Analysis Of NAChR β1Subunit Polymorphism And Associate With Imidacloprid Resistance In The Brown Planthopper

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. As a representative of neonicotinoid insecticides, imidacloprid was widely used to control BPH in most Asian countries and areas. However, since2005, imidacloprid resistance developed sharply and reached a peak level over500-fold in2006. In China, imidaclprid was not recommended to use singly for BPH control from2007. Nicotinic acetylcholine receptors (nAChRs) are targets of neonicotinoid insecticides. In order to understand the imidacloprid resistance mechanisms in BPH and apply the effective control of BPH by insecticides, the current imidacloprid resistant status and the diversities of nAChR β1subunit 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. Imidacloprid resistance levels in different field populationsIn order to reveal the imidacloprid 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, all12BPH populations show high resistance to imidacloprid, in which10populations were with resistance ratios (RR) over110-fold. Nanjing population showed the highest level of imidacloprid resistance (RR155.9-fold). Two populations from Fuzhou and Jiujiang showed lower levels of imidacloprid resistance, but still above70-fold. The reasons for high imidacloporid resistance in field populations might from two aspects. Although imidacloprid was not recommended to control BPH, imidacloprid was still widely used in many areas in China due to the low price of this insentcide. Imidacloprid was widely used in other countires, such as Vietnam and Thailand, and BPH with high imidacloprid resistance from these countries immigrated into China.2.Sequence polymorphisms in nAChR β1functional regions of field populationsIn order to know the polymorphisms of β1subunit in different populations, the extracellular regions, contributing to imidacloprid binding, were sequenced in28populations collected from different areas, and40individuals were sequenced in each population. Results showed that, in28field populations from different countires, polymorphism sites were found in16populations, in which K75Q and R81Q were located in loop D and K188E and N189D in loop F.3. Sequence polymorphisms in (31functional 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 imidacloprid. The extracellular regions of β1subunit were sequenced in the artificially mutated and selected individuals. One amino aicd deletion (A21) was found in individuals from the colchicine treatment and one amino acid substitution (R81Q) was found in individuals from the UV radiation treatment. R81Q was located in loop D of β1subunit, which was important for imidacloprid binding.4. Influence of amino acid substitution on imidacloprid sensitivityLoop D or F of BPH β1subunit were introduced into the corresponding sites and to repleace loop Dor F of rat P2subunit construct the chimera β2LD and β2LF. Matations, K75Q and R81Q in loop D and K188E and N189D in loop F, were introduced into the corresponding sites in the chimera subunits. Rat β2, chimera subunits or sununit mutants were co-expressed with BPH α1subunit in Xenopus oocytes. The influence of different amino acid substitutions found above on imidacloprid sensitivity was evaluated. All mutations, K75Q, R81Q, K188E and N189D, were found to significantly affect the imidacloprid response and sensitivity. In addition, the influence of the double mutations K188E and N189D on imidacloprid snesitivities was significantly higher than any single mutation, which indicated the double mutations should be with more influence on imidacloprid resistance, if they really existed in field populations.