Pesticide detoxifying mechanism in field population of Spodoptera litura (Lepidoptera: noctuidae) from South India

The army worm Spodoptera litura (fabricius) is one of the most damaging pests of ground nut and castor in India. We determine the susceptibility of S. litura reared on a castor leaves, to three insecticides (Temephos, Dichlorvos, and lambdacyhalothrin) under laboratory conditions, associated with the enzyme activities of acetylcholinesterase (AChE), carboxylesterase (CarE), glutathione S-transferase (GST) and glutathione Sreductase (GSH) in this larvae. Among treatments Dichlorvos showed high mortality then lambda cyhalothrin and Temephos in whole body of the insect. The AChE activities of Spodoptera litura treated with lambda cyhalothrin, Temephos at 10ppm were almost 1.5 fold high. The CarE activity at 10ppm was almost 3 fold high. The GSH and GST activity were low then control (0.2 ± 0.4, 1.5 μM mg protein min1). The present study suggests that esterase and acetylcholine esterase may play a role in detoxification of synthetic pyrethroid and organophosphates in Spodoptera litura from South India.


Spodoptera litura (fabricius) (Lepidoptera:
Noctuidae) is a polyphagous insect pest (Holloway, 1989).It is an indigenous pest of a variety of crops in South Asia and was found to cause more than 26-100% yield loss in groundnut (Dhir et al., 1992).It is variously known as Indian Leaf worm and tobacco cutworm (Rao et al., 1993).The control of this pest has depended on application of chemical insecticides; as a result which may develop resistance (Armes et al., 1997;Kranthi et al., 2002) and subsequent control failures.Insecticide resistance in lepidopteran population involves mainly two mechanisms, enhanced detoxification, and target site insensitivity.In many cases detoxification enhancement causes metabolic resistance and involves three major groups of enzymes like esterases, glutathione complex and cytochrome P450 (Hemingway, 2004).
Various detoxifying enzymes confer insecticide resistance to insects.In insect AChE is the major target for organophosphate (OP) and carbamates insecticides, which inhibit enzyme activity by covalently phosphorylating or carbamylating the serine residues with in active site (Corbett, 1974).Resistance to organophosphate and carbamates pesticides has been reported in Helicoverpa armigera and the army worm Spodoptera litura in India (Kranthi et al., 2001).
Insecticide detoxifying enzyme, glutathione s-transferase (GST) belongs to a protein family involved in detoxification of xenobiotics, protection from oxidative damage and intracellular transport of hormones, endogenous metabolites and exogenous chemicals.
Carboxylesterase (CarE) is the cytoplasmic enzyme which plays an important role in neutralizing xenobiotics (Tang et al., 1990).These esterases detoxify organophosphate (OP) and carbamates (CB) pesticides and synthetic pyrethroid (SPs) by two main ways, hydrolysis of the ester bond and binding of the pesticide (OP) to the active site of CarE (Crow et al., 2007).
The goal of this present study is to understand the detoxification mechanism of Spodoptera litura in field population from South India.

Insects
Spodoptera litura populations used in this study were obtained from groundnut field at Salem district, Tamil Nadu (India) during 2009-2010; the larvae were periodically collected, with no contact with any kind of insecticide.Samples were brought to the laboratory, caster leaves were replaced on alternate days as a food source.Larvae was maintained at 26±1°C and 55±10 %RH with 16:8(L: D) photoperiod.

Bioassay
Bioassays were performed on 3 rd instars larvae using the standard topical application procedure followed (Robertson and Preisler, 1992).Three pesticides were used namely: Temephos 65% EC (emulsifiable concentrate), Dichlorvos 50% EC, and λ-cyhalothrin 5% EC purchased from commercial suppliers in Tamilnadu.Different concentration of pesticide was prepared and control with water.After treatment the larvae was released in 9mm diameter Petri plate.Six third instar larvae were released and each treatment as replicated three times.Observations for mortality were recorded for 24hours.Larvae were considered dead if unable to move in a coordinated way when prodded with a fine haired brush.The corrected mortality was calculated by using (Abbott's, 1925) formula data were subjected to probit analysis as described by (Finney, 1971).

Sample preparation
Intoxicated insects from selected bioassay experiments were weighed, were rinsed with acetone (2 X 5ml) to remove surface residues, the whole larval homogenate was prepared by grinding twenty 3 rd instar larvae in ice-cold 50mM sodium phosphate buffer (pH 7.5).The homogenate was centrifuged at 10, 000 rpm for 20 min at 4ºC.The supernatant were stored at -20ºC and used as enzyme source.The protein content of the enzyme extract was estimated by using the (Lowry et al., 1951) method, using bovine serum albumin (BSA) as a standard protein to construct the standard curve.

Enzyme assays Carboxylesterase Assay
Carboxylesterase activity was measured by the method of (Kranthi, 2005).100µl of enzyme solution from untreated control (water) and treated larvae were added to the tubes containing (100µl 0.3 mM α-napthyl acetate as a substrate, 4.8ml of 40mM PB pH 6.8) was added to the test tubes and incubated in dark for 20 minutes at room temperature.After gentle shaking, 1ml of staining solution (1% fast blue BB salt in phosphate buffer [40mM pH 6.8] with 5% sodium dodecyl sulphate (SDS) was added to each tube and incubated at 20ºC for 30 minutes the absorbance was recorded at 590 nm.The enzyme activity was calculated from α-napthol standard curve.Each sample was measured in triplicate to minimize error.

Glutathione S-transferase Assay
Activity of Glutathione Stransferase (GST) was carried using the method of (Habig et al., 1974).Fifty micro liters of 50mM (CDNB) and 150µl of reduced glutathione (GSH) were added to 2.79 ml phosphate buffer (40mM pH 6.8).Ten microliters of enzyme stock was then added.The mixture were gently shaken and incubated for 2-3 minutes at 20ºC and then transferred in the sample cuvette slot of a UV spectrophotometer.The change in absorbance was measured at 340nm up to 5 min and the enzyme the enzyme activity in terms of µmol of CDNB conjugated min --1 mg of enzyme protein -- 1 was calculated using the extinction coefficient of 9.6 mM -1 cm -1.

Acetylcholinesterase Assay
Acetylcholine esterase (AChE) activity was measured using acetylcholine-iodide as a substrate according to (Ellman et al., 1961).Two hundred micro liters of enzyme stock and 100µl of (0.075 M acetylthiocholineiodide), 240µl of 0.1M phosphate buffer (pH 7.4) were added and incubated for 15 min at 27ºC, and then 500µl of 0.1M eserine was added and mixed.The change in absorbance was measured at 412 nm.

Glutathione S-reductase Assay
The activity of GSH was measured as per the method (Beutlar et al., 1963) with slight modification.Thirty microliters of enzyme stock and1.7 ml of phosphate buffer (pH 7.4) were added then the tubes were incubated for 3min at room temperature.After gentle shaking 100µl of 10mM 5, 5 dithiobis 2nitrobenzoic acid (DTNB), 50µl of 50mM reduced glutathione, 50µl of 4.3mM Nicotinamide adenine diphosphate (NADPH) was added then the mixture was Incubate for 2-3 minutes at 20ºC.The change in absorbance was measured at 412 nm.

Statistical analysis
The percentage of larval mortality was corrected and the data's were subjected to probit analysis.Variation in the activities of AChE, CarE, GST and GSH in each treatment and control were analyzed using Analysis of Variance (ANOVA) with Bonferronii multiple comparison tests.

Bioassay:
The toxicity of Spodoptera litura to organophosphate and synthetic pyrethroid is given in (Table 1).Among tested Dichlorvos shows high mortality (75%) at 9 ppm.Synthetic pyrethroid showed maximum mortality at 10ppm.Whereas Temephos at 31.6ppm.Care activity: Esterase activity was 3 fold higher in λ-cyhalothrin treatment as compared to dichlorvos treatment (500 µM mg protein -1 min -1) at 10ppm, whereas at 100ppm the significant reduction was recorded in control and other treatments (Fig 1).

AChE activity:
The activity of AChE was significantly increased at 10ppm in λcyhalothrin (1.5 µM mg protein -1 min -1) treatment.But the activity was decreased in both treatments at 100ppm as compared to control (Fig 3).

GSH activity
GSH activity was increased in Dichlorvos treatment at both concentrations 10ppm, 100ppm (0.2 ± 0.4 µM mg protein -1 min -1 ), however the activity decreased in all treatments as compared to control (Fig 4).

Insecticide
resistance in lepidopteran is a major concern throughout the world.Several species of lepidopteran viz Helicoverpa armigera, Spodoptera litura, and Plutella xylostella have been reported to have developed resistance to several classes of insecticides (Denholm et al., 1998).In the present study dichlorvos was found to be more toxic as compared to temephos and lambda-cyhalothrin.Similar observations were done by (Hassan et al., 2009).Bioefficacy of insecticides on Spodoptera exigua showed that Bifenethrin was highly effective as compared to other classes of insecticides (Jian-Long and Toscano 2005).
Carboxylesterases are important hydrolyses for the detoxification of various endogenous and exogenous substances.This large family of enzymes can be characterized based on their electrophoresis nobilities or inhibitor and  substrate specificities (Dauterman, 1985;Soderlund, 1997).Esterase activity in treated samples at lower pesticide concentration and control samples showed an increased activity.The activity was decreased high dose.This indicates that xenobiotic elicit increased activity of esterases at lower concentrations in Spodoptera litura.In supporting this suggestion (Yang et al., 2004;Gao et al., 1998;Xu et al., 1999) reported a high esterase activity is normally correlated with development of resistance in insects.Glutathione Stransferase activity was higher in control as compared to treatments, showing that organophosphate and synthetic pyrethroid interfere with GST mediated detoxification at low and high concentrations.GSH being a free radical scavenger is known to be an important component of pesticide detoxification in several insects (Buyukguzel, 2009).
However Decreased GSH content indicates that organophosphate and synthetic pyrethroid may consume GSH through detoxification reactions of glutathione-dependent enzymes (Jovanovic-Galovic et al. 2004).
Acetylcholinesterase is a key enzyme in the insect nervous system, terminating neurotransmission by the hydrolysis of the neurotransmitter acetylcholine.AChE is the target-site of inhibition by organophosphate and carbamate insecticides, and if this hydrolysis does not take place, build-up of acetylcholine occurs and leads to repeated firing of neurons and ultimately death of the insect (McCaffery, 1999;Gunning and Moores, 2001).
Most of the organophosphate induces excitotoxicity by phosphorylating the serine of active site in the AChE blocking the hydrolysis of the neurotransmitter acetylcholine (Hirvonen et al.1993).These metabolically activated intermediates inhibit not only AChE but also impair enzymatic and non enzymatic activity that actually functions to detoxify contaminants (Malik & Summer 1982).AChE activity was higher in pyrethroid treatment as compared to control and organophosphates; this suggests an increased expression level of AChE enzyme in response to pesticide exposure showed tolerance and increased AChE activity, target site insensitivity which seems to be dominant mechanisms conferring resistance in lepidopteran pest (Gao, 1992).The present study shows that esterase and acetylcholine esterase enzymes may play an important role in detoxification of synthetic pyrethriods and organophosphates in Spodoptera litura.Further studies are carrying out to understand the gene expression patterns in field population.

Figures:
Figures: Detoxification enzyme profile of 3 rd instar Spodoptera litura larvae exposed to organophosphate and synthetic parathyroid insecticides.

Fig. 1 :
Fig. 1: Esterase activity of 3 rd instar Spodoptera litura exposed to organophosphate and synthetic parathyroid.Mean (±SD) values are expressed based on One Way ANOVA (Bonferronii post-hoc test).Significant difference among treatments represented by *(P<0.05) with respect to control.

Fig. 2 :Fig. 3 :
Fig. 2: GST activity of 3 rd instar Spodoptera litura exposed to organophosphate and synthetic pyrethroid.Mean (±SD) values are expressed based on One Way ANOVA (Bonferronii post-hoc test).Significant difference among treatments represented by *(P<0.05) with respect to control.

Table 1 :
LD 50 Values of 3 rd Instar Spodoptera litura exposed to organophosphate and synthetic pyrethroid, after 24 hours observation.