Identification of rumen ciliates using small subunit ribosomal RNA (18S‐rRNA)‐targeted oligonucleotide probes and fluorescence in situ hybridization (FISH)

Abstract

Up to half of the biomass in the rumen can be represented by ciliates, which play an important role in the digestion processes of their hosts. In the literature, very little information can be found on determination of the diversity of complex rumen ciliate communities. The identification of these fast moving protists is mainly based on live observations and comparisons of their highly variable cell morphologies. It makes accurate identification and quantification of rumen ciliates very difficult if not impossible. The development of fluorescence in situ hybridization (FISH), established as a technique to identify prokaryotes and eukaryotes using ribosomal RNA‐targeted fluorescently labeled oligonucleotide probes, is a promising approach to identify and investigate rumen ciliates. The present study, part of CIMES (ciliates as monitors for environmental safety), a project sponsored by the European Commission, shows the problems of applying FISH on rumen ciliates and how to solve them. Tests resulted in a new protocol, which recommends para‐formaldehyde and formaldehyde at 1–2% final concentration to preserve the ciliates before applying FISH. Furthermore, seven new oligonucleotide probes could be developed and successfully be tested to identify different rumen ciliate taxa of the order Entodiniomorphida (class Litostomatea) by applying FISH. It is also shown, how FISH together with confocal laser scanning microscopy can improve analyses of ciliate cell morphologies. Thanks to Peter Pristas, Peter Javorsky, Ralf Einspanier, Susanne Ulbrich (providing rumen samples), Seung Yeo Moon‐van der Staay, Georg van der Staay (providing unpublished 18S‐rDNA sequences), the European Commission (financial support, project QLK3‐CT‐2002‐02151).