Somasundaram Sripoorna, Yu Zhongtang
Department of Animal Sciences, Animal Science Building, 2029 Fyffe Road, Columbus, OH 43210, United States.
Center of Microbiome Science, 352K Wiseman Hall, 400 W 12th Ave, Columbus, OH 43210, United States.
ISME Commun. 2025 Feb 11;5(1):ycaf024. doi: 10.1093/ismeco/ycaf024. eCollection 2025 Jan.
Traditional morphology-based ciliate classification is often time-consuming and inaccurate, necessitating molecular approaches. Although 18S rRNA gene sequencing is widely used for taxonomic analyses of ciliates, its high degree of conservation makes it challenging to achieve species-level resolution. This study explores the potential of internal transcribed spacers (ITS1 and ITS2) and the 28S rRNA gene to improve taxonomic resolution beyond that offered by 18S rRNA gene in free-living and host-associated ciliates. A comparative analysis of ITS, the 18S, and 28S rRNA gene sequences retrieved from public databases indicated that ITS regions exhibit greater inter- and intra-specific sequence dissimilarity compared to 18S rRNA gene, supporting existing literature. We then designed universal primers targeting the ITS and 28S rRNA gene for freshwater and rumen ciliates. These primers were rigorously evaluated for their inclusiveness, specificity, and amplification efficiency using - PCR, experimental PCR, followed by sequencing and metataxonomic analyses of the ciliate communities. - analyses revealed inclusiveness exceeding 80%, while experimental analyses validated their specificity. Metataxonomic analyses of ciliates demonstrated that the ITS and 28S rRNA gene captured significantly greater taxonomic diversity than 18S rRNA gene. Also, ITS1 offered superior taxonomic resolution by detecting the most ciliate species that went unnoticed by the 18S rRNA gene. These findings underscore the superiority of ITS1, and to a lesser extent ITS2, as taxonomic markers for enhancing the resolution of freshwater and rumen ciliate communities. We recommend ITS1 as an alternative marker to overcome the limitations of 18S rRNA gene-based approaches in free-living and host-associated ciliate taxonomy.
基于传统形态学的纤毛虫分类通常既耗时又不准确，因此需要采用分子方法。尽管18S rRNA基因测序广泛用于纤毛虫的分类分析，但其高度保守性使得实现物种水平的分辨率具有挑战性。本研究探讨了内转录间隔区（ITS1和ITS2）以及28S rRNA基因在提高自由生活和宿主相关纤毛虫分类分辨率方面的潜力，该分辨率超过18S rRNA基因所提供的分辨率。从公共数据库检索到的ITS、18S和28S rRNA基因序列的比较分析表明，与18S rRNA基因相比，ITS区域在种间和种内表现出更大的序列差异，这支持了现有文献。然后，我们针对淡水和瘤胃纤毛虫设计了靶向ITS和28S rRNA基因的通用引物。使用PCR、实验性PCR对这些引物的包容性、特异性和扩增效率进行了严格评估，随后对纤毛虫群落进行了测序和宏分类分析。分析显示包容性超过80%，而实验分析验证了它们的特异性。纤毛虫的宏分类分析表明，ITS和28S rRNA基因捕获的分类多样性明显高于18S rRNA基因。此外，ITS1通过检测到18S rRNA基因未注意到的大多数纤毛虫物种，提供了更高的分类分辨率。这些发现强调了ITS1以及在较小程度上ITS2作为分类标记在提高淡水和瘤胃纤毛虫群落分辨率方面的优越性。我们建议将ITS1作为替代标记，以克服基于18S rRNA基因的方法在自由生活和宿主相关纤毛虫分类中的局限性。
