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INSECTICIDE RESISTANCE IN LEAFHOPPER, AMRASCA BIGUTTULA
BIGUTTULA (ISHIDA) OF MAJOR COTTON GROWING DISTRICTS OF
KARNATAKA, INDIA
D. Sagar, R. A. Balikai and B. M. Khadi1
Department of Ag. Entomology, 1Directorate of Research, University of Agricultural Sciences, Dharwad -580 005, India
e-mail: garuda344@gmail.com
(Accepted 5 October 2013 )
ABSTRACT : Laboratory experiments were carried out during 2011-12 and 2012-13 to study the toxicity of
organophosphates against leafhopper population of major cotton growing districts of Karnataka, India. Raichur and
Yadgir districts leafhopper population recorded higher LC50 values to almost all insecticides bioassayed, viz.,
monocrotophos, acephate, oxydemeton methyl and dimethoate. Haveri, Dharwad and Belgaum districts leafhopper
population recorded medium range LC50 values and Mysore district leafhopper population recorded lower LC50
values to the insecticides bioassayed. Relatively more mixed function oxidases activity values corresponding to the
higher LC50 values of organophosphates in Raichur and Yadgir districts leafhopper population indicated the role of
mixed function oxidases enzyme in detoxification of insecticides and development of insecticide resistance in cotton
leafhopper against the commonly used insecticides.
Key words : Toxicity, LC50, mixed function oxidases, insecticide resistance, Amrasca biguttula biguttula.
INTRODUCTION
Cotton (Gossypium spp.) popularly known as “White
Gold” is a major commercial crop unanimously designated
as “King of Fibres” and has a global significance which is
grown for its lint and seed. After the introduction of Bt
cotton, there was a check only to the bollworm complex,
but not to the sucking pest population (Mohan and Nandini,
2011). Among the sucking pest complex of Bt cotton, the
cotton leafhopper, Amrasca biguttula biguttula (Ishida)
(Homoptera: Cicadellidae) is an alarming pest causing both
quantitative and qualitative losses.
Inspite of repeated use of insecticides, it is becoming
difficult to manage this pest and control failures have been
experienced by the cotton growers at times. Though control
failure may be due to many factors, one of the major
factors is the development of resistance to insecticides
(Jeya Pradeepa and Regupathy, 2002). The development
of resistance against insecticides by insects is either due to
pre-adaptation or post adaptation process. In pre-
adaptation, the genetic differences are already present in
the insect population and insecticides acts as selective agent
by eliminating the susceptible population favouring the
resistant genotype or population. While in post adaptation,
the resistance is physiological by the presence of detoxifying
enzymes which breakdown the toxicant much faster
(Srivastava and Saxena, 2000).
As there is little documented evidence regarding
insecticide resistance acquired by the leafhopper to
commonly used insecticides, there is a need to assess the
degree of resistance acquired by the pest following
conventional bioassay to advanced biochemical analysis.
Keeping these points in view, the present study was
undertaken.
MATERIALS AND METHODS
The present investigations were undertaken during
2011-12 and 2012-13 in the Department of Agricultural
Entomology, UAS, Dharwad. The population of cotton
leafhopper nymphs was collected from farmer’s fields of
Dharwad, Belgaum, Haveri, Mysore, Raichur and Yadgir
districts during early morning hours along with leaves and
was used for bioassay. The leafhopper nymphs collected
from each location were exposed to graded concentrations
of test insecticides viz., monocrotophos 36 SL, acephate
75 SP, oxydemeton methyl 25 EC and dimethoate 30 EC.
Each replication consisted of 10 nymphs and there were
three replications for each concentration of test insecticide.
A control was maintained at each time of experimentation
where in the leaves were dipped in the water. Bioassays
were conducted following the procedure based on the
standard Bemesia tabaci susceptibility test Insecticide
Resistance Action Committee (IRAC) method No. 8
developed and recommended by the IRAC with slight
modification.
The mortality of leafhopper was recorded at 48 hours
Biochem. Cell. Arch. Vol. 13, No. 2, pp. 261-265, 2013 ISSN 0972-5075
after treatment, moribund leafhopper nymphs which did
not respond to probing were considered as dead. Percentage
of mortality for each concentration of test insecticide and
control were computed and corrected per cent mortality
was calculated by Abbot’s formula (Abbott, 1925). The
corrected mortality data of each test insecticide of each
location was subjected to probit analysis using EPA probit
analysis program used for calculating LC/EC values version
1.5 for calculation of LC50and LC90 for the leafhopper
populations of Dharwad, Belgaum, Haveri, Mysore,
Raichur and Yadgir districts of Karnataka to the test
insecticides used. The relative resistance ratio for each
insecticide and location was calculated using the formula
as given below.
Relative resistance LC50 of particular location leafhopper population
ratio (RRR) = ____________________________________________________________________________
LC50 of relatively susceptible location leafhopper population
Mixed function oxidases (MFO’s) activity assay
Biochemical basis of insecticide resistance in cotton
leafhoppers was determined by estimating the activity of
MFO enzyme. Randomly 80-120 nymphs of A. biguttula
biguttula were collected from Bt cotton fields of Dharwad,
Belgaum, Haveri, Mysore, Raichur and Yadgir districts of
Karnataka for estimation of MFO’s activity.
The estimation of mixed function oxidases activity
involved microsomes isolation followed by protein
estimation from microsomal fraction and measurement of
O-dealkylation of ethoxyresorufin by direct fluorometric
method. Microsomes were isolated following methodology
as described by Lee and Scott (1989). Protein estimation
was carried out by following the standard method as given
by Bradford (1976). The activity O-dealkylation of
ethoxyresorufin was determined by the by direct
fluorometric method as suggested by Prough et al (1978)
with slight modification. Mixed function oxidase activity
was presented as nanomoles of resorufin formed per
minute per milligram of protein.
Enzymatic activity ratio for mixed function oxidase
enzyme of each location leafhopper population was
calculated using the formula as given below.
Enzymatic activity in leafhopper population of particular location
Enzymatic = _____________________________________________________________________________________
activity ratio (EAR) Enzymatic activity in leafhopper population of relatively
susceptible location
The results of biochemical investigations were
correlated with the insecticide resistance pattern results of
cotton leafhopper population of major cotton growing
districts of Karnataka.
RESULTS AND DISCUSSION
Log dose probit assay was carried out for
monocrotophos, acephate, oxydemeton methyl and
dimethoate across six different geographic populations of
A. biguttula biguttula representing major cotton growing
ecosystem of Karnataka. For calculating the relative
resistance ratio and enzymatic activity ratio, Mysore district
leafhopper population was considered as relatively
susceptible, as this population recorded lower LC50 values
for the insecticides bioassayed and lower mixed function
oxidases activity among six districts leafhopper population.
Monocrotophos 36 SL: Among the chemical
insecticides bioassayed, monocrotophos registered higher
LC50 values (225.66 and 262.10 ppm) during both the
years. The LC50 values of monocrotophos against the
leafhopper population of major cotton growing districts of
Karnataka varied from 225.66 (Raichur) to 80.30 ppm
(Mysore) during 2011-12 and 262.10 (Raichur) to 89.22
ppm (Mysore) during 2012-13 (Table 1). Higher LC50
and LC90 values to monocrotophos during both the years
were noticed in the leafhopper population of Raichur
followed by population from Yadgir, Dharwad, Belgaum,
Haveri and Mysore. The relative resistance folds as
compared to Mysore district leafhopper population varied
from 2.81 (Raichur) to 1.34-folds (Haveri) during 2011-
12 and 2.93 (Raichur) to 1.40-folds (Haveri) during 2012-
13. Similar studies were done by Chalam and Subbaratnam
(1999) and reported that the leafhopper population of
Guntur district was relatively more resistant than the
populations of Warangal and Kurnool districts to
endosulfan, monocrotophos and cypermethrin.
The increased LC50 and LC90 values for
monocrotophos in the leafhopper population of major
cotton growing districts of Karnataka including Mysore
location is because of intensive use of monocrotophos by
the cotton growers in state from the past three decades.
The results of the present findings corroborate with the
reports of Jhansi et al (2004) who opined that indiscriminate
use of insecticides on cotton for the past two decades in
the state have created undue selection pressure on insects
resulting in acquisition of higher degree of insecticide
resistance.
Acephate 75 SP : The LC50 and LC90 values along
with relative resistance ratio to acephate was highest in
the leafhopper population of Yadgir, followed by Raichur,
Dharwad, Belgaum and Haveri, while lowest was observed
in the population from Mysore during both the years. The
LC50 values of acephate against the leafhopper population
of major cotton growing districts of Karnataka varied from
183.34 (Yadgir) to 78.14 ppm (Mysore) during 2011-12
and 209.48 (Yadgir) to 84.16 ppm (Mysore) during 2012-
13 (Table 2). It was reported that LC50 values of acephate
ranged from 0.0008 (Bhatinda) to 0.1622 ml/l (Nagpur)
262 D. Sagar et al
Table 1 : Toxicity of monocrotophos 36 SL against Amrasca biguttula biguttula in major cotton growing districts of Karnataka.
2011-12 2012-13
Location LC50 Fiducial limits LC90 Relative LC50 Fiducial limits LC90 Relative
(ppm) Lower Upper (ppm) resistance (ppm) Lower Upper (ppm) resistance
ratio ratio
Dharwad 153.69 112.51 195.67 793.79 1.91 181.37 137.78 227.96 882.12 2.03
Belgaum 129.50 93.92 164.56 600.99 1.61 149.33 109.74 189.93 736.64 1.67
Haveri 108.16 75.87 139.27 494.47 1.34 125.59 88.24 162.11650.82 1.40
Mysore 80.30 54.47 104.77 321.40 1.00 89.22 58.55 118.26 434.29 1.00
Raichur 225.66 174.79 285.78 1152.07 2.81 262.10 209.96 326.73 1132.64 2.93
Yadgir 195.04 149.77 244.71 941.86 2.42 231.40 182.66 288.52 1044.96 2.59
Table 2 : Toxicity of acephate 75 SP against Amrasca biguttula biguttula in major cotton growing districts of Karnataka.
2011-12 2012-13
Location LC50 Fiducial limits LC90 Relative LC50 Fiducial limits LC90 Relative
(ppm) Lower Upper (ppm) resistance (ppm) Lower Upper (ppm) resistance
ratio ratio
Dharwad 124.65 83.66 159.53 393.78 1.59 143.24 101.04 279.50 451.57 1.70
Belgaum 113.29 76.09 144.80 324.39 1.44 126.16 85.84 160.54 389.49 1.49
Haveri 95.91 58.33 127.28 284.37 1.22 110.25 71.71 142.82 331.37 1.31
Mysore 78.14 41.40 107.86 221.95 1.00 84.16 46.20 115.66 256.79 1.00
Raichur 158.82 116.69 195.48 485.07 2.03 187.83 145.67 225.50 543.94 2.23
Yadgir 183.34 140.48 221.39 545.72 2.34 209.48 165.97 249.05 604.44 2.48
Table 3 : Toxicity of oxydemeton methyl 25 EC against Amrasca biguttula biguttula in major cotton growing districts of Karnataka.
2011-12 2012-13
Location LC50 Fiducial limits LC90 Relative LC50 Fiducial limits LC90 Relative
(ppm) Lower Upper (ppm) resistance (ppm) Lower Upper (ppm) resistance
ratio ratio
Dharwad 88.26 61.95 113.11338.21 1.58 99.54 71.52 126.21 385.41 1.62
Belgaum 61.12 38.92 81.69 227.18 1.09 68.38 43.92 91.16 275.85 1.11
Haveri 74.75 49.96 97.95 291.65 1.37 84.36 57.63 109.48 340.56 1.37
Mysore 55.57 35.13 74.27 190.43 1.00 61.15 39.72 80.97 215.63 1.00
Raichur 106.59 77.79 134.00 405.46 1.91 119.91 88.43 150.31 482.68 1.96
Yadgir 91.19 64.04 116.86 357.95 1.65 103.43 73.65 131.74 426.59 1.69
(Anon., 2012). Similarly, Jeya Pradeepa and Regupathy
(2002) reported that the LC50 and LC95 values
of acephate for F1 and F6 generations varied from 114.79
to 46.02 and 3981.10 to 831.76 ppm, respectively.
The reasons attributed for higher LC50 and LC90
values in acephate might be due its intensive usage in
cotton growing ecosystem especially in Raichur and Yadgir
districts as a tank mixture along with monocrotophos and
imidacloprid (Sagar et al, 2013) and also due to cross
resistance development to organophosphates by the
leafhopper population.
Insecticide resistance in leafhopper 263
Oxydemeton methyl 25 EC : Among the
organophosphates bioassayed, oxydemeton methyl has
recorded relatively lower LC50 and LC90 values. The LC50
values of oxydemeton methyl to six different geographic
populations of A. biguttula biguttula varied from 106.59
(Raichur) to 55.57 ppm (Mysore) during 2011-12 and
119.91 (Raichur) to 61.15 ppm (Mysore) during 2012-13
(Table 3). Similar studies were carried out by Kalra et al
(2001) to study the toxicity of various insecticides viz.,
oxydemeton methyl, dimethoate and monocrotophos
against the okra leafhopper and reported that LC50 values
were 0.126, 0.178 and 0.063 per cent, respectively.
Lower LC50 and LC90 values of oxydemeton methyl
might be due to reduced usage of oxydemeton methyl in
the cotton ecosystem in the recent past. Present reasoning
is in agreement with Forrester (1990) who noticed that
resistance levels rose when insecticides were used, but
fell significantly when they are with held. Thus, pesticides
are creating high pressure for evolution of resistant
genotypes.
Dimethoate 30 EC: The LC50 values for dimethoate
against cotton leafhopper population of major cotton
growing districts of Karnataka during 2011-12 and 2012-
13 varied from 127.23 (Dharwad) to 66.47 ppm (Mysore)
and 139.72 (Dharwad) to 72.17 ppm (Mysore), while
computed relative resistance ratio at LC50 in comparison
with relatively susceptible strain (Mysore district leafhopper
population) varied from 1.91 to 1.16-folds and 1.93 to
1.15-folds (Table 4). Jeya Pradeepa and Regupathy (2002)
reported that the LC50 and LC95 values of dimethoate for
F1 and F7 generations varied from 153.90 to 41.03 and
2722.70 to 812.83 ppm, respectively.
In comparison to monocrotophos and acephate, the
usage of dimethoate in cotton growing ecosystem of
Karnataka is less; because of this reason dimethoate has
recorded lower LC50 and LC90 values as compared to
monocrotophos and acephate. These findings are in
agreement with Kshirsagar et al (2012) who reported that
continuous selection pressure of neonicotinoids against the
cotton leafhopper in the last decade forced the population
towards relatively resistance against the neonicotinoids and
comparatively less use of dimethoate during the same
Table 4 : Toxicity of dimethoate 30 EC against Amrasca biguttula biguttula in major cotton growing districts of Karnataka.
2011-12 2012-13
Location LC50 Fiducial limits LC90 Relative LC50 Fiducial limits LC90 Relative
(ppm) Lower Upper (ppm) resistance (ppm) Lower Upper (ppm) resistance
ratio ratio
Dharwad 127.23 97.07 153.83 363.71 1.91 139.72 107.43 168.40 419.03 1.93
Belgaum 77.66 52.97 98.02 192.66 1.16 83.33 57.75 104.81 215.94 1.15
Haveri 89.98 63.80 112.24 237.94 1.35 97.32 70.74 120.17 258.83 1.34
Mysore 66.47 40.65 87.13 170.77 1.00 72.17 46.61 93.02 185.78 1.00
Raichur 108.08 79.57 132.79 303.44 1.62 121.22 51.77 174.23 343.85 1.64
Yadgir 98.61 71.68 121.78 265.31 1.48 108.08 79.57 132.79 303.44 1.49
Table 5 : Protein content and mixed function oxidases activity in field population of cotton leafhopper in major cotton growing
districts of Karnataka.
Sl. No. District Protein content in microsomes Mixed function oxidases activity Enzymatic activity ratio
(mg/ml of microsomal fraction) (nmol/min/mg protein) (Fold increase in mixed function
(Mean ± Standard deviation) oxidases activity)
1Dharwad 1.76 2.05 ± 0.03 2.11
2Belgaum 2.29 1.38 ± 0.009 1.42
3Haveri 1.34 1.99 ± 0.01 2.05
4Mysore 1.31 0.97 ± 0.04 1.00
5Raichur 1.44 2.22 ± 0.05 2.28
6Yadgir 1.20 2.09 ± 0.06 2.15
264 D. Sagar et al
period made the leafhopper population relatively susceptible
to dimethoate. For insecticidal resistant management
strategies, neonicotinoids can be rotated with dimethoate
or any conventional insecticides to delay the process of
resistance development in cotton leafhopper.
Mixed function oxidases (MFO’s) activity : The
mixed function oxidase enzyme activity of field collected
cotton leafhopper population of major cotton growing
districts of Karnataka varied from 2.22 nmol/min/mg
protein (Raichur district) to 0.97 nmol/min/mg protein
(Mysore) (Table 5). Enzymatic activity ratio of leafhopper
population as compared to Mysore district leafhopper
population varied from 2.28 (Raichur) to 1.42 folds
(Belgaum). Higher mixed function oxidase enzyme activity
and enzymatic activity ratio was noticed in the leafhopper
population of Raichur followed by the population from
Yadgir, Dharwad, Haveri and Belgaum while, lower mixed
function oxidase enzyme activity was noticed in Mysore
leafhopper population.
The increased MFO’s activity in field collected
leafhopper population indicated the role of mixed function
oxidase in detoxification of insecticides resulting in the
development of insecticide resistance against the commonly
used insecticides, which is evident from the inventory of
insecticide resistance results. The insecticide resistance
results are in association with the results of biochemical
basis of insecticide resistance and the present results are
in line with the reports of Chalam et al (2001) who reported
that MFO’s played a predominant role in
imparting resistance to cypermethrin and monocrotophos,
which was indicated by high synergistic ratios. On the
other hand, in endosulfan and phosphamidon, MFO acted
as one of the mechanisms of resistance, which was evident
in the low to moderate synergistic ratios. In leafhopper,
MFO’s plays a predominant role in imparting resistance
to insecticides (Regupathy and Ayyasamy, 2004).
CONCLUSION
The present study concludes that organophosphates
should be rotated with insecticides having IGR action or
alternate chemistry and any conventional insecticides to
delay the process of resistance development in cotton
leafhoppers. Mixed function oxidase enzyme activity plays
a major role in development of insecticide resistance in
cotton leafhopper against the commonly used insecticides.
ACKNOWLEDGEMENT
With utmost pleasure and sincerity, the senior author
thankfully acknowledge whole heartedly INSPIRE
PROGRAM, Department of Science and Technology,
Ministry of Science and Technology, Government of India
for providing financial assistance by awarding INSPIRE
fellowship to pursue Ph. D. in Agricultural Entomology at
the University of Agricultural Sciences, Dharwad,
Karnataka, India.
REFERENCES
Abbott W S (1925) A method of computing of effectiveness of an
insecticide. J. Econ. Entomol. 18, 256-267.
Anonymous (2012) Annual Report (2011-12), Central Institute for
Cotton Research, Nagpur, Maharashtra, India, pp. 7-11.
Bradford M M (1976) A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principles of protein-
dye binding. Anal. Biochem. 72, 248–254.
Chalam M S and Subbaratnam G V (1999) Insecticide resistance in
cotton leafhopper, Amrasca biguttula biguttula (Ishida) in Andhra
Pradesh. Pest Manage. Econ. Zool. 7, 105-110.
Chalam M S V, Subbaratnam G V and Rao G R C (2001) Role of mixed
function oxidases in imparting resistance to the cotton leafhopper,
Amrasca biguttula biguttula (Ishida). Pest Manage. Econ.
Zool. 9(1), 49-53.
Forrester N W (1990) Designing, implementing and servicing an
insecticide resistance management strategy. Pestic. Sci. 28,167-
179.
Jeya Pradeepa S and Regupathy A (2002) Generating base line data for
insecticide resistance monitoring in cotton leafhopper, Amrasca
devastans (Distant). Resistant Pest Manage. Newslett. 11(2),
4-5.
Jhansi K, Radhika P and Subbaratnam G V (2004) Insecticide resistance
in insect pests of cotton in Andhra Pradesh. Paper presented in:
Int. Symp. on “Strategies for Sustainable Cotton Production
– A Global Vision” - Crop Protection, 23-25, November 2004,
University of Agricultural Sciences, Dharwad, Karnataka, India.
pp.198-200.
Kalra V K, Singh Ram, Saini R K, Rohilla H R, Jaglan R S, Chauhan R
and Sharma S S (2001) Occurrence of insecticide resistance in
leafhopper, Amrasca biguttula biguttula (Ishida) on Okra. J.
Ent. Res. 25(4), 263-265.
Kshirsagar S D, Satpute N S and Moharil M P (2012) Monitoring of
insecticide resistance in cotton leafhopper, Amrasca biguttula
biguttula (Ishida). Ann. Pl. Protec. Sci. 20(2), 283-286.
Lee S S T and Scott J G (1989) An improved method for preparation,
stabilization and storage of housefly (Diptera: Muscidae)
microsomes. J. Econ. Entomol. 82(6), 1559-1563.
Mohan S and Nandini S (2011) A promising entry for cotton leafhopper.
Pestology 35(6), 11-13.
Prough R A, Burke M D and Mayer R T (1978) Direct fluorometric
methods for measuring Mixed-Function Oxidase activity. Methods
in Enzymology 52, 372-377.
Regupathy A and Ayyasamy R (2004) Insecticide resistance in sucking
pests of cotton and resurgence. In: Proc. Int. Symp. on
“Strategies for Sustainable Crop Production-A Global
Vision”- Crop Protection, 23-25, November 2004, University of
Agricultural Sciences, Dharwad, Karnataka, India. pp. 42.
Sagar D, Balikai R A, Patil R R, Udikeri S S and Bheemanna M (2013)
Insecticide usage pattern in major Bt cotton growing districts of
Karnataka, India. J. Exp. Zool. India 16(2), 461-466.
Srivastava R P and Saxena R C (2000) A Textbook of Insect
Toxicology. Himanshu Publications, New Delhi, p.166.
Insecticide resistance in leafhopper 265
