西方蜜蜂工蜂肠道菌群定殖起始点和定殖稳态点的结构比较分析

Comparison of intestinal flora structure between starting point and steady point of colonization in workers (Apis mellifera)

摘要:

[目的] 目前，针对昆虫肠道细菌定殖规律的研究还未见报道。探索西方蜜蜂（Apis mellifera）肠道菌群定殖过程两个重要时间节点（起始0日龄和稳态7日龄）间肠道细菌群落（菌群）结构组成的差异，加深对蜜蜂及昆虫肠道菌群定殖规律的认识。[方法] 分别采集两个化蛹后工蜂发育阶段的个体各5只，分别解剖并提取其肠道菌群DNA。使用Illumina技术对肠道菌群16S rDNA高变区进行高通量测序。通过生物信息学的分析方法对肠道菌群进行多样性分析，并对两个时间点相对丰度最高的肠道菌群进行统计分析，比较肠道菌群相对丰度和组成的差异。[结果] 共获得515156条高质量序列，长度为227904953 bp，平均长度为442 bp。基于OTUs的分类表明，工蜂肠道细菌分别隶属于34个门82个纲221个目405个科799个属。此外，工蜂肠道菌群定殖起点和终点间的Alpha多样性指数存在显著差异（ACE，P=0.0014；Chao，P=0.0013；Shannon，P=0.0003；Simpson，P=0.0028，Student'st检验）。此外，相较0日龄工蜂，7日龄工蜂肠道中的乳酸杆菌Lactobacillus、Gilliamella、双歧杆菌Bifidobacterium、Snodgrassella 4个属的相对丰度显著增加；相反，不动杆菌Acinetobacter、大肠杆菌志贺氏菌Escherichia-Shigella、鞘脂单胞菌Sphingomonas、类杆菌Bacteroides、涅斯捷连科氏菌Nesterenkonia、栖热菌Thermus 6个属的细菌相对丰度显著降低（P<0.05）。[结论] 出房（0日龄）成年工蜂的肠道菌群多样性显著高于菌群定殖完成（7日龄）工蜂的肠道菌群多样性，且成年工蜂肠道菌群定殖完成前后部分类群的相对丰度显著改变。本研究的结果不仅可增加我们对蜜蜂肠道菌群定殖规律的认识，也能够为研究其他昆虫肠道菌群的定殖规律提供重要的参考信息。

关键词:西方蜜蜂工蜂;肠道菌群定殖;16S rDNA;多样性;相对丰度

Abstract:

[Objective] Now, there is no reports on colonization of intestinal bacteria in insects. Exploration for difference of intestinal bacterial structure and composition between two key time points (The starting point is 0-days and the steady point is 7-days after pupation) of intestinal bacterial colonization in Apis mellifera, deepening understanding of intestinal flora colonization in bees and even insects.[Methods] After pupation, five worker individuals were collected from each of the two time points. Samples of workers were dissected and then DNA of intestinal flora was extracted. High-throughput sequencing for the highly variable region of 16S rDNA of intestinal flora was implemented using illumine platform, and then diversity of intestinal flora was analyzed by bioinformatics. Intestinal flora showing the highest relative abundance at the two time points was statistically analyzed, as well as the relative abundance and composition of intestinal flora were compared. [Results] A total of 515156 quality sequences were obtained, with a total length of 227904953 bp and an average length of 442 bp. According to OTUs-based classification, intestinal bacteria of workers were classified into 34 phylum, 82 classes, 221 orders, 405 families and 799 genera, respectively. In addition, there was a significant difference between Alpha diversity index from the starting point and that from the end point of intestinal flora colonization in workers (ACE, P=0.0014; Chao, P=0.0013; Shannon, P=0.0003; Simpson, P=0.0028). It is found that the relative abundance of Lactobacillus, Gilliamella, Bifidobacterium and Snodgrassella increased significantly, and the relative abundance of Acinetobacter, escherichia-shigella, Sphingomonas, Bacteroides, Nesterenkonia and Thermus decreased significantly (P<0.05) in 7-days workers by comparing to 0-days workers. [Conclusion] Intestinal bacterial diversity of the newly emerged (0-day) adult A. mellifera was significantly higher than that of fully colonized (7-day) workers. The relative abundance of several intestinal bacteria in adult worker bees changed significantly after colonizing. Results of the study not only increase our understanding for colonization rules of intestinal bacteria in bee, and also provide important references for studying colonization rules of intestinal bacteria in other insects.

Key words:Apis mellifera;intestinal flora colonization;16S rDNA;diversity;relative abundance