INTRACELLULAR PROKARYOTES IN RUMEN CILIATE PROTOZOA - DETECTION BY CONFOCAL LASER-SCANNING MICROSCOPY AFTER IN-SITU HYBRIDIZATION WITH FLUORESCENT 16S RIBOSOMAL-RNA PROBES

In situ hybridization of rumen ciliate protozoa with 16S ribosomal RNA fluorescent oligonucleotide probes specific for Archaea and Bacteria provided semi-quantitative indication of the location, type and extent of prokaryotic colonization of various protozoal species. The isotric hid holotrich ciliates generally carried a smaller load of intracellul ar microorganisms than did the entodiniomorphid species. Thus, the vas t majority of the Dasytricha ruminantium population had neither bacter ial not archaeal endosymbionts, although a very small minority of thes e ciliates (only 11 out of 447: < 3%), contained numerous Bacteria cel ls. Their food vacoules contained only Bacteria. Thirty per cent of Is otricha spp. were without endosymbionts. Polyplastron multivesiculatum invariably had large numbers (> 250 cells per ciliate) of intracellul ar Bacteria, bur no Archaea. Only some Epidinium spp. had intracellula r prokaryotes, whereas Entodinium spp. and Ent. simplex almost always had. Many of the larger entodiniomorphid genera were heavily colonized by both bacterial and archaeal species. Eudiplodinium maggii had no o bvious bacterial associates, although the autofluorescence of ingested plant material made difficult the detection of possible endosymbionts .