Biochemical detection of pyrethroid resistance mechanism in Aedes

aegypti

in Ratchaburi province, Thailand.

Pungasem Paeporn1, Kasin Supaphathom1, Raweewan Srisawat 2, Narumon

Komalamisra2, Vanida Deesin 2, Phubeth Ya-umphan 1, Somjai Leeming sawat 2
1

Chemical Control Section, Department of Medical Science, Mintstry of Public

Health, Nonthaburi 11000, Thailand; 2Departmentment of Medical Entomology,

Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.

Abstract. The emergence of insecticide resistance in mosquito vectors was an

important issue to be considered as one of factors influencing the success of vector
control. The early detection of resistance could help the health personnel to plan and
select

appropriate alternative control measures or insecticide for effective control.

Therefore biochemical assay of enzymes in mosquito was conducted to detect the

emergence of insecticide resistance and to define the machanisms involved in
pyrethroid resistance. Adults of Aedes aegypti from two localtities in Ratchaburi
province were subjected to permethrin and deltamethrin selection in laboratory. After
three generations of selection, permethrin -selected and deltamethrin -selected strains
were established. Their LT

50

increased to 7.46 and 1.18 folds in the F3 strains that

were selected with permethrin and deltamethrin respectively. The enzymes of these
mosquitoes were assayed biochemically to study the mechanisms of resistance. The
results revealed significant increase of esterase activity and monooxygenase levels in
both strains when compared with labolatory susceptible strain. Glutathione -Stransferase activity was found to increase in permethrin-selected strain but not in
deltamethrin- selected strain. This suggested that not only esterase and
monooxygenase but also glutathione-S-transferase were associated with permethrin
resistance in Ae. aegypti. The exposing of permethrin -selected and deltamethrin selected mosquitoes to diagnostic concentration of permethrin (0.75%) and
deltamethr in (0.05%) indicated no cross resistance for permethrin to deltamethrin
while slight cross resistance from deltamethrin to permethrin was evident. It seemed
that glutathione S-tranferase was not associated with cross resistance since its activity
in deltamethrin-selected strain remained unchanged as compared with that of
l a b o r a t o r y

s u s c e p t i b l e

s t r a i n .

INTRODUCTION
Dengue haemorrhagic fever is still one of the major mosquito borne diseases in
Thailand with Aedes aegypti as the principal vector. As long as an effective, safe and
effordable vaccine is not available, no adequate prevention or control measure other
than control of vector are available. Especially during epidemics of the disease, the
use of insecticides are needed. For example, application of temephos for larval control,
thermal fogging or ULV spray of certain organophosphates and pyrethroids for adult
control. Insecticide resistance could develope and would thus be a major problem in
controlling

the

vectors

and

other

pest

insects.

The majo r metabolic enzymes involved in resistance against pyrethroids in

insects include P450 mediated monooxygenases, elevated non-specific esterases, and
reduced sensitivity of sodium ion channels along nerve axons (Oppenoorth, 1985;
Georghiou,1986; Nelson et al., 1996; Roberts & Andre, 1994; Scott et al., 1998;
Feyerisen, 1999). Moreover, increased levels of glutathione-S-tranferase (GSTs) have
been associated in Ae.

aegypti (Grant & Matsumura, 1988).

In this study, we conducted biochemical assays of enzymes integrated with

dose-mortality bioassays for detection of resistance and to define the underlined
mechanisms involved in pyrethroid resistance in Ae. aegypti which were selected for
insectcide resistance under laboratory conditions.

ATERIALS AND METHODS

Test populations
A susceptible colony of Ae. aegypti from the Department of Medical Sciences,
Ministry of Public Health, Thailand has been used as a laboratory susceptible strain.
Aedes aegypti from Tambon Pongsawai and Tambon Wat KhuBua were
established as permethrin-selected strain and deltamethrin-selected strain through a
series of laboratory selection with permethrin and deltamethrin.
S

Aedes aegypti from Tambon Pongsawai, Ratchaburi province, was subjected to

permethrin selection and Ae. aegypti from Wat KhuBua; Tumbon KhuBua,
Ratchaburi province was as subjected to deltamethrin selection respectively. The
procedure was as in WHO(1981), as previously described by Paeporn et al., (2004).
These two strains were continually selected for 3 generations by exposing non-blood

fed females aged 2-3 days old for a certain time that produced 60-70 % mortality to
t

D i a g n o s t i c

s u s c e p t i b i l i t y

a s s a y

Non-blood fed females aged 2-3 days old of the Tambon Pongsawai and Wat KhuBua;
Tambon KhuBua strains were exposed to the diagnostic concentrations of
permethrin(0.75%) and deltamethrin(0.05%) impregnated paper respectively for 1
hour and the mortality was recorded at the end of 24 hours holding period. The cross
resistance between the se two insecticides was studied.
Biochemical test
Individual mosquitoes were homogenized in 200 l distilled water. Each of 10 l of
the homogenate was used for monooxygenase, glutathion S-transferase and protein
assay. The other twenty l of homogenate was used for esterases assay. The protocol
for each assay followed WHO(1998); technique to detect insecticide resistance
mechanisms (field and laboratory manual). The details of each assay is as followed;
P

The total protein content of individual mosquitoes was determined using the Bio -Rad
Protein Assay Kit (Bio -Rad Laboratories) in order to detect the differences in size
among individuals that might require correction factors for the enzyme assays .
Esterase assay
Twenty l of homogenated were placed in separate wells of microtitre plate. Two
hundred l of 0.3 mM - napthyl acetate were added to each well. Leave the plate at
room temperature for 1 min and then added 50 l of fast blue strain solution. After 5
minutes, enzyme activity was de termined as an OD value by microplate reader at 450
nm. Esterase activity was expressed as : OD/min/mg protein
Monooxygenase (Cytochrome p450) assay
Ten l of homogenate were placed in separate of microtitre plate followed by addition
of 80 l 0.625 M pota ssium phosphate buffer (pH 7.2). Ten mg of 3,3,55,Tetramethyl Benzidine(TMBZ) in 5 ml methanol was prepared and a 15 ml of 0.25 M
sodium acetate buffer(pH 5.0) was prepared. Two hundred l of the above TMBZ
solution was added in to each well followed by 25 l of 3% hydrogen peroxide. The
p l a t e

w a s

r e a d

a f t e r

h o u r s

a t

6 3 0

n m .

This assay measures the haem content of insect based on that of Brogdon et
al., (1997). It is therefore a rough means of monooxygenase content. It is not a

measure of monooxygenase activity in the insect. Monooxygenase level was

e x p r e s s e d

a s

O D

m g

p r o t e i n

Glutathione -S-transferase (GST) assay

Ten l of each homogenate was transferred to a microplate well followed by 200 l of
the GSH/CDNB working solution which was prepared by adding 125 l of 63 mM 1chloro-2,4-dinitrobenzene (CDNB) to 2.5 ml 10 mM glutathione solution(GSH). The
plates were read after 5 mins with the ELISA plate reader at a wavelength of
340 nM. GST activity was expressed
D

as: mMoles/ min / mg protein

Abbotts formula was used to correct the observed mortality in adult susceptibility test
(WHO 1981) the LT 50 value was analyzed by probit analysis using a Basic program
(Raymond,1985). A one way analysis of variance (ANOVA) was used to compare the
enzyme expression levels between population by using SPSS 10.0 program. All levels
of statistical significant were determined at P < 0.05.

RESULTS
Aedes aegypti were selected for permethrin and deltamethrin resistance. Resistance
ratios after 3 generations of permethrin selection of the Ae. aegypti from Tambon
Pongsawai were increased to 513.93 and 7.46-fold when compared with laboratory
susceptible strain and with non selected strain (Table1).
The resistance ratios after 3 generations of delta methrin selection of Ae.
aegypti from Wat KhuBua; Tambon KhuBua increased to 62.29 and 1.18-fold when
compared with laboratory susceptible strain and with non selected strain (Table 2).
Diagnostic dose test
The adults of laboratory susceptible strain showed complete
diagnostic

concentration

of

permethrin

(0.75%)

and

mortality to the

deltamethrin

(0.05%)

impregnated papers. While the mosquitoes from Tambon Pongsawai non-selected

strain and Tambon Pongsawai F3 permethrin-selected strain were exposed to 0.75%
permethrin, the mortalities of 22 % and 7.27% were observed. And these two strains
showed the same mortality (31%) when exposed to 0.05% deltamethrin.
On the other hand, the adults from Wat KhuBua non-selected and F 3
deltamethrin selected

strains showed 17% and 16% mortality respectively when

exposed to diagnostic concentration of deltamethrin. The test also showed 46% and

30% mortality

respectively

when

exposed

to

permethrin

at

diagnostic

concentration(Table 3).
Biochemical test
The results of the biochemical assay showed significant elevation of esterase activity
and monooxygenase content in permethrin and deltamethrin selected as compared
with laboratory susceptible strains (Table 4 and 5).
The Pongsawai strain after 3 generations of permethrin selection revealed an
increase of glutathione-S-transferase activity when compared with Wat KhuBua
deltamethrin selected strain and laboratory strain (Table 6). While no evidence of
elevated glutathione-S -transferase activity was found in Wat KhuBua deltamethrin
s

DISCUSSION
The selection of two strains of Ae.aegypti with permethrin and deltamethrin for 3
consecutive generations revealed increase in resistance ratio to 7.46 and 1.18 in
permethrin-selected and deltamethrin-selected strains respectively. When the
permethrin selected strain was exposed to diagnostic concentration of permethrin the
mortality was 7.27% as compared with 22% of the non-selected strain (Table 3)
which was compatible with the increase of resistance ratio to permethrin. The
development of permethrin resistance was suggested. When exposed to deltamethrin
diagnostic concentration the cross resistance to deltamethrin was not observed as
shown by the same percentage mortality (31%) as observed in non selected strain.
While the resistance ratio of deltamethrin-selected strain was almost unchanged from
the non-selected (1.18 : 1) strain . This indicated probably resistance to deltamethrin
has not developed yet. Cross resistance to permethrin was slightly observed since its
mortality decreased from 46% to 30% when exposed to diagnostic concentration of
p

Biochemical analysis showed significant increase of esterases, glutathione -Stranferase activities and monooxygenase content in permethrin-selected strain. It
revealed the association of these enzymes to the development of resistance to
permethrin. While the glutathione S tranferase activity seemed to be not associated
with cross resistance to deltamethrin. In contrast a low level of cross resistance to
permethrin was observed in deltamethrin-selected strain eventhough the level of
g l u t a t h i o n e - S - t r a n f e r a s e
5

w a s

n o t

e l e v a t e d .

In deltamethrin -selected strain only esterases activity and monooxygenase

content were found to increase. It might be due to glutathione-S-transferase activity
was not associated with resistance to deltamethrin or the level of resistance ratio to
deltamethrin was still very low. Hence, further selection pressure to obtain
deltamethrin resistant strain is needed for verification this phenomenon.
As the elevated glutathione -S-transferase activity was related to DDT resistance
(Mourya et al.,1993,1994,1994) and continuous use of permethrin could induce
higher activity of this enzyme. The cross resistance between these insecticides would
probably become a problem in the area where DDT was used for malaria control.
Acknowledgement. Our work was supported by the Department of Medical Sciences
for research fund. We would like to thank to the staff of the Provincial Health Office
in Ratchaburi Province, the staff of the Vector-borne Disease Control unit No.4,
Ratchaburi Province for providing data of Dengue cases and chemical control usage,
the staff of Chemical Control Section, Department of Medical Sciences for collecting
larvae, Ms. Mariam Santithipong for taking care of mosquito colonies and conducting
t

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