POTENTIAL DEVELOPMENT OF TEMEPHOS RESISTANCE IN AEDES

AEGYPTI RELATED TO ITS MECHANISM AND SUSCEPTIBILITY TO

DENGUE VIRUS
Pungasem Paeporn1,2, Narumon Komalamisra1, Supatra Thongrungkiat1, Vanida Deesin1,
3
1
Yuki Eshita and Yupha Rongsriyam
1

Insecticide Research Unit, Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol
2
University, Bangkok; Chemical Control Section, Department of Medical Sciences, Ministry of Public Health,
Nonthaburi, Thailand; 3Department of Infectious Diseases, Oita Medical University, Japan
Abstract. The addition of temephos to water containers as a larvicide against Aedes aegypti was commonly used
as a part of DHF control programs. The widespread, or long-term, application of insecticides can lead to the
development of mosquito resistance to the insecticides through selection pressure. This presents a problem for
disease control. Therefore, this study was conducted in the laboratory to observe the potential development of
resistance to temephos and the mechanism involved in Ae. aegypti, and to study the significance for dengue
infection. The larvae were selected in consecutive generations. The level of resistance to temephos was detected
by WHO assay technique. After 19 generations of selection, a low level of resistance was found. The resistance
ratio at LC50 was 4.64 when compared with the non-selected group. The assay for major enzyme-based resistance
mechanisms was done in a microtiter plate to detect elevated non-specific esterases, monooxygenase, and
insensitive acetylcholinesterase in the temephos-selected and non-selected groups. It revealed a significant increase
in esterase activity when compared with the non-selected group. There was no elevation of monooxygenase or
insensitive acetylcholinesterase activities. However, when an esterase inhibitor (S, S, S-tributyl phosphorotrithioate,
or DEF) was added to temephos and the susceptibility in the selected group was studied, the resistance ratio was
reduced from 16.92 to 3.57 when compared with a standard susceptible strain (Bora Bora). This indicates that
the esterases play an important role in temephos resistance.
Dengue-2 virus susceptibility was studied by oral feeding to females of the temephos-selected (S19) and the
non-selected groups. The dissemination rates, when the titer of virus in the blood meal was 7.30 MID50/ml, were
11.11% and 9.38% for the selected and non-selected groups, respectively. When the titer of virus in the blood
meal was 8.15 MID50 /ml, the dissemination rates increased to 24.24% and 33.33%, respectively. A statistical
difference in viral susceptibility was not found between the two groups. This suggested that the low level of
temephos resistance might not affect oral susceptibility. However, this needs further study.

INTRODUCTION
Dengue hemorrhagic fever first appeared as an
epidemic in Bangkok in 1958. The epidemic pattern has
changed from one of alternate years to an irregular
pattern (Kantachuvessiri, 2002), with more than 20,000
deaths per year. The mosquito Aedes (Stegomyia)
aegypti (L.), the main vector of dengue virus, was
presumably introduced to Thailand and other parts of
Southeast Asia by vessels sailing across the Indian Ocean
from Africa, where this species originated (Mattingly,
1957; Laird, 1994). Ae. aegypti was primarily urban,
with the highest densities in cities and towns.
Until an effective, safe and affordable vaccine
Correspondence: Miss Pungasem Paeporn, Chemical
Control Section, Department of Medical Sciences,
Ministry of Public Health, Nonthaburi 11000, Thailand.
Tel: +66 (0) 2951-0000 Ext 9252;
Fax: 66 (0) 2591-5449
E-mail: pungasem@hotmail.com
136

becomes available, no adequate prevention or control

measures, other than control of the vector, Ae. aegypti,
are available to deal with epidemics of disease (Gratz,
1993). The only effective approach to Ae. aegypti
control has been the elimination of larval habitats from
the domestic environment, or source reduction (Soper
et al, 1943; Schliessman and Calheiros, 1974).
However, difficulty with control has been encountered
due to the emergence of insecticide-resistant
mosquitos. Data on insecticide susceptibility, resistance
detection and characterization of the mechanisms of
Ae. aegypti will provide base-line data for planning
control programs and making decisions about
insecticide usage. In this study, the differences in
insecticide susceptibility, and susceptibility to dengue2 virus in a temephos-selected group were examined.
MATERIALS AND METHODS
Mosquitos
Three groups of mosquitos were used in this
study. The Nonthaburi colony was derived from the
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TEMEPHOS RESISTANCE IN AE. AEGYPTI AND ITS SUSCEPTIBILITY TO DENGUE VIRUS

collection of Ae. aegypti larvae indoors and outdoors

at Anurajprasit Kindergarten School and the area
around the school. After one generation in the
laboratory, this colony was divided into 2 groups;
the first was subjected to temephos selection
(Nonthaburi-Sel group) and the other was maintained
without selection (Nonthaburi-Non-sel group). The
third group, Ae. aegypti Bora Bora strain (WHO
susceptible strain), which was obtained from Prof
Yap Han Heng, Universiti Sains Malaysia, Penang,
was used as the reference susceptible group.
Insecticides
Technical grade (90% purity) temephos (Abate),
an organophosphate insecticide, was obtained from
Cyanamid Co.
Bioassay procedures
The late third or early fourth instar larvae of all
groups were used for bioassay test. The procedures
were as recommended by WHO (1963). The results
were analyzed for median lethal concentration (LC50)
and LC95 by probit analysis using a Basic program
(Raymond, 1985).
Selection procedures
The Nonthaburi group was used for selection. The
groups of 25 late third or early fourth instar larvae were
exposed to temephos in 250 ml of dechlorinated tap
water for 24 hours. The concentrations used for
selection were 0.0025 mg/l for S1 to S4 generations,
0.005 mg/l for S5 to S7, 0.01 mg/l for S7 to S14 and
0.02 mg/l for S15 to S19 generations. The surviving
larvae from each exposure were reared for further
selection.
Synergism test for confirmation of the defence
mechanism
This test was similar to the bioassay tests, except
that 0.5 ml of the maximum sublethal concentration
of an esterase inhibitor, S,S,S-tributyl phosphorotrithioate, (0.5 g/ml) was added to each cup with
0.5 ml of insecticide.
Microtiter plate assay
Esterase assay. Total esterase activity in
individual, frozen mosquito larvae (late third or early
fourth instar) from Nonthaburi-Non-sel, NonthaburiSel and Bora Bora strains were dertermined according
to the method of Lee (1991). Enzyme activity was
determined as an OD value by microplate reader at
450 nm.
Monooxygenase assay. To measure the activity
Vol 34 (Suppl 2) 2003

of monooxygenases from individual larvae, the

procedure described by Vulule et al (1999) was adopted
with a single modification, by using the same buffer
(potassium phosphate buffer) as esterase assay in
preparing the larval homogenate. Enzyme activity was
determined by a microplate reader at 620 nm.
Acetylcholinesterase assay. Homogenates of the
mosquito larvae were tested for insensitive AChE using
the method of Lee et al (1992), which was modified
from the Ellman test (Brogdon et al, 1988). Enzyme
activity was determined by a microplate reader at
410 nm.
Protein concentration determination
The protein in each larva was determined by the
method of Bradford (1976), in order to detect the
differences in size among individuals that might require
correction factors for the enzyme assays, as in the case
of esterase and monooxygenase assays.
Virus susceptibility
After 19 generations of selection with temephos,
the susceptibilities to dengue 2 virus by oral feeding
(Sucharit et al, 1997) were compared among the
temephos selected-group, the non-selected group and
the standard susceptible strain (Bora Bora). The method
for determining susceptibility to dengue virus is as
follows:
Preparation of dengue virus antigen. Den-2
infected mosquitos, Toxorhynchites splendens, were
ground with phosphate-buffered saline (PBS) pH 7.5
containing 30% inactivated fetal calf serum (FCS) and
o
clarified by centrifugation at 7,000 rpm at 4 C for 1
hour.
Oral feeding. Infectious blood meals were
prepared by mixing 2 parts virus suspension, 1 part
washed red cells, and 2 parts 10% sugar solution. Three
to five days old, fasted female mosquitos were forced
to feed on infectious blood meals using an artificial
membrane feeder for 30 minutes. Fully engorged
mosquitos were kept, provided with 10% sugar solution
o
and maintained at 32 C, 70-80% RH for 14 days.
Detection of dengue antigen. After 14 days
incubation, squashed heads of Aedes aegypti were
checked by direct immunofluorescence (DFAT), using
fluoro-isothiocyanate (FITC) conjugated to determine
the infection.
Virus assay. Virus titration was done by
intrathoracic inoculation of 10-fold serial dilution of
virus suspension into Tx. splendens. The mosquito
infectious dose 50 (MID50) was calculated from the
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SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

positive mosquito head squash after inoculation for 14

days, by the method of Reed and Meunch (1938).
RESULTS
In generation 19 of temephos selection, the LC50
in the Nonthaburi-Sel group increased to 0.0154. It
showed low level resistance by an increase of nearly
5-fold at the LC 50 , when compared with the
Nonthaburi-Non-selected strain (Table 1).
In the absence of selection pressure, the temephos
resistance ratio of the Nonthaburi-Non-selected group,
compared with the Bora Bora strain was 3.65, at LC50.
After 19 generations of selection, the temephos
resistance ratio at LC50 increased to 16.92 (Table 3).

Table 1
a
LC 50 of temephos in selected generations of Aedes
aegypti.
Generation

S0 (F1)
S2
S4
S6
S8
S9
S10
S15
S19
a

Nonthaburi-Sel

0.00332
0.003
0.006
0.005
0.010
0.010
0.013
0.013
0.0154

The addition of the esterase inhibitor, DEF, to the

temephos resulted in a reduction in the resistance ratio,
as shown in Table 3. The resistance ratio in the
Nonthaburi-Sel group was reduced to 3.57 at LC50.
Biochemical testing revealed the presence of
elevated esterase activity in the Nonthaburi-Sel group
(Table 2). No changes were found for monooxygenase
activity (Table 4) and no evidence of insensitive
acetylcholinesterase in the Nonthaburi-Sel group
(Table 5). This suggested that the resistance was not
associated with monooxygenases or insensitive
acetylcholinesterase.
The susceptibility of mosquitos to dengue-2 virus
showed the dissemination rates, when the titer of virus
in the blood meal was 7.30 MID50/ml, were 11.11%,
9.38% and 3.13% for the selected, non-selected groups
and Bora Bora, respectively. When the titer of virus
in the blood meal was 8.15 MID 50 /ml, the
dissemination rates increased to 24.24%, 33.33% and
8.57%, respectively (Table 6). However, there was no
significant difference in virus susceptibility between
each group.

Resistance
ratio

DISCUSSION
Selection for temephos resistance showed that Ae.
aegypti had the potential to develop resistance to this
insecticide. A low level of resistance was shown as an
almost 5-fold increase in resistance ratio after 19
generations of selection (Table 1) when compared with
the non-selected group. However, in comparison with
the WHO susceptible strain (Bora Bora) it showed a
marked increase in resistance ratio (Table 3). It was
noted that Ae. aegypti developed resistance to this
insecticide slowly, probably due to the low selection
pressure used.

1.00
0.90
1.81
1.51
3.01
3.01
3.92
3.92
4.64

Median lethal concentration (mg/liter).

Table 2
Average esterase activities in the larvae of Aedes aegypti from Bora Bora, Nonthaburi-Non-sel and temephosselected groups.
Mean esterase activitya
Group

Ae. aegypti Bora Bora

Nonthaburi-Non-sel
Nonthaburi-Sel (S19)
a
b

Number

Mean + SDb

25
25
25

0.132 + 0.031
0.189 + 0.042b
0.275 + 0.044c

Minimum
value

Maximum
value

0.069
0.124
0.210

0.181
0.294
0.376

Esterase activity expressed as absorbance / minute / mg protein.

Means followed by the same letter are not significantly different (p = 0.05, LSD test).

138

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TEMEPHOS RESISTANCE IN AE. AEGYPTI AND ITS SUSCEPTIBILITY TO DENGUE VIRUS

Table 3
Effect of temephos and temephos with esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF), on
resistance levels to temephos of Aedes aegypti groups in comparison with the susceptible strain (Bora Bora).

Insecticide
Temephos

Temephos
+ DEF

Group
Bora Bora
Nonthaburi-Non-sel
Nonthaburi-Sel (S19)
Bora Bora
Nonthaburi-Non-sel
Nonthaburi-Sel (S19)

LC50(mg/l)

LC95(mg/l)

0.00091
0.00332
0.01540
0.00042
0.00044
0.00150

0.00248
0.00985
0.03880
0.00092
0.00097
0.00310

Resistance ratio
LC50

LC95

1
3.65
16.92
1
1.05
3.57

1
3.97
15.65
1
1.05
3.37

Table 4
Average monooxygenase activities in the larvae of Aedes aegypti from Bora Bora, Nonthaburi Non-sel and
temephos-selected groups.
Mean monooxygenase activitya
Group

Ae. aegypti Bora Bora

Nonthaburi-Non-sel
Nonthaburi-Sel (S19)
a
b

Number

Mean + SDb

25
25
25

0.188 + 0.052
0.187 + 0.091a
0.189 + 0.066a

Minimum
value

Maximum
value

0.111
0.107
0.093

0.277
0.420
0.360

Monooxygenase activity expressed as absorbance / minute / mg protein.

Means followed by the same letter are not significantly different (p = 0.05, LSD test).

Table 5
Propoxur-inhibited acetylcholinesterase (AChE) activity expressed as a percentage of uninhibited AChE activity
in larvae of Aedes aegypti from Bora Bora, Nonthaburi Non-sel and temephos-selected groups.
Percentage of uninhibited AChE activity
Group

Ae. aegypti Bora Bora

Nonthaburi-Non-sel
Nonthaburi-Sel (S19)
a

Number

Mean + SD

10
10
10

64.16 + 8.42a
a
67.54 + 16.53
61.49 + 16.20 a

Minimum
value

Maximum
value

49.02
33.00
39.62

75.86
90.90
93.33

Means followed by the same letter are not significantly different (p=0.05, LSD test).

When the resistance to temephos had developed,

the biochemical assay for enzymes revealed elevations
in esterase activity in the selected group. It showed
significantly higher esterase activity than the nonselected group (Table 2). In order to confirm the
Vol 34 (Suppl 2) 2003

association of esterase activity with temephos

resistance, the esterase inhibitor (S, S, S,-tributyl
phosphorotrithioate, or DEF) was added to the
temephos. The selected group became more
susceptible to temephos by reducing the resistance ratio
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SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

Table 6
Dissemination rates in Aedes aegypti after oral infection with Den-2 viruses.
Titer of blood meal
Log10 MID50/mla
7.30
8.15
a
b

No. infected/No. tested (%)

Bora Bora

Nonthaburi-Non-sel

Nonthaburi-Sel

1/32 (3.13)
3/35 (8.57)

3/32 (9.38)
8/24 (33.33)

4/36 (11.11)
8/33 (24.24)

MID50 = 50% mosquito infectious dose.

Determined by direct fluorescent antibody test (DFAT) on head squashes.

nearly 4-fold, than the WHO susceptible strain and

the Nonthaburi-Non-sel group (Table 3). This indicated
that esterases play a significant role in temephos
resistance. Elevated esterase activity associated with
temephos resistance was also reported in Ae.aegypti
from Tortola, British Virgin Islands (Wirth and
Georghiou, 1999) and from Trinidad (Vaughan et al,
1998).
Study of other enzymes showed no change in
monooxygenase activity (Table 4) and no evidence of
insensitive acetylcholinesterase (Table 5) in the
selected group. This suggested that the temephos
resistance was not associated with monooxygenase and
insensitive acetylcholinesterase.
Following the identification of the resistance
mechanism, it may be useful to be aware of possible
cross-resistance to other insecticides conferred by this
mechanism. Brown (1986) also reported crossresistance between temephos and chlorpyrifos in a
strain of Ae. nigromaculis (Ludlow) from California.
It is evident that this important vector species, Ae
aegypti, has the potential to develop temephos
resistance, which may result in control problems.
Continuous monitoring of insecticide susceptibility in
Aedes populations is critical for decisions on insecticide
usage. Source reduction, environmental manipulation
and self-protection must be emphasized in order to
reduce insecticide use, and to delay the further
development of organophosphate resistance.
Regarding the susceptibility of the resistant
mosquito to dengue-2 virus, a statistical difference in
dissemination rate was not found between each group.
This is supported by the work of Gokhale et al (2000),
where their selection of mosquito strain with
malathion showed increased esterase activity but no
increase in dengue virus susceptibility. However,
this needs further study for higher levels of temephos
resistance.
140

ACKNOWLEDGEMENTS
The authors would like to thank Associate
Professor Dr Somjai Leemingsawat and staff of the
Department of Medical Entomology, Mahidol
University, for their help and kindness, the Center of
Vaccine Development, Mahidol University for
providing dengue antigen, Assist Prof Kasinee
Buchachart for her kind suggestions on statistical
analysis, Dr Lee Han Lim and Ms Nazni Wasi Ahamad,
Institute of Medical Research, Malaysia, Dr Mir S
Mulla and Dr Margaret C Wirth, Department of
Entomology, University of California Riverside, USA
and Dr William G Brogdon, Center for Disease Control
and Prevention, USA, Dr Anna-Bella Failloux, Institut
Pasteur, France for their kind helpful guidance, Mr
Paul Adams for reviewing the manuscript.
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