Biochemical And Molecular Mechanisms Of Resistance To Alpha-cypermethrin And Abamectin In Bemisia Tabaci

1. Biotype identification of the NJ strain of whitefly, Bemisia tabaciThe biotype of the NJ strain of whitefly collected in Nanjing, China, was identified with biological, biochemical and molecular approaches. The NJ strain can induce squash silverleaf symptom by infecting. The native PAGE banding pattern of esterase is obviously different between the NJ strain and a Q-type En-Q strain. The mitochondrial cytochrome oxidaseâ… gene sequence of the NJ strain was 98% similar to that of B-type strains. These results confirmed that the NJ strain of B. tabaci is B-type.2. A mutation in sodium channel gene associated with pyrethroid resistance and its detection in Bemisia tabaciA mutation, leucine to isoleucine at position 925(L925I) in theâ…¡S4-6 linker of the para-type sodium channel protein of the whitefly, Bemisia tabaci from NJ strain was identified to be associated with pyrethroid resistance. The DNA-based genotyping technique PASA(PCR Amplification of Specific Allele) for rapid detection of the L925I mutation was established. The NJ strain showed 266-fold resistance to alpha-cypermethrin compared with the susceptible SUD-S strain. The resistance to alpha-cypermethrin in the selected Nanjing strain(NJ-R1) increased to 785-fold after several selections with alpha-cypermethrin. PASA genotyping results indicated that 100% individual females from NJ-R1 strain had L925I mutation(61.1% homozygotes and 31.9% heterozygotes), whereas 75% individual females from the unselected NJ strain had L925I mutaion(35% homozygotes, 40% heteozygotes and 25% wild type), SUD-S strain individuals had not L925I mutation. These results indicated that L925I mutation was tightly associated with alpha-cypermethrin resistance in the whitefly from Nanjing. 3. The relative importance of kdr mutation and detoxifying metabolism to alpha-cypermethrin resistance in B-type whitefly, Bemisia tabaciThe field-collected NJ strain has 266-fold resistance to alpha-cypermethrin compared to the susceptible SUD-S strain. The NJ-R1(785-fold) and NJ-R2(2634-fold) strains were derived from the NJ strain using mass selection and single pair selection with alpha-cypermethrin respectively. The synergism ratios of piperonyl butoxide(PBO) to alpha-cypermethrin in these three strains were similar(about 20-fold), which suggested metabolic detoxification probably confers about 20-fold inherent resistance in the B-type whitefly, as PBO is a mixed function oxygenase(MFO) and esterase(EST) inhibitor and glutathione S-transferase(GST) is not affinity to resistance in B. tabaci. The synergism ratio of triphenyl phosphate(TPP) to alpha-cypermethrin in the NJ strain was 12-fold, which implied the synergism of PBO in the B-type whitefly is mainly due to inhibiting esterase activity. The remaining resistance after PBO inhibiting conferred by kdr mutation was 149-, 40-and 14-fold in the NJ-R2, NJ-R1 and NJ strains respectively, which suggested that kdr mutation is a determinant factor for alpha-cypermethrin resistance in B. tabaci and would confer about 150-fold resistance at most because NJ-R2 strain was homozygous for L925I mutation.4. Cross-resistance and biochemical mechanisms of abamectin resistance in the B-type Bemisia tabaciTo better understand resistance risk and possible mechanisms to abamectin in B-type Bemisia tabaci, a resistant strain of B. tabaci was selected in laboratory and cross-resistance pattern and resistance mechanisms to abamectin were investigated. The NJ-Abm strain of B. tabaci was derived from a field population(NJ) collected in Nanjing of China in 2002 with 18 generations of selection with abamectin in the laboratory. Compared with the unselected NJ strain, the selected NJ-Abm strain developed 14.5-fold resistance to abamectin and showed significant cross-resistance to emamectin benzoate(4.4-fold) and imidacloprid(3.4-fold), but no cross-resistance to fipronil. The oxidase inhibitor PBO and glutathione S-transferase inhibitor diethyl meteate(DEM) produced significant synergism on abamectin in the NJ-Abm strain(with synergistic ratios of 3.9-and 4.1-fold respectively); however the esterase inhibitor TPP did not give any synergism. Biochemical analysis confirmed that P450 monooxygenase activity and glutathione S-transferase activity of the NJ-Abm strain were elevated to 2.1-and 2.0-fold respectively compared with that of the NJ strain. This indicated enhanced metabolism mediated by P450 monooxygenase and glutathione S-transferase is likely involved in abamectin resistance and cross-resistance to imidacloprid and emamectin benzoate in the NJ-Abm strain.5. Cloning and sequence analysis of glutamate-gated chloride channel receptor gene in Bemisia tabaciBy using RT-PCR and RACE methods, a complete cDNA of glutamate-gated chloride channel receptor gene(BtGluCla1) was cloned and sequenced from Bemisia tabaci. The full-length cDNA of BtGluCla1 was 1353 base long and encoded a protein of 450 amino acid residues. Gene tree analysis demonstrated that BtGluCla1 was close to glutamate-gated chloride channel receptor genes of insects, and far from that of nematodes. Putative protein of BtGluCla1 has one neurotransmitter-gated ion-channel signature, nine phosphorylation sites and four extracellular cysteine residues. Four transmembrane domains near the C-terminal of the protein were predicted by TMHMM. The identity of deduced amino acid sequence of BtGluClal was 81% to that of GluClalpha of Drosophlila melanogaster(GenBank accession no. NP₇32447). There were eight amino acid phylmorphism sites in the resistant NJ-Abm strain and only three amino acid phylmorphism sites in the susceptible NJ strain. A truncated BtGluCla1 lacking the transmembraneâ…£domain(named as dBtGluCla1) was detected at a frequency of 20% in the NJ strain and 83% in the NJ-Abm strain. Analysis of the genomic DNA sequences of a single whitefly showed that BtGluCla1 and dBtGluCla1 may be encoded by different genes.