Indian J Med Res 117, January 2003, pp 30-38

Pyrethroid susceptibility & enzyme activity in two

malaria vectors, Anopheles stephensi (Liston) & A.
culicifacies
(Giles) from Mysore, India
K.N. Ganesh, J. Urmila & V.A. Vijayan
Department of Studies in Zoology, University of Mysore, Mysore, India
Received May 15, 2002
Background & objectives: Anopheles stephensi and A. culicifacies are the two major
vectors of malaria in Karnataka. These mosquito populations are continuously
being exposed directly or indire ctly to different insecticides including the most
effective pyrethroids. Therefore, there is a threat of insecticide resistance
development. We subjected these vectors to larval bioassay using two popular
pyrethroids viz deltamethrin and permethrin. An attempt was also made to
correlate the activities of certain detoxifying enzymes such as A- esterase, Besterase, glutathione-S transferase (GST) and glucose-6-phosphate dehydrogenase
(G6PD) with the tolerance levels of the two vectors.
Methods: Larval bioassay was carried out following the standard WHO procedure
on field-collected larvae. The LC 50 and LC 90 values were calculated following
Probit analysis. Biochemical estimations were done with a U V spectrophotometer
and the isozyme studies employing nati ve polyacrylamide gel electrophoresis
(PAGE).
Results: The results of the larval bioassay revealed that A. stephensi has more
tolerance to deltamethrin than A. culicifacies and vice versa for permethrin.
Biochemical estimations revealed significantly (P<0.05) higher levels of A-esterase
and GST activity in A. stephensi whereas A. culicifacies showed significantly higher
(P<0.05) levels of B-esterase and G6PD activity. The total larval protein assayed
was found to be more (P<0.05) in A. stephensi. The isozyme profiles also revealed
difference in mobility, intensity and the number of bands.
Interpretation & conclusion: As these malaria vectors are exposed to different kinds
of insecticides, they develop increased enzyme activities to overcome the insecticide
pressure. This has enhanced the tolerance level against the pyrethroids tested.
Thus, A. stephensi was found to be tolerant to deltamethrin depicting a higher
activity of A-esterase and GST enzymes, whereas the higher activity of B-esterase
and G6PD has resulted in the development of tolerance to permethrin in A.
culicifacies.
Key words Anopheles culicifacies - A. stephensi - A-esterase - B-esterase - detoxifying enzymes - insecticide
resistance

The National Malaria Eradication Programme (NMEP) has been reporting 2.5 to 3 million
malaria cases every year in India 1. Malaria is one of the major communicable diseases in
Karnataka state, especially in the irrigated districts of the plains 2. Anopheles stephensi and A.
culicifacies are the major malaria vectors found in and around Mysore city. These vectors are
exposed to different insecticides especially due to intense agricultural activities, and the
possibility exists of development of insecticidal resistance. Synthetic pyrethroids, popularly
known as emergency insecticides have been reported to be more effective than other
insecticides and natural pyrethrins 3. However, continuous and indiscriminate use of such
chemicals can lead to the development of resistance/tolerance by the insect pests. Reports of
resistance in A. gambiae to pyrethroids, deltamethrin and permethrin4,5 and alphacy-

permethrin 6 are available. A. sinensis has also been found to be tolerant to deltamethrin7.
Reports are also available for the susceptibility of mosquito vectors to pyrethroids. It is
hence, necessary to monitor local populations of Anopheles vectors against these insecticides.
The primary routes of insecticide resistance in all insects are alterations in the insecticide
target site or changes in the rate at which the insecticide is detoxified8. So far esterases,
glutathione
S-transferases and monooxygenases are known to be involved in the detoxification of the four
major groups of insecticides8. It has been found that the increased activity of these enzymes
will help in the development of physiological resistance to insecticides9. Esterases and
multifunction oxidases are involved in the resistance to pyrethroids 10. Since glucose-6phosphate dehydrogenase (G6PD) is NADP generating system for microsomal
monooxygenases, increase in the monooxygenase activity will be reflected in the G6PD
activity10. Reports correlating high levels of GST with high resistance to pyrethroids are
available for several insect species including mosquitoes11,12.
The present study was undertaken to know the susceptibility status of two malaria vectors
viz.
A. stephensi and A. culicifacies against deltamethrin and permethrin. Investigations were also
conducted to correlate the qualitative and quantitative activities of detoxifying enzymes such
as esterases, GST and monooxygenases with the tolerance levels of these two vectors.
Material & Methods
A. stephensi larvae were collected from fountains and tanks from Mysore city and
immatures of A. culicifacies from pools/streams and paddy fields around Mysore city. The
larvae were brought to the Vector Biology Laboratory of the Department of Zoology and
maintained at 28 1oC and 80 5 per cent relative humidity in enamel trays (32x32 cm)
employing dechlorinated water. Hardness and pH of the rearing water were measured
periodically and maintained at permissible range. The immatures were provided with finely
ground dog biscuit and yeast in the ratio of 3:1. Early fourth instar larvae were used for
bioassay and biochemical studies.
Insecticides and chemicals: Deltamethrin: (S)-- Cyano-3- phenoxybenzyl cis-(IR)-3 (2,2dibromovinyl)-2, 2-dimethyl cyclopropane carboxylate, was obtained from M/s AgrEvo India
Ltd., Mumbai and permethrin [(3 phenoxyphenyl) methyl () cis, trans-3 (2,2dichloroethenyl)-2,2-dimethylc yclopropane - carboxylate] from M/s Rousell Ucalf Ltd, UK.
Fast
blue
RR
salt
[4-Benzoylamine-2,5-dimethoxybenzedene-diazonium chloride hemi(zinc chloride) salt] was
procured from Sigma Chemicals (USA). All other chemicals employed for biochemical assay
and gel electrophoresis were of analytical grade (AR), procured from M/s. SRL Chemicals,
Mumbai and M/s. SD Fine Chemicals, Mumbai.
The biochemical assays were carried out using Systronics (India) UV spectrophotometer.
The gel electrophoresis was performed with Broviga (Chennai, India) vertical slab (10x11cm)
gel electrophoretic system. The documentation of the gel and densitometric graphs were
carried out using UV doc (UK) automated gel documentation system. Centrifugation was
carried out in Hermle (Germany) ultracentrifuge.
Larval bioassay: Insecticide susceptibility tests were performed on the early fourth instar
larvae, following the standard WHO13 procedure. Technical grade pyrethroids were used to
prepare different concentrations of insecticides employing ethanol as solvent. A minimum of
five different concentrations (deltamethrin 0.002-0.03mg/l and permethrin 0.04-5 mg/l), were
used to get the mortality up to 90 per cent after 24 h exposure. Each concentration had at
least three sets of replicates comprising 25 early fourth instar larvae. Control experiments
were done by adding 1 ml of ethanol to 249 ml of dechlorinated water.

Biochemical analysis:
(i) Sample preparation Batches of 25 early fourth instar larvae were homogenized
individually in 200l of double distilled water using a glass homogeniser immersed in ice
cubes. The homogenates were transferred to a 1ml Eppendorf tubes and spun at 10,000g for
3min at 4o C in an ultracentrifuge. The supernatant was used as crude enzyme extract for
esterase assay. However, for GST and G6PD assays, 100 l homogenate was transferred
separately into two Eppendorf tubes and spun at 10,000g for 3 min and at 860g for 30 min
respectively at 4oC. The supernatant was used as enzyme samples. Twenty five replicates
were used for each enzyme assay.
(ii) Esterase assayThe carboxyl esterase assay was carried out following the method of
Penilla et al14 with a slight modification. To 200l of each replicate of the homogenized
sample, 2ml of the / naphthyl acetate solution was added. The enzyme reaction was
allowed to run for 30 min at room temperature. To this reaction mixture 500 l of the fast
blue stain solution containing 22.5 mg fast blue RR salt in 2.25 ml distilled water and
5 per cent SDS in 0.2 M phosphate buffer (pH 7.2) was added. The fast blue helps to stain the
mixture and SDS in it will stop the reaction. Replicate blanks contained 200 l of distilled
water, 2 ml of / naphthyl acetate solution and 500 l of the fast blue stain solution.
Enzyme activity was read at 570 nm. Absorbance level for individual larva was compared
with the help of a standard curve of absorbance for known concentrations of and naphthol
respectively. The enzyme activity was expressed as g of / naphthol produced / min / mg
larval protein.
(iii) Glutathione S-transferase (GST) assayGST activity was estimated following the
procedure of Habig et al15 with slight modification. To 100 l of the larval homogenate 0.1
ml of 30mM chlorodinitrobenzene (CDNB) was added and the volume adjusted to 2.9 ml with
distilled water. After preincubation of the reaction mixture for 5 min at 37oC, 0.1 ml of
30mM reduced glutathione (GSH) was added. The change in the absorbance level was noted
at 340 nm for 5 min after every 30 sec in the UV spectrophotometer. Reaction mixture
without the enzyme was used as blank.
(iv) Glucose-6- phosphate dehydrogenase assay The G6PD activity was estimated
according to the modified method of Kumar et al10. One hundred micro litre of the larval
homogenate was taken in 1 ml microcuvette and 0.4ml of 0.15M Tris HCl (pH7.5)
containing 3.8x 10-14M NADP, 0.01 ml of 0.3M MgCl2 and 0.5 ml of 0.03M D-glucose 6
phosphate was added to it. The reaction mixture in the microcuvette was read at 340 nm in an
UV spectrophotometer. The amount of NADP reduced ( mole/mg larval protein) was
calculated from the molar extinction coefficient, 6.22106.
(v) Protein assay Quantification of the total protein of the early fourth instar larva was
done according to the standard procedure of Lowry et al16. A known concentration of bovine
serum albumin (BSA) was used as the standard protein.
Polyacryla mide gel electrophoresis (PAGE): Continuous nondenaturing polyacrylamide gel
electrophoresis (Native PAGE) was conducted to detect the carboxylesterase profiles of the
two malaria vectors according to the procedures of Gopalan et al17 and Bonning et al18. A
small vertical slab (1011 cm) gel electrophoresis system was used for the present
experiment. Individual larval samples were homogenized using 30 l of 40 per cent sucrose
solution and spun at 2400g for 5 min at 4oC in an ultracentrifuge. An equal volume of
supernatant sample was loaded to each well. Gels were initially run at 40 V for nearly 30 min
and then increased to 60V for about 4 h. The separated gel was immersed in 0.1 M phosphate
buffer containing / naphthyl acetate solution (pH 6.0 at 37oC) for 10 min and then stained
with 0.025 per cent fast blue RR salt in order to trace the isozyme patterns. The gels were
washed with distilled water and preserved in 7 per cent acetic acid for further analysis.

The LC50 and LC90 values for the two pyrethroids were calculated by Probit analysis 19. The
per cent mortality was corrected using Abbots formula 20 when the control mortality was less
than 20 per cent. The difference in enzyme activities in the two species were analyzed using
Students t test.
Results
The results of the bioassay with the two pyrethroids are provided in Table I. The LC50 and
LC90 values for the two species indicate differential tolerance levels. A two-fold increase was
seen in the tolerance level to deltamethrin in A. stephensi compared to A. culicifacies. In
contrast
A. culicifacies showed a 18.76 fold increase in tolerance against permethrin compared to
A. stephensi. Further, deltamethrin was found to be 12.5 and 47 times more effective than
permethrin in A. stephensi and A. culicifacies respectively. Per cent mortality data for the two
species against various concentrations of the two pyrethroids are depicted in Fig. 1.

Table I. Susceptibility status of A. stephensi and A. culicifacies, against deltamethrin and permethrin at Mysore
Species

Chemicals
used

A. stephensi
Deltamethrin

A. culicifacies

A. stephensi

Permethrin
A. culicifacies

Concentrations in
mg/1

Per cent
mortality

0.002

34

0.004

52

0.008
0.01

66
76

0.03

88

0.002

52

0.004

70

0.006
0.008

80
86

0.01

96

0.04

36

0.06

58

0.08
0.2

72
84

0.4

94

0.9

50

1.0

56

2.0
3.0

62
74

4.0

82

LC50 *

0.00418

Fiducial limits**
LCL
UCL

0.0030

0.0051

LC90 *
mg/l

0.03358

Fiducial limits**Fold
LCL
UCL

0.0186

0.0606
2.10

0.00204

0.0014

0.0029

0.00893

0.0065

0.0123

0.05032

0.0382

0.0662

0.25703

0.1846

0.4546
18.76

0.93803

0.6477

1.3298

6.68975

4.0170

5.0
92
LCL, lower concentration limit; UCL, upper concentration limit; *by probit analysis; **at 95% confidential limit
no.

increase
in LC50

of larvae used for each concentration and respective replicates = 25. Control mortality in all experiments = zero

11.115

Fig. 1. Bioassay profile of A. stephensi and A. culicifacies larvae treated with deltamethrin and permethrin
Anst/del, A. stephensi treated with deltamethrin; Anst/per, A. stephensi treated with permethrin; Ancu/del, A.
culicifacies treated with deltamethrin; Ancu/per, A. culicifacies treated with permethrin.

The spectrophotometric analysis of the carboxylesterases revealed significantly higher

levels of A-esterase and GST activity (P<0.05) in A. stephensi compared to A. culicifacies. In
contrast B-esterase and G6PD activity were found to be significantly high (P<0.05) in A.
culicifacies (Table II). Total larval protein estimated was significantly higher (P<0.05) in A.
stephensi.

Table II. Enzyme and protein variation between A. stephensi and A. culicifacies
Species
A. stephensi
A. culicifacies

A-esterase*

B-esterase*

GST**

0.54660.03

0.31250.003

12.61850.004

0.3020.05

1.440.047

11.07950.07

G6PD** Total protein***

0.00910.002 0.0530.046
0.01310.0004

0.0370.006

Values are mean SD. Total number of larvae used for enzyme = 25. P < 0.05 compared to A. culicifacies
* activity expressed as g napthol produced/min/mg larval protein
** activity expressed as Mole/min/mg larval protein
*** mg/individual larva

A-esterase

A.stephensi

A.culicifacies

B-esterase

A.stephensi

A.culicifacies

Fig. 2. Isoenzyme bands of A-esterase and B-esterase with differential electrophoretic mobility and intensity in A.
stephensi & A. culicifacies.

Fig. 3. Densitometric profile of A-esterase and B-esterase showing differential mobility and activity in A. stephensi
and A. culicifacies.

Electrophoretic profiles of A-esterase and B-esterase in the two species are shown in Fig. 2.
A-esterase in both the species showed three bands, which were clearly demarcated in the
densitometric scan. However the two bands in case of A. stephensi had more intensity,
displaying higher peaks in densitometric graph as well, supporting the higher activity (Fig. 3).
Similarly, in case of B-esterase there were three darkly stained bands followed by a lighter
band in A. culicifacies in contrast to A. stephensi which showed one dark band followed by
two light ones. This was further evidenced by the densitometric graph (Fig. 3) depicting
differential peaks. In addition, the mobility of these bands were varied for the respective
enzymes indicating the probability of genetic variation with respect to A-and B-esterase
alleles between the two species at Mysore.
Discussion
Chemical insecticides play a major role in vector control. However, the continuous and
indiscriminate use of insecticide in a population will lead to the development of physiological
resistance in the insects. The present results clearly suggest the differential effect of the same
class of insecticides on two species belonging to different habitats. Earlier, Revanna and
Vijayan21, and Vijayan et al22 had shown differential susceptibility status in a few species of
Culex mosquitoes from the same district. Bansal and Singh23 studied the susceptibility levels
of some anophelines, such as A. culicifacies, A.annularis, A. stephensi and A. subpictus from
Rajasthan, India and found that all these species were resistant to DDT and dieldrin, but were
susceptible to fenitrothion and permethrin. This is in contrast to the present observations at
Mysore with regard to the differential impact of pyrethroids in anophelines. Chandre et al24
tested the field samples of A. gambiae from Cote d Ivoire using five pyrethroids and found
them to be cross resistant. The present study has also revealed a significant two-fold increase
in the A-esterase activity in A.stephensi, which could be correlated with the two-fold
increase in the deltamethrin tolerance compared to A. culicifacies. However, the tested
enzymes have not shown increased activity to the extent of increased LC50 level in A.
culicifacies against permethrin. But there was a significant increase in B-esterase activity

(P<0.05) in A. culicifacies, which could be correlated with the permethrin tolerance status.
This may suggest species specific biochemical mechanism for detoxification.
The increased detoxification is a common mechanism of resistance to pesticides25. In
Culex pipiens,such a mechanism is often involved in resistance to organophosphates.
However, the low levels of organophosphate and pyrethroid resistance could be conferred by
either the elevated esterase or monooxygenase enzymes14. Elevation in the A- and B-esterases
are commonly found in the organophosphate resistant strains 26. In some cases, overproduction
is seen only in one type of esterase, i.e. esterase-A or esterase-B27, in others both the types are
overproduced simultaneously 28,29. In the present study we have also observed an increased Besterase activity in A. culicifacies, which is relatively tolerant to permethrin. A. culicifacies
being a rural vector that breeds in the paddy fields and irrigation canals might have been
exposed to various agricultural organophosphate pesticides, prior to the introduction of
pyrethroids. This might have triggered the overproduction of B-esterase through genetic
selection/gene amplification in the field. Therefore, increased tolerance to permethrin in A.
culicifacies might be a result of organophosphate cross-resistance, resulting in elevated Besterase activity. The isozyme profile of B-esterase has further supported this. In addition,
the significantly higher level of G6PD might be the result of recently introduced agricultural
pyrethroids leading to a higher level of monooxygenase activity. Thus the elevated pyrethroid
tolerance
in
A. culicifacies might be because of increased level of esterase and monooxygenase activity.
Several earlier workers stated that pyrethroids do not serve as substrates for GST11,12. So
other enzymes systems have been proposed as being responsible for conferring metabolic
detoxification of pyrethroids. However, induction of GST activity has been reported not only
after exposure to organophosphates and organochlorides but also against pyrethroid 30,31.
Reports correlating the elevated levels of GST with resistance to pyrethroids do exist for
Tribolium castaneum11 and Aedes aegypti12. Therefore, the significantly higher level of GST
activity might play a role in pyrethroid tolerance in A. stephensi along with A-esterase
activity.
As the mosquito populations of Mysore area are exposed to different class of insecticides
in their respective habitats, tests conducted on the tolerance level and the enzymes involved in
detoxification mechanisms are important. The present study in respect of bioassay and
biochemical estimations have revealed the probable mechanism developed by the local
malaria vectors to combat the insecticides. Further, the study of enzymes involved in the
detoxification mechanism will help us to introduce appropriate control measures such as
combinations of insecticides and synergists for a better and effective control programme
during outbreaks of malaria.
Acknowledgment
The authors thank the Chairman, Department of Studies in Zoology, University of Mysore, for the facilities
and Prof.Vishnu Hebbar, Department of Studies in Statistics, University of Mysore, for the help in statistical
analysis.

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Reprint requests : Dr V.A. Vijayan, Reader, Department of Studies in Zoology, University of Mysore
Manasagangotri, Mysore 570006, India