Sensitivity Of Striped Flea Beetle, Phyllotreta Striolata To Chlorpyrifos And Cloning Of Related Enzyme Genes

Striped flea beetle (SFB), Phyllotreta striolata, is a significant pest in cruciferous vegetables. Chemical treatment plays a major role in the control and management of SFB. However, the resistance problem has emerged due to the mass application of insecticides. In this paper, susceptibility difference among field and laboratory populations of SFB to chlorpyrifos were determined, and the genes of two resistance-related enzymes, esterase (Est) and acetylcholinesterase (AChE), were cloned and analyzed, to provide a solid and scientific basis for the resistance management of SFB.Bioassay was conducted to examine and compare LC₅₀ values of a field natural population (FNP) and two laboratory populations fed with Rorippa indica (RiP) and Brassica campestris (BcP) of SFB exposed to chlorpyrifos. The results indicated that LC₅₀ value of RiP to chlorpyrifos was the lowest with 30.3459 mg·L^-1, while FNP had the highest of 77.8448 mg·L^-1. The sensitivity index of FNP was 0.39. The enzymatic activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutothine-transferase (GST) were measured to show difference of biochemical indexes of the SFB populations. The lowest AChE activity was observed in FNP, significantly different from those in RiP and BcP. The highest GST activity was found in FNP, while the lowest and highest CarE activities in RiP and FNP, respectively. Our study suggested that chlorpyrifos had an inhibitory effect on the AChE activity, and the resistance of SFB to chlorpyrifos might be associated to some extent with higher CarE and GST activities.Using a pair of primers based on the conserved sequences of Est genes in insects, RT-PCR was conducted and one Est gene fragment of 281 bp and one AChE gene fragment of 406 bp were cloned and characterized.The 3'-sequence (1468 bp, GenBank accession number: EU166919) of Est gene was obtained by 3'-RACE and found to encode 442 amino acids. Homology analysis revealed that the similarity of nucleotide sequence between the obtained fragment and Est1 genes of Tribolium castaneum and Tribolium confusum was the highest, with the nucleotide identity of 71 % (211/297). The cloned fragment also shared 70% (156/222) identity to the alpha-esterase5 gene of Drosophila melanoqaster. And the amino acid sequence had 43% (188/434), 43 % (188/434), 33 % (144/432) similarities with the esterase genes of Tribolium confusum, Tribolium castaneum and Drosophila melanoqaster, respectively. Based on these results, it could be inferred that the obtained fragment should be a partial nucleotide sequence of Est gene of SFB.Homology analysis revealed that the obtained AChE nucleotide sequence (406 bp, GenBank accession number: EU169456) in SFB shared the most similarity 75% (303/402) to AChE gene of Leptinotarsa decemlineata, and was similar to AChE gene of Nilaparvata lugens with 73% (300/407) nucleotide identity. The fragment also showed more than 68% nucleotide identity to AChE genes in other 20 insect species. And the amino acid sequence had 87% (119/136), 83% (114/136), 83% (114/136) similarities with the AChE genes of Leptinotarsa decemlineata, Helicoverpa armigera and Bombyx mori, respectively. The results indicated that the cloned cDNA fragment in SFB was a partial nucleotide sequence of its AChE gene.