Internal transcribed spacers enable species-level Metataxonomic analysis of ciliated protozoa.

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 in-silico PCR, experimental PCR, followed by sequencing and metataxonomic analyses of the ciliate communities. In-silico 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.