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:

63

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.

64

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

65

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.

66

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

)

Table 2 : Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in Setapak, Kuala Lumpur ( Block H – WETTABLE

POWDER )

PERCENTAGES OF LARVAL MORTALITY

1st Treatment 2nd Treatment 3rd Treatment 4th Treatment 5th Treatment 6th treatment 7th treatment 8th Treatment

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 hours

1 week

24 hours

1 week

24 hours

1 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

PERCENTAGES OF LARVAL MORTALITY

1st Treatment 2nd Treatment 3rd Treatment 4th Treatment 5th Treatment 6th treatment 7th treatment 8th Treatment

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 hours

1 week

24 hours

1 week

24 hours

1 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

67

Table 3 : Field Trials Using TMOF-Bti Formulations against Aedes aegypti at High-Rise Flats in Setapak, Kuala Lumpur ( Block H – RICE

HUSK )

PERCENTAGES OF LARVAL MORTALITY

1st Treatment

2nd Treatment

3rd Treatment

4th Treatment

5th Treatment 6th treatment 7th treatment

8th Treatment

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 Hours

1 Week

24 hours

1 week

24 hours

1 week

24 hours

1 week

BLOCK F ( 1st trial ) 100 100 100 100

98.1

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

68

i) Figure 1 Natural Population of Aedes mosquito in PPR Air Panas, Setapak (1st

evaluation)

0

200

400

600

800

1000

1200

1400

1600

1st week

3rd week

5th week

7th week

9th week

11th week

13th week

15th week

17th week

19th week

21th week

23th week

25th week

27th week

29th week

Block A (Control) Block H (Wettable powder) Block F (Rice husk)

No. of eggs

69

ii) Figure 2 Natural Population of Aedes mosquito in PPR Air Panas, Setapak

(2nd evaluation)

No of eggs

70

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 )

71

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

72

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.