Kangetsu Hirase

The A2′N Mutation of the RDL γ-Aminobutyric Acid Receptor Conferring Fipronil Resistance in Laodelphax striatellus (Hemiptera: Delphacidae)

ABSTRACT The planthopper Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) is a serious insect pest of rice, Oryza sativa L., and has developed resistance to fipronil in Japan. Sequence analysis of L. striatellus RDL &ggr;-aminobutyric acid (GABA) receptor subunit (LS-RDL) genes from a fipronilresistant population and a fipronil-susceptible strain identified the A2′N mutation (index number for M2 membrane-spanning region), that was previously implicated in fipronil resistance in the planthopper Sogatella furcifera (Horváth) (Hemiptera: Delphacidae). Nineteen of 21 fipronil-resistant L. striatellus individuals were genotyped as heterozygous for the A2′N mutation, suggesting that this mutation is associated with fipronil resistance and that most fipronil-resistant L. striatellus express wild-type and A2′N mutant LS-RDL simultaneously. To confirm the role of the A2′N mutation of LS-RDL, Drosophila Mel-2 cells were transfected with wild-type and A2′N mutant LS-RDL genes, either individually or together. A membrane potential assay showed that fipronil had no inhibitory effect at 10 &mgr;M on cells transfected with the A2′N mutant LS-RDL gene with or without the wild-type LS-RDL gene. By contrast, the IC50 value of fipronil for wild-type LS-RDL homomers was 14 nM. These results suggest that the A2′N mutation of the RDL GABA receptor subunit confers fipronil resistance in L. striatellus as well as S. furcifera.

Comparison between the modes of action of novel meta-diamide and macrocyclic lactone insecticides on the RDL GABA receptor

Macrocyclic lactones, avermectins, and milbemycins are widely used to control arthropods, nematodes, and endo- and ectoparasites in livestock and pets. Their main targets are glutamate-gated chloride channels. Furthermore, macrocyclic lactones reportedly interact with insect RDL γ-aminobutyric acid (GABA) receptors, but their modes of action on insect RDL GABA receptors remain unknown. In this study, we attempted to better understand the modes of action of macrocyclic lactones on RDL GABA receptors. We observed that ivermectin and milbemectin behaved as allosteric agonists of the Drosophila RDL GABA receptor. G336A, G336S, and G336T mutations had profound effects on the activities of ivermectin and milbemectin, and a G336M mutation abolished the allosteric agonist and antagonist activities of these macrocyclic lactones. These results suggest that G336 in TM3 of the Drosophila RDL GABA receptor is important for the binding of macrocyclic lactones. Recently, it has been suggested that a novel RDL GABA receptor antagonist, 3-benzamido-N-(2-bromo-4-perfluoroisopropyl-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7), binds to the transmembrane intersubunit pocket near G336 in the Drosophila RDL GABA receptor. Thus, we compared the effects of mutations around G336 and A302 mutations in TM2 on the activities of macrocyclic lactone and meta-diamide 7. The effects of L281C, V340Q, V340N, A302S, and A302N mutations on the activity of meta-diamide 7 differed from those on ivermectin and milbemectin. Molecular modeling studies showed that macrocyclic lactones docked in the intersubunit pocket near G336 in the Drosophila RDL GABA receptor in the open state. In contrast, meta-diamide 7 docked into the Drosophila RDL GABA receptor in the closed state. This suggests that the modes of action of macrocyclic lactone binding to the wild-type Drosophila RDL GABA receptor differ from those of meta-diamide binding.

Concentration-Dependent Effects of GABA on Insensitivity to Fipronil in the A2′S Mutant RDL GABA Receptor From Fipronil-Resistant Oulema oryzae (Coleoptera: Chrysomelidae)

ABSTBACT The beetle Oulema oryzae Kuwayama (Coleoptera: Chrysomelidae), an important pest of rice, has developed fipronil resistance in Japan. Molecular cloning and sequence analysis of O. oryzae RDL &ggr;-aminobutyric acid (GABA) receptor subunit (OO-RDL) genes from fipronil-susceptible and -resistant O. oryzae identified the A2′S mutation (index number for the M2 membrane-spanning region). To investigate the effect of the A2′S mutation on fipronil resistance, we stably expressed the wild-type and mutant OO-RDL homomers in Drosophila Mel-2 cells. A membrane potential assay exhibited that the IC50 values of fipronil for inhibition of the response to EC80 GABA of the wild-type and A2′S mutant OO-RDL homomers were 0.09 µM and 0.11 µM, respectively. However, the IC50 values of fipronil for inhibition of the response to EC95 GABA of the wild-type and A2′S mutant OO-RDL homomers were 0.11 µM and ≈5 µM, respectively. These results suggest that the GABA concentration is an important factor affecting fipronil resistance in O. oryzae carrying the A2′S mutation in OO-RDL.

Purification and properties of propanil hydrolase in Pseudomonas pickettii

Abstract Rice is resistant to propanil by virtue of an aryl acylamidase I enzyme which carries out detoxification of the herbicide. In order to obtain other crop plants resistant to propanil a bacterial source cally to transform plants, thus conferring herbicide resistance. For the primary stage, the purification and properties of a propanil hydrolase, isolated from a soil bacteria, Pseudomonas pickettii , was carried out. This enzyme was purified to homogeneity by ammonium sulfate fractionation, hydrophobic interaction chromatography, ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. The relative molecular weight was estimated to be 102,000 by HPLC equipped with gel filtration column. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 49,000. These results indicate that the propanil hydrolase from P. pickettii is a homodimer with subunit molecular weight of 49,000. The purified enzyme was stable for 42 days at 4°C. Amino acid composition of this enzyme was also determined.

Physiological role of the propanil hydrolyzing enzyme (aryl acylamidase I) in rice plants

Abstract With the view of studying the physiological role of the propanil hydrolyzing enzyme (aryl acylamidase I), a comparison was made between the propanil susceptible rice mutant, which lacks the enzyme activity, and normal rice. Also, the properties of rice treated with a carbamate insecticide, a strong aryl acylamidase I inhibitor, were examined. The growth of the rice mutant was repressed approximately twice as much as that of the normal one by ammonium sulfate and urea. The mutant rice accumulated twice as much free ammonia as the normal one in the shoots under a high nitrogen supply. m-Tolyl methylcarbamate (MTMC) was used as the inhibitor of the enzyme. The growth of normal rice treated with MTMC was repressed 20% by ammonium sulfate but that of untreated rice was not affected at all. The propanil susceptible rice mutant, which lacks the activity of aryl acylamidase I and has higher susceptibility to ammonium sulfate by nature, showed no changes after MTMC treatment. The rice mutant is also more susceptible to a microb-originated herbicide bialaphos, l -2-amino-4-[(hydroxy)(methyl)phosphinoyl]butyryl- l -alanyl-alanine, of which the mode of action is assumed to be ammonia accumulation, than the normal one. The mutant was controlled 50% by the foliar application of 40 ppm bialaphos but normal rice was not affected at all at this concentration. Furthermore, twice as much free asparagine was found in the mutant rice than in the normal rice. Accordingly, it is suspected that aryl acylamidase I plays an important role in the normal nitrogen metabolism relating asparagine in rice plants.