FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates

Abstract

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.

Using digital PCR to predict ciliate abundance from ribosomal RNA gene copy numbers

“…Therefore, the next step would be to rigorously test the applicability of the here presented dPCR protocol for ciliate species quantification in natural samples. One possible approach could be a comparison of the results obtained from dPCR with established microscopy‐based approaches such as catalyzed reporter deposition‐fluorescence in situ hybridization (CARD‐FISH) and QPS (Dirren‐Pitsch et al, 2022).…”

“…Investigating microbial eukaryotes (protists) is important for our understanding of ecosystem functioning Worden et al, 2015). Numerous studies have already revealed the enormous diversity of protists in all major environments and demonstrated that many of them provide central functional roles (Caron et al, 2011;De Vargas et al, 2015;Filker et al, 2015;Foissner, 2008;Forster et al, 2016;L opez-García et al, 2001;Oliverio et al, 2018;Singer et al, 2021;Šlapeta et al, 2005;Worden et al, 2015). By contrast, fewer studies have been conducted that deal with the abundances and dynamics of protists in those same environments (Costas et al, 2007;Foissner, 2008;Logares et al, 2015;Massana et al, 2006;Nolte et al, 2010;Pitsch et al, 2019;Stoeck, Breiner, et al, 2014;Weisse, 2014).…”

“…In the past 20 years, major advances have been made that resulted in novel molecular tools for protistan research, including quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (HTS). These approaches use hypervariable genetic regions such as the small subunit of the ribosomal RNA (SSU-rRNA) as barcodes to detect protists (De Vargas et al, 2015;Nolte et al, 2010;Stoeck, Przybos, & Dunthorn, 2014). Compared to morphological approaches, they allow greater sampling volumes that can be processed faster.…”

“…The extensive morphologies of ciliates allowed researchers to characterise them, and investigate their abundances using various microscopy‐based methodologies. Some of the most common quantification approaches that have been extensively used throughout the last few decades include the use of counting chambers (e.g., the Utermöhl method; Kemp et al, 1993; Utermöhl, 1931) the Quantitative Protargol Staining (QPS) (Foissner et al, 2008; Gifford & Caron, 2000; Lynn & Montagnes, 1991) and fluorescence in situ hybridization (FISH), which simplified the enumeration by using a target‐specific probe and fluorescence microscopy (Dirren‐Pitsch et al, 2022; Filker et al, 2015). Relatively recent advances introduced an automated imaging and cell counting of diverse protistan groups (Mangot et al, 2018), however, the typical bias of microscopy‐based methods remains also in this automated solution: weaker fluorescence signals are easily overlooked, the fluorescence of non‐target particles may lead to false positives, and only small volumes of water can be screened at a time, making the detection of low‐abundant target organisms very difficult.…”

Changes in the Epilimnion Ciliate Assemblage Reflect the Reduction of the Nutrient Load of the Reservoir for Two Decades

“…Quantitative Protargol Stain (QPS; [22][23][24]) enabled the direct application of protargol stain to samples concentrated on membrane filters. The method has been routinely applied since the original publication (e.g., [18,[25][26][27][28][29]) and become worth combining modern molecular methods [30,31].…”