Imidacloprid Resistance And Its Mechanisms In Field Populations Of Brown Planthopper, Nilaparvata Lugens (St(?)l)

The brown planthopper (BPH), Nilaparvata lugens Stal, is one of the most important migratory rice pests, and its control is emphasized and largely relied on insecticids. Though resistance to organochlorines, organophosphates, carbamates and pyrethroids had been reported from time to time in this pest, no high resistance widely occurred and resulted in failture in field control. The only exception is the resistance to imidacloprid recently occured. In order to seek efficient substitute of imidacloprid and achieve a good control of BPH, the development and the mechanism of imidacloprid resistance in field population were purchased based on the previous studies with laboratory selected imidacloprid resistance.1. Establishment of the relative susceptible baseline data to common insecticides and resistance monitoring in field populations of BPHThe susceptibility of BPH to 9 insecticides of five different categories was determined with laboratory susceptible strain and the base line data were obtained. Based on these data, four field populations (Anqing, Jiangpu, Wujiang and Guilin) collected from paddy field in 2005 and 2008 were monitored for resistance to various insecticides. The results revealed that dinotefuran, imidacloprid, carbosulfan, pymetrozine, acetamiprid and fipronil have high relative toxicity index to susceptible strain among the nine insecticides. Various field populations usually showed similar resistance to the same insecticide, except for resistance to imidacloprid in 2005. But it is sure that resistance of field populations varied with different insecticides and monitoring time. BPH of field populations in 2005 had developed moderate to high level of resistance to imidacloprid (RR 26.6-147.8) and low level to acephate(RR<10), but no resistance to fipronil, carbosulfan, and buprofezin. Whereas in 2008, multiple resistances had been developed. Beside the high level of resistance to imidacloprid was maintained (61.1-87.3), moderate level of resistance to pymetrozine, acetamiprid and buprofezin (12.2-23.8), moderate to high level of resistance to carbosulfan (24.9-64.5), and low level of resistance to acephate and Chlorpyrifos(4.9-14.3) were developed, leaving only dinotefuran and fipronil as sensitive ones among the insecticides tested.2. Susceptibility recovery of field resistant population and further selectionTo declare the stability and dynamics of imidacloprid resistance in BPH, the hoppers from WJ field resistant population (RR 147.8) were separated into two groups and subjected to parallel treatments, susceptibility recovery and further resistance selection with imidacloprid. The results showed that susceptibility recovery by breeding without contacting any insecticides had a dramatic decline in its imidacloprid resistance (RR from 147.8 to 21.9 in 19 generations), especially in the first several generations. On the other hand, the further selection made the resistance increased (from 147.8-fold to 381.0-fold in 19 generations) and a high resistant strain (R19) was developed. However, after nineteen generations selected, it seemed to be difficult to select a higher imidacloprid resistance. Selections in every two generations made a slow decline in resistance and stopping selection made a quick decline (RR from 380.9-fold to157.7-fold in 6 generations). So, all of these results indicated that imidacloprid resistance was not stable, especial the resistance at high level.3. Cross-resistance in field population of BPH resistant to imidaclopridThe cross-resistance to various insecticides was analysized by testing of the toxicity of eight insecticides of four different categories on the susceptible and resistant strains and the field resistant population of BPH. The results indicated that the BPH with high resistance to imidacloprid did not show obvious cross-resistance to all the neonicotinoid insecticides. Only pymetrozine and acetamiprid amongthe insecticides tested were confirmed to be cross-resistant. To Dinotefuran and other non-neonicotinoid insecticides, such as acephate, fipronil, buprofezin and carbosulfan, no significant cross-resistance was found. In conclusion, imidacloprid might have a narrow of cross-resistance spectrum. Thus, it means that the alternate and mixed use of substitutes with little cross-resistance could be selected to delay the resistance development inthis pest.4. The relative fitness of BPH resistant and susceptible to imidacloprid The relative fitness of resistant BPH has been studied by comparing the population trend index, which was obtained through constructing life tables of susceptible, susceptibility-recovery and resistant strains. The results showed that the resistant strain (RR 380.96) had very lower population trend index (25.6) with dramaticly poor fecundity and hatchability, which were less than half that of susceptible strain. The relative fitness index calculated was only 0.191. However, the population trend and relative fitness index was 119.5 and 0.889, respectively in susceptibility recovery strain (RR 21.92). Among the tested indexes, only two (survival rate and emergence rate) were found decreased. These indicated that susceptible BPH had advantages over resistant ones under the circumstance without imidacloprid selection pressure, and further revealed that imidacloprid resistance in heterozagous populations was not stable. So, the susceptibility of BPH could recovery if they were kept from contacting with imidacloprid or other insecticides exhibiting cross-resistance.5. Biochemical mechanisms for imidacloprid resistance in field population of BPHThe synergisms of TPP, PBO and DEM, the inhibitors of esterase (EST), P450 monooxydase and glutathione S-transferase (GST), on imidacloprid in the susceptible, susceptibility recovery and resistant strains were first tested. The results revealed that the enhanced P450-detoxification could be an important biochemical mechanism for imidacloprid resistance in field populations, EST could also play some roles for super high level resistance, but GST seemed not. For further demonstration of the biochemical mechanisms, the changes in the activity of P450 and EST among different strains were analyzed. Of which, the correlation index of the P450 activity and imidacloprid resistance among different strains is as high as 0.95 (P<0.01, DPS6.50), but the EST activity did not correlate with imidacloprid resistance level. It implied that the enhancement of P450 activity was the key factor for imidacloprid resistance in BPH field populations. Furthermore, the P450 activity in four field strains collected in 2005 and the activity of three detoxification enzymes (P450, EST and GST) in the strains collected in 2008 were also tested, respectively. These results also proved that higher activity of P450 was the main biochemical mechanism for imidacloprid resistance in BPH and also for multiple resistances in the populations tested in 2008.6. Cloning of the functional extracellular regions of nAChR a-subunit genes and amino acid polymorphisms analysis With degenerate primers designed for nAChR a-subunit genes, half nest PCR was carried out and four cDNA fragments of the nicotinic acetylcholine receptor (nAChR) genes were cloned from BPH. Further sequence analyzed and comparison with previously reported sequences of nAChR genes confirmed that these fragments had a typical characteristic of nAChR a-subunit and high similarity with other insect a-subunits (75-92%). So, the four cloned putative nAChR subunits were designated as Nlal-like (FJ628418), Nlα2-like (FJ557108), Nlα3-like (FJ557109) and Nlα4-like (FJ557110). For checking the resistance mutation previously reported in laboratory selected strains and studying the mechanism for target resistance in BPH field population, the functional regions of the four nAChR a-subunits were cloned and the cDNA sequences from resistant and susceptible individuals were compared. The results revealed that each gene contained some amino acid polymorphisms. However, most of them occurred in very low frequence and no mutation was found to be associated with imidacloprid resistance.7. Quantitative expression of cytochrome P450 genes and the relationship with imidacloprid resistance in Nilaparvata lugensAccording to the published sequences of Actin, CYP6AX1 and CYP6AY1 in GenBank, and other related gene sequences (CYP3A4, CYP4C1, CYP6A2, CYP4G1 and CYP6A20) obtained in our laboratory, primers were designed for real-time quantitative PCR. Then considered Actin as the housekeeping gene and sensitive CYP3A4 gene expression as control, the relative mRNA expression of various CYP450 genes in resistant and susceptible strains of BPH were estimated by CT comparison method. It was found that most CYP450 genes had higher level of relative mRNA expression in resistant strain. Especially, the gene of CYP6AY1 was 21.7 folds higher in resistant strain than that in susceptible strain. Combination of the previous results in biochemical mechanism analysis revealed that the over-expression of CYP6AY1 might associate with the high resistance to imidacloprid in field populations of BPH.