Table_2_FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates.DOCX

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

浮游纤毛虫类群具有多种营养功能群，是淡水食物网的核心组成部分。分子分析技术的进步为纤毛虫群落组成的研究带来了全新视角。然而，纤毛虫典型的18S rDNA拷贝数高且波动幅度大，使得通过扩增子测序实现可靠定量的难度极高。若要精准测定种群丰度，经典的基于形态学的定量蛋白银染色法仍是首选方法。但形态型分析不仅耗时较长，还需要具备专业的分类学知识储备。催化报告沉积荧光原位杂交（Catalyzed reporter deposition fluorescence in situ hybridization，CARD-FISH）可将分子鉴定与显微定量相结合，是分析浮游纤毛虫的极具潜力的技术手段。本研究以9种实验室培养的纤毛虫为实验材料，验证了CARD-FISH技术的适用性：基于这些纤毛虫的18S rRNA基因，研究人员设计了8条物种特异性寡核苷酸探针与3条属特异性寡核苷酸探针。随后，本研究对CARD-FISH实验流程进行了优化，并验证了所有探针的特异性。本研究进一步针对单一纯培养体系与模拟群落样本，检测了CARD-FISH定量的精准度。针对湖水样本的成功测试证实，CARD-FISH可用于野外环境样本中浮游纤毛虫的鉴定与定量。通过双重杂交实验，本研究还可探究两种纤毛虫之间的种间捕食-被捕食相互作用。综上，本研究证实，使用物种特异性探针的CARD-FISH技术，能够助力高采样频率下近缘、小型或隐存类群的种群动态研究。