【作者】 王洪秀；

【导师】 张宏宇；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 橘小实蝇(Bactrocera dorsalis)是一种极具危害性的检疫性害虫,其食性广、寄主杂,可危害柑桔、番石榴、杨桃、芒果和瓜类等40多个科250多种水果、蔬菜和花卉,对农业生产造成了很大的威胁。通过研究橘小实蝇成虫肠道细菌多样性,肠道可培养细菌群落结构组成,及肠道细菌对寄主成虫的引诱作用、存活率和寿命的影响,可为探讨肠道菌群对寄主的生理功能和生态学意义奠定基础,为进一步了解寄主与其共生菌的协同进化、物种进化途径提供新的认知,最终为利用微生物防治此类害虫提供新思路。本研究通过利用16S rRNA基因的PCR-DGGE分析技术研究了橘小实蝇三个种群(实验室正常喂养种群,实验室无菌糖水喂养种群和野生种群)成虫肠道细菌多样性及肠道可培养细菌的群落结构组成；研究了肠道细菌对寄主成虫的引诱作用；肠道细菌通过饲喂菌悬液的方式对寄主实蝇死亡率和寿命的影响。主要研究结果如下：从橘小实蝇三个种群成虫肠道分离到150种不同细菌遗传型,分属于γ-变形菌纲(Gammaproteobacteria)、放线菌纲(Actinobacteria)、α-变形菌纲(Alphaproteobacteria)、δ-变形菌纲(Deltaproteobacteria)、拟杆菌纲(Bacteroidetes)、黄杆菌纲(Flavobacteria)和厚壁菌门(Firmicutes)等7个不同的细菌纲。在三个种群中,γ-变形菌纲都超过了肠道细菌总数的70%,是肠道的绝对优势细菌种类。此外,我们把每一种群所占比例大于10%的类群也归为优势细菌种类。因此,我们发现放线菌纲为实验室种群的优势细菌种类(所占比例为10.1%),厚壁菌门为野生种群的优势细菌种类(所占比例为12.3%)。而δ-变形菌纲、拟杆菌纲和a-变形菌纲在三个种群中只占据很少的克隆子(每种类群都少于8个克隆子)。在三个种群的150个细菌遗传型中,有3个分类组(文中标记为Unclassified)无法归属于目前已知的细菌类群,这是目前认识不足的一类微生物,可能与橘小实蝇肠道特殊的微环境有关。以序列相似性大于97%的克隆归为相同的细菌种类为标准,三个种群存在一些隶属于肠杆菌科(Enterobacteriaceae)的共有菌种,如Klebsiella, Citrobacter, Enterobacter, Pectobacterium和Serratia。进一步对三个种群的多样性指数计算及群落结构组成分析表明,三个种群的细菌群落存在显著性差异。其中实验室无菌糖水喂养种群的细菌多样性最低,只有很少的细菌遗传型与另外两个种群共存。而实验室种群和野生种群的细菌多样性相对较高。从橘小实蝇三个种群成虫肠道600株可培养细菌得到53种不同细菌遗传型,分属于肠杆菌科(Enterobacteriaceae)、肠球菌科(Enterococcaceae)和芽孢杆菌科(Bacillaceae)等三个科。其中肠杆菌科是肠道可培养细菌最优势的细菌种类。同样以序列相似性大于97%的菌株归为相同的细菌种类为标准,找到了三个种群可培养细菌的共有菌种,结合菌落形态观察和生理生化特征鉴定,确定共有菌种为肠杆菌属5株,克雷伯氏菌属2株,柠檬酸杆菌属1株,泛菌属1株,肠球菌属2株,以及芽孢杆菌属4株。室内引诱实验发现,所有供试菌株的不同处理发酵液对橘小实蝇均有显著的引诱作用,其中以Bacillus cereus, Enterococcus faecalis, Enterobacter cloacae和Citrobacter freundii四种菌的引诱效果最好。菌悬液离心后的上清液高压灭菌的引诱效果要好于上清液的过滤除菌液和发酵原液。室内引诱效果最好的六种菌进行了野外引诱实验。结果表明：六种菌的引诱效果都好于对照,其中B. cereus和C. freundii引诱到了最多的雌性、雄性和雌性加雄性实蝇,并且除Klebsiella oxytoca(引诱到的雌蝇和雄蝇一样多)外其余五种菌引诱到的雌蝇都多于雄蝇,六种菌的野外引诱结果与室内引诱结果一致。利用饲喂菌悬液的方法,研究了肠道可培养细菌占多数比例的肠杆菌科细菌和占少数比例的芽孢杆菌对橘小实蝇存活率和寿命的影响。结果表明：实蝇寿命(无论雌雄)不受喂食抗生素的影响。实蝇对上述两类菌的摄取明显地影响自身的死亡率和寿命。总体而言,饲喂蜡样芽孢杆菌对寿命有不利影响,而饲喂肠杆菌科细菌则有利。对比只饲喂20%蔗糖溶液的实蝇,饲喂蜡样芽孢杆菌的实蝇死亡时间显著加快且寿命明显缩短。相反,饲喂肠杆菌科细菌的实蝇则比只饲喂20%蔗糖溶液的实蝇死亡时间平缓,寿命更长。更多还原

【Abstract】 Bactrocera dorsalis Hendel is a polyphagous, cosmopolitan and invasive pest at home and abroad, and it can infect fruits, vegetables and flowers of more forty families and two hundred-fifty species, such as mangoes, oranges, tangerines, carambolas, guavas and sorts of gourds. In our study, we investigated the diversity of the intestinal bacteria community of the gut of adult B. dorsalis, the structure of the cultured bacterial community, the attraction function of cultured bacteria and it’s influence to host’s mortality and longevity. These results contributed to explore intestinal bacteria on the impact of the physiological function and ecological significance of B. dorsalis, to understand coevolution between B. dorsalis and its symbiotic bacteria, and to provide new knowledge for species evolution ways. Further studies will be focused on identifying the function of each representative species and establishing whether these species could play important roles in the future as biocontrol agents.In this study, we employed traditional culture techniques and PCR-DGGE molecular methods to study the diversity of the intestinal bacteria community and the structure of the culturable bacterial community of adult B. dorsalis from three different populations (lab-reared (LR), lab sterile sugar-reared (LSSR) and field-collected (FC)). We also studied the attraction function of intestinal cultured bacteria to adult B. dorsalis and it’s influence to host’s mortality and longevity. The main results were shown as follows:150 different DGGE profiles of bacteria clones were obtained from the adult gut of all three populations of B. dorsalis. By sequence similarities and phylogenetic analyzing, seven major phyla of bacteria were observed. These bacteria respectively belonged to Gammaproteobacteria, Actinobacteria, Alphaproteobacteria, Deltaproteobacteria, Bacteroidetes,. Flavobacteria and Firmicutes. In all three populations, Gammaproteobacteria which was the preponderant bacterium in the gut represented 70% of the total clones. Besides this phylum, the following phyla were judged to be major components of each population since they constituted 10% or more of the total clones of a given population:Actinobacteria (10.1%) was the major component of LR and Firmicutes (12.3%) was the major component of FC. Flavobacteria, Deltaproteobacteria, Bacteroidetes, and Alphaproteobacteria appeared in various populaitons with a few clones (less than 8 per phylum). Among the 150 sequences of bacterial 16S rDNA, three distinct clusters were identified (labeled as unclassified groups). This was the kind of microbial flora which presents lacking of knowledge, might be related to the special microenvironment of B. dorsalis gut. The closely related sequences (>97% sequence similarity) which had been retrieved from three populaitons were grouped as one common species. Three oriental fruit fly populations shared common representatives of the Enterobacteriaceae, including Klebsiella, Citrobacter, Enterobacter, Pectobacterium, and Serratia. According to the analysis of diversity indices, phylotype composition and phylogenetic distribution of the 16S rDNA clones, we found that the bacterial communities of three populations were significantly different from each other. The LSSR populaiton was the least diverse and had fewer phylotypes in common with the other libraries, whereas the populaitons of LR and FC were relatively more diverse.600 cultured bacteria in the intestinal tract of adult B. dorsalis from three populations were classified into 53 unique phylotypes. All sequenced bacteria strains were grouped into three families:Enterobacteriaceae, Enterococcaceae and Bacillaceae. The Enterobacteriaceae was dominated in all the populations (72.0%-82.5%). The closely related sequences (>97% sequence similarity) which had been retrieved from culturable bacteria of three populaitons were grouped as one common species. By colony morphology, physiological and biochemical characteristics test, we determined the common species:five strains of bacteria as Enterobacter, two strains of Klebsiella, one strain of Citrobacter, one strain of Pantoea, two strains of Enterococcus, and four strains of Bacillus.The attractancy bioassays results in the laboratory showed that all bacteria strains were significantly more attractive to B. dorsalis adults than the tryptic soy broth control. Among them, Bacillus cereus, Enterococcus faecalis, Enterobacter cloacae and Citrobacter freundii were the most attractive bacteria. The autoclaved supernatants were significantly more attractive than the filtered supernatants or whole beer. Furthermore, the results of the field test showed that the six bacteria strains were significantly more attractive than the control. B. cereus and E. faecalis caught significantly more males, females, and males+females than any other of the bacterial metabolites. All the baits except Klebsiella oxytoca caught more females than males. These results were consistent with that of laboratory bioassays.By feeding bacterial suspension solution, we assessed the effect of the dominant Enterobacteriaceae community and the minor Bacillus community on the oriental fruit fly’s survival and longevity. The results showed that fly longevity (of both males and females) was not affected by the presence of antibiotics. Ingestion of bacteria significantly affected mortality rate and average fly longevity in both sexes, regardless of previous exposure to antibiotics. In general, feeding on Bacillus had a negative effect on longevity, while feeding on the Enterobacteriaceae mixture had a beneficial effect. Compared to flies fed on sugar alone, flies fed on Bacillus died at a significantly faster rate and their average longevity was significantly reduced. Conversely, flies fed on Enterobacteriaceae died more gradually and lived significantly longer, on average, than flies fed on sugar.更多还原