松墨天牛肠道共生细菌的多样性及其在松材线虫侵染循环中的功能研究

Insects construct various symbiotic relationships with a wide diversity of microorganisms during their co-evolution, and gut bacteria are specially proved to confer a variety of benifits to the host, such as direct or indirect nutrition, assistance in overcoming host defenses, protection from natural enemies, improved development and reproduction, and communication. Based on our previous research accumulations and by means of Entomotomy, Entomological physiology, Microbiology, Molecular biology, Bioinformatics and Biostatistics techniques, the purpose of this research was to characterize the gut bacterial communities associated with the pine sawyer beetle Monochamus alternatus. We will examinefourth-instar larvae, pupae, and newly emerged adults using both culture-based and culture-independent methods. We also want to make comparisons among fore-, mid-, and hindguts of selected beetles and environmentally derived samples to identify and distinguish bacterial taxa that are transmitted from the adult to larval and pupae stages. By identifying and comparing bacterial community members among different life stages of the insect and with bacteria it encounters in the environment to determine the source of the bacterial community harbored by Bursaphelenchus xylophilus.Through megagenome sequencing we compile a suite of candidate genes found in the gut bacteria community whose annotations are consistent with cellulose-degrading capabilities and other genes that have roles in nutrient synthesis and detoxification. Additionally, we isolate and identify dominant cellulose-degrading bacteria and terpene-detoxificating bacteria and test their nutritional and metabolic potential to host insect. Finally we will separately and jointly examine gut bacteria and terpenes influences on survival, development and reproduction of B. Xylophilus, so as to confirm their prominent functions on interaction between M. Alternatus and B. Xylophilus. These results are crucial to focus future research on particular bacterial species and functional gene that are more likely to be of critical importance to the insect and may help explore transmission and pathogenic mechanism of this plant-parasitic nematode.

昆虫在长期协同进化过程中与肠道细菌相互选择，相互适应，形成了密切的共生关系。本项目以松墨天牛肠道共生细菌为研究对象，采用昆虫解剖学、昆虫生理学、微生物学、分子生物学、生物信息学、统计学等原理和方法，研究松墨天牛幼虫、蛹和成虫的肠道共生细菌组成和群落结构，进而测定分析与不同虫态偶联的松材线虫及寄主植物的蛀道、蛹室及取食和产卵部位的细菌群落特征，探明松材线虫共生菌来源及其与松墨天牛肠道共生细菌组成和群落结构的相关性；分离、筛选具有分解纤维素和降解树脂萜类的优势菌，验证其对纤维素和单萜、二萜的分解能力，阐释肠道共生细菌对松墨天牛的营养作用和松材线虫抵御寄主化学防御的功能；通过生物测定天牛肠道共生细菌和树脂萜类对松材线虫转型发育，繁殖的协同作用，探究松墨天牛肠道共生菌在松材线虫与媒介昆虫互作中的作用，为进一步以媒介昆虫的肠道共生细菌为切入点探讨和揭示松材线虫的传播扩散机制和致病机理提供理论依据。

为探究肠道共生菌在松墨天牛生活史和松材线虫侵染循环中的作用，本研究采用昆虫学、微生物学、分析化学、分子生物学、生物信息学等技术和方法，系统研究了松墨天牛不同虫态以及幼虫蛀道、蛹室内细菌组成和群落结构特征，分析了松墨天牛肠道共生细菌的多样性，分别筛选出降解纤维素和单萜的菌株并研究了其功能。（1）明确了松墨天牛成虫和幼虫肠道中可培养共生细菌的群落结构。发现藤黄微球菌、产气肠杆菌和腐生葡萄球菌为林间成虫肠道中优势菌群；乳酸乳球菌、产气肠杆菌、产酸克雷伯氏菌和粘质沙雷氏菌为室内成虫肠道中优势菌群；枯草芽孢杆菌为林间幼虫肠道中优势菌群，产气肠杆菌为室内幼虫肠道中优势菌群。不同虫态也具有特有种群，如成虫肠道中的荧光假单胞菌、蜡状芽孢杆菌。（2）发现粘质沙雷氏菌、Pseudomonadaceae sp.、Chryseobacterium sp为幼虫蛀道和蛹室共有菌群，产气肠杆菌（E. aerogenes）、荧光假单胞菌、Dyella yeojuensis、Enterobacter sp.为幼虫蛀道中优势菌群，解鸟氨酸拉乌尔菌、杓兰果胶杆菌、Yersinia sp.、Burkholderia sp.为蛹室内优势菌群，分析了松墨天牛肠道共生细菌与其栖息环境共生细菌的群落结构的相关性。（3）利用高通量测序结合生物信息学方法，分析了不同分类水平上成虫和幼虫肠道细菌的多样性，预测了松墨天牛肠道共生细菌的纤维素和萜类代谢酶基因。（4）筛选出7株具有纤维素降解能力的菌株，即克雷伯氏菌Q8、欧文氏菌Z1、甲基营养型芽孢杆菌Z3、欧文氏菌H4、伯克氏菌F7、肠杆菌M7和肠杆菌M9。测定了菌株的代谢分解能力，明确了肠道细菌对宿主昆虫的营养功能。（5）筛选出6株具有降解（±α）蒎烯和β蒎烯的菌株，即芽孢杆菌Z3、芽孢杆菌Z4、芽孢杆菌Z5、泛菌H2、涅斯捷连科氏菌H4和芽孢杆菌H6。测定了对单萜的降解效率，明确了肠道共生细菌对克服寄主植物化学防御的功能。生物测定了高降解率菌株及其与单萜联合对松材线虫繁殖的影响。本研究为进一步探究肠道共生菌帮助松墨天牛获得营养以及松材线虫克服寄主防御机理奠定了基础。