Carboxylamidases in Spodoptera exigua properties and distribution in the larval body

P hytoparasitica 21:1, 1993
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ቤተ መጻሕፍቲ ባይዱ
benzoylphenylurea (BPU) insecticides diflubenzuron and tellubenzuron are also recommended for controlling S. exigua in the Netherlands. However, the beet armyworm has shown some tolerance to both of these BPUs, while it was still susceptible to some of the recently developed BPUs such as chlorfluazuron and hexaflumuron (18,19). Although insects are capable of resisting toxic effects of insecticides through a variety of defense mechanisms, the most important route is in many cases enhanced detoxification. A metabolic study showed that hydrolytic cleavage of diflubenzuron and teflubenzuron is the major route of detoxification in S. exigua (18) and other Spodoptera species (9,11). A number of amino-containing organophosphate insecticides are metabolized to their corresponding carboxylic acid derivates (7). Whereas this reaction has been reported to occur in mammals and plants, little or no carboxylamidase - - responsible for hydrolysis of amidecontaining insecticides - - has been demonstrated in insects. The objective of this study was to characterize carboxylamidases activity in S. exigua using a sensitive colorimetric method. The biochemical properties of this enzyme system, along with the effect of various insecticide groups on its activity, were studied. MATERIALS AND METHODS Insects. The S. exigua strain was originally obtained from P. Smits (Wageningen, the Netherlands). The insects were reared on castor bean leaves (Ricinus communis L.) under standard laboratory conditions of 23~ 65% r.h., and a photoperiod of 16:8 (L:D). Fifth instar larvae were used in all assays. Chemicals. 4-Nitroaniline and 4-nitroacetanilide were purchased from Janssen Chimica (Beerse, Belgium). Diflubenzuron (Dimilin, 250 g 1-1 WP formulation) and technical diflubenzuron (99.5%) were supplied by Duphar BV ('s-Graveland, the Netherlands). Teflubenzuron (Nomolt, 150 g /-1 EC formulation) and technical teflubenzuron (98%) were obtained from Celamerck (Ingelheim, Germany). Hexaflumuron (Consult, XRD-473, 50 g /-1 EC formulation) was supplied by Dow Chemicals (Norfolk, UK). Methomyl (Lannate, 250 g 1-1 WP formulation), technical methomyl (99%) and dimethoate (Perfekthion, 500 g 1-1 EC formulation) were gifts from the Laboratory of Crop Protection Chemistry, Faculty of Agricultural Sciences, University of Gent, Belgium. Enzyme Preparation. Last instars were selected on a daily basis to provide 2-day-old larvae for enzyme preparations. Twenty to 50 midguts were dissected on ice, split longitudinally, and their gut contents removed. They were then washed in 1.15% KC1 and homogenized with a motor-driven Teflon pestle for 30 sec in ice-cold 0.05 M phosphate buffer, pH 7.5. The homogenate was centrifuged at 10,000g and 4~ for 15 min in a Martin Christ Ultracentrifuge. The pellet was discarded and the supernatant was recentrifuged at 105,000g for
K. Van Laecke and D. Degheele (1993) Phytoparasitica 21(1):9-21
Carboxylamidases in Spodoptera exigua: Properties and Distribution in the Larval Body Kristiaan Van Laecke and Danny Degheele1
Resistance to many insecticides demonstrated by the beet armyworm, Spodoptera exigua (Hiibner), can be caused by the action of carboxylamidases. A colorimetric method, based on the hydrolysis of 4-nitroacetanilide to 4-nitroaniline by carboxylamidases, was used for evaluating biochemical properties of these detoxifying enzymes in beet armyworm. The optimum pH and temperature were 7.5 and 38~ respectively. K (Michaelis constant) .and Vmax (maximal velocity) at 28~ were 2.3 X 10-4 M and 2.0r~ nmol min-1 mg protein- , respectively. The enzyme activity was evaluated in several body parts and located mostly (66.2%) in the midgut. The soluble fraction (supematant of 105,000g) contained the highest enzyme activity relative to the total (69.3%), and exhibited the highest specific activity. Carboxylamidase activity was totally inhibited in vitro at a concentration of 10-6 M methomyl. The analysis of the in vitro inhibition kinetics indicated the ability of methomyl and diflubenzuron to inhibit carboxylamidases noncompetitively. Over 95% inhibition in vivo was obtained when the larvae were fed with castor bean leaves dipped in 250 mg/-1 of methomyl. The in vivo enzyme activity could be reduced to half with a pretreatment of 15 mg/-1 diflubenzuron. KEY WORDS: Insecta; Spodoptera exigua; detoxitication; carboxylamidases; in vitro inhibition; methomyl; dimethoate; diflubenzuron; teflubenzuron; hexafinmuron. INTRODUCTION T h e beet armyworm, Spodoptera exigua (Hiibner), is a serious, polyphagous pest o f agricultural and floricultural crops (5). The insect has developed resistance to various insecticides, including some chlorinated hydrocarbons, organophosphates (OPs), carbamates and pyrethroids. Yoshida and ParreUa (21) detected resistance to m e t h o m y l in beet armyworms collected from floricultural crops in Florida. A population o f S. exigua in Guatemala has developed resistance to deltamethrin (6). Alava and Lagunes Tejeda (1) reported that in selection experiments with methyl parathion, the beet a r m y w o r m developed resistance to malathion, endrin, and methomyl. Accidental introduction o f an insecticide-tolerant strain into Dutch greenhouses in 1976 along with chrysanthemum cuttings from Florida resulted in great losses (16). The Received June 16, 1992; received in final form Dec. 8, 1992. x Laboratory of Agrozoology, Faculty of Agricultural Sciences, University of Gent, B-9000 Gent, Belgium. [Telefax: 32-91-646239]