CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology

Abstract

Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances

“…Recent studies conducted mostly in freshwater systems have been dealing with characterizing the growth rates of major bacterivorous taxa (Grujcic et al ., 2018 ; Šimek et al ., 2018 , 2020 ). So, for a better understanding of the bacterial grazing and its impact on microbial food web structure, it is still necessary to investigate the physiological parameters of the dominant marine HF taxa (Piwosz et al ., 2021 ).…”

Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions

“…Another method that can directly enumerate specific taxa is fluorescence in situ hybridization (FISH), which recruits a fluorescently labeled probe that can hybridize the complementary sequences in the targeted cells. Because of its high sensitivity, it has been used to detect and/or quantify low abundance microbes and those with “low ribosome content” [ 66 , 67 ]. Although FISH provides only a relative abundance measurement, it can be combined with flow cytometry and microscopy to determine the absolute abundance of each taxon of interest independently of all bacterial members [ 63 , 68 , 69 ].…”

“…Kuo et al, 2021 designed four different probes using dyes Cy3, FAM, Texas red, and Cy5, respectively, and successfully detected four coliform bacteria ( E. coli , K. pneumoniae , E. aerogenes , and C. freundii ) in water samples [ 70 ]. Detecting rare taxa in a community using FISH requires a great number (500–1000) of counted cells to maximize the precision of the targeted count [ 67 ].…”

“…Catalyzed reporter deposition (CARD)-FISH is an improved version of FISH that has a 200-fold greater sensitivity than FISH and can be used to analyze physiology, metabolic activity, and ecological interactions [ 67 , 68 , 97 , 98 ]. Besides the benefits of specific taxa detection, double CARD-FISH using multiple fluorescent tracers allows the exhibition of the processes of interactive activities directly among communities, such as predator-prey interactions [ 99 ].…”

Protist diversity and metabolic strategy in freshwater lakes are shaped by trophic state and watershed land use at a continental scale

“…Efforts to study protists from a variety of biomes have unearthed a vast diversity and begun to map their distributions at a global scale (22)(23)(24). Such investigations have brought insights into protist environmental preferences and community assembly processes (25)(26)(27)(28), food web dynamics (29)(30)(31)(32)(33), symbioses (34)(35)(36), viruses (37), functional traits (38), and bioindicator values (39,40). Large-scale lake surveys have shown that protist assemblages are shaped by broad biogeographic patterns (41)(42)(43) but are also influenced by local environmental factors and interactions with bacteria (44).…”