基于DNA复合条形码技术的蝗虫肠道共生真菌多样性研究

利用DNA复合条形码技术,研究了11个样本的蝗虫肠道共生真菌的多样性。结果显示:ITS在所研究的物种中鉴定了5门16纲29目40属2786 OTU真菌。肠道真菌群落组成分析结果显示:所有物种肠道真菌类群中含量最高的是木耳菌目和银耳目,其中斑翅蝗科的真菌类群多样性相对最高,斑腿蝗科的真菌类群多样性相对最低,表明各蝗虫肠道之间存在着明显的菌群多样性变化。α多样性分析结果显示:斑翅蝗科的共生真菌群落丰富度和多样性最高,斑腿蝗科的则最低。β多样性分析结果显示:(1)同科的各个种的肠道真菌群落结构差异性较小,不同科的种的肠道真菌群落结构差异性较大;(2)剑角蝗科的肠道真菌群落结构与其他物种的相似性均相对较低,而且在两个不同取样地得到的中华剑角蝗的真菌群落结构相似性也相对较低。聚类分析结果显示:(1)同科的蝗虫肠道真菌首先聚到一起,且群落相似性也相对较高;(2)布勒掷孢酵母属、内疣衣属和外瓶霉属3个属在蝗虫肠道真菌中是优势菌属。

Study of the biodiversity in intestinal symbiotic fungi in grasshoppers species by using DNA meta-barcoding

DNA meta-barcoding technology, which is a combination of DNA barcoding and high-throughput sequencing, is a highly efficient method for monitoring microbial diversity. In this study, we investigated symbiotic fungi in the gut of 8 grasshopper species collected from Qinling Mountain and campus of Shaanxi Normal University with respects of alpha diversity, beta diversity, and cluster analyses, and evaluated the differences in the microbial diversity of the samples collected from the two locations. A total of 8 individuals from 8 species collected from Qinling Mountain (namely, Trilophidia annulata, Atractomorpha sinensis, Acrida cinerea, Chorthippus chinensis, Pedopodisma tsinlingensis, Fruhstorferiola omei, Euchorthippus unicolor, and Xenocatantops brachycerus) and 3 individuals from 3 species collected from the campus of Shaanxi Normal University (namely, Trilophidia annulata, Atractomorpha sinensis and Acrida cinerea) were sampled. The grasshoppers were starved overnight and dissected, and the intestinal gut was fixed in 100% ethanol for DNA extraction. Internal transcribed spacers (ITS) were selected as barcoding sequences. After DNA extraction and PCR amplification using fungus ITS universal primers, the amplicons were sequenced using the 454 FLX+ platform. Two software, Qiime and Mothur, were used to analyze the raw data, and to obtain an operational taxonomic unit (OTU) list. Ecological analysis was subsequently performed using Excel, R, and Qiime software. Analysis of the fungal species composition revealed a total of 2786 OTUs, 40 genera, 29 orders, 16 classes, and 5 phyla. With respect to community composition, analysis of the ITS sequences revealed that the orders Tremellales and Auriculariales contained the highest number of species, and fungus diversity was the highest in Oedipodidae and lowest in Catantopidae. These findings indicated that the diversity of fungi in the gut of the grasshoppers was significantly different. Moreover, the alpha diversity analysis showed that Oedipodidae had relatively high values of community richness and diversity when compared with the other families. The beta diversity analysis demonstrated that the intestinal fungal community structure of the grasshoppers showed no significant difference within a family; in contrast, exhibited more differences among species of different families. However, the gut fungal community structure of Acrida cinerea in different environments showed a relatively low similarity. Finally, the cluster analysis showed that grasshoppers from the same family primarily clustered together and their community similarity was relatively high. Bullera, Ochrolechia, and Exophiala were the dominant genera among the grasshoppers'' intestinal fungi. This also showed that the fungal populations in the intestines of the grasshoppers were highly diverse in terms of the number of species and their community structure composition, although the differences in the fungal communities varied with the grasshopper host species and surrounding environment.