INTRODUCTION
Spodoptera litura (F.) is harmful for plant and can cause economic loss for farmers. According to Tengkano & Suharsono (2005), the agriculture crops located at 22 provinces in Indonesia, like west java, east java, south Sumatra etc., were attacked by S. litura with an area of 11,163 ha/year.
This research was carried out at Garut and West
Bandung districts, well known as the production centres of vegetables in West Java (BPS, 2016).
Control of armyworm on farm level are generally still using synthetic insecticides (Laoh, Puspita, &
Hendra, 2003; Razak, Santhakumar, Mageswari, &
Santhi, 2014) which is in long intensive use, it may trigger the occurance of S. litura resistance in both regions.
ARTICLE INFO Keywords:
Acetylcholinesterase Armyworm
Botanical insecticide Esterase
Glutathione s-transferase Article History:
Received: March 14, 2022 Accepted: August 25, 2022
*) Corresponding author:
E-mail: [email protected]
ABSTRACT
This study aimed to obtain resistance information of S. litura from Karangpawitan and Lembang districts (West Java, Indonesia) against chlorpyrifos insecticides (200 g/l) and their sensitivity to oil mixture of A. indica and C. nardus (1:1) using feeding and topical assay. The activity of acetylcholinesterase, esterase, and glutathione s-transferase from S. litura larvae in both populations were tested to determine their role in insect resistance. Results showed that S. litura population from Lembang had a higher level of resistance to chlorpyrifos compared to Karangpawitan. The sensitivity of the two S. litura populations had a relatively similar resistance ratio (RR) value of less than 1 to botanical insecticide. These indicated that resistant population could be controlled by a mixture of this botanicals insecticide. Enzyme activity test indicated that chlorpyrifos at a concentration of 0.26%
could inhibit the acetylcholinesterase activity of insect population from Karangpawitan by 98.66% while those from Lembang, it was only 35.31%. Specific activity of esterase from Karangpawitan was 13.37 units/mg while Lembang population was 119.65 units/
mg. The specific activity of the Glutathione S-Transferase (GST) of Karangpawitan population was 1140.82 units/mg while Lembang population was 793.73 units/mg. The high activity of the three enzymes could be responsible for resistance of S. litura larvae to chlorpyrifos.
ISSN: 0126-0537Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia, Decree No: 30/E/KPT/2018
Cite this as: Ramadhan, R. A. M., Widayani, N. S., Dono, D., Hidayat, Y., & Ishmayana, S. (2022). Resistance level and enzyme activity of Spodoptera litura F. to chlorpyrifos and their sensitivity to the oil formulation of Azadirachta indica Juss. and Cymbopogon nardus (L.) Rendl.. AGRIVITA Journal of Agricultural Science, 44(3), 419-430. http://doi.
org/10.17503/agrivita.v41i0.3729
Resistance Level and Enzyme Activity of Spodoptera litura F. to Chlorpyrifos and Their Sensitivity to the Oil Formulation of Azadirachta indica Juss. and Cymbopogon nardus (L.) Rendl.
R. Arif Malik Ramadhan1,2), Neneng Sri Widayani2), Danar Dono3*), Yusup Hidayat3) and Safri Ishmayana4)
1) Agrotechnology Study Program, Universitas Perjuangan, Tasikmalaya, West Java, Indonesia
2) Agronomy Study Program, Faculty of Agriculture, Universitas Padjadjaran, Bandung, West Java, Indonesia
3) Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran, Bandung, West Java, Indonesia
4) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Bandung, West Java, Indonesia
The resistance of S. litura has been reported in various countries in Asia. The high level of selection and the ability of S. litura genes to evolve rapidly in response to synthetic insecticides became a major problem for farmers in many countries (Ahmad, Denholm, & Bromilow, 2006). S. litura has resistance to pyrethroid and organophosphate insecticides in Hunan province (China) and Punjab (Pakistan) (Abbas et al., 2014; Saleem, Hussain, Ghouse, Abbas, & Fisher, 2016; Tong, Su, Zhou, &
Bai, 2013). S. litura in India has developed resistance to chlorpyrifos, quinaolphos, lambda-cyhalothrin, and Chlorantraniliprole (Kiran, Patil, & Srujana, 2016; Muthusamy, Vishnupriya, & Shivakumar, 2014). These cases indicate that S. litura resistance has occurred in various active ingredients in these countries. Unfortunately, there have not been reports of S. litura resistance in Indonesia. One of the reported research on genus of Spodoptera was Spodoptera exigua Warnasari population (Brebes, Indonesia) that has a higher level of resistance to methoxyfenozide caused by intensive use of the active ingredient (Wibisono, Trisyono, Martono, &
Purwantoro, 2007). According to the latest record in Arthropod Pesticide Resistance Database (APRD), it has been reported that 55 cases of S. litura were reported resistant to chlorpyrifos throughout the world (Mota-Sanchez & Wise, 2022). Chlorpyrifos as contact insecticide has mode of action as Acetylcholinesterase (AChE) inhibitors, meanwhile methoxyfenozide (diacylhydrazines class) act as ecdysone receptor agonists (IRAC, 2022)growers, advisors, extension staff, consultants and crop protection professionals with a guide to the selection of insecticides or acaricides for use in an effective and sustainable insecticide or acaricide resistance management (IRM.
This resistance can be influenced by the activity of enzymes in S. litura body. According to Yu (2006), the activity of the acetylcholinesterase (AChE) enzyme from field strains is less sensitive to inhibition of carbamate and organophosphate insecticides. The activity of the esterase enzyme and the Glutathione S-transferase enzyme also influence the level of resistance to these insecticides (Xu, Zou, Zhang, Feng, & Zheng, 2015; Xue, Pang, Li, & Liu, 2010).
Formulation of mixture botanical insecticide of A. indica and C. nardus oil is one potential insecticide to serve as an alternative means to control this pest. The application of a single A. indica
oil at dose of 2500 ppm can cause hight mortality and affect various aspects of S. litura biology (Malik et al. 2017). Application of C. nardus oil reduces oviposition, egg hatch ability of fruit damage due to Helicoverpa almigera (Setiawati, Murtiningsih, &
Hasyim, 2011). Pinheiro et al. (2013) reported that C. nardus oil was toxic to M. persicae and have LC50 value of 0.36% and LC90 of 0.66%. A mix of both botanical insecticides is expected to control S. litura effectively, efficiently, and to minimize the occurrence of resistance. Crocidolomia pavonana F. resistant to profenofos indicated susceptible to Nicotiana tabacum leaf extract (Dono, Natawigena,
& Kharismansyah, 2014). Therefore, this study was aimed to obtain information on the level of resistance and activity of the enzyme acetylcholinesterase, esterase, and glutathione S-transferase from S.
litura from Karangpawitan and Lembang Subdistricts against chlorpyrifos insecticides and their sensitivity to the botanical insecticide mixtures of A. indica and C. nardus oil.
MATERIALS AND METHODS
This research used chlorpyrifos 200 g/l and botanical insecticide formulation from a mixture of A. indica and C. nardus oil (1:1) with emulciable concentrate (EC) formulation. S. litura from Lembang (West Bandung regency, West Java, Indonesia) served as the tested population while those from Karangpwaitan (Garut Regency, West Java, Indonesia) subdistrict was considered as the susceptible population. The concentrations used were five levels which were expected to result in the mortality of test insects between 0 < x < 100% and control. Each treatment was repeated 3 times and each treatment contained 30 insects. The control treatment used 0.05 ml/l emulsifier of alkyl aryl polyglycol ether 400 mg/l (Agristik R). Bio-assay was conducted at Laboratory of Pesticide and Environmental Toxicology - Faculty of Agriculture, and enzyme analysis was conducted at Laboratory of Biochemistry - Faculty of Mathematic and Natural Sciences, Universitas Padjadjaran. The research was carried out from April to August 2018.
Feeding Assay (Dipping) Method
The experiment was conducted by dipping a circular leaf with a diameter of 3 cm into an insecticide solution for 20 seconds. Then, the leaves were dried for 3 hours and given to S. litura (3rd instar) in a petri dish. The leaves treatment was
given for 2 x 24 hours. The insecticide-reted leaves were then replaced with untreated leaves (Dono, Natawigena, & Kharismansyah, 2014).
Contact Method (Topical Test)
The larval activity was reduced by cooling treatment for 4-5 minutes. The insecticide was tested by dripping the larvae up to the dorsal thorax level on 1 µl insecticide solution using microsyringe.
Observation on the effect of chlorpyrifos insecticide to mortality of S. litura was carried out after 48 hours treatment. LC50 values were calculated using probit analysis. The value of the resistance level was obtained from the results of the mortality test.
The resistance ratio was calculated based on the following equation (Dono, Ismayana, Idar, Prijono,
& Muslikha, 2010) :
Determination of resistance level is refers to the standard test of pest resistance to pesticides in the scope of agriculture. According to Winteringham (1969), insects are determined to have resistance if insects have a ratio of resistance > 4, while an indication of resistance has occurred if the ratio of resistance > 1.
Testing of S. litura Susceptibility to Botanical Insecticide
S. litura population of Karangpawitan and Lembang populations were tested for their susceptibility to mixture of A. indica and C. nardus oil (formulation 50 EC). The experiment used a food dipping method (residual method on plants) as already been described ealier in the testing level of S. litura resistance. Observation of the death insect was carried out after 24-hour for 14 days.
To obtain values of LC50 and LC95, the relationship between botanicals insecticide concentration and mortality was analyzed using Polo Plus program.
Susceptibility of the tested insects against mixture of this botanicals oils was indicated by the resistance ratio (RR). Insects are sensitive to mixture of this insecticide if they have RR < 1.
Homogenate Preparation and Analysis of Protein Levels of S. litura Larvae
Sample of S. litura (3rd instar) for enzyme and protein analysis was prepared according to Burden (2012) and Dono, Ismayana, Idar, Prijono,
& Muslikha (2010).
Measurement of protein levels of S. litura larvae was performed based on Lowry method
(Kresze 1983). The absorbance of the solution was measured using spectrophotometer (λ = 750 nm). The alkaline copper and Folin ciocalteau were were used as the reagent. Alkaline copper reagents consist of a mixture 1% CuSO4.5H2O, 2% NaK- Tartrate, and 2% Na2CO3 in 0.1 N NaOH. As a blank, aquades was used. The standard protein used was BSA (bovine serum albumin) at various levels of concentration (Dono, Ismayana, Idar, Prijono, &
Muslikha, 2010).
Acetylcholinesterase Activity (AChE) Test The method for AChE assay was conducted according to Dono, Ismayana, Idar, Prijono, &
Muslikha (2010) and Ellman, Courtney, Andres Jr.,
& Featherstone (1961). The homogenate of test insect 3rd larvae instar was used as enzyme source.
Enzyme activity was calculated using equation (2). Then, the percentage of inhibition of acetylcholinesterase activity by insecticides was determined using equaiton (3).
Where: AU = Activities Unit; As = Absorption of samples tested; Ak = Control absorption (Boiled samples); ti = Incubation time (30 minutes); Ve = Volume of enzyme tested: AchE (0.2 ml), Esterase (0.05 ml) and GST (0.02 ml).
Where: A0 = Control of acetylcholinesterase (M substrate/minute/mg protein) control (without insecticide); A1 = Acetylcholinesterase activity with insecticide treatment.
Esterase Activity Assay
The esterase activity was measured using α-naphthyl acetate as substrate (Dono, Ismayana, Idar, Prijono, & Muslikha, 2010; Yu, Nguyen, &
Abo-Elghar, 2003). The homogenate of test insect was used as enzyme source. Enzyme activity was calculated using equation (2).
Glutathione S-Transferase Activity Assay
CDNB (1-chloro-2,4-dinitrobenzene) was used as substrate for measure of Glutathione S-Transferase activity (Dono, Ismayana, Idar, Prijono, & Muslikha, 2010; Habig, Pabst, & Jakoby, 1974; Shen et al., 2020). The activity of GST was calculated based on equation 2.
...1)
...2)
...3)
RESULTS AND DISCUSSION
Level of S. litura Resistance to Chlorpyrifos Insecticide 200 g/l
The resistance level of S. litura in the contact and feeding assay methods to chlorpyrifos exhibited various values (Table 1 and Table 2). LC50 values of the S. litura of the Karangpawitan and Lembang populations were 0.0081 ml/l and 0.0257 ml/l (feeding assay method) and 0.1075 ml/l and 0.1334 ml/l (contact method). The results of the analysis of the resistance ratio of S. litura in the population of Karangpawitan to the population of Lembang were 3.17 (feeding assay method) and 1.24 (contact methods). When referring Winteringham (1969), the S. litura population of Lembang showed indications of resistance to chlorpyrifos. The difference in toxicity values between the two populations showed that selection pressure on S. litura was different in each region. Che, Shi, Wu, & Yang (2013) explained that local selection caused differences in the level of insect resistance. Development of metabolism and insect excretion in each region may also affect the toxicity of an insecticide.
The LC50 value in the contact method was higher than the feeding assay method (Table 1 and
Table 2). The development of resistance may be influenced by the higher inhibition of chlorpyrifos insecticide penetration into insect integument. The resistant insects has thicker cuticule and make the insecticide penetration become slower, and the ability of excretion is high (Ahmad, Denholm, &
Bromilow, 2006; Nannan, Fang, Qiang, Pridgeon,
& Xiwu, 2006). Cuticle thickening is present within a pyrethroid-resistant strain of Cimex lectularius L.
and cuticle thickness varies according to time-to- knockdown upon exposure to an insecticide (Lilly, Latham, Webb, & Doggett, 2016). Similar evidence reported of a possible association between cuticle thickness and resistance in Cimex hemipterus (F) against fenitrothion and imidacloprid (Soh &
Singham, 2021). However, chlorpyrifos has value of oral LD50 for rats ranging from 95 to 270 mg/kg, and dermal > 2,000 mg/kg. It means, chlorpyrifos is more toxic by oral application than dermal (NPIC, 2022).
It is known that resistance involves many factors, therefore further researched are needed to prove the role of the cuticle in insect resistance to insecticides.
The resistance level of S. litura from Lembang population was 3.172 fold (feeding assay method) and 1.241 fold (contact method) if compared to Karangpawitan population. The difference in the Table 1. Probit regression parameters of chlorpyrifos 200 g/l against S. litura on 48 hours after treatment (feeding assay)
Population a±SE b±SE LC50 CI95% LC95 CI95% RR
Karangpawitan (Kp) 12.024 ± 0.965 3.450 ± 0.460 0.008 0.007 – 0.010 0.024 0.019 – 0.037 3.172 Lembang (L) 8.573 ± 0.537 2.248 ± 0.314 0.026 0.015 – 0.539 0.139 0.062 – 1.964
Remarks: a: Intercept, b: Slope, SE: Standard Error, LC: Lethal concentration (ml/l), CI: Confidence interval, RR:
Resistance Ratio (LC50L/LC50Kp)
Table 2. Probit regression parameters of chlorpyrifos 200 g/l against S. litura on 48 hours after treatment (contact assay)
Population a±SE b±SE LC50 CI95% LC95 CI95% RR
Karangpawitan (Kp) 7.998 ± 0.467 3.096 ± 0.434 0.108 0.078 – 0.161 0.366 0.220 – 1.252
1.241 Lembang (L) 7.927 ± 0.434 3.347 ± 0.451 0.133 0.095 – 0.199 0.414 0.256 – 1.397
Remarks: a: Intercept, b: Slope, SE: Standard Error, LC: Lethal concentration (ml / l), CI: Confidence interval, RR:
Resistance Ratio (LC50L/LC50Kp)
Table 3. Probit regression parameters of the mixture of A. indica and C. nardus oil against S. litura
Population a±SE b±SE LC50 CI95% LC95 CI95% RR
Karangpawitan (Kp) 4.769 ± 0.131 4.285 ± 0.573 1.132 0.986 – 1.307 2.739 2.183 – 3.939
0.998 Lembang (L) 4.788 ± 0.128 4.010 ± 0.530 1.129 0.976 – 1.316 2.904 2.281 – 4.262
Remarks: a: Intercept, b: Slope, SE: Standard Error, LC: Lethal concentration (ml/l), CI: Confidence interval, RR:
Resistance Ratio (LC50L/LC50Kp)
value of the resistance ratio can be influenced by different regional, genetic, and environmental factors (Gong et al. 2013). As in this study, S. litura from Karangpawitan and Lembang populations have different susceptibility to chlorpyrifos insecticides.
When comparing their RR values, the detoxification enzymes play as a mechanism of resistance in S.
litura populations. Baek et al. (2005) stated that metabolic enzymes contribute to detoxification in Lepidoptera insects. Influential enzymes such as the acetylcholinesterase, esterase, and GST enzymes were tested in this study.
The Sensitivity of S. litura to Mixture of A. indica and C. nardus Oil
S. litura with feeding assay method showed that both populations have a similar level of sensitivity to the mixture of A. indica and C. nardus oil. The value of resistance ratio of Lembang population to Karangpawitan population was 0.998 fold. The LC50 and LC95 values of S. litura of Karangpawitan and Lembang populations were 1.132 ml/l; 1.129 ml/l and 2.739 ml/l; 2.904 ml/l, respectively (Table 3). S. litura sensitivity to botanical insecticides was tested as one of the solutions to manage resistance. The mixture of A. indica and C. nardus oil was used because their main compound had a different mode of action from chlorpyrifos insecticide. Oils of these plants contain many classes of compound, therefore, further research is needed regarding the effect of the pure main compound on insect resistance biochemicals.
Mixture of A. indica and C. nardus oil showed a fairly good response to the tested insects. The ratio value of resistance to the mixture formulation of A.
indica and C. nardus was close to 1 (one) indicating the sensitivity values of the two populations can be considered the same (Table 3). Azadirachtin, a main bioactive compound of neem oil, can cause instant mortality to insects and gradual death due to interference with the hormone ecdysone of the insects (Samsudin, 2011). Azadirachtin resulted in inhibition of the release of protoraxicotrophic hormone (PTTH) from the corpora cardiaca glands and inhibited the release of ecdysone from the brain ring glands complex (BRC’s) resulting in development disruption of the insect (Koul, 1996; Mordue (Luntz) &
Blackwell, 1993). Bioactive compound extracted from A. indica can reduce the level of insect detoxification enzymes, and make insecticides more effective when applied to resistant insects (Lowery & Smirle, 2000;
Mordue (Luntz) & Nisbet, 2000) aside from growth disruption (Govindachari, Suresh, Gopalakrishnan, &
Wesley, 2000). Some researcher also reported that
the extract or bioactive compound of A. indica act as growth and development disruption and feeding inhibition (Bezzar-Bendjazia, Kilani-Morakchi, & Aribi, 2016; Dawkar et al., 2019). Koul (1996) reported that azadirachtin inhibit the release of prothoraxicotrophic hormone (PPTH) from corpora cardiaca gland and ecdysone that cause development disruption. While, citronella (Cymbopogon oil) can cause significant morphological and histological damage and be effective to control T. castaneum with LC50 of 9.3%
v/v (de Oliveira, 2021). Isman (2006) reported that C. nardus oil can affect digestive and neurological enzymes of insect. C. nardus extract is toxic and has antifeedant and antioviposition effects on insects from the order Lepidoptera (Hernández-Lambraño, Caballero-Gallardo, & Olivero-Verbel, 2014). Ajayi, Oladipupo, & Ojo (2018) report that enzyme (α amylase and β amylase) activities increase along with the increase of Cymbopogon oil dosage as a physiological response to adapt with abnormal condition. In contrast, Chlorpyrifos act as inhibitor of acetylcholinesterase, that degrade of acetylcholine, a neurotransmitter (IRAC, 2022).
These different mode of action between neem and Cymbopogon oils with chlorpyrifos as a basic of the oils can be used as alternative to manage of S.
litura resistance to chlorpyrifos. Botanical insecticide can be used as alternative insecticide or an option in rotating insecticide. Rotation of insecticides that have different modes of action is recommended for resistance management to slow the evolution of resistance in S. exigua (Ahmad, Farid, & Saeed, 2018; Che, Huang, Guan, Wu, & Yang, 2015).
Enzyme Activity of Acetylcholinesterase, Esterase and Glutathione S-transferase
Acetylcholinesterase Activity
Protein levels of Karangpawitan population was 1.3163 mg/ml and Lembang population was 2.197 mg/ml. The application of chlorpyrifos 200 g/l insecticide with a concentration of 0.003% to 0.026%
against S. litura Karangpawitan population could inhibit the activity of the acetylcholinesterase enzyme by 46.17% to 98.66% (Fig. 1). The high value of the inhibitory activity of the acetylcholinesterase enzyme indicated that the Karangpawitan population had a high sensitivity to the chlorpyrifos 200 g/l insecticide.
The similar treatment to S. litura Lembang population shows different results. Chlorpyrifos 200 g/l insecticide with a concentration of 0.003% to 0.026% could only inhibit the activity of the acetylcholinesterase enzyme of 17.24% to 35.31%. on the S. litura population from Lembang.
Fig. 1. Inhibition to the activity of the acetylcholinesterase enzyme in S. litura from Karangpawitan and Lembang populations
Fig. 2. Esterase specific enzyme activity of the S. litura in Karangpawitan and Lembang populations
The population of S. litura from Lembang had a lower inhibition value than those from Karangpawitan.
The inhibition of acetylcholinesterase due to insecticide at the concentration of 0.026% were 35.3% at S. litura from Lembang and 98.66% at those from Karangpawitan (Fig. 1). The sensitivity of S. litura from Karangpawitan to inhibition by chlorpyrifos insecticide was 2.7 fold than Lembang.
The higher the value of inhibition of insecticides, the fewer molecules of acetylcholine can be broken down into molecules of choline, lactic acid, and water. The comparison indicated that the acetylcholinesterase activity of the Lembang population was not well inhibited by chlorpyrifos insecticides. Lembang population of S. litura was more resistant than the Karangpawitan population.
Esterase Activity
Esterase enzyme activity analyzed from Karangpawitan and Lembang populations had different activity values. The specific esterase activity values were 13.37 units/mg in the Karangpawitan population and 119.65 units/mg in the Lembang population (Fig. 2). The high specific activity of the Esterase enzyme in the Lembang population has indicated resistance to chlorpyrifos insecticides.
Glutathione S-Transferase Activity
Analysis of Glutathione S-Transferase (GST) enzyme activity of S. litura larvae from Karangpawitan and Lembang populations showed relatively similar results. The specific activity of GST in both populations was very high. There are 1140.82 units/mg for the Karangpawitan population and 793.733 units/mg for the Lembang population (Fig. 3). The specific activity of the GST enzyme in Karangpawitan populations was higher than Lembang population. The high activity of the Glutathione S-Transferase enzyme shown that both populations have been indicated to be resistant to organophosphate insecticides.
Inhibition to acetylcholinesterase enzyme activity at S. litura Karangpawitan (Fig. 1) was higher than Lembang population. This showed there was a decrease in the sensitivity of acetylcholinesterase in S. litura in the Lembang population. So it can be stated that the asetylcholinesterase insensitive plays a role in the level of resistance to the chlorpyrifos insecticide. Acetylcholinesterase from field strains can reach 2 to 85 fold compared to susceptible strains, and its activity correlates with to resistance to organophosphate insecticides (Charpentier & Fournier, 2001; You et al., 2020; Yu, Fig. 3. The Specific activity of GST enzyme activity the S. litura in Karangpawitan and Lembang populations
Nguyen, & Abo-Elghar, 2003; Zabel et al., 2017).
The esterase specific enzyme activity also showed that its activity was higher in S. litura from Lembang population (Fig. 2). The high activity of this enzyme as a detoxification enzyme influenced the level of resistance on organophosphate insecticides (Darvishzadeh & Sharifian, 2015; Mulyaningsih, Umniyati, & Hadianto, 2017; Tiwari, Stelinski, &
Rogers, 2012; Xue, Pang, Li, & Liu, 2010). The level of carboxylesterase activity in insects decreases sensitivity to organophosphates (Karuppaiah, Srivastava, & Subramanian, 2017). Further research of genetic resistance of this insect needed to be conducted because of many cases of resistance controlled genetically as reported by Boaventura, Martin, Pozzebon, Mota-Sanchez, & Nauen (2020) that S. frugiperda from Indonesia indicated genetically resistance for Acetylcholinesterase inhibitor insecticides.
In this study, GST enzyme activity seems likely to play a role in resistance to the insecticide chlorpyrifos. High GST activity was detected in both populations which S. litura from Karangpawitan showed the highest GST (Fig. 3). According to Xu, Zou, Zhang, Feng, & Zheng (2015), the increased activity of the GST enzyme can be triggered by exposure to heavy metal compounds and various insecticide compounds, one of which is chlorpyrifos. GST plays an important and varied role in insecticide detoxification, oxidative stress response, or cellular metabolism (Li, Schuler, &
Berenbaum, 2007; Pavlidi, Vontas, & Van Leeuwen, 2018). The possible role of GST in insecticide resistance was in GST cytosolic class delta and epsilon. Here, GST is divided into grade class:
Delta, Epsilon, Omega, Sigma, Theta, and Zeta (Enayati, Ranson, & Hemingway, 2005; Sheehan, Meade, Foley, & Dowd, 2001). The detoxification activity of SlGSTE1 GST epsilon in recombinant S.
litura has high binding activity against chlorpyrifos, deltamethrin, malathion, phoxim and dichloro- diphenyl-trichloroethane (DDT) insecticides (Xu, Zou, Zhang, Feng, & Zheng, 2015). The insecticide induces co-expression of glutathione S-transferases through the ROS / CncC pathway in S. exigua (Hu et al., 2019). So that even though the GST activity of S. litura from Lembang is lower than Karangpawitan, it is possible that the GST enzyme still plays a role in the insensitivity of S. litura to the chlorpyrifos insecticide.
CONCLUSION
S. litura population from Lembang subdistrict has a higher level of resistance to chlorpyrifos compared to Karangpawitan population. Resistance ratio value for the feeding assay method was 3.172 fold and for the contact method was 1.241 fold. The two populations showed relatively similar susceptibility to botanical insecticide mixtures of A.
indica seed oil and C. nardus (RR <1).
The chlorpyrifos concentration of 0.26% can inhibit the activity of the S. litura acetylcholinesterase from Karangpawitan and Lembang populations as much as 98.66% and 35.31%, respectively. The specific activity of the Esterase of S. litura from Karangpawitan population was 13.37 units/mg while the Lembang population was 119.65 units/mg. The specific activity of the Glutathione S-Transferase of S. litura from Karangpawitan population was 1140.82 units/ml while the population of Lembang was 793.73 units/ml. The high activity of the three enzymes affects the level of resistance of S. litura larvae to chlorpyrifos insecticides. Therefore, it is important to know the biochemical of resistance mechanism to manage the insects resistant to insecticide.
ACKNOWLEDGEMENT
This research was funded by the Universitas Padjadjaran internal grant program (HIU) through Competency Research Scheme (Number 014/UN6/
EP/PL/2018-2019). Thank you to The Rector of Universitas Padjadjaran for this funded.
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