肠道微生物促进宿主桔小实蝇成虫抵抗低温胁迫的分子机制
发布时间：2021-03-12 22:20
桔小实蝇Bactrocera dorsalis（Hendel）是最具入侵性和多食性的害虫之一,为害250多种水果和蔬菜作物,造成严重损失。桔小实蝇的高入侵性原因取决于多方面因素,如寄主范围广、繁殖力强、扩散能力强、对环境胁迫压力的快速适应性等。对寒冷环境的适应可能是其地理扩张的一个关键因素之一。在环境胁迫条件下,昆虫肠道共生菌被证明可显著提高宿主的环境适应性。然而,在长期的低温胁迫下,肠道微生物对宿主适应性的作用仍不清楚。本文旨在研究明确肠道微生物在协助宿主桔小实蝇抗10℃低温胁迫中的功能,筛选鉴定其关键肠道共生细菌,并通过转录组学和代谢组学关联分析和功能研究,阐明肠道微生物影响宿主桔小实蝇成虫抵抗低温胁迫的分子机制。1.肠道微生物增强宿主桔小实蝇成虫对低温胁迫的抵抗力首先研究了肠道共生菌在桔小实蝇成虫抵抗低温胁迫中的作用。结果表明,通过抗生素处理去除肠道菌群后,将桔小实蝇暴露于10℃的低温胁迫中,中位生存时间显著降低至正常种群组桔小实蝇的约68%。通过回补筛选,我们发现Klebsiella michiganensis BD1 77是影响桔小实蝇成虫抗寒性的一种关键共生细菌,通过回补实...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：226 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of Abbreviations
Chapter 1: General introduction and literature review
1.1 Background of oriental fruit fly
1.2 Management strategies of oriental fruit fly
1.2.1 Chemical control
1.2.2 Cultural control
1.2.3 Attractant cue-lures
1.2.4 Male annihilation technique
1.2.5 Biological control
1.2.6 Sterile insect technique
1.2.7 Possible applications of gut bacteria for the management of insect pests
1.3 Fruit Fly Gut Microbiome Diversity
1.3.1 Tephritidae Fruit fly microbiota structure
1.3.2 Factors influencing gut microbial diversity
1.4 Gut microbe functions for the fitness improvement of host fruit flies
1.4.1 Gut bacteria provide nutrients to the host
1.4.2 Development and reproduction
1.4.3 Gut symbionts assist to overcome host plant defences
1.4.4 Resistance to pathogens
1.4.5 Symbiont-mediated insecticide resistance
1.5 Practical applications of gut microbiota in fruit flies
1.5.1 Probiotics used in mass-reared SIT
1.5.2 Attractant cue-lures
1.6 Knowledge gaps
1.6.1 Combination of incompatible insect technique （ⅡT） and SIT
1.6.2 Harnessing gut bacteria to promote the efficiency of mass-reared flies
1.6.3 Symbiont-mediated stress resistance
1.6.4 Possible applications of gut bacteria for the management of fruit flies
1.7 Identification and characterization of stress related genes stimulated by gut microbiota
1.7.1 Functions of molecular chaperones under extreme temperature stress
1.7.2 Importance of molecular cryoprotectants during extreme temperature stress
1.7.3 Symbiont-mediated stimulation of molecular cryoprotectant genes during low-temperature stress
1.8 Research objectives
Chapter 2: Gut microbiota enhances the host resistance to low-temperature stress in adultBactrocera dorsalis
2.1 Introduction
2.2 Materials and Methods
2.2.1 Fly culture
2.2.2 Isolation and identification of cultivable bacteria
2.2.3 Antibiotic treatment
2.2.4 Fitness parameter tests
2.2.5 Real-time quantitative PCR（RT-qPCR）
2.2.6 Statistical analysis
2.3 Results
2.3.1 Effect of the gut microbiota on the survival of B. dorsalis under low-temperature
2.3.2 A key symbiotic bacterium, K. michiganensis, enhances the resistance of B. dorsalis tolow-temperature stress
2.3.3 K. michiganensis maintenance in the gut improves host health throughout lifepostexposure to 10℃
2.3.4 K. michiganensis enhances the major nutrients level of B. dorsalis during low-temperature stress
2.3.5 Gut symbionts does not promote survival under UV stress treatment
2.4 Discussion
Chapter 3: Impact of gut microbiota on the metabolomic and transcriptomic responses of the hostto low-temperature stress in Bactrocera dorsalis
3.1 Introduction
3.2 Materials and Methods
3.2.1 Insect culture
3.2.2 UPLC/MS analysis
3.2.3 RNA sequencing
3.2.4 Primer designing
3.2.5 Real-time quantitative PCR（RT-qPCR）
3.3 Results
3.3.1 Influence of gut bacteria on the metabolomic response of the host to low-temperaturestress
3.3.2 Impact of gut bacteria on the transcriptomic response of the host to low-temperaturestress
3.4 Discussion
Chapter 4: Gut microbiota promotes host resistance to low-temperature stress by stimulating itsarginine and proline metabolism pathway in adult Bactrocera dorsalis
4.1 Introduction
4.2 Materials and Methods
4.2.1 Insect culture
4.2.2 Strains and plasmids
4.2.3 Laboratory reagents and kits
4.2.4 The Experimental equipment
4.2.5 Antibiotics, reagents, medium and buffer preparation
4.2.6 Microinjection
4.2.7 ATP assay
4.2.8 Real-time quantitative PCR（RT-qPCR）
4.2.9 dsRNA synthesis and delivery by injection
4.2.10 Transmission electron microscopy （TEM）
4.2.11 Quantification of distorted mitochondria percentage
4.2.12 Statistical analysis
4.3 RESULTS
4.3.1 Effect of gut microbiota on the regulation of the transcriptomic and metabolomicpathways of the host
4.3.2 Gut bacteria aids the host to stimulate gene expression levels of the arginine andproline metabolism pathway during low-temperature stress
4.3.3 RNAi mediated silencing of arginine and proline genes reduce the survival ofconventional flies under low-temperature stress
4.3.4 Combined effect of key genes silencing on the survival of conventional fliespostexposure to low-temperature stress
4.3.5 Functional validation of arginine and proline for the improvement in low-temperaturestress resistance
4.3.6 Gut bacteria maintains mitochondrial morphology and ATP levels during low-temperature stress
4.4 Discussion
Chapter 5: Characteristics and expression analysis of arginine and proline metabolism genes inBactrocera dorsalis
5.1 Introduction
5.2 Materials and Methods
5.2.1 Insect culture
5.2.2 Characteristics, sequence alignment and phylogenetic analysis
5.2.3 Developmental stages and tissues
5.2.4 Temperature and 20E treatment
5.2.5 Total RNA extraction and reverse transcription
5.2.6 Real-time quantitative PCR（RT-qPCR）
5.2.7 Statistical analysis
5.3 Results
5.3.1 Identification and characterization of five APMs
5.3.2 Expression of APMs at different developmental stages
5.3.3 Expression levels of APMs in different tissues
5.3.4 Expression of APMs in response to thermal stress
5.3.5 Expression regulation of APMs by 20E
5.4 Discussion
Chapter 6: Summary, innovations and future perspectives
6.1 Summary
6.2 Innovations
6.3 Future perspectives
References
Appendices
Appendix A. Significantly Enriched metabolites from metabolomics analysis
Appendix A1. Differentially expressed metabolites in the conventional/ antibiotic treated（ABX） fruit flies
Appendix A2. Differentially expressed metabolites in Klebsiella michiganensis/ antibiotictreated （ABX） fruit flies
Appendix B.Gene ontology （GO） enrichment analysis of DEGs from transcriptomics data
Appendix B1. Overrepresented GO terms in the conventional/ antibiotic treated （ABX） fruitflies
Appendix B2. Overrepresented GO terms in Klebsiella michiganensis/ antibiotic treated（ABX） fruit flies
Acknowledgements
【参考文献】：
期刊论文
[1]The proline synthesis enzyme P5CS forms cytoophidia in Drosophila[J]. Bo Zhang,?mür Y.Tastan,Xian Zhou,Chen-Jun Guo,Xuyang Liu,Aaron Thind,Huan-Huan Hu,Suwen Zhao,Ji-Long Liu. Journal of Genetics and Genomics. 2020(03)
[2]橘小实蝇植物源引诱活性物质的生物测定[J]. 王波,韩英,黄居昌,陈家骅. 应用昆虫学报. 2012(06)
[3]橘园常用杀螨剂对巴氏钝绥螨和柑橘全爪螨的选择毒性[J]. 肖顺根,余丽萍,舒畅,钟玲,李爱华,夏斌. 植物保护. 2010(03)
[4]前裂长管茧蜂对桔小实蝇的寄生效能研究[J]. 邵屯,刘春燕,陈科伟,曾玲. 华南农业大学学报. 2009(02)
[5]广东橘小实蝇寄生蜂调查[J]. 姚婕敏,谢翠红,何衍彪,邱波,陈华燕,许再福. 环境昆虫学报. 2008(04)
[6]布氏潜蝇茧蜂对橘小实蝇幼虫寄生作用的研究[J]. 吕增印,黄居昌,季清娥,杨建全,陈家骅. 华东昆虫学报. 2007(03)
[7]桔小实蝇不同发育阶段过冷却点的测定[J]. 侯柏华,张润杰. 昆虫学报. 2007(06)
[8]长尾潜蝇茧蜂对橘小实蝇幼虫的寄生效能[J]. 林玲,黄居昌,陈家骅,季清娥,杨建全. 华东昆虫学报. 2006(04)
[9]阿里山潜蝇茧蜂对橘小实蝇卵的寄生效能[J]. 郭庆亮,黄居昌,季清娥,杨建全,陈家骅. 华东昆虫学报. 2006(04)
[10]球孢白僵菌对桔小实蝇致病力的测定[J]. 潘志萍,李敦松,黄少华. 华中农业大学学报. 2006(05)
本文编号：3079081
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：226 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of Abbreviations
Chapter 1: General introduction and literature review
1.1 Background of oriental fruit fly
1.2 Management strategies of oriental fruit fly
1.2.1 Chemical control
1.2.2 Cultural control
1.2.3 Attractant cue-lures
1.2.4 Male annihilation technique
1.2.5 Biological control
1.2.6 Sterile insect technique
1.2.7 Possible applications of gut bacteria for the management of insect pests
1.3 Fruit Fly Gut Microbiome Diversity
1.3.1 Tephritidae Fruit fly microbiota structure
1.3.2 Factors influencing gut microbial diversity
1.4 Gut microbe functions for the fitness improvement of host fruit flies
1.4.1 Gut bacteria provide nutrients to the host
1.4.2 Development and reproduction
1.4.3 Gut symbionts assist to overcome host plant defences
1.4.4 Resistance to pathogens
1.4.5 Symbiont-mediated insecticide resistance
1.5 Practical applications of gut microbiota in fruit flies
1.5.1 Probiotics used in mass-reared SIT
1.5.2 Attractant cue-lures
1.6 Knowledge gaps
1.6.1 Combination of incompatible insect technique （ⅡT） and SIT
1.6.2 Harnessing gut bacteria to promote the efficiency of mass-reared flies
1.6.3 Symbiont-mediated stress resistance
1.6.4 Possible applications of gut bacteria for the management of fruit flies
1.7 Identification and characterization of stress related genes stimulated by gut microbiota
1.7.1 Functions of molecular chaperones under extreme temperature stress
1.7.2 Importance of molecular cryoprotectants during extreme temperature stress
1.7.3 Symbiont-mediated stimulation of molecular cryoprotectant genes during low-temperature stress
1.8 Research objectives
Chapter 2: Gut microbiota enhances the host resistance to low-temperature stress in adultBactrocera dorsalis
2.1 Introduction
2.2 Materials and Methods
2.2.1 Fly culture
2.2.2 Isolation and identification of cultivable bacteria
2.2.3 Antibiotic treatment
2.2.4 Fitness parameter tests
2.2.5 Real-time quantitative PCR（RT-qPCR）
2.2.6 Statistical analysis
2.3 Results
2.3.1 Effect of the gut microbiota on the survival of B. dorsalis under low-temperature
2.3.2 A key symbiotic bacterium, K. michiganensis, enhances the resistance of B. dorsalis tolow-temperature stress
2.3.3 K. michiganensis maintenance in the gut improves host health throughout lifepostexposure to 10℃
2.3.4 K. michiganensis enhances the major nutrients level of B. dorsalis during low-temperature stress
2.3.5 Gut symbionts does not promote survival under UV stress treatment
2.4 Discussion
Chapter 3: Impact of gut microbiota on the metabolomic and transcriptomic responses of the hostto low-temperature stress in Bactrocera dorsalis
3.1 Introduction
3.2 Materials and Methods
3.2.1 Insect culture
3.2.2 UPLC/MS analysis
3.2.3 RNA sequencing
3.2.4 Primer designing
3.2.5 Real-time quantitative PCR（RT-qPCR）
3.3 Results
3.3.1 Influence of gut bacteria on the metabolomic response of the host to low-temperaturestress
3.3.2 Impact of gut bacteria on the transcriptomic response of the host to low-temperaturestress
3.4 Discussion
Chapter 4: Gut microbiota promotes host resistance to low-temperature stress by stimulating itsarginine and proline metabolism pathway in adult Bactrocera dorsalis
4.1 Introduction
4.2 Materials and Methods
4.2.1 Insect culture
4.2.2 Strains and plasmids
4.2.3 Laboratory reagents and kits
4.2.4 The Experimental equipment
4.2.5 Antibiotics, reagents, medium and buffer preparation
4.2.6 Microinjection
4.2.7 ATP assay
4.2.8 Real-time quantitative PCR（RT-qPCR）
4.2.9 dsRNA synthesis and delivery by injection
4.2.10 Transmission electron microscopy （TEM）
4.2.11 Quantification of distorted mitochondria percentage
4.2.12 Statistical analysis
4.3 RESULTS
4.3.1 Effect of gut microbiota on the regulation of the transcriptomic and metabolomicpathways of the host
4.3.2 Gut bacteria aids the host to stimulate gene expression levels of the arginine andproline metabolism pathway during low-temperature stress
4.3.3 RNAi mediated silencing of arginine and proline genes reduce the survival ofconventional flies under low-temperature stress
4.3.4 Combined effect of key genes silencing on the survival of conventional fliespostexposure to low-temperature stress
4.3.5 Functional validation of arginine and proline for the improvement in low-temperaturestress resistance
4.3.6 Gut bacteria maintains mitochondrial morphology and ATP levels during low-temperature stress
4.4 Discussion
Chapter 5: Characteristics and expression analysis of arginine and proline metabolism genes inBactrocera dorsalis
5.1 Introduction
5.2 Materials and Methods
5.2.1 Insect culture
5.2.2 Characteristics, sequence alignment and phylogenetic analysis
5.2.3 Developmental stages and tissues
5.2.4 Temperature and 20E treatment
5.2.5 Total RNA extraction and reverse transcription
5.2.6 Real-time quantitative PCR（RT-qPCR）
5.2.7 Statistical analysis
5.3 Results
5.3.1 Identification and characterization of five APMs
5.3.2 Expression of APMs at different developmental stages
5.3.3 Expression levels of APMs in different tissues
5.3.4 Expression of APMs in response to thermal stress
5.3.5 Expression regulation of APMs by 20E
5.4 Discussion
Chapter 6: Summary, innovations and future perspectives
6.1 Summary
6.2 Innovations
6.3 Future perspectives
References
Appendices
Appendix A. Significantly Enriched metabolites from metabolomics analysis
Appendix A1. Differentially expressed metabolites in the conventional/ antibiotic treated（ABX） fruit flies
Appendix A2. Differentially expressed metabolites in Klebsiella michiganensis/ antibiotictreated （ABX） fruit flies
Appendix B.Gene ontology （GO） enrichment analysis of DEGs from transcriptomics data
Appendix B1. Overrepresented GO terms in the conventional/ antibiotic treated （ABX） fruitflies
Appendix B2. Overrepresented GO terms in Klebsiella michiganensis/ antibiotic treated（ABX） fruit flies
Acknowledgements
【参考文献】：
期刊论文
[1]The proline synthesis enzyme P5CS forms cytoophidia in Drosophila[J]. Bo Zhang,?mür Y.Tastan,Xian Zhou,Chen-Jun Guo,Xuyang Liu,Aaron Thind,Huan-Huan Hu,Suwen Zhao,Ji-Long Liu. Journal of Genetics and Genomics. 2020(03)
[2]橘小实蝇植物源引诱活性物质的生物测定[J]. 王波,韩英,黄居昌,陈家骅. 应用昆虫学报. 2012(06)
[3]橘园常用杀螨剂对巴氏钝绥螨和柑橘全爪螨的选择毒性[J]. 肖顺根,余丽萍,舒畅,钟玲,李爱华,夏斌. 植物保护. 2010(03)
[4]前裂长管茧蜂对桔小实蝇的寄生效能研究[J]. 邵屯,刘春燕,陈科伟,曾玲. 华南农业大学学报. 2009(02)
[5]广东橘小实蝇寄生蜂调查[J]. 姚婕敏,谢翠红,何衍彪,邱波,陈华燕,许再福. 环境昆虫学报. 2008(04)
[6]布氏潜蝇茧蜂对橘小实蝇幼虫寄生作用的研究[J]. 吕增印,黄居昌,季清娥,杨建全,陈家骅. 华东昆虫学报. 2007(03)
[7]桔小实蝇不同发育阶段过冷却点的测定[J]. 侯柏华,张润杰. 昆虫学报. 2007(06)
[8]长尾潜蝇茧蜂对橘小实蝇幼虫的寄生效能[J]. 林玲,黄居昌,陈家骅,季清娥,杨建全. 华东昆虫学报. 2006(04)
[9]阿里山潜蝇茧蜂对橘小实蝇卵的寄生效能[J]. 郭庆亮,黄居昌,季清娥,杨建全,陈家骅. 华东昆虫学报. 2006(04)
[10]球孢白僵菌对桔小实蝇致病力的测定[J]. 潘志萍,李敦松,黄少华. 华中农业大学学报. 2006(05)
本文编号：3079081
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