Available online at www.sciencedirect.

com

Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76

AcE-Bs 2011 Bandung

ASEAN Conference on Environment-Behaviour Studies, Savoy Homann Bidakara

Bandung Hotel, Bandung, Indonesia, 15-17 June 2011

Aedes Mosquitoes Resistance in Urban Community Setting

Farah Ayuni Shafie*, Mohd Pozi Mohd Tahir & Nor Muniza Sabri
Environmental Health Department, Faculty of Health Sciences, Universiti Teknologi MARA, 42300 Puncak Alam, Malaysia

Abstract

In response to high incidence of Dengue cases in the less affordable settlements of Malaysia, the susceptibility of
Aedes mosquitoes should be assessed to identify the 50% knockdown time (KT50) of adult Aedes mosquitoes to
malathion. Wild strain mosquitoes from Shah Alam has shown potential to insecticide resistance with the resistance
ratio of 2.2 with 95% mortality. In contrast, wild strain from Pantai Dalam may have developed insecticide resistance
with the resistance ratio of 2.4 with 78% mortality. Health education and community should be propagated to
promote healthy environment by developing positive attitude and practice towards Dengue prevention.

© 2012
© 2011Published
Publishedbyby Elsevier
Elsevier B.V.Ltd. Selection
Selection andpeer-review
and/or peer-review under
under responsibility
responsibility of Centre
of Centre for Environment-
for Environment-Behaviour
Studies(cE-Bs), Faculty
Behaviour Studies of Architecture,
(cE-Bs), Planning & Surveying,
Faculty of Architecture, Planning &Universiti
Surveying,Teknologi MARA,
Universiti Malaysia
Teknologi MARA, Malaysia
Open access under CC BY-NC-ND license.
Keywords: Aedes; insecticide resistance; health risks

1. Introduction

In Malaysia, dengue fever was first reported in 1902 and is now one of the major public health threats
in Malaysia, especially with the emergence of dengue haemorrhagic fever (DHF) and the subsequent
dengue shock syndrome. Since there is no vaccine for preventing dengue, the World Health Association
(WHO) tries to eliminate the vector for the past 30 years by controlling Aedes mosquitoes. The dengue
fever arise due to higher densities os Aedes mosquitoes arising from the various sources of breeding
places. The usual breeding places are the roof gutters, flower pots, flower pot plates and roadside drains.

*
Corresponding author. Tel.: +603-3258-4450; fax: +603-3258-4599.
E-mail address: farahayuni@salam.uitm.edu.my

1877-0428 © 2012 Published by Elsevier B.V. Selection and/or peer-review under responsibility of Centre for Environment-Behaviour Studies(cE-Bs),
Faculty of Architecture, Planning & Surveying, Universiti Teknologi MARA, Malaysia Open access under CC BY-NC-ND license.
doi:10.1016/j.sbspro.2012.03.008
 Farah Ayuni Shafie et al. / Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76 71

The breeding places may also be in unexpected places such as the plant axils, tree holes, air-conditioner,
canvas sheets, and discarded receptacles.. Malaysia has been using malathion since 1970 for adult
mosquitoes control. The extensive use of malathion mainly to combat Aedes mosquitoes during outbreak
have indirectly contributed to the resistance to this organophosphate in Culex quinquefasciatus.
After almost 40 years, the insecticide that was supposed to kill the Aedes mosquitoes has become less
effective to Aedes and indirectly caused resistance to other breed of mosquitoes (Nazni et al, 2005). The
detection of resistance can actually help public health personnel to formulate appropriate steps in
encountering the reduction in effectiveness of Aedes control efforts. Furthermore, life span of Aedes
mosquitoes is short and development of insecticide resistance become more rapid after each generation.
Aedes mosquito develops resistance via two major routes; speed at which the insecticide is detoxified and
alteration of target site to reduce sensitivity to poisoning.
This study aims to determine the susceptibility status of the adult Aedes mosquitoes based on mortality
and killing time (KT50) value in two localities; Shah Alam and Pantai Dalam. Shah Alam is a residential
surrounding where the city has been well-planned with good road connections and ample infrastructures.
Pantai Dalam on the other hand, is a now rapidly growing area but in the midst of the development, a
number of low cost houses mainly flats and less affordable settlements are still intact. These two localities
are selected to represent two different localities in terms of the environment of the area as a whole.

2. Literature Review

Resistance can develop in every class of insecticide, including microbial drugs and insects’ growth
regulators (Brogdon & McAllister, 1998). A study on mosquito resistance to insecticide shows true or
incipient resistance to at least one of the three organophosphates tested (Jose et al, 2003). Another study
in Thailand to determine the effects of 0.75 % permethrin and 0.05 % deltamethrin impregnated paper
selection on the strain having the highest lethal time (LT 50) level arrived with a conclusion that
mosquitoes become resistant towards insecticide after a few generation. Resistance Ratio (RR) was found
to vary from 23.19 to 68.92. When bioassay was done on the third generation of the mosquitoes, the study
proved that the resistance ratio in those generations increased to 513.93 and 7.46 fold when compared
with laboratory susceptible strain. Also, previous study conducted in South Vietnam supported the
finding that Aedes mosquitoes were found to be susceptible to the malathion (Huong & Ngoc, 1999).
Therefore, it is proven that the evolution of resistance can be developed by generation. A study conducted
in Thailand on the other hand depicted that mosquitoes started developing resistance at the first and after
the third generation, and the resistance ratio increased about seven fold from the earlier detection
(Paeporn et al, 2002).

3. Methodology

1.1. Sample collection

A number of 50 Mosquitoes Larvae Trapping Device (MLTD) were used to collect Aedes larvae from
Shah Alam and Pantai Dalam. Larva of Aedes mosquitoes were collected from the MLTDs was then
transferred into bottles containing dechlorinated water. Matured pupa was transferred into a cage to let the
mosquitoes to emerge later. The larva and pupa were fed with a mixture of fine yeast, blended cow liver,
milk and cat food. Adult mosquitoes were fed with diluted honey soaked onto cotton wool.
72 Farah Ayuni Shafie et al. / Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76

1.2. WHO Adult Bioassay

The bioassay procedure of WHO was employed against all the mosquitoes species. Sugar-fed less than
7 days old adult female mosquitoes were used. Bioassays to detect resistance of adult were performed
according to the methodologies recommended by the WHO. WHO recommends that the test is more
viable by using non-blood fed, 2 to 5 day old adult mosquitoes to assess the resistance and susceptibility
status of a natural Aedes population. Batches of 20 adult mosquitoes were exposed to insecticide-
impregnated papers in standard WHO test tubes lined with the papers.
All tests were undertaken at temperature 26 ± 2 °C and relative humidity 60 ± 20 rh. The mosquitoes
were exposed to the diagnostic dosages at the respective exposure period. Cumulative mortality counts
were recorded at every 5 minutes for the respective exposure periods. After the exposures periods, the
mosquitoes were transferred into holding tube and provided with diluted honey. The test mosquitoes and
the controls were held for 24 hours recovery period and the mortality was recorded. If the control
mortality was between 5% and 20%, the percentage mortalities should be corrected by Abbott’s formula
(WHO, 1981).
% test mortality - % control mortality x 100
- % control mortality

All data were subjected to a probit analysis computer program and KT50 was obtained (Raymond,
1985). Resistance ratio was then calculated.

1.3. Insecticides

The insecticide used in this study is organophosphate with technical grade 5% malathion. The rationale
of using this selected insecticide was because malathion has been frequently used for dengue outbreaks
control especially in Malaysia. The insecticide used in the adult susceptibility test was diagnostic dosages
of WHO impregnated papers obtained from the Vector Control Research Unit in University of Science,
Penang. For control group, the adult mosquitoes were exposed to OP-Carbamate Control.

1.4. Observation

A checklist was developed to observe dwellers behaviour in prevention and control of Aedes
mosquitoes. The checklist is to survey the presence of the following; 1. potential breeding places (indoors
and outdoors), 2. use of repellant spray 3. schedule of fogging activity. A number of 30 houses in Pantai
Dalam were selected through simple random sampling and observed.

2. Results and Discussions

4.1 Resistance outcome

Table 1 shows the mosquitoes’ knockdown after being exposed to the insecticide for an hour. The
number of knocked down mosquitoes was recorded every 5 minutes. The 45 th minute was used as the
breakpoint as all the lab strain mosquitoes were knocked down.
 Farah Ayuni Shafie et al. / Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76 73

Table 1. Total number of mosquitoes’ knockdown in every 5 minutes interval

Time Shah Alam % Pantai Dalam % Lab strain %

strain strain
(minute)
5 2 1 0 0 0 0
10 7 2 1 0 0 0
15 19 7 12 4 22 7
20 40 13 22 7 71 24
25 68 23 48 16 179 60
30 80 27 60 20 234 78
35 100 33 83 28 267 89
40 115 38 99 33 289 96
45 124 41 115 38 300 100
50 139 46 126 42 300 100
55 155 52 144 48 300 100
60 167 57 154 51 300 100

It is evident that for Pantai Dalam mosquito strain, 38% were knocked down while 41% of the Shah
Alam mosquito strain was wiped out. It is also worth noting that Pantai Dalam mosquito strain took
longer time (only after 15 minutes) to be initially knocked down compared to Shah Alam mosquito strain.
After 1 hour, the percentage of knocked down mosquitoes from Pantai Dalam strain compared to Shah
Alam strain was 51% and 57% respectively. After the one-hour exposure, the mosquitoes were kept for
observation for another 24 hours. After 24 hours, 21% of the remaining mosquitoes from Pantai Dalam
still survived the exposure compared to mosquitoes from Shah Alam with only 5% survival (refer Table
2).

Table 2. Mortality rate after 24 hours and insecticide resistance criteria

Strain Total After 24 hours Category*

Mortality Percentage
Shah Alam 300 286 95 Possible to insecticide resistance
Pantai Dalam 300 234 79 Resistance to insecticide
Laboratory 300 300 100 Susceptible to insecticide resistance

* Susceptible criteria: 98%-100% Susceptible to insecticide, 80%-97% Possible to insecticide resistance, <80% Resistance to
insecticide (Davidson & Zahar, 1973).

The resistance standing of the strain is then categorized according to the criteria from Davidson &
Zahar, 1973. With 95% mortality, strain from Shah Alam is considered having possibility to be resistant
to the insecticide while strain from Bangsar is considered as resistant to insecticide with 79% mortality.
The mortality data were then analyzed using probit analysis to identify the value of KT 50, KT95 and KT99.
From the analysis, the identified KT50, KT95 and KT99 were shown in Table 3:
74 Farah Ayuni Shafie et al. / Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76

Table 3. KT50,KT95, and KT99 of Aedes mosquitoes from different origins exposed to malathion

Strain KT50 KT95 KT99

(95 % CI) (95 % CI) (95 % CI)
Shah Alam 52.814 226.458 413.937
(50.132,56.001) (193.261,273.573) (336.440,530.307)
Pantai Dalam 56.689 195.088 325.543
(53.949,59.984) (168.934,231.856) (270.122,407.428)
Labaratory 23.792 37.87 45.914
(23.289,24.285) (36.754,39.145) (44.132,47.998)

Table 3 summarizes the value of KT50, KT95 and KT99 defined from probit analysis which is then used
to calculate the resistance ratio. Resistance ratio (RR) was calculated by:

KT50, KT95, KT99 (wild strain)

KT50, KT95, KT99 (lab strain)

If the value of RR is greater than 1 it is indicative of resistance and value less than or equal to 1 are
considered susceptible (Chen et al, 2005).

4.2 Residents behaviour and environmental factor

Through observation using the checklist, it was found that 75% and 70% of the houses in Pantai Dalam
harbour potential outdoor and indoor breeding places respectively. In the context of sanitation, the
residents seem not to care to keep their houses and compound clean and tidy. It was also discovered that
72% of the household seek for supplementary mosquito control by using various repellent spray. These
findings could indicate that high potential breeding places simultaneously introduce high density of Aedes
mosquitoes. When the Aedes mosquitoes density become overwhelming, use of household repellant spray
become habitual. In some cases, the public would urge the local authority to conduct fogging, as if it is
the only and final solution to reduce the density of Aedes mosquitoes. Therefore, the extensive use of
repellent spray together with uncontrolled fogging activities by the local authority may eventually
contribute to the resistance of insecticide. According to Guidelines for Community Fogging, a fogging
activity may only be carried out as control measure when there is an outbreak (usually defined by more
than 2 cases in a locality within the incubation period), and as preventive measures when Aedes Index
(AI) is more than 1% and Breteau Index is more than 5. Aedes Index is defined by the percentage of
premises with Aedes breeding while Breteau Index is the number of containers with Aedes breeding
within 100 premises (Ministry of Health Malaysia, 2005).
It can also be observed in some other areas such as in private condominiums where fogging is carried
out indiscriminately by the managing authority. Ministry of Health and Local Government primary
dengue-control strategy has been source reduction, that is, to reduce or remove all potential mosquito-
breeding sites. Fogging should be carried out only when there are dengue-fever cases or when the adult
Aedes mosquito population is high so as to prevent more people from contracting dengue fever. The
public should be advised to move away from the mentality of carrying out fogging as a routine to control
mosquitoes but to focus on preventing mosquito breeding. A broad approach to the prevention of dengue
outbreak involves integration of the measures. Environmental management of dengue vector can be
successfully combined with health education and public health communication. The complete cooperation
 Farah Ayuni Shafie et al. / Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76 75

between non-health sectors such as non-governmental organizations, civic organizations and community
understanding and involvement in implementation is essential to achieve sustainability of successful
vector control programs. Public participation to combat Dengue would be the biggest challenge. The
effectiveness of anti-dengue programs was associated with the public’s attitude rather than with
knowledge or practice. Most of the people were aware of the campaign, but to practice it, they tend to
take it lightly. To overcome this, latest information need to be disseminated to the public especially on the
insecticide resistance so that they are aware that destroying Aedes mosquitoes is the end-of-the-pipe
solution. The findings from this study are hoped to have a wide impact on dengue prevention and control
programs in Malaysia. Instead of letting health authority shoulder a large part of the responsibility to
control the dengue epidemic, individuals and members of communities also need to play their part in
reducing mosquito breeding sites.

3. Conclusion

Fogging with insecticide alone would not be able to control dengue, for as long as there was Aedes
breeding, new mosquitoes could emerge after fogging and spread the disease. Instead of reducing the
Aedes density by short term and rapid destruction of Aedes mosquitoes, the drive should be focusing to
preventing emerging of the mosquitoes itself. The health authority could actively promote community
participation in dengue control through Communication for Behavioural Impact (COMBI) approach
because it was still the best approach and strongly recommended by the WHO. The public is made known
of the importance to keep their surrounding in good sanitation. Gotong-royongs should also include the
no man’s land (such as abandoned land, houses etc) which could also harbor breeding. Hence, to prevent
and control disease mosquito, the health authority should emphasize the control in changing human
behaviour to search and destroy potential breeding places and ultimately to control Dengue fever. This is
achievable through adequate knowledge on Aedes as a biological vector for Dengue transmission;
subsequently develop positive attitude and practice to keep their house and compound free from
receptacles that may offer breeding ground for Aedes mosquitoes.

Acknowledgements

The authors would like to thank UiTM Excellence Fund for the great support towards this study. The
supports from residents of studied areas are also appreciated.

References

Rodriguez, M. M., Bisset, J. A. & Fernandez, D. (2007). Levels of insecticide resistance mechanisms in Aedes aegypti from some
Latin American countries, Journal of the American Mosquito Control Association, 23(4): 420-429.
Lima, J.B.P., Da-Cunha M. P., Da Silva Júnior R.C., Gallardo A.K.R., Soares S.D.S, Braga, I.A., Ramos, R.P. & Valle, D. (2007).
Resistance of Aedes aegypti to organophosphates in several municipalities in the state of Rio de Janeiro and Espírito Santo,
Brazil. The American Society of Tropical Medicine and Hygiene. 68(3), 2003, pp. 329-333.
Nazni, W. A., Lee, H.L., & Azahari, A. H. (2005). Adult and larval insecticide susceptibility status of Culex quinquefasciatus (Say)
mosquitoes in Kuala Lumpur, Malaysia. Tropical Biomedicine, 22(1): 63-68
Ministry of Health, Malaysia. (2005). Garis Panduan Semburan Kabus Komuniti.
Chen, C. D., Nazni, W. A., Lee, H. L., & Sofian-Azirun, M. (2005). Susceptibility of Aedes aegypti and Aedes albopictus to
temephos in four study sites in Kuala Lumpur City Centre and Selangor State, Malaysia. Tropical Biomedicine 22(2): 207-216.
Liu, H., Cupp, E. W., Guo, A. & Liu, N. (2004). Insecticide Resistance in Alabama and Florida mosquito strains of Aedes
albopictus, Journal of Medical Entomology, 41(5): 946-952.
76 Farah Ayuni Shafie et al. / Procedia - Social and Behavioral Sciences 36 (2012) 70 – 76

Paeporn, P., Ya-umphan, P., Supaphathom, K., Savanpanyalert, P., Wattanachai, P. & Patimaprakorn, R. (2002). Insecticide
Susceptibility and Selection For Resistance In A Population Of Aedes aegypti from Ratchaburi Province, Thailand.
Brogdon, W.G. & McAllister, J.C. (1998). Insecticide resistance and vector control. Emerging Infectious Diseases, 4(4).
Huong, V. D. & Ngoc, N. T. B. (1999). Susceptibility of Aedes aegypti to insecticides in South Vietnam. Dengue Bulletin 23.
World Health Organization (1981). Instructions for determining the susceptibility or resistance of adult mosquitoes to
organochlorine, organophosphate and carbamate insecticide. Establishment of the base line. WHO/VBC/81.805
Davidson, G., & Zahar, A., R. (1973). The practical implications of resistance in malaria vectors to insecticides. Bulletin World
Health Organization, 49(5): 475-4