【作者】 赵丽坤；

【导师】 张锋； 吕志堂；

【作者基本信息】 河北大学 ， 动物学， 2023， 博士

【摘要】 蜘蛛是重要捕食性天敌,在害虫生物防治中发挥着关键作用。蜘蛛的肠道、体表和生殖系统等部位都存在着微生物群落,这些微生物与蜘蛛形成密切的共生关系,并相互协作。本研究综合运用扩增子测序、培养组学、宏基因组学和代谢组学等技术,研究了白额巨蟹蛛Heteropoda venatoria细菌群落组成和功能,以及共生菌与宿主解毒代谢之间的关系。研究总结如下:1)揭示了白额巨蟹蛛四种组织共生菌的群落多样性组成与群落功能特征。通过对16S rRNA序列V3-V4可变区进行高通量测序,结果显示,白额巨蟹蛛不同组织共生细菌分属于34门、72纲、170目、277科和458属。其中,毒腺、丝腺和卵巢中芽孢杆菌属相对丰度最高,肠道细菌群落则以寡养单胞菌属、不动杆菌属和鞘氨醇单胞菌属为优势。基于扩增子测序结果,设计了21种分离培养条件,对卵巢、肠道、毒腺和丝腺中的细菌进行分离培养。共得到119株细菌,分属于4门25属,其中以芽孢杆菌属和沙雷氏菌属为主。在13个培养组学目标菌属中,有6个属被成功分离鉴定出来,而分离结果中有10个属在扩增子测序中没有被检测到。根据共生菌功能预测,代谢功能具有最高丰度,从中选取三个通路进行验证,测定了共生菌农药降解,抑菌以及肿瘤细胞抑制活性。结果表明:5株菌具有较高的联苯菊酯和阿特拉津降解效率,有28株菌对至少一种供试敏感菌表现出抑菌活性。此外,菌株GL312、PL211和PL316对人类胃癌细胞株MGC-803有明显的细胞毒性,尤其是菌株PL316的发酵粗提物对MGC-803细胞凋亡具有诱导作用。2)明晰了白额巨蟹蛛不同发育阶段蜘蛛共生菌群落多样性及肠道细菌功能演替规律。采用16S rRNA测序分析技术,研究了白额巨蟹蛛不同发育阶段和饵料的微生物群落组成和多样性。结果表明:在卵巢中,红球菌属和叶杆菌属为优势属;1龄蜘蛛中主要由葡萄球菌属组成;到了3龄期,芽孢杆菌属,产碱杆菌属和沙雷氏菌属成为主要优势属;5龄和8龄期两组样本显示较高的物种丰富度,优势属分别是短杆菌属和葡萄球菌属。12龄期,假单胞菌属成为优势菌属,其丰度比例为55.5%。此外,肠道中的优势菌属为红球菌,所占比例为53%。还对5龄期和12龄龄期的粪便样本进行宏基因组测序分析。结果显示:白额巨蟹蛛肠道微生物组的主要功能包括碳水化合物代谢、氨基酸代谢、能量代谢、辅因子和维生素代谢以及异生物质降解和代谢。3)探究了共生菌对白额巨蟹蛛解毒代谢的影响机制。通过给白额巨蟹蛛饲喂四环素、利福平和头孢他啶等抗生素,并结合实时荧光定量PCR检测共生菌含量,发现这三种抗生素均能够清除蜘蛛共生菌。随后,进一步检测抗生素处理对蜘蛛解毒代谢酶活力变化,结果显示抗生素处理4天后,蜘蛛解毒相关酶活性下降约40%。药物敏感性实验表明,利福平处理后蜘蛛对联苯菊酯的敏感性明显提高。为了进一步研究农药和抗生素对蜘蛛肠道微生物的影响,采用宏基因组技术进行分析。结果表明,农药处理后,厚壁菌门丰度增加;而抗生素和农药复合处理后,软壁菌门丰度增加。此外,KEGG功能注释结果表明,在肠道微生物与宿主解毒代谢相关的异生素降解和代谢功能上,三个组之间存在显著差异。最后采用LC-MS非靶向代谢组学技术对三组样品的肠道代谢物进行检测,结果验证了抗生素和联苯菊酯对蜘蛛代谢化合物的影响。整合宏基因组学和代谢组学数据,发现差异微生物和差异代谢物之间存在一定对应关系,抗生素处理对微生物群落丰度和代谢物表达量上产生的影响趋于一致。综合以上数据分析,白额巨蟹蛛共生菌群落可能通过影响宿主体内药物代谢,特别是细胞色素P450介导宿主解毒代谢。综上所述,本研究对白额巨蟹蛛细菌群落的组成和功能进行了全面分析,证实了共生细菌在宿主解毒代谢中的关键作用,为节肢动物和微生物之间的共生关系提供了新的思路。更多还原

【Abstract】 As a vital natural predator,spiders play an essential role in controlling pests.Microbial communities exist in the gut,body surface,and reproductive system of spiders,forming a symbiotic relationship with the host and collaborating with each other.This study focuses on the spider Heteropoda venatoria,utilizing amplification sequencing,culturomics,metagenomics,and metabolomics techniques to comprehensively analyze the composition and function of the spider bacterial community and the relationship between symbiotic bacteria and host detoxification metabolism.The research results provide a theoretical foundation for spider protection and resource development.The summarized findings of the research are presented below:1.Integration of high-throughput sequencing and culturomic approaches reveals diversity and functionality of bacterial symbionts in various tissues of spider H.venatoriaThe community diversity and functional characteristics of four symbiotic bacteria in different tissues of H.venatoria,were revealed using a combination of amplicon sequencing and culturomics.High-throughput sequencing of the V3-V4 variable region of the 16 S r RNA gene showed that the symbiotic bacteria in different tissues of the spider belonged to 34 phyla,72 classes,170 orders,277 families,and 458 genera.Among them,the most abundant bacteria genus in the venom gland,silk gland,and ovary were Bacillus,while the dominant bacterial genera in the gut microbiota were Stenotrophomonas,Acinetobacter,and Sphingomonas.Based on the amplicon sequencing results,21 different cultivation conditions were designed using culturomics to isolate bacteria from the ovary,gut,venom gland,and silk gland,resulting in a total of 119 bacterial strains belonging to 4 phyla and 25 genera,with Bacillus and Serratia as the main genera.Six out of 13 targeted bacterial genera were successfully isolated and identified,and 10 genera were not detected by amplicon sequencing.According to functional predictions,metabolism was the most abundant,and pesticide degradation,inhibition of bacterial growth,and inhibition of tumor cell growth were selected for in vitro validation experiments.The results showed that five strains had high efficiency in degrading bifenthrin and atrazine,while 28 strains showed antimicrobial activity against at least one sensitive strain tested.Additionally,the strains GL312,PL211,and PL316 had significant cytotoxicity against the human gastric cancer cell line MGC-803,and the fermentation crude extract of strain PL316 was an effective inducer of MGC-803 cell apoptosis.2.Reveal the diversity and functional succession of symbiotic bacteria in different life stages of the spider using amplicon sequencing and metagenomic sequencing techniquesThe microbiota composition and diversity in different life stages and food sources were analyzed.The results showed that the ovary primarily contained the genera Staphylococcus and Bacillus,while the WA larva mainly consisted of Staphylococcus.The WB stage had predominant genera such as Bacillus,Alkalibacillus,and Serratia,while the WC and WD stages showed high species richness with predominant genera such as Corynebacterium and Staphylococcus.In the WE stage;Pseudomonas was the dominant genus with an abundance ratio of 55.5%.Furthermore,the dominant genus in the gut was Staphylococcus,accounting for53% of the proportion.Metagenomic sequencing analysis was performed on the fecal samples of the 5th(FC)and 12th(FE)instars,and the results showed that carbohydrate metabolism,amino acid metabolism,energy metabolism,co-factor and vitamin metabolism,and xenobiotic degradation and metabolism were the main metabolic pathways of spider gut microbiome.3.Integrated metagenomics-metabolomics analysis was conducted to investigate the impact of symbiotic bacteria on the detoxification metabolism of the spider host.After administering antibiotics such as tetracycline,rifampicin,and ceftazidime to spiders and using real-time fluorescence quantitative PCR to detect the content of symbiotic bacteria,it was observed that all three antibiotics were effective in eliminating symbiotic bacteria in spiders.Further testing was carried out to determine the impact of antibiotics on the detoxification metabolism enzyme activity of spiders.Results indicated that after four days of antibiotic treatment,spider enzyme activity decreased by approximately 40%.To better understand the impact of pesticides and antibiotics on intestinal microorganisms in spiders,metagenomic techniques were employed for analysis.Results revealed an increase in Firmicutes abundance following pesticide treatment,while combined treatment with antibiotics and pesticides resulted in an increase in Tenericutes abundance.Furthermore,KEGG functional annotation results showed significant differences among the three groups in the degradation and metabolic functions of intestinal microorganisms related to host detoxification metabolism.Finally,LC-MS metabolomics was utilized to detect the intestinal metabolites of the three groups of samples,and the results validated the influence of antibiotics and bifenthrin on spider metabolic compounds.Integrating metagenomics and metabolomics data demonstrated a correlation between differential microorganisms and differential metabolites,and the impact of antibiotic treatment on microbial community abundance and metabolite expression appeared to be consistent.Based on the above data analysis,it is suggested that the symbiotic bacteria community of spider can affect drug metabolism in the host via cytochrome P450-mediated detoxification metabolism.In summary,this study provided a comprehensive analysis of the composition and function of the bacterial community in the spider H.venatoria and highlighted the crucial role of symbiotic bacteria in host detoxification metabolism.The study tries to find a new idea for the symbiotic relationship between arthropods and microorganisms.更多还原