Effect of Neem Pesticide (Achook) on Midgut Enzymatic
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JOURNAL OF PLANT PROTECTION RESEARCH Vol. 53, No. 3 (2013) DOI: 10.2478/jppr-2013-0036 EFFECT OF NEEM PESTICIDE (ACHOOK) ON MIDGUT ENZYMATIC ACTIVITIES AND SELECTED BIOCHEMICAL COMPOUNDS IN THE HEMOLYMPH OF LESSER MULBERRY PYRALID, GLYPHODES PYLOALIS WALKER (LEPIDOPTERA: PYRALIDAE) Roya Khosravi, Jalal Jalali Sendi* Department of Plant Protection, College of Agriculture, University of Guilan, 41635–1314 Rasht, Iran Received: August 01, 2012 Accepted: July 21, 2013 Abstract: Synthetic organic compounds and those of plant origin used in insect pest control are known to affect digestive enzymes and biochemical compounds. The lesser mulberry pyralid Glyphodes pyloalis Walker is a monophagous and dangerous pest of mulber- ry that has been recently observed in Guilan province, northern Iran. In this study the effect of the neem formulation, Achook (0.03% azadirachtin) was studied on nutritional physiology and gut enzyme activity of the lesser mulberry pyralid G. pyloalis. The LC25, LC50, and LC90 values on 4th instar larvae were estimated as 113.6, 256.84, and 1,210.02 ppm, respectively. The neem insecticide exhibited a significant antifeedant activity when used at the highest concentration. When G. pyloalis larvae were provided with mulberry leaves treated with the neem extract, all nutritional indices, except approximate digestibility, decreased. Neem was found to affect digestive enzyme activities in the midgut of treated larvae. When larvae were fed on treated leaves, biochemical compounds in the hemolymph, such as protein, lipid, and glucose decreased but the amount of uric acid increased compared with the control. Key words: Achoock, antifeedant, biochemical compounds, enzyme activity, mulberry pyralid, nutritional indices INTRODUCTION produce yellow oval shaped fruits once or twice per year. Pesticides of biological origin have been intensive- Although several potentially insecticidal active ingredi- ly investigated for the past 30 years. An effort has been ents occur in neem seed extract, the principal active in- made to find an alternative to conventional insecticides. gredient in most formulations is tetranortriterpene azadi- The alternative should be able to reduce health and envi- rachtin (AZA). Azadirachtin is active in nearly 550 insect ronmental impacts (Copping and Menn 2001). There has species (Schmutterer 1990; Mordue and Blackwell 1993), been a worldwide interest in the development of alterna- The lesser mulberry pyralid, Glyphodes pyloalis Walker tive strategies, including the re-examination of using plant (Lepidoptera: Pyralidae) is a specialist insect on mulberry. derivatives against agriculturally important insect pests. This insect is widely distributed throughout Asia and the Plant-derived materials are more readily biodegradable. northern province of Iran. This pest has caused severe Some have low toxicity to humans as well as natural en- damage to farm fields of mulberry in northern Iran and emies, and are more selective in action (Keita et al. 2001; has posed a serious concern to silkworm growers. The Tapondjou et al. 2002; Rahman and Siddiqui 2004). amount of food eaten by the first and second instar larvae Among the plant families, Meliaceae and Rutaceae is negligible, but feeding increases in later instars. Fourth have attracted most of the entomologists’ and phytochem- and fifth instar larvae secrete fine threads to fold the leaf ists’ attention because they produce chemicals known as and feed on the mesophyll inside the folds. Fifth instar lar- triterpenes (Connolly 1983). Many of these compounds vae feed on the whole leaf until only the ribs remain. Each have been demonstrated to affect insect growth and be- generation takes 25 days and it can have five generations havior, acting as antifeedants, toxins, and insect growth in a year so that damage from the fourth and fifth genera- regulators (Champagne et al. 1992; Senthil-Nathan et al. tions is more important (Khosravi and Jalali Sendi 2010). 2005a, 2005b, 2006a, 2006b). The aim of this study was to identify the effect of aza- In the Meliaceae family, the neem tree is native to the dirachtin on the food consumption of the fifth instar lar- Indian sub-continent and also grows in many countries vae of G. pyloalis and its effects on digestive enzymes and in the world, including subtropical countries such as Af- biochemical compounds in the hemolymph. In this study rica, Central and South America, and Australia (Schmut- we tried to throw some light on the possible mechanisms terer 1990). The insecticidal effect of the compound aza- involved in azadirachin’s effect on this noxious pest of dirachtin was first reported by Ruscoe (1972). The trees mulberry. *Corresponding address: [email protected] Effect ot ffet fectetift (ec okt tetidgctf nzemctetmectitctfetm itefefecfit t ecfetemete e g ies 239 Probit analysis was used to determine FD50 (concen- MATERIALS AND METHODS tration required to produce a 50% feeding deterrence compared to untreated discs). Laboratory mass culture of G. pyloalis The larvae of G. pyloalis were collected from mulberry Quantitative food utilization orchards near the city of Rasht in northern Iran. They The newly molted fifth instar larvae were used for were reared on fresh mulberry leaves (Kenmochi vari- this experiment. After measuring the initial weight of the ety) in the laboratory at 24±1°C, 75±5% relative humidity larvae, they were individually introduced into separate (RH) and a 16:8 (L:D) hour photoperiod. The larvae were containers. The larvae were starved for 4 h (10 larvae/con- kept in transparent plastic boxes (18x15x7 cm). The boxes centration) and then allowed to feed on weighed quanti- were covered with muslin cloth for aeration. As adults ties of treated and untreated leaves for 24 h. Four repli- emerged, they were separated and placed in transpar- cates were carried out. The uneaten leaves were removed ent plastic boxes (18x7 cm) with a 10% honey solution on after 24 h, and replaced with fresh untreated leaves. The cotton wool for feeding, and mulberry leaves for ovipo- experiment was continued for four days. Dry weights of sition. Using moist cotton wool, leaves with eggs were larvae and leaves at the outset of the experiment were cal- transferred to the transparent plastic boxes (18x15x7 cm). culated using a wet weight/dry weight conversion, estab- lished using a separate cohort of larvae and leaves. Bioassays Nutritional indices were calculated in a traditional For bioassay experiments, Azadirachtin-rich neem manner such as: Relative consumption rate (RCR) = E/ formulation, Achook (Registered No. CIR-23, 925/96, TA, Relative growth rate (RGR) = P/TA, Approximate di- 0.03% azadirachtin, India), was used and diluted with gestibility, (AD) = 100(E – F)/E, Efficiency of conversion of distilled water. Four concentrations of azadirachtin (800, ingested food (ECI) = 100 P/E, Efficiency of conversion of 400, 200, 100 ppm) in distilled water with emulsifier (0.5% digested food (ECD) = 100 P/(E – F), where: A – mean dry of Triton X-100) were used for the evaluation of LC50 val- weight of animal during T, E – dry weight of food eaten/ ues, with one distilled water and emulsifier as the control. number of alive larvae, F – dry weight of faeces produced, A leaf dip experiment was carried out using freshly cut P – dry weight gain of insect, and T – duration of experi- leaves of mulberry that were dipped in a solution of aza- mental period (Waldbauer 1968). dirachtin (treatment) or distilled water (the control) for 10 s, and dried for 20 minutes at room temperature. New- Preparation of enzyme extract ly molted fourth instar larvae were starved for 4 h. Then Fourth instars of treated G. pyloalis were used to quan- these larvae were allowed to feed on azadirachtin treated tify the enzyme and biochemical compound. Individuals leaves for 24 h. The tests were conducted in four repli- were dissected under a stereomicroscope (OLYMPUS cates. There were 10 larvae for each replicate. Mortality SZX12, Japan) in ice-cold saline buffer (6 µmol/l NaCl). was recorded after 24 h. The LC25, LC50, and LC90 values, The entire digestive tract was dissected out, in ice-cold and 95% confidence limit were calculated from probit re- insect Ringer’s solution. The gut was homogenized for gressions using the Polo-PC program (LeOra 1987). 3 min at 4°C in ice-cold citrate-phosphate buffer (pH 6.8) using a tissue grinder. The homogenate was centrifuged Antifeedant activity at 13,000 rpm for 15 min and the supernatant was used as The antifeedant activity of azadirachtin was carried the enzyme source. out by a leaf-disc choice test. For this purpose four con- centrations (800, 400, 200, 100 ppm) of azadirachtin were (eemzt otα-mezemeftmectitcz prepared. The leaf discs (R = 5 cm) were dipped for 10 s α-amylase activity was assayed by the dinitrosalicylic in the desired concentrations. The control leaf disc was acid (DNS) procedure (Bernfeld 1955), using 1% soluble dipped in water. One treated leaf disc and one control starch (Merck, Darmstadt, Germany) as the substrate. which was dried at room temperature, were randomly Twenty micro liters of the enzyme were incubated for placed in a transparent plastic box 21x15x5 cm over moist 30 min at 35°C with 100 µl universal buffer and 40 µl sol- filter paper to avoid dryness. This experiment was repli- uble starch. The reaction was stopped by adding 100 µl cated 4 times by 10 fifth instar larvae starved for 4 h be- DNS and heating in boiling water for 10 min. Since DNS fore the start of the experiments. The dishes were simi- is a color reagent, the reducing groups released from larly placed in a controlled room as above. Consumption starch by α-amylase action were measured by the reduc- was recorded by using a digital leaf area meter (ADC, tion of DNS. The boiling water stops α- amylase activity, bioscientific Ltd., England) after 24 h.