Impacts of Transgenic Bt Rice on Non-Target Brown Planthopper ——Nilaparvata Lugens Stal(Homoptera:Delphacidae)

论文摘要

Rice （Oryza saliva L.） is one of the most important food crops grown widely throughout the world, providing a staple food for nearly half of the global population. Pest and disease problems are major constraints for increasing rice production in almost all parts of the world. The increasing awareness of detrimental effects on the reliance of chemical based management practices in the past has transformed the direction of research efforts towards developing a more environmental friendly and economically feasible management practices on crop production. As a novel approach, Bt rice plants containing cry genes have been successfully developed for the management of lepidopteran pests of rice. Although transgenic rice varieties have not been approved for commercial cultivation any part of the world yet, China is likely to soon commercialization of Bt rice. However, the impact of Bt rice on non-target herbivorous pest insects are necessary parts of an environmental risk assessment studies, that should be undertaken before commercialization to ensure safety consideration for Bt rice. For example, although the brown planthopper （BPH）, Nilaparvata lugens （Stal）, is an important pest of rice worldwide, the impacts of transgenic Bt rice on the non-target BPH are poorly understood. Therefore, non-target impacts of three transgenic Bt rice lines （expressing the crylAb/crylAc, crylCa and cry2Aa proteins） on the BPH, were evaluated.Cellular immune system responses were assayed with a detailed analysis of total hemocyte count （THC）, differential hemocyte count （DHC） and histopathology of hemocytes of BPH across three consecutive generations. Given that the morphological characteristics of BPH hemocytes were unknown before this study, an attempt was made to investigate the detailed microscopic analysis of the hemocytes of BPH. Expression of the phenoloxidases activation system in BPH fed on Bt rice plants were studies as phenolxidases served an indicator of intensity of the insects immune reactions. In particular, abundance of the yeast-like symbiotes （YLS） in BPH fed on different Bt rice and non-Bt parental control was monitored. Moreover, the molecular responses of digestion, detoxification and immune response genes of BPH were elucidated for the better understanding the ability of the BPH to adapt the Bt rice plants. Fitness consequences of BPH fed on Bt rice plants were observed by monitoring fecundity, survival, growth and development, indicating a significant cost to the adaptation of BPH to Bt rice. In addition, proteomic analysis was performed to identify the Bacillus thuringiensis toxin binding activity in the BPH fed on Bt rice plants. Examination of hemocytes from BPH indicated the presence of six types of hemocyte: prohemocytes （PRs）, plasmatocytes （PLs）, granulocytes （GRs）, adipohemocytes （ADs）, oenocytoids （OEs） and spherulocytes （SPs） and to the best of our knowledge, this is the first study to detect the hemocytes of BPH. It was estimated that the mean numbers of total haemocyte count are varied from6096.4to7393.6cells per mm3of haemolymph with the different physiological stages of the insects and it was statistically increased in the aged adults. PLs, GRs and PRs are the most abundant cells in the haemolymph in all development stages of BPH while SPs is the scarcest type.The results revealed that transgenic Bt rice expressing Cry1Ab/Cry1Ac, Cry2Aa and Cry1Ca had no significant adverse effects on total hemocyte count, differential hemocyte count and phenoloxidases activation of BPH, indicating that Bt rice could not affect the hemocytes and PO activities of BPH. No significant differences were observed on the population abundances of YLS in BPH, which were fed on Bt rice compared to the non-Bt parental rice.The results revealed that the tested Bt rice strains have no unfavorable effect on fecundity, survival and growth of BPH. Furthermore, no differences were observed in tested gene expression among the BPHs fed on Bt rice lines and their non-transgenic parental cultivar, thus, provides the conclusion that Bt rice have no detrimental effects on the physiological processes of digestion, detoxification and immune responses of BPH due to our data. The the western blot analysis showed that the materials reacting with cry1Ac and cry1Ca antibodies were present in crude homogenates from adult BPH, which had been fed on Bt rice lines, but, the interaction between cry toxins and gut receptors of BPH are complex and need further investigation at molecular levels to confirm the functional receptors for cry toxins in BPH.This study enhances understanding of the complex interaction between transgenic rice and BPH. The new findinds would be useful for understanding the effects of Bt rice on non-target sap sucking insects and it is hoped that this will serve as a more reliable proofs in determining environment safety assessment whether to proceed with the commercialization of Bt rice in the future.

论文目录

• ACKNOWLEDGEMENTS
• ABSTRACT
• LIST OF ABBREVIATIONS
• LIST OF FIGURES
• LIST OF TABLES
• AIM OF THE STUDY
• CHAPTER 1: LITERATURE OVERVIEW
• 1:1 Brown Planthopper - Threat to Rice Production
• 1:1:1 Brown Planthopper problem in China
• 1:1:2 Brown Planthopper problem in Sri Lanka
• 1:2 Genetically Modified Rice
• 1:2:1 Insect-Resistant Genetically Modified Rice
• 1:2:1:1 Crystal delta endotoxins from Bacillus thuringiensis
• 1:2:1:2 Protease inhibitors
• 1:2:1:3 Plant lectins
• 1.3 Effect of transgenic Bt rice on BPH
• 1.4 Safety consideration of Bt rice
• 1.4.1 Food safety of Bt Rice
• 1.4.2 Environmental concerns
• 1.4.3 Socio-economic concerns
• 1.5 The immune system in insects
• 1.5.1 Cellular immune responses
• 1.5.1.1 Types of hemocytes
• 1.5.1.2 Immune functions of hemocytes
• 1.5.2 Humoral immune responses
• 1.5.2.1 Antimicrobial peptides （AMPs）
• 1.5.2.2 Prophenoloxidase activating system
• CHAPTER 2: CHARACTERIZATION OF HEMOCYTES OF THE BROWNPLANTHOPPER; Nilaparvata lugens Stal（Hemiptera:Delphacidae）
• 2.1 Introduction
• 2.2 Materials and methods
• 2.2.1 Experimental insects
• 2.2.2 Hemocyte morphotypes:Observation of hemocytes on stained
• 2.2.3 Hemocyte morphotypes: Observation of the hemocytesimmediately after bleeding
• 2.2.4 Differential hemocyte count
• 2.2.5 Total hemocyte count
• 2.2.6 Statistical Analysis
• 2.3 Results
• 2.3.1 Morphological observations
• 2.3.1.1 Prohaemocytes
• 2.3.1.2 Plasmatocytes
• 2.3.1.3 Granulocytes
• 2.3.1.4 Oenocytoids
• 2.3.1.5 Adipohaemocyte
• 2.3.1.6 Spherulocytes
• 2.3.2 Differential hemocyte count
• 2.3.3 Total hemocyte population
• 2.4 Discussion
• CHAPTER 3: IMPACTS OF TRANSGENIC BT RICE ON HEMOCYTES OF BROWNPLANTHOPPER; Nilaparvata lugens Stal （Homoptera:Delphacidae）
• 3.1 Introduction
• 3.2 Materials and methods
• 3.2.1 The experimental population of N. lugens
• 3.2.2 Test plants
• 3.2.3 Rearing the experimental populations of BPH
• 3.2.4 Total hemocyte count
• 3.2.5 Differential hemocyte count
• 3.2.6 Histopathology of Hemocytes
• 3.2.7 Statistical Analysis
• 3.3 Results
• 3.3.1 Alterations onTHC of BPH after espouse to Bt toxin
• 3.3.2 Alterations on DHC of BPH after espouse to Bt toxin
• 3.3.3 Hemocyte abnormalities
• 3.4 Discussion
• CHAPTER 4: PHENOLOXIDASE ACTIVITY IN BROWN PLANTHOPPER;Nilaparvata lugens Stal （Homoptera:Delphacidae） FEEDING ON BT RICE
• 4.1 Introduction
• 4.2 Materials and methods
• 4.2.1 The experimental population of N.lugens
• 4.2.2 Experimental plant materials
• 4.2.3 Rearing of N. lugens on experimental plants
• 4.2.4 Hemocyte Lysate Supernatant（HLS）Preparation
• 4.2.5 Assay of PO Activity
• 4.2.6 Kinetic parameters（Vmax and Km）of PO
• 4.2.7 Statistical Analysis
• 4.3 Results
• 4.3.1 Phenoloxidase activity（PO）
• 4.3.2 Determination of kinetic parameters of PO
• 4.4 Discussion
• CHAPTER 5: DYNAMICS OF YEAST-LIKE SYMBIOTES IN BROWNPLANTHOPPER Nilaparvata lugens Stal（Hemiptera:Delphacidae）TO BT RICE
• 5.1 Introduction
• 5.2 Materials and methods
• 5.2.1 Insects
• 5.2.2 Plants
• 5.2.3 Feeding of insects on experimental plants
• 5.2.4 Quantification of YLS in BPH
• 5.2.5 Statistical Analysis
• 5.3 Results
• 5.4 Discussion
• CHAPTER 6: DIFFERENTIAL GENE EXPRESSION IN RESPONSE TO BT RICE INBROWN PLANTHOPPER Nilaparvata lugens Stak（Hemiptera:Delphacidae）
• 6.1 Introduction
• 6.2 Materials and methods
• 6.2.1 Test plant materials
• 6.2.2 Test insects
• 6.2.3 Total RNA Isolation and cDNA Synthesis
• 6.2.4 Quantitative real-time PCR（qRT-PCR）analysis
• 6.2.5 Statistical Analysis
• 6.3 Results
• 6.3.1 Immune-related genes
• 6.3.2 Detoxification related genes
• 6.3.3 Digestion-related gene
• 6.4 Discussion
• CHAPTER 7: BT RICE ON THE PHYSIOLOGICAL DEVELOPMENT OF BROWNPLANTHOPPER Nilaparvata lugens Stal（Hemiptera:Delphacidae）
• 7.1 Introduction
• 7.2 Materials and methods
• 7.2.1 Stock colonies of BPH
• 7.2.2 Plant materials
• 7.2.3 Oviposition preference of BPH
• 7.2 4 Survival,growth and development of BPH
• 7.2.5 Statistical analysis
• 7.3 Results
• 7.3.1 Oviposition preference opf BPH
• 7.3.2 Survical,growth and development of BPH
• 7.4 Discussion
• CHAPTER 8: PROTEOMIC ANALYSIS OF BROWN PLANTHOPPER;Nilaparvatalugens Stal（Homoptera:Delphacidae）FEEDING ON BT RICE:IDENTIFICATION OFBacillus thuringiens BINDING PROTEINS
• 8.1 Introduction
• 8.2 Materials and methods
• 8.2.1 Insects rearing
• 8.2.2 Purification of Cry toxin-binding proteins and Cry-binding assays
• 8.2.2.1 Crude Protein extraction from BPH
• 8.2.2.2 One dimensional-polyacrylamide gel electrophoresis（1DE-PAGE）
• 8.2.2.3 Two dimensional-polyacrylamide gel electrophoresis（2DE-PAGE）
• 8.2.2.4 Detection of Cry toxin by Western blot analysis
• 8.2.2.5 Cry binding assays
• 8.2.2.6 Silver staining for visualizes the protein spots
• 8.3 Results
• 8.3.1 Detection of Cry toxin by Western blot analysis
• 8.3.2 BPH Binding of Cry toxin to BHP
• 8.4 Discussion
• CHAPTER 9: GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES
• REFERENCES