be due to
a weakened chitinous mouth parts
that was insufficiently rigid to effect
hatching. Ovicidal activity of the
tested CSIs in the present study could
be due to the disturbance in cuticle
formation of the embryo. Ivan et al.,
(2011) also reported that reduced
hatchability resulted from numerous
changes occurring in the course of
embryonic development.
Table 5. Percent Unhatched Eggs of RTW when Treated
with Lufenuron and Teflubenzuron under
Laboratory Condition
Treatments
Means(
+
SE)
Teflubenzuron (112.5g a.i./ha)
36.25
+
1.25b
Lufenuron (4
0g a.i./ha)
91.25
+
1.25a
Endosulfan(700g a.i./ha)
92.5
+
1.45a
Untreated control
17.5
+
1.45c
CV=4.55%
Means followed by the same letters are not significantly
different by Student Newman Keuls (SNK) test (P<0.001)
From ANOA results, significant
differences (P<0.001) between
treatments in affecting the egg
hatchability were observed. However
all treatments were significantly
different from the untreated control,
there was no significant differences
between lufenuron (91.25%) and
endosulfan (92.5%) in inhibiting the
hatchability of the egg of RTW. On the
other hand, lufenuron is highly
significant difference from
teflubenzuron (36.25%) to affect egg
hatchability.
Reports from previous studies found
that the exposure of diamondback
moth eggs to different concentrations
of teflubenzuron led to significant
inhibition of egg hatching when
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
compared with other IGRs
(Karimzadeh et al.,2007 ; Hayens and
Smith, 1993;Perng et al., 1988) in
contrary, the present study indicated
that teflubenzuron was inferior to
lufenuron and endosulfan to inhibit
egg hatchability. Osman and
Mahmoud (2008) observed that 88.3%
reductions of cotton leafworm eggs 24
h after treatment with lufenuron when
compared with control. Sammour et
al. (2008) also reported 73.2%
reduction in egg hatchability of the
same insect. Therefore, the results
justified that eggs of RTW were highly
affected by lufenuron and endosulfan;
however, teflubenzuron is
alternatively preferable than
untreated control to inhibit the egg
hatchability of RTW.
Figure1. A, B and C are the Effect of Lufenuron, Teflubenzuron and Endosulfa, Respectively on Egg Hatchability of RTW
Conclusions
The comparative effectiveness of
lufenuron and teflubenzuron on 3rd
larval instar of RTW showed that
lufenuron was more effective than
teflubenzuron, as it has lower LD50
(9.88) and LD90 (24.79) values. The
relative potency values indicated that
lufenuron was more effective than
teflubenzuron with 1.21 and 12.15
times great potency against 3rd instar
A B
C
Tariku Tesfaye et al. [22]
Journal of Science and Sustainable Development (JSSD), 2013, 1(2) 13-24
larvae of RTW at the LD50 and LD90
level, respectively than teflubenzuron.
Lufenuron caused highly significant
egg hatchability inhibition of RTW.
Generally, the total efficiency for
laboratory and green house
experiments indicated that lufenuron
and teflubenzuron caused mortality of
RTW larvae and inhibited egg
hatchability. However, lufenuron
caused high mortality at lower dose. It
can therefore be concluded that,
because of its safety to environment
and other beneficial organisms,
lufenuron can be used at dose of 40g
a.i./ha for further study under open
and large field conditions for the
control of RTW.
Acknowledgements
The authors wish to thank and express
their heartfelt gratitude to Agrisher
Ethiopia PLC for their assistance for
providing the samples of Insect
Growth Regulators. Ambo Plant
Protection Research Center is also
highly acknowledged for proving the
necessary materials and place of
working.
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