Preliminary Proteomic Analysis Of The Bactrocera Dorsalis (Hendel)Under Spinosad Stress

The Bactrocera dorsalis (Hendel) is an important pest on fruits and vegetables in south China. It can damage more than250kinds of fruit including citrus, mango, guava and carambola. The Bactrocera dorsalis is ranked in the quanrantine pest in many countries and regions. The spinosad is a kind of pollution-free biological macrolide pesticides that extracted from the fermentation of the Saccharopolyspora spinosa, which acts on the nicotinic acetylcholine receptor (nAChR) and gamma aminobutyric acid receptor (GABAR), it can effectively control the pests of Lepidoptera, Diptera and Thysanoptera etc. Spinosad is one of the important pesticides to control the Bactrocera dorsalis at present, but the Bactrocera dorsalis have developed a certain degree of resistance risk of spinosad. Therefore, in order to rationally use the spinosad to prevent the damage of the Bactrocera dorsalis and prolong the life of spinosad, this study combined the insect toxicology, bioinformatics and proteomics to research the Bactrocera dorsalis proteomics under the spinosad stress. The two-dimensional gel electrophoresis conditions of the crude proteins in the larvae Bactrocera dorsalis were established, and the difference proteins before and after spinosad stressed were separated and identified, witch laid the foundation for the in-depth study of mechanism of resistance formation of spinosad. The main results are summarized as follows:1. Established and optimized the two-dimensional electrophoresis conditions of the crude proteins in the3th instar larvae of Bactrocera dorsalis. The results showed that separating the crude proteins in the larvae of Bactrocera dorsalis extracted by the BPP method with the24cm pH4-7IPG strips, and staining the2-DE gels with the coomassie brilliant blue was the most suitable method for the Bactrocera dorsalis proteomics research, which laid the foundation for the preliminary proteomics research to the Bactrocera dorsalis under spinosad stress.The BPP and three TCA-acetone methods to extract the crude proteins in the3th instar larvae of Bactrocera dorsalis were compared, the results showed that the BPP method was the best and1187±42protein spots were attained with this method. The13and24cm IPG strips were used to separate the crude proteins in the larvae of Bactrocera dorsalis, the results showed that1129±46protein spots were obtained with24cm IPG strips, representing an increase of483spots with13cm IPG strips. The similar results were come out when coomassie brilliant blue staining and silver staining were used, but background of coomassie brilliant blue staining was cleaner and clearer. The10specific protein spots on BPP gel were identified by mass spectrometry, and9protein spots were successfully retrieved in Metazoa (Animals) and6in Drosophila (fruit flies), searching database of Metazoa (Animals) can be obtained more comprehensive information than Drosophila (fruit flies).2. The difference proteins of Bactrocera dorsalis before and after5mg/mL spinosad stressed in a shorter period of time were separated and identified. The results showed that the identified proteins were mainly involved in material and energy metabolism, immune response, detoxification function and cytoskeletal formation, witch provided basic information support for the in-depth study of its response mechanism of lower concentration of spinosad stress.Using dipping method treated the3th instar larvae of Bactrocera dorsalis, soaking for10s and placed in the soil. Treated with acetone as the control group and sampled after placed in the soil for5min, with spinosad as the experimental group and sampled after placed in the soil for5min and2h respectively.58different protein spots whose expression levels changed2times were observed and identified by mass spectrometry.44spots were identified successfully, among which23spots were up-regulated and21spots were down-regulated. The successfully identified proteins can be divided into material and energy metabolism related proteins, immune response related proteins, detoxification proteins, cytoskeletal proteins and others according to the functions analyzed by bioinformatics, in witch,43%were taken part in material and energy metabolism.3. The difference proteins of Bactrocera dorsalis before and after10mg/mL spinosad stressed in a longer period of time were separated and identified. The results showed that the identified proteins were material and energy metabolism related proteins, immune response related proteins, nervous system development associated proteins, cytoskeletal proteins and others, witch provided basic information support for the in-depth study of its response mechanism of higher concentration of spinosad stress.Using dipping method treated the3th instar larvae of Bactrocera dorsalis, soaking for10s and placed in the soil. Treated with acetone as the control group and sampled after placed in the soil for6h, with spinosad as the experimental group and sampled after placed in the soil for6h and12h respectively. The crude proteins were separated by2-DE and identified by mass spectrometry,54different protein spots were observed and42spots were identified successfully, among which17spots were up-regulated and25spots were down-regulated. The successfully identified proteins were classified into5groups according to the functions analyzed by bioinformatics, including67%material and energy metabolism related proteins,2%immune response related proteins,7%nervous system development associated proteins,2%cytoskeletal proteins and22%other proteins.