5/20/2010

DELAYING THE DEVELOPMENT OF RESISTANCE

TO BT CORN IN THE POPULATION OF ASIAN
CORN BORER , OSTRINIA FURNACALIS

Project Leaders:

Belen Morallo-Rejesus, Ph. D.

(Nov. 2005 – Feb. 2007)

Pio A. Javier Ph. D.

(March 2007 – April 2008)

Project Duration
Date Started: November 2, 2005
Date Completed: April 30, 2008

Period covered by this report

November 1, 2005 – April 30, 2008

Project Funding
Amount approved for the three years= PhP 3,258,754.48

Amount released in first year = PhP 1,034,881.18

Amount approved for the second year = PhP 300,000.00

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INTRODUCTION

Approval of the commercialization of transgenic Bt-corn in

the Philippines in December 2002 introduces a new
technology to control ACB in an ecologically benign way.
 Transgenic crops significantly increase the efficacy of the
Cry toxins compared to Bt sprays, thus, are more
attractive to use than the conventional Bt formulations.

 Since the Cry protein engineered into corn is basically

the same as those found in natural Bt organisms, and
Bt toxins are expressed in high amounts throughout the
growing season, the risk of resistance development is
more likely to occur.

INTRODUCTION
 Several species of insects including ACB and the
European corn borer, were reported to developed
resistance to Bt corn.

 Baseline data on the susceptibility of ACB poplns

in some areas where BT corn were grown in the
country and the rate of resistance devt to Bt corn
of ACB poplns. from Bt corn growing areas, are
therefore important in designing proper
resistance management tactics adapted to
Philippine conditions.

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INTRODUCTION
Transgenic Bt plants typically provide continuous
protection against damage from susceptible insect
species, thereby, increasing the potential of insect
selection for resistance and the probability of negative
effects on natural enemies.

• Very few studies have been reported on the effect of Bt

corn plant on predators that were fed with Bt-fed preys.

General Objectives:
1. Survey the occurrence of resistant ACB poplns in
selected growing areas (Bt corn planted for 2 years)

2. Determine the rate of development of resistance to

Cry1Ab of ACB popl’ns in the laboratory

3. Determine the effect of Bt protein on biology of earwigs

fed with ACB larvae reared on artificial diet, ACB
treated with Bt-protein and dog food.
4. Determine the delaying effect of augmentative releases
of predatory earwigs on the increase of ACB population
resistant to Bt corn in the field.

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Study 1. Survey of Resistance Development and Rate

of Development of Resistance to Bt-corn in
Asian Corn Borer (ACB) Population
Study Leaders:
Dr. Barbara L. Caoili - Nov. 2005 – Oct. 2006
Dr. Pio A. Javier - Nov. 2006 – April 2008

Study 2. Effect of Bt Corn on the Biology of Predatory

Earwig, Euborellia annulipes and on the
Development of Resistant Asian Corn Borer
Population
Study Leaders:
Dr. Belen Morallo-Rejesus - Nov. 2005 – Feb. 2007

Dr. Pio A. Javier - March 2007 – April 2008

Study 1. Survey of Resistance Devt and Rate of Devt of

Resistance to Bt corn in ACB Population.
STUDY OBJECTIVES
% Accom-
Objectives: plishment

1. Determine the susceptibility of ACB

popln from Bt corn growing areas to 75
Cry1-Ab and establish the LC99 for
monitoring resistance.
2. Determine the rate of devt of resistance
of ACB popln from Bt corn growing 10
areas by selection in the laboratory.

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METHODOLOGY:

1. Determined planting history in each site - interview

2. Collected ACB larvae & pupae (250 from each site):
 Bt-corn areas:
- Alcala, and Santa Maria (Pangasinan)
- Candon City (Ilocos Sur)

 Non-Bt corn areas:

- San Felix (Sto. Tomas, Batangas)
- BIOTECH and CES, UPLB (College, Laguna)
- Tranca (Bay, Laguna)
- Bunggo (Calamba City)
- Sto Tomas (Calauan, Laguna)

3. ACB reared in artificial diet

4. Larvae used for bioassay/maintaining culture.

METHODOLOGY……………….
Bioassay studies using lyophilized non-Bt and
Bt-corn leaves

While sourcing purified Cry1Ab protein from other

institution, lyophilized Bt-CL (25-day old ) were used.

Lyophilization of Bt and non-Bt leaves done at

BIOTECH, UPLB.

Cry1Ab protein concentration (freeze-dried sample)

estimated through ELISA , at BIOTECH, U.P. Los
Baños. Cry1Ab antibody and ELISA reagents by Drs. V.
Exconde and T. M. Espino.

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METHODOLOGY………..

Estimation LC values for Cry1Ab on ACB neonates

 Stock solution 1 mg/ml Cry1Ab prepared in 0.1M CAPS

buffer, pH 11.2, stored in a -20ºC freezer until use.
 Diet-incorporation bioassay
 Initial test used 4 to 5 concentrations diluted into 18 μL
artificial diet.
 Control group given an artificial diet with 0.1M CAPS buffer.
 Treatments replicated 3X; 10 larvae/rep.
 Mortality (moribund & dead) observed after 7 days.
 Data analyzed with PriProbit ver. 1.63 program.

Accomplishment & Major Findings - Study 1

 ACB larvae reared in the laboratory for several

generations were tested on lyophilized Bt corn leaf,
exposed on a detached Bt corn leaf in the laboratory and
on the Bt-corn potted plants in the screen house.

 Use of powdered lyophilized Bt-corn leaves (laborious)

may offer an alternative method in the estimation of LC
values in the event that purified Cry1Ab protein is
unavailable.

The Cry1Ab content in the powdered lyophilized Bt-corn

leaves was estimated to be 1.25 ng/mg leaves by ELISA.

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Accomplishment and Major Findings- Study 1

Table 1. Percent mortality of ACB neonates treated with Cry1Ab protein.

CONC’N %
POPULATION
(ng/uL) MORTALITY
San Felix, Sto Tomas, Batangas 0.10- 2.5 17.20ab
Tranca, Bay, Laguna 0.10- 2.5 14.70c
Bungo, Calamba, Laguna 0.10- 2.5 16.88bc
Sto Tomas, Calauan, Laguna 0.10- 2.5 18.80ab
Pili Drive, CES, UPLB 0.10- 2.5 19.50a
BIOTECH, UPLB 0.10- 2.5 19.60a
 Purified Cry1Ab protein obtained from Dr. William Moar, Professor,
Auburn University, Alabama, USA.
 All populations of ACB neonates showed comparable susceptibility
against Cry1Ab toxin except Tranca, population.

Accomplishment and Major Findings- Study 1

Table 2. Percent weight reduction on retarded growth of ACB neonates
treated with Cry1Ab protein1,2.

POPULATION CONC’N % WEIGHT

(ng/uL) REDUCTION1,2
San Felix, Sto Tomas, Batangas 0.10- 2.5 85.88a
Tranca, Bay, Laguna 0.10- 2.5 82.56ab
Bungo, Calamba, Laguna 0.10- 2.5 72.28c
Sto Tomas, Calauan, Laguna 0.10- 2.5 77bc
Pili Drive, CES, UPLB 0.10- 2.5 82.72ab
 Significantly highest weight
reduction in Sto Tomas, A- Retarded larva.
B- normal larva
Batangas, and lowest
in Calamba & Calauan Lag.
1Weight reduction was based only on larvae
treated with 0.10 to 1.0 ng/uL (amount on Bt-corn leaves)
A B
2All larvae are dead at 2.5 ng/uL treatment.

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Table 2. Susceptibility of the ACB neonates collected from non-Bt corn

field to Cry1Ab protein by surface contamination method.
LC(ng/cm2)
POPULATION Fiducial Fiducial Fiducial
LC25 LC50 LC99
Limits Limits Limits
San Felix, Sto Tomas, 0.034 0.004 - 0.084 0.138 0.044-0.238 6.191 3.045-29.75
Batangas
Tranca, Bay, Laguna 0.089 0.019 – 0.174 0.356 0.189-0.569 41.33 9.86 – 1,684

Bungo, Calamba, Lag. 0.060 0.012 – 0.122 0.216 0.099 – 0.339 17.46 5.35 - 321

Sto Tomas, Calauan, Lag 0.059 0.005 – 0.147 0.216 0.058 – 0.385 19.39 6.43 – 377.92

Pili Drive, CES, UPLB 0.020 0.002 – 0.057 0.091 0.021 – 0.177 17.28 5.28 – 310.33

BIOTECH, UPLB 0.124 0.038 – 0.216 0.298 0.152 – 0.436 6.191 3.045 – 29.75

1Based on five trials; 30 insects per replicate, replicated 3X (n=150)

2Observation was done after 7 days.
The lower the LC values the more susceptible.
The higher the LC values the more tolerant

Table 3. Susceptibility of the ACB neonates collected from Bt corn growing

areas to Cry1Ab protein by surface contamination method.
MEAN MORTALITY LC RESIS-
POPULATION TANCE
Treated Treated
LC50 LC99 RATIO
with LC50 with LC99
Asingan, Pangasinan 14.20 26.20 0.258 7.08 1.145
Sta Barbara, Pangasinan 19.2 28 0.180 6.63 1.071
Sta Maria, Pangasinan 15.6 27.6 0.35 6.73 1.07

LC50 = 0.216 ng/cm2 LC99 = 6.191 ng/cm2

 Based on resistance ratio,
all populations collected
Resistance ratio = __LC99 population_______
from three sites in
LC99 susceptible population Pangasinan are still
susceptible to Cry1Ab
RR = 1.0 – 1.9 = susceptible toxin.
= 2.0 - 9.9 = tolerant
= 10 - 20 = moderately resistant
> 20 = resistant

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Study 2. Effect of Bt corn on the Biology of

Predatory Earwig, Euborellia annulipes
and on the Development of Resistant
Asian Corn Borer Population

Study Leaders:

Dr. Belen Morallo- Rejesus -

Pio A. Javier -

Project Staff:
Evangeline G. Punzalan - University Res. Associate I:
Jonathan Dechitan - Laborer

Objectives % Accom-
Specific Study Objectives plishment
1. Determine the effect of feeding ACB larvae with Bt corn leaf, Bt 100
corn leaf extracts and Bt corn potted plants.
2. Determine the LD50 & LD99 of Cry1AB on 3rd instar ACB larvae 100
3. Determine the effect of feeding on tassel w/ pollen & decaying Bt
corn leaves/organic matter (additional objectives after the 100
inception meeting).
4. Determine predatory consumption of earwig feed w/ ACB larvae
reared on artificial diet only & those fed w/ ACB larvae imbibed 100
w/ Bt-protein & from those fed w/ dog food-corn cob diet.
5. Determine the effect of integrating earwigs & Bt corn in delaying
development of resistant ACB popln (budget was not released). 30
6. To determine popl’n of natural enemies in Bt corn plants &
ordinary hybrid corn with & without earwig releases. 30
7. Determine effect of Cry1Ab protein on the life cycle, fecundity,
egg viability, survival, sex ratio & longevity of earwigs fed w/
100
ACB larvae reared on artificial diet, fed w/ ACB larvae imbibed
w/ Bt-protein and from those feed with dog food-corn cob diet .

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METHODOLOGY-STUDY 2
Effect of feeding ACB larvae with Bt- CL and Bt corn leaf extracts
(Bt-LE)
 ACB reared on artificial diet (Ceballo); predatory earwigs on dog
food-corn cob combination (Punzalan & Moralllo-Rejesus, 2004).

 Indirectly, portion of corn leaf (MON 818) that contained the

highest Bt conc’n was determined by allowing the 3rd instar
larvae to feed on the detached lower, middle and tip portion.
 Third instar larva individually imbibed with the Bt-LE from the
lower, middle and tip portion by droplet method. Tests replicated
3X, 10 larvae/rep, mortality recorded at 24 and 48 hours.
 Another expt., five larval instars of ACB were allowed to feed
separately on detached Bt-CL (on top of moistened tissue) placed
inside a pan. Test replicated 3X , 10 larvae/rep. Mortality noted at
24, 48, 72, 96 and 120 hrs.

METHODOLOGY- STUDY 2

Screen house

Neonate 2nd and 3rd instar

larvae were allowed to feed
on Bt corn plant enclosed
in Mylar cage (Fig. 2).

Larvae were placed on the

leaf.

Mortality and behavior of

larvae noted at 12 and 48
Fig. 2. Set-up on the feeding of ACB
hrs, thereafter every 24 hrs larvae on Bt corn plants.
for 3 days, a total of 7 days
observation.

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METHODOLOGY – STUDY 2

Predatory capacity
 Four nymphal instars of earwigs starved for 48 hrs were
introduced with different ACB larval instars 24, 48, and
72 hours.

 One earwig released/acrylic pan and fed with varying

prey densities of 150 neonates, 100 2nd instar, 60, 40,
and 20 for the 3rd, 4th, 5th instars ACB larvae, respectively.
Same number of prey was given every six hours.

 Predatory consumption of the female earwigs were

separately done from the males, expt. replicated 3X.

 Number of preys killed/consumed was recorded at 6, 12,

18, 24, 48 and 72 hours.

METHODOLOGY – STUDY 2
Insect rearing
Mass rearing of ACB and Predatory earwigs, Euborellia annullipes
Treatments:
Prey A = earwigs fed with ACB larvae imbibed with LD25 (300 ng)
Prey B = earwigs fed with ACB larvae imbibed with LD50 (600 ng)
Prey C = earwigs fed w/ ACB larvae imbibed w/ pure Bt corn leaf extracts

Prey D = earwigs fed with ACB larvae imbibed 1,250ng (amount of Bt

protein expressed in Bt corn leaf obtained by ELISA)
Prey E = earwigs fed with untreated ACB larvae (control)
Prey F = earwigs fed with DFC diet (control)

P1 earwigs were reared until adult stage using artificial diet (dog food &
corn cob combination) & soil MC was maintained at 27 to 30%.

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METHODOLOGY – STUDY 2
 Bt & non- Bt corn (IPB 911) tassel & leaves
were collected from pots planted with corn
in the screen house.
 Materials were wet, stored in a covered
plastic container for 15 days.
 200 g each were placed inside an acrylic A
pan & one pair of 3rd nymphal instar
earwig was released & allowed to feed
on it .
 20 containers/replicate, replicated 3X (60
pairs/medium)
 Artificial diet served as the control. B

 % survivors computed and compared Fig 3. A- Bt corn tassel with

from the control. pollen & decaying Bt leaves.

METHODOLOGY – STUDY 2

Fig. 4. Different varieties of corn planted at CES, UPLB.

 Field expt. was set-up July – Oct. 2006 in an area of 5,000 sq. m. at
CES, divided into 9 blocks with 6 m. border between
each block.
 Mon 818 (Bt corn), IPB 911 and IPB supersweet (Asukar) were
planted/block at a planting distance of 75 cm between rows
and 50 cm between hills.
 Expt. was arranged RCBD with three reps ( 250 sq. m/ block),
with 1,500 plants.

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METHODOLOGY – STUDY 2
Earwigs and other NE’s monitored at
pre-vegetative, whorl & tassel stages
by putting traps and by sweepings B

Initial collection done 5 days after A

setting up the traps, thereafter every A B

day for 7 days.
Collected earwigs/block were
brought to the lab, fed w/ dog food-
corncob diet & then returned in each
block from where it was collected &
were deducted from the 250 earwigs
that were released/ block.
Earwigs collected at harvest/ block
were pooled according to variety &
brought to the laboratory for the
determination of their biological
differences.
D
C
Fig. 5. Trapping of predatory earwigs:
A- plastic container containing dog food
& surrounded w/ corn cob,
B- covered with corn husk,
C- collection of earwig in the corn husk,
D- collection of earwig in the plastic
container).

ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2

Table 4. The lethal time (LT50) that caused 50% mortality of the third instar
ACB larvae fed w/ the different portions Bt corn leaf & imbibed
with its leaf extract1.

LT50 (Hours)
Parts of the leaf
Imbibed (pure) Feed
Lower 48.95 80.35
Middle 31.27 63.42
Tip 11.68 23.12
1Ten insects per replicate; replicated 3X (n=90)

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ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2

Table 5. The lethal time (LT50) that caused death of different ACB larval
instars fed with whole Bt –corn leaf kept inside an acrylic pan.

Larval instar LT50 (Hours)

Neonate (1st instar < 24 hr) 20.35
2nd instar 50.87
3rd instar 85.08
4th instar 191.27
5th instar 260.31

1Ten larvae per replicate; replicated 3X (n=90)

ACCOMPLISHMENT AND MAJOR FINDINGS - STUDY 2

200

1st instar nymph

150
Average Number of Preys Consumed by the Earwig

2nd instar nymph

3rd instar nymph

4th instar nymph

100

Adult Male

Adult Female
50
0

6h 12h 18h 24h 6h 12h 18h 24h 6h 12h 18h 24h 6h 12h 18h 24h 6h 12h 18h 24h 6h 12h 18h 24h

eggmass 1st instar 2nd instar 3rd instar 4th instar 5th instar
-50

Stages of Preys (ACB)

Fig. 6. Average number of the different stages of ACB consumed by the different stages of
predatory earwigs in 24 hour.

All the nymphal instars & adult earwigs prefer to fed on early instar prey
especially neonates
Due to the bigger size of 4th & 5th instar ACB larval preys, adult earwigs have
difficulty in paralyzing the larvae through their forceps.
Some of the adult earwigs that persist on preying had their notal membrane
split, consequently resulting to their death.

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ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2

120 Bt corn (MON 818) 120 Bt corn (MON 818)
Non-Bt corn (IPB 911) Non-Bt corn (IPB 911)
100 Artificial Diet (Dog Food-Corn Cob mixture) 100 Artificial Diet (Dog Food-Corn Cob mixture)
Percent Survivor

Percent Survivor
80 80

60 60

40 40

20 20

0 0
3 6 9 12 15 18 21 24 27 30 3 6 9 12 15 18 21 24 27 30
Days of Counting Days of Counting

Fig. 7. % earwig survivor when fed with A – Bt corn tassel & B –Bt corn decaying eaves.

 No difference in % earwig survivor when the predator was fed with both Bt &
non-Bt corn tassel w/ pollen & decaying leaves for 30 days (Fig. 7).
 Tassel, pollen and decaying Bt corn leaves were not detrimental to the
earwigs indicating that the continuous planting of Bt corn will not reduce the
popln of predatory earwig in the field.

ACCOMPLISHMENT & MAJOR FINDINGS - STUDY 2

Predatory earwigs fed with Prey A, B, C, & D had

an average of three egg batches per female with
80 to 90 eggs per batch, which is comparable with
Prey E (control) (Table 7).

 No differences on the life cycle of earwigs was

observed when the predator was fed with
Cry1Ab-treated and -untreated ACB larvae but
feeding them with Prey F showed a slightly l
longer life span ( Table 8).

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ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2

Table 7. Fecundity and percent survival of predatory earwigs fed with Cry1Ab-
treated third instar ACB larvae.
PREYS
Parameters
A B C D E F
No. of egg batches laid
per female 3 3 3 3 3 4

Ave. no. of eggs laid

90 85 80 84 85 110
per batch
Percent hatchability 100 100 100 100 100 100
Percent survival
85 84 78 82 84 100
1st to 2nd instar
Percent survival
81 80 88 85 87 97
2nd to 3rd instar
Percent survival
81 82 84 88 87 92
3rd to 4th instar
Percent survival
4th instar to adult 78 78 71 73 78 95

Sex Ratio F:M 3:2 3:1 3:2 3:1 3:1 3:2

ACCOMPLISHMENT AND MAJOR FINDINGS - STUDY 2

Table 8. Comparative life cycle1 (days) of predatory earwigs fed with Cry1Ab
treated 3rd instar ACB larvae.
DIETS
Stages/Period
A B C D E F
Eggs
7 7 7 7 7 8
Incubation
Nymphal Stages 7 7 7 7 7 5
First Instar 7 7 7 7 7 7
Second Instar 9 9 9 9 9 8
Third Instar 8 8 8 8 8 8
Fourth Instar
Total 31 31 31 31 31 28

Adult emergence to first

10 10 10 10 10 10
egg laying
Adult longevity 63 65 63 65 65 72
Egg 101 103 101 103 103 108

1Based on 90 females (5 trials); Prey A - ACB larvae imbibed with LD25 (300 ng); Prey B - ACB larvae
imbibed with LD50 (600 ng); Prey C - ACB larvae imbibed with pure Bt corn leaf extracts ; Prey D - ACB
larvae imbibed 1,250 ng (amount of Bt protein expressed in Bt corn leaf obtained by ELISA) ; Prey E -
untreated ACB larvae (control); Prey F – Dog food –corn cob combination diet (control)o death of an adult

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 Abundance of earwig in Bt corn MON 818 was comparable to

hybrid and Asukar variety (Table 8).

Table 8. Mean number of predatory earwigs collected from the trap 1

installedd in the cornfield (July – October2006; CES, UPLB).

VARIETY EARLY WHORL LATE WHORL TASSEL STAGE

(30-35 DAP) (36-40 DAP) (46 – 50 DAP)

Bt corn (MON 818) 20.66 3.33 2

Hybrid corn (IPB-911) 27.67 3.67 1

Green corn (Asukar) 23.67 3.67 0.67

1Based on 50 plants/replication; 25 plants in X-pattern were examined.

ACCOMPLISHMENT AND MAJOR FINDINGS - STUDY 2

Bt corn was totally free from ACB damage throughout the duration of
the expt (32 to 60 DAP) while slight damage was noted in IPB 911.

In contrast, 16.67% damage was recorded in supersweet corn at 32

DAP andgradually increased towards maturity. An ETL of 25% was
reached at 53 DAP and slightly increased at 32 DAP.

Table 9. Mean percent Ostrinia furnacalis infestations on three corn varieties

(July – October 2006; CES, UPLB).

VARIETY/ PERCENT DAMAGED PLANT1

HYBRID 32 DAP 39 46 DAP 53 DAP 60 DAP
DAP
Bt corn (MON 818) 0.00 0.00 0.00 0.00 0.00
Hybrid (IPB 911) 8.00 13.33 13.33 16.00 16.00
Supersweet corn 16.67 20.67 23.33 26.67 31.33
(Asukar)

1Based on 50 plants/replication; 25 plants in X-pattern were examined.

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SUMMARY AND CONCLUSION

 The Cry1ab content of the powdered lyophilized Bt corn leaves was

estimated to be 1.25 ng/mg leaves by ELISA.

 Collected ACB populations from non-Bt corn growing areas, Calauan

and Calamba populations were the most susceptible while Tranca was
the least.

 The established baseline concentration (LC50) was 0.216 ng/cm2 while

the established discriminating concentrations (LC99) was 6.191 ng/cm2.

 Based on resistance ratio, all populations collected from three sites in

Pangasinan are still susceptible to Cry1Ab, although these sites are
near the Monsanto Bt corn planting area.

Based on the life cycle, percent survival and fecundity, earwigs preyed
with Cry1Ab-treated and –untreated ACB larvae showed no differences.
In effect, Cry1Ab is not detrimental to earwigs.

RECOMMENDATION

 Yearlymonitoring on the resistance development of ACB

from Bt corn growing areas.

 Intensive study on the determination of the rate of

development of resistance of ACB population from Bt corn
growing areas should be undertaken.

 More field experiments should be conducted to determine

the effect of integrating earwigs and Bt corn in delaying the
development of resistant ACB population.

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ACKNOWLEDGEMENT

The authors gratefully acknowledge the Department

of Agriculture-Bureau of Agricultural Research (DA-
BAR) for funding the project.

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