1. END OF PROJECT REPORT
NN-001-2008
Evaluation of Proprietary Formulations of Insect Repellent Containing
EGX-101 and Testing Mosquito Larvicide products Based on Trypsin
Modulating Oostatic Factor (TMOF) Against Dengue Vectors
PROJECT LEADER: PROF DR SALLEHUDIN BIN SULAIMAN
PROJECT TEAM MEMBERS: PROF MADYA DR HIDAYATULFATHI OTHMAN
NORASHIQIN BINTI MISNI
MOHD SAIFUL AZLAN BIN NORDIN
LAU MING SEONG
SITI FATIMAH JOHARRY
Project Duration: 24 (months)
Project Start Date: November (month) 2008 (year)
Project End Date: November (month) 2010 (year)
Collaberations:
1

2. EVALUATION OF TMOF-BTI FORMULATIONS AGAINST Aedes aegypti Linnaeus AT
HIGH RISE FLATS IN KUALA LUMPUR, MALAYSIA
By Saiful Azlan Bin Nordin, Biomedical Sciences, FSKB, UKM
Supervised by Prof. Dr. Sallehudin Sulaiman & PM. Dr. Hidayatulfathi Othman
5th November 2010
INTRODUCTION
The incidence of dengue fever and dengue haemorrhagic fever has increased
dramatically over the last decades. It has become endemic in more than 100 countries and
more than 2.5 billion people are at risk mainly in Africa, the Americas, the Western
Mediterranean, South and South and East Asia and the West Pacific(WHO 2008). Hence, some
control activities should be taken in order to reduce the dengue incidence. Larvicides which kill
mosquito larvae include biological insecticides such as Bacillus thuringiensis israelensis (Bti), a
microbial larvicide. Bti is a naturally occurring soil bacterium registered for mosquito larvae
control which is commonly used as larvicidal agent. Trypsin modulating oostatic factor (TMOF),
a peptide hormone originally isolated from the ovaries of adult Aedes aegypti, is currently under
commercial development as a new pesticide with a novel mode of action for the control of larval
mosquitoes. This hormone stops mosquitoes from producing a crucial digestive enzyme called
trypsin, preventing them to draw nutrients from food and therefore causing them to starve to
death.
METHODOLOGY
Field Trial at High Rise Flats in PPR Kampung Baru Air Panas, Setapak
Two formulations of TMOF-Bti were evaluated against first instar larvae of Aedes aegypti
at high-rise flats in Kuala Lumpur viz: wettable powder and rice husk formulations. Both
formulations were supplied by EntoGeneX. Three blocks of 17 storey high-rise consisted of 136
units of flats in Setapak, namely Block A, H and F in PPR Kampung Baru Air Panas were
selected in order to test the efficiency of TMOF-Bti against larvae of Aedes aegypti in the field.
Each block was located at least 500 m apart. Block H was treated with TMOF-Bti in wettable
powder formulation sprayed by ULV. Block F was treated with TMOF-Bti in rice husk formulation
distributed manually. While Block A acted as control.
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3. Six weeks before the field trial, ovitrap survey was conducted to obtain the baseline
information of Aedes aegypti ovipositions. 36 ovitraps per high-rise flats were distributed on
the 1, 3, 5, 7, 9, 11, 13, 15, 17 levels, with four ovitraps on each floor. These ovitraps were
placed at the corridor and inside of each of the flats. Ovitraps were replaced weekly. Paddles of
the ovitraps were taken to laboratory and eggs were counted. The larvae that hatched from
eggs were identified and counted. The first block which was block H was treated with WP
TMOF-Bti and sprayed using ULV. TMOF-Bti in wettable powder formulation was dissolved in 3
liters of water and sprayed at 0.01 g a.i/m2 (30 g / 3000 m2) using LECO/Model 1600 cold
aerosol generator mounted on the vehicle was used for spraying. The head nozzle of the
sprayer was adjusted at an angle of 45° to the ground. The flow rate of ULV application was
estimated at 104 ml/min and velocity of the vehicle approximately 6 km/ hr. Spraying was
conducted in the evening at 1700 hr. The second block , block F was treated with TMOF-Bti in
rice husk formulation distributed manually (50 mg) into 4 liters containers. The third block,
block A was sprayed with 3 liters of water only as the control. All the windows and doors
remained open during ULV spraying.
Each of the three blocks was distributed with 36 buckets (filled with 4 liters of water for
each bucket) each containing 20 1st instar Ae. aegypti larvae . Four buckets were distributed on
each floor at levels 1, 3, 5, 7, 9, 11, 13, 15, 17 of each block. These buckets were placed at the
corridor and inside each of the pre-determined flats. Crushed dry leaf powder were supplied as
source of food for the larvae. All larvae in buckets at the control block and treated blocks with
wettable powder and rice husk were counted after 24 hours of spraying for mortality. The
buckets were covered by mosquito netting. (* in case of any dengue cases occuring and
required fogging in that area, buckets should be covered by lids provided during fogging
operation.). A new batch of 20 1st instar larvae were introduced into the buckets in every two
weeks. Larval mortality in the buckets were recorded after 24 hours and weekly after spraying.
Treatments were repeated in every two weeks. At the same time, 36 ovitraps with paddles were
placed besides the buckets to monitor field population. Ovitraps were replaced weekly. Paddles
of the ovitraps were taken to laboratory for further eggs counting.
For the second trial which had started on 14th July 2010 until now at the high rise flats
at Setapak, the first block, was sprayed with WP TMOF-Bti using ULV. TMOF-Bti in wettable
powder formulation was dissolved in 3 liters of water and sprayed at 0.01 g a.i/m2 (60 g / 3000
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4. m2) using LECO/Model 1600 cold aerosol generator mounted on the vehicle .The concentration
used for spraying was twice higher than the first trial.
Block 2 was treated with TMOF-Bti in rice husk formulation and distributed manually (25
mg) into 4 liters containers which had been reduced halved in weight from the first evaluation .
The third block was sprayed with 3 liters of water only as the control. All the windows and
doors remained open during ULV spraying. Larval mortalities and eggs ovipositions in ovitraps
were counted.
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5. RESULTS
1) Field trial at high rise flats in PPR Kampung Baru Air Panas, Setapak
Table 1: Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in Setapak, Kuala Lumpur ( Block A – CONTROL
)
PERCENTAGES 1st Treatment 2nd Treatment 3rd Treatment 4th Treatment 5th Treatment 6th treatment 7th treatment 8th Treatment
OF LARVAL 24 1 24 1 24 1 24 1 24 1 24 1 24 1 24 1
MORTALITY Hours Week Hours Week Hours Week Hours Week Hours Week hours week hours week hours week
BLOCK A ( 1st
trial ) 56.94 87.22 24.72 33.33 40.56 49.44 50.55 70.14 47.92 57.36 38.89 42.64 31.25 45.56 34.31 47.08
BLOCK A ( 2nd
trial ) 24.52 33.61 21.25 27.63 40.69 47.5 26.39 40.69 10.2 36.11 11.38 42.36 6.67 34.31 10.67 51.21
Table 2 : Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in Setapak, Kuala Lumpur ( Block H – WETTABLE
POWDER )
1st Treatment 2nd Treatment 3rd Treatment 4th Treatment 5th Treatment 6th treatment 7th treatment 8th Treatment
PERCENTAGES 24
OF LARVAL Hour 1 24 1 24 1 24 1 24 1 24 1 24 1 24 1
MORTALITY s Week Hours Week Hours Week Hours Week Hours Week hours week hours week hours week
BLOCK H ( 1st
trial ) 64.77 75.83 31.67 55 33.66 57.08 31.67 52.22 31.67 78.47 44.44 74.03 45.42 90.69 54.72 80.69
BLOCK H ( 2nd
trial ) 50.83 78.1 73.89 83.08 56.57 81.94 54.08 65.4 50 73.61 54.36 75.42 49.43 72.28 50 73.5
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6. Table 3 : Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in Setapak, Kuala Lumpur ( Block H – RICE
HUSK )
1st 2nd 3rd 4th 5th 8th
Treatment Treatment Treatment Treatment Treatment 6th treatment 7th treatment Treatment
24 1 24 1 24 1 24 1 24 1 24
PERCENTAGES OF Hour Wee Hour Wee Hour Wee Hour Wee Hour Wee 24 1 24 1 hour 1
LARVAL MORTALITY s k s k s k s k s k hours week hours week s week
98.1
BLOCK F ( 1st trial ) 100 100 100 100 9 100 100 100 100 100 99.72 100 100 100 100 100
BLOCK F ( 2nd trial) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
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7. i) Figure 1 Natural Population of Aedes mosquito in PPR Air Panas, Setapak (1st
evaluation)
No. of eggs
1600
1400
1200
1000
800
600
400
200
0
we k
9 th e k
th e k
dw k
5 th e e k
th e k
th k
th e k
th e k
th k
th e k
th e k
th k
th e k
ek
7 th e e
3r ee
15 w ee
21 w ee
27 w ee
11 w e
13 w e
17 w e
19 w e
23 w e
25 w e
29 w e
we
w
tw
1s
Block A (Control) Block H (Wettable powder) Block F (Rice husk)
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8. ii) Figure 2 Natural Population of Aedes mosquito in PPR Air Panas, Setapak
(2nd evaluation)
No of eggs
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9. Figure 3: Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in
Setapak, Kuala Lumpur ( Block A – CONTROL )
Figure 4 : Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in
Setapak, Kuala Lumpur ( Block H – WETTABLE POWDER )
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10. Figure 5 : Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in
Setapak, Kuala Lumpur ( Block F – RICE HUSK )
Discussion
From the graphs of Figures 1 and 2, showing the natural population of the eggs of
Aedes aegypti on the first and second trials. The data indicated that there were
difference between both trials.The second trial recorded that a lower number of eggs
oviposited compared to the first trial This was due to the effectiveness of the TMOF-Bti
formulation used in this study to control the population of Ae. aegypti.
Figure 3, the graph shows the data at the control block of the experiment from
the first and second trials. The percentage of larval mortality at both trials shows a
difference whereby the percentage of larval mortality of the second trial was much
lower compared to that of the first trial..
At block H ( Figure 4 ), showa a different between the first trial to that of the
second trial. The percentage of larval mortality for the first four treatments of the
second trial was much more higher. However, the percentage of both trials became
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11. similar on the following weeks. This mighr be due to the effect of the weather in the
field ,either it was calm, windy or raining. Furthermore, the concentration of wettable
powder used were twice much more higher compared to the first trial which causing
the percentage of larval mortality was much more higher on the second trial.
Figure 5 shows the result of the efficacy of rice husk formulation in block F. The
second trial was much better than the first trial whereby the percentage of larval
mortality was 100 % in all treatments throughout the study period. Although the
concentration of rice husk used decreased from 50gm for 4 litre of water to 25 gm, the
formulation still killed all larvae effectively.
CONCLUSION
From the study which had been conducted in the field, we can strongly conclude that
TMOF – Bti in rice husk formulation which was distributed directly was much more
effective compared to the TMOF- Bti in wettable powder formulation sprayed using ULV.
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