Pakistan Entomologist EFFECT OF INSECTICIDES ON SUSCEPTIBILITY AND ESTERASE ACTIVITY OF SPODOPTERA LITURA (NOCTUIDAE:LEPIDOPTERA)

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1 Pakistan Entomologist Journal homepage: EFFECT OF INSECTICIDES ON SUSCEPTIBILITY AND ESTERASE ACTIVITY OF SPODOPTERA LITURA (NOCTUIDAE:LEPIDOPTERA) 1 1* Sana Barkat, Asim Gulzar, Muhammad Tariq, Muhammad Javaid Asad, Raja Tahir Mahmood, Asad Ali and Kaleem Tariq 1 Department of Entomology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 2 Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 3 Department of Agriculture, Abdul Wali Khan University, Maan ARTICLE INFORMATION Received: January 16, 2016 Received in revised form: May 25, 2016 Accepted: June 05, 2016 *Corresponding Auor: Asim Gulzar asim@uaar.edu.pk ABSTRACT Spodoptera litura is e major pest of economically important crops and many vegetables. It has more an 120 host plant species and is e major pest of cauliflower, cotton, strawberry, tomato, apple and many oer crops. Many insecticides have been used for e management of S. litura but e continuous use of insecticide leads to e development of resistance against ese insecticides. S. litura has developed resistance against e major insecticides including pyreroids and carbamates. Present study was conducted to determine e susceptibility and esterase activity of e 3 and 5 instar larva of Spodoptera litura against ree insecticides. Esterase is one of e enzymes which cause e detoxification of e insecticides leading to e resistance development. Three different concentrations i.e. LC 40, LC 50 and LC 60 of each insecticide were used. Esterase activity against each insecticide was determined after 30 min, 2, 24 and 48 hrs. The results revealed at highest activity was found against fenvelrate and lowest activity was found against flubendamide in 3 and 5 instar of S.litura. On e basis of toxicity and enzyme activity among all e tested insecticides, flubendamide found to be very effective against S.litura and can be used for e control of is pest. Keywos: Insecticide, Esterase, Spodoptera litura INTRODUCTION Spodoptera litura is e major pest of economically important crops and many vegetables in Sou Asian countries (Qin et al., 2004; Mallikarjuna et al., Moist and warm field conditions are important for its multiplication and development (Dhir et al., 1992). It is notorious leaf feeding caterpillar and has ability to travel long distance, erefore it is difficult to control it in case of outbreak situation (Sayyed et al., 2008). It has more an 120 host plant species and is e major pest of cauliflower, cotton, strawberry, tomato, apple and many oer crops (Rao et al., 1993). Many insecticides have been used for e management of S. litura but e continuous use of insecticide lead to development of resistance against ese insecticides (Zhai and Robinson, 1992). S. litura had developed resistance against e major insecticides including pyreroids and carbamates (Ahmad et al., 2007). There are number of factors responsible for e development of resistance against e major insecticides in S. litura. Insecticide resistance involves ree major mechanisms i.e., enhance detoxification, decreased penetration and target site insensitivity (Ahmad and Mccaffery, 1999). Enzyme detoxification is important mechanism against e synetic insecticides. These enzymes include esterases and cytochrome, P450, monooxygenases enzymes (Ahmad et al., 2007). The detoxification mechanism of enzyme is divided into metabolic transformation and reaction (Visetson, 1991). It is reported at enzymes are accumulated in intestine and adipose cells. When insects are exposed to chemicals ey may change eir behavior and decreases e chemical effect wiin e insect body and is behavior is called behavior avoidance. Insects have several detoxification and behavior avoidance system. Insects use detoxification enzymes to decrease e toxicity of poison (Visetson and Milne, 2001). Esterase is large group of hydrolase, which hydrolyze non ester and ester compound, and play a vital role in conferring detoxification of insecticides in arropods species and insect (Mouches et al., 1986). It has two types, arylesterases which does not inhibit by e organophosphate and e second type is Cite is article as: Barkat, S., A. Gulzar, M. Tariq, M.J. Asad, R.T. Mahmood, A. Ali and K. Tariq, Effect of insecticides on susceptibility and esterase activity of Spodoptera litura (Noctuidae: Lepidoptera). Pak. Entomol., 38(1):

2 carboxylesterase cholinesterase and aliesterase which are inhibited by organophosphate due to e irreversible phosphorylation of active serine site (Dauterman, 1985). The main objective of e present study was to determine e susceptibility and activities of esterase enzymes of S. litura under e application of different insecticides for better results. Enzyme Activity Enzyme activity was measured as follows. Enzyme Activity = Absorbance Standa factor DF 20 Statistical analysis LC 40, and values of e insecticide were determined wi R version (R Development Core Team, 2009). MATERIALS AND METHODS Field collection and rearing of Spodoptera litura The larvae of S. litura were collected from cauliflower field crops of National Agriculture Research Centre (NARC). Larvae were reared accoing to e meod of Smi (1996). Larvae were placed in six-holed Petri dishes and artificial diet was provided to each larvae. Diet was changed after 3-4 days. The pupae were kept in separate boxes till e adult emergence. Adults were transferred to e plastic jars and provided 10 % sugar solution as food. Egg batches were collected daily and were transferred to e new six-holed Petri dishes. Insecticides Three different insecticides, Flubendiamide (Belt 48SC, Bayer Crop Sciences Pvt Pakistan), Fenvalerate (Fencur) and Deltamerin (Decis 25 EC, Bayer Crop Sciences Pvt Pakistan) were used to evaluate e esterase activity of S. litura. Toxicity bioassays Leaf dip Bioassays were conducted wi e 3 and 5 instar larvae of S. litura (Sayyed et al., 2008). Test solutions were prepared in distilled water wi Triton X-100 (50 μg/ml). Leaf discs (4.8 cm dia.) were immersed in a test solution for 10 s, allowed to dry at room temperature and placed in Petri dishes (5 cm dia.) containing a moistened filter paper. Five larvae were placed in each dish, and each treatment was replicated 7 times. The mortality was assessed after 48 h. Three different concentrations e.i. LC 40, and were used to measure e esterase enzyme activity of S. litura. Sample preparation For esterase extraction, S. litura treated wi LC 40, and LC60 of insecticides were ground in 3 ml of ice-cold sodium phosphate buffer (0.04 M, ph 7.0) in a tissue grinder. The homogenates were centrifuged at 10,000 rpm for 15 min at 4 ºC. The resulting supernatants were used as e enzyme source. S. litura place on leaves treated wi water were used as control. The samples were taken after 30 min, 2h, 24 h and 48h. Esterase assay Esterase assay was performed by mixing 1 ml of enzyme stock solution. (10 µl of supernatant of S. litura) homogenate in 990 µl phosphate buffer (40 mm, ph 6.8) into 5 ml of substrate soln. (30mM α-naphtyhl acetate). Mixture was o incubated at 30 C for 20 min and en 1 ml of staining solution (Fast blue BS salt (1%), phosphate buffer (0.04 M) and SDS 5% w/v) was added and again incubated for 20 min. After at absorbance was taken spectrophotometrically at 590 nm to measure esterase activity. RESULTS Susceptibility of insecticide against S. litura The susceptibility of ree insecticides was evaluated against 3 and 5 instars larvae of S. litura. The higher LC values were found for deltamerin against 3 instar larva of S. litura and lower LC value was observed for flubendamide (Table 1). Leal concentration values (LC 40,, and ) were also determined for 5 instar larvae (Table 2). Esterase activity of S. litura larvae after application of deltamerin The mean esterase activity in 3 and 5 instar larvae of S. litura after e application of deltamerin was given in table3. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 40 in 3 instar was , , and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 40 in 5 instar was , , and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 50 in 3 instar was , , and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 5 instar was , , and 345 IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 3 instar was , , and IU/ml/min. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 5 instar was 435.6, , and 613 IU/ml/min respectively. Highest mean esterase activity ( IU/ml/min) was recoed against 5 instar at LC 40 after 24 hrs. While lowest mean esterase activity ( IU/ml/min) was recoed wi LC40 after 30 min. Esterase activity of S. litura larvae after e application of fenvelrate The mean esterase activity in 3 and 5 instar larvae of S. litura after e application of fenvelrate was given in table3.the mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 40 in 3 instar was , , and IU/ml/min. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 40 in 5 instar was , , and 244 IU/ml/min. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 3 instar was , , 252 and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 5 instar was , , and 210 IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 3 instar was , , and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs 34

3 application of in 5 instar was , , and IU/ml/min respectively. The highest mean esterase activity (834 IU/ml/min) was recoed in 5 instar wi LC50 after 30 minutes and lowest mean esterase activity (237 IU/ml/min) was recoed in 3 instar larvae wi LC 40 after 30 min. Esterase activity of S. litura larvae after e application of flubendamide The mean esterase activity in 3 and 5 instar larvae of S. litura after e application of flubendamide was given in table 3. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 40 in 3 instar was , 425.5, and IU/ml/min. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of LC 40 in 5 instar was , , and 244 IU/ml/min. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 3 instar was , , and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 5 instar was 275, 401, 301 and 210 IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 3 instar was , , and IU/ml/min respectively. The mean esterase activity after 30 min, 2 hr, 24 hrs and 48 hrs application of in 5 instar was 331.3, 374.2, and IU/ml/min respectively. Highest esterase activity (413 IU/ml/min) was recoed in 3 instar wi after 30 min and lowest esterase activity (164 IU/ml/min) was recoed in 3 wi after 30 min. Table 1 Leal concentration values of Deltameirn, Flubendamide and Fenvelrate against 3 instar larvae of S. litura. Insecticides LC Slope± SE N LC 40 (95%CI) Deltamerin Flubendamid Fenvelrate Table 2 91 ( ) 19 ( ) 95 (52-162) 188 ( ) 31 (21-48) 17 ( ) 390 ( ) 53 (36-96) 306 ( ) 0.803± ± ±177.9 Leal concentration values of Deltameirn, Flubendamide and Fenvelrate against 5 instar larvae of S. litura. Insecticides LC Slope± SE N Deltamerin LC 40 (95%CI) 236 ( ) 348 ( ) 511 ( ) 1.515± Flubendamid 31 ( ) 54 ( ) 95 ( ) 1.052± Fenvelrate ( ) ( ) ( ) 1.045±

4 Table 3 Esterase activity in 3 and 5 instar larvae of S. litura after e application of Deltamerin. Time Control LC 40 Instars min h h h Table 4 Esterase activity in 3 and 5 instar larvae of S. litura after e application of Flubendamide. Time Control LC 40 Instars min h h h Table 5 Esterase activity in 3 and 5 instar larvae of S. litura after e application of Fenvelrate. Time Control LC 40 Instars min h h h

5 DISCUSSION The results of e present study revealed at e flubendamide is more toxic to e 3 and 5 instar larvae of S. litura an deltamerin and fenvelrate at LC 40, and. The toxicity of e insecticide depends upon e larval instars and e chemical nature of e insecticides. Toxicity of different insecticide was evaluated against different insect pests by many researchers. Sheikh et al. (2012) conducted experiment on 4 instar larvae of S. litura to evaluate e effect of chlropyrifos and LC values were reported ppm. In anoer study dichlorovos was used against 3 instar larvae of S. litura and LC values recoed were 9.04 ppm 50 (Muusamy et al., 2011). Extract of A. annua used against 3 instar larvae and adult of S. litura showed of 48 ppm and 19.14ppm respectively (Shekari et al., 2008). Deltamerin showed value of 25ppm on 3 instar larvae of army worm (Cho et al., 1999). The ineffectiveness of insecticide in controlling insect pests and subsequently e development of insecticide resistance are due to e action of enzymes which are eier insensitive to e insecticide or able to degrade it to less toxic metabolites (Biddinger, et al., 1996). There is a relationship between e increase of insecticide resistance and e activity of detoxification enzymes (Xin-Ju and Hui-Min, 2011). Esterases are large and diverse group of hydrolases at hydrolyze numerous substrates (Walker and Mackness, 1983). Esterase is one of e enzymes which cause e detoxification of e insecticides leading to e resistance development against e insecticides. The level of e esterase is directly related to e resistance against e insecticides (Huang and Han, 2007). Increased esterase activity is a major mechanism of insecticide insensitivity or even resistance in many insect species (Zhou, et al., 2002). In e present study, esterase activities in 3 and 5 instar larvae of S. litura, treated wi ree insecticides at LC, LC and was measured after 30 min, 2h, 24 h and 48 h. The result revealed at highest activity was found against fenvelrate and lowest activity was found against flubendamide in 3 and 5 instar of S.litura. Previously, Xue et al., (2010) evaluated e susceptibility of four host plants againts larvae of S. litura and also reported at e glutaione S-transferase activities of larvae were highest when ey fed on tobacco, followed by Chinese cabbage and cowpea, and e lowest activities were observed when larvae fed on sweet potato. The extract of A. annua affects e biochemical properties of e elm leaf beetles (Shekari et al., 2008). Su et al. (2012) reported at esterase activities of most of e field populations were higher an e susceptible populations of S. litura. The larvae of S. litura treated wi indoxacarb for 24 hour showed alpha esterase activities of 517 nd and 945 IU/ml/min in 2 and 4 instars respectively. The beta nd esterase activity was 786 and 450 IU/ml/mint in 2 and 4 instar respectively (Gamil et al., 2011). Conclusion/recommendations Esterase enzyme activity responded towas e insecticide application. Enzyme activity fluctuated up and down from normal count due to destruction caused by application of insecticides. Higher activity indicates resistance against insecticides and decrease esterase activity shows susceptibility against insecticides. It is concluded at based 37 on toxicity and enzyme activity among all e tested insecticides, flubendamide found to be very effective against S. litura and can be used for e control of is pest. REFERENCES Ahmad, M., A. R. Maaaffery, Penetration and metabolism of trans cypermerin in a susceptible and a pyreroid resistant strain of Helicoverpa armigera. Pestic. Biochem., 65: Ahmad, M., M. I. 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