Studies On The Resistance Mechanism Of Laodelphax Striatellus (Fallen) To Chlorpyrifos And Buprofezin

The small brown planthopper Laodelphax striatellus (Fallen) is a kind of pests that bring serious harm to grain crops. It is not only sucking the gramineous plants, but also spreading the virus between crops. This pest broke out only intermittently in China, and it was not an important pest for pest controlling before2000years. Since the early2005, however L. striatellus has reAChEd large number in china, especially in Jiangsu province. Long-term and unreasonable using of chemical insecticides in a wide range of rice-growing areas is the important reason for resistance of L. striatellus to the insecticides, and in recent years, insecticide resistance has been associated with frequent occurrence of the L. striatellus. Therefore, insecticide resistance management strategies must be developed to prevent further increase in resistance of L. striatellus. Lacking of the knowledge of resistance mechanism is the barrier for implementation of resistance management and efficient control. In this paper, efforts have been made to declare its biochemical and molecular mechanism of resistance to two types of synthetic insecticides, chlorpyrifos and buprofezin, which have been frequently used for controlling L. striatellus and other crop insect pests in fields for years, based on the lab-selected resistance strains. The results obtained are summarized as follows.1. The resistance selection and biochemical mechanism of chlorpyrifos resistance in L. striatellusCompared with the susceptible strain (YN), the chlorpyrifos resistance strain (YN-CPF) of L. striatellus developed213.49-fold resistance by continuous selection with chlorpyrifos for36generations on the strain YN in the laboratory. The chlorpyrifos resistance recession strain (YN-DPF) of L. striatellus came from the strain YN-CPF and was reared without contacting with any insecticides for12consecutive generations, and the resistance levels of strain YN-DPF recovered to83.08-fold relative to the strain YN. The biochemical mechanisms of chlorpyrifos resistance in L. striatellus were first studied by synergism test and detoxifying enzyme activity. The synergistic effects of PBO, TPP and DEM on chlorpyrifos in strain YN were compared with those in strains YN-CPF and YN-DPF. The resultes indicated that the oxidase inhibitor PBO showed3.77-and2.28-fold synergism with chlorpyrifos in strains YN-CPF and YN-DPF respectively, but no synergism of chlorpyrifos efficacy in strain YN, P450monooxygenase activity in strain YN-CPF was lower (only68%of the YN level) than in strain YN; The glutathione depleter DEM showed2.82-and1.79-fold synergism with chlorpyrifos in strains YN-CPF and YN-DPF respectively, but no synergism of chlorpyrifos efficacy was also found in strain YN, while the glutathione S-transferase activity towards CDNB was not significantly different between the distinct strains; The esterase inhibitor TPP synergized chlorpyrifos both in strains YN-CPF (4.31-fold) and YN-DPF (2.59-fold), but not in strain YN, while esterase activity using α-naphthyl acetate as substrate was significantly higher both in strains YN-CPF (4.15-fold) and YN-DPF (1.92-fold) than in strain YN. In addition, this study also found the maximum reaction rate (Vmax) of acetylcholinesterase (AChE) in strain YN-CPF was2.56-fold higher than that in strain YN. Affinity (Km) and inhibition concentration (Ⅰ10) experiment of AChE on chlorpyrifos-oxon also showed AChE insensitivity in the chlorpyrifos resistance strain. The results revealed that AChE insensitivity and elevated esterase activities may play important roles in conferring chlorpyrifos resistance in L. striatellus, the specific action mechanism of P450monooxygenase and glutathione S-transferase in L. striatellus on chlorpyrifos resistance remained to be further elucidated in future.2. The resistance selection and biochemical mechanism of buprofezin resistance in L.striatellusCompared with the strain YN, the buprofezin resistance strain (YN-BPF) of L. striatellus developed82.08-fold resistance by41generations of continuous selection with buprofezin on the strain YN in the laboratory; After12generations of continuous selection with buprofezin on the nanjing field strain (NJ), we obtained another buprofezin resistance strain (NJ-BPF) of L. striatellus which separately showed4.46-and96.58-fold resistance in comparison with the strains NJ and YN. The biochemical mechanisms of buprofezin resistance in L. striatellus were studied by synergism test and detoxifying enzyme activity. The synergistic effects of PBO, TPP and DEM on buprofezin in strain YN were compared with those in strains YN-BPF and NJ-BPF. The results showed that they all had no synergistic effects on strain YN, but the oxidase inhibitor PBO had1.77-and1.69-fold synergism with buprofezin in strains YN-BPF and NJ-BPF respectively, and P450 monooxygenase activity in strains YN-BPF (1.39-fold) and NJ-BPF (1.89-fold) was higher than that in strain YN. The glutathione depleter DEM showed1.02-and1.03-fold synergism with buprofezin in strains YN-BPF and NJ-BPF respectively, and the glutathione S-transferase activity towards CDNB was not significantly different among three strains. The esterase inhibitor TPP synergised buprofezin both in strains YN-BPF (1.50-fold) and NJ-BPF (1.77-fold), while esterase activity using a-naphthyl acetate as substrate was significantly higher in strains YN-BPF (1.69-fold) and NJ-BPF (2.16-fold) than that in strain YN. In addition, this study also found the activity of acetylcholinesterase (AChE) in strains YN-BPF and NJ-BPF were separately1.79-and1.53-fold higher than that in strain YN. The results indicated that enhanced detoxification that was mediated by P450monooxygenase and esterase could contribute to buprofezin resistance to some extent, moreover, the elevated AChE activity as an additional mechanism of buprofezin resistance cannot be ruled out.3. The sequence identification and expression analysis of carboxylesterase genes in L. striatellus31carboxylesterase-like genes (CarEs) were identified from the transcriptome database of L striatellus by using RT-PCR technique (tentatively named by Ls.CarEl-Ls.CarE31). Real-time quantitative PCR (qPCR) was performed for the relative expression analysis of31CarEs. Compared with that in strain YN, Ls.CarEl and Ls.CarE2showed significantly overexpression with32.06-and8.52-fold in strain YN-CPF, respectively. Then, qPCR of Ls.CarEl and Ls.CarE2in strain YN-DPF was conducted to verify their correlation with chlorpyrifos resistance. Compared with that in strain YN, the expression levels of Ls.CarEl and Ls.CarE2separately showed8.6-and1.09-fold in strain YN-DPF, which indicated a significant linear relationship between the expression level of Ls.CarEl and the level of phenotypic resistance (LC50), but didn’t in the case of Ls.CarE2. In addition, this study also found some amino acid mutations of Ls.CarE9, Ls.CarEl6, Ls.CarE24and Ls.CarE25in YN-CPF individuals. The results indicated that overexpression of Ls.CarEl might play important roles in conferring chlorpyrifos resistance in strain YN-CPF, and the amino acid mutants of carboxylesterase genes as an additional resistance mechanism cannot be ruled out.4. The sequence identification and expression analysis of P450genes in L. striatellusA total of112unique sequences annotated as cytochrome P450genes (P450s)(partial or full length cDNA, from100bp to over2000bp) were identified from the transcriptome database of L. striatellus. After the shorter and repeated sequences were deleted, we finally obtained55P450s which ranging from279bp to2001bp in length, of which25P450s had the complete open reading frame (ORF), the55P450s were named by Dr. David Nelson in accordance with the P450nomenclature committee convention (http://drnelson.uthsc.edu/cytochromeP450. html). Then qPCR was used to analyze the relative expression levels of the55P450s among those resistant and susceptible strains of chlorpyrifos and buprofezin. Of these genes, CYP6CW1showed9.72-fold higher expression in strain YN-BPF than in strain YN. Among the55P450s, the expression of CYP4C, CYP4DE1and CYP6AX decreased82.01%,89.22%and92.25%in strain YN-CPF compared with that in strain YN. These results suggested that overexpression of CYP6CW1might play important roles in conferring buprofezin resistances, and chlorpyrifos resistance might be partially achieved through decrease chlorpyrifos activation in L. striatellus.5. The functional analysis via RNAi and prokaryotic expression of carboxylesterase gene to elucidate the chlorpyrifos resistance in L. striatellusThe Rapid Amplification of cDNA Ends (RACE) technique was employed to clone the full-length sequence of Ls.CarEl from L. striatellus. The results showed that the ORF of Ls.CarEl was1839bp, encoding sequences of474aa in size. The5’and3’untranslated regions (UTR) were87bp and111bp, respectively. Analysis of the Ls.CarEl sequence revealed that it had these characteristics shared in many insect carboxylesterase genes, including the catalytic triads Ser215, Glu345and His466; oxyanion hole Gly134, Gly135and Ala217; the conserved cysteines Cys92and Cys110. Homologous analysis of amino acid sequences indicated that Ls.CarEl shared72%high identity with the carboxylesterase gene of Nilaparvata lugens (GenBank accession no. CAZ65617.1), so we could conclude that the Ls.CarEl was carboxylesterase gene in L. striatellus. Furthermore, Ls.CarEl was chosen for RNA interference (RNAi) followed by chlorpyrifos bioassay. The dsRNA concentrations for RNAi were separately designated as doses of80ng/ul and200ng/μL. qPCR was performed to analyze the relative expression level of Ls.CarEl after ingestion of the dsRNA on the sixth day. The results showed that the transcript level of Ls. CarEl was significantly decreased (60%for80ng/ul and70%for200ng/μL) as compared with those in the controls. At the same time, mortalities of the YN-CPF nymphs by RNAi increased from33.7%to72.31%(for80ng/ul) and31.7%to69.56%(for200ng/μL), respectively. These results provided a believable evidence for the role of Ls.CarEl in the resistance of L. striatellus to chlorpyrifos. Overexpression of gene does not always result in increasing of protein content. Therefore, we obtained the fusion protein of Ls.CarEl through prokaryotic expression technology, which laid a foundation for the following experiment on specific antibody preparation and western-blot to detect the difference of Ls.CarEl quantity between the chlorpyrifos resistance and susceptive strain.