| Nilaparvata lugens Stal (Brown planthopper. BPH) is one of the most important pests on rice and chemical control is the main way to manage this insect pest. BPH has developed high resistance to many insecticides commonly used. Fipronil is one of the most important insecticides for rice pest control in2006-2009. However, it was reported that BPH in field lost its sensitivity to fipronil significantly and resistance levels were high in some areas. In order to apply the effective control of BPH by insecticides, the current fipronil resistant status and the diversities of y-aminobutyric acid (GABA) receptors (GABARs) Rdl subunit were investigated in field populations from different coutries and artificially mutated populations from a susceptible strain. The influence of some interesting amino acid subsitutions on fipronil sensitivity was also determined.1. Firponil resistance levels in different field populationsIn order to reveal the fipronil resistance status,12field populations were collected around China and fipronil resistance was monitored. Toxicities of insecticide were measured using the rice-stem dipping method. The results showed that five populations from Fuzhou. Jiujiang. Taizhou, Nanjing and Anqing showed significant higher resistance (resistance ratios44.0-to65.7-fold) to fipronil than other populations. Nanjing population showed the highest level of fipronil resistance with the resistance ratio (RR) of65.7-fold, and the populations collected from Nanning showed the lowest level of fipronil resistance (RR=13.5-fold), which indicated the quick development of fipronil resistance in field populations.2. Sequence polymorphisms in Rdl functional regions of field populationsIn order to know the polymorphisms of Rdl proteins in different populations. Rdl transmembrane regions (TM1-TM3), the binding region of fipronil in GABA receptors, were sequenced in28populations collected from different areas, and40individuals were sequenced in each population. As a result, several polymorphism sites were found in21filed populations. After selection with super-high concentration of fipronil in some field populations, the increase of polymorphism frequency at3loci was found, such as Y258S. R282Q and A284S/G, in which A2084S/G was corresponding to A302S/G/N in other insect species. It suggested that these polymorphisms might be associated with fipronil resistance.3. Sequence polymorphisms in Rdl funetional regions of artificially mutated populationsTo find the association of resistant mechanism between field and laboratory selected populations, the artificial mutation was performed on individuals from a susceptible population in laboratory using both UV radiation and colchicine mutagenesis methods, which were then selected with high concentration of fipronil. Rdl transmembrane regions (TM1-TM3) were sequenced in the artificially mutated and selected individuals. Two polymorphism sites were found in the second transmembrane domain (TM2) of Rdl subunit, such as R282Q and M293I, in which R282Q was also found in some field populations mentioned above.4. Influence of amino acid substitution on fipronil sensitivityBy expressing of RDL wide-type or RDL mutants with different mutations in Xenopus oocytes and detection by the voltage-clamp, the influence of different amino acid substitutions found above on fipronil sensitivity was evaluated. All mutations, Y258S, R282Q and A284S/G, were found to significantly affect the inhibition of fipronil on GABA response. In addition, the·influence of the double mutations R282Q and A284S on the fipronil inhibition effect was significantly higher than any single mutation, which indicated the double mutations should be with more influence on fipronil resistance, if they really existed in field populations. |
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