Distinctive gut microbioata of honey bees assessed using deep sampling from individual worker bees

Adult workers of the honey bee, Apis mellifera, appear to possess a normal gut microbiota dominated by about eight distinctive bacterial species, based on several non-culture based studies. However, previous studies have been based on relatively few sequences from pooled samples, so the extent of variation inmicrobiota among individuals within and between colonies and locations has not been clear. We surveyed the gut microbiota of 40 individual adult workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Of these, over 99% of the resulting sequences belonged to clusters for which the first blastn hits inGenBank were members of known bee phylotypes. Four of the phylotypes, belonging to Gammaproteobacteria, Betaproteobacteria and two Lactobacillus species, were present in every bee, though their frequencies varied. Statistical tests for differences in microbiota community profile among colonies and sites revealed significant differences, and these differences were largely due to the presence of two species of Enterobacteriaceae in some Arizona colonies; these two species have not been retrieved previously from bees and potentially represent pathogens or perturbation of the normalmicrobiota. Overall, these results show that the species typical in the normal microbiota are widespread but that their relative abundances vary among worker bees.

针对西方蜜蜂（Apis mellifera）的成年工蜂，多项非培养依赖型研究表明其肠道菌群以约8种独特的细菌类群为核心构成。然而，此前的相关研究多基于混合样本的少量测序序列，因此蜂群内部、不同蜂群间以及不同采样地点间，工蜂肠道菌群的个体差异程度仍不明确。本研究对来自美国亚利桑那州与马里兰州两个采样点的40只成年工蜂的肠道菌群进行了调查，每个采样点各选取4个蜂群进行采样。本研究使用通用引物扩增16S核糖体RNA基因（16S ribosomal RNA genes）的特定区域，并通过454焦标签测序法（454 pyrotag）对扩增子进行测序，最终获得约33万条细菌读段用于后续分析。其中，超过99%的序列可聚类为多个类群，这些类群在基因银行（GenBank）数据库中通过BLASTn（核苷酸局部比对工具）比对得到的首个匹配结果均为已知的蜜蜂系统发育型（phylotypes）菌株。其中4种系统发育型分别隶属于γ-变形菌纲（Gammaproteobacteria）、β-变形菌纲（Betaproteobacteria）以及2种乳杆菌属（Lactobacillus）物种，在所有受试工蜂体内均有检出，不过它们的相对丰度存在差异。对不同蜂群及采样点间菌群群落结构差异的统计学检验结果显示存在显著差异，这类差异主要源于亚利桑那州部分蜂群中检出的2种肠杆菌科（Enterobacteriaceae）物种；这两种物种此前从未在蜜蜂体内被分离到，可能为蜜蜂致病菌或可引发正常肠道微生物组失衡的外来菌。综上，本研究结果表明，正常肠道微生物组中的典型菌群物种在工蜂中普遍存在，但它们的相对丰度在不同工蜂个体间存在差异。