Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that contain microtubule-like structures are bacteria related to Verrucomicrobia

Abstract

Epixenosomes, ectosymbionts on hypotrich ciliates (genus Euplotidium) defend their host against the ciliate predator Litonotus lamella. Although here only Euplotidium itoi and Euplotidium arenarium from tide pools along a rocky shore near Leghorn (Ligurian sea) were studied in detail, these epibionts are certainly present on specimens of E. itoi and on other Euplotidium species in similar north coastal habitats. The complex life history of epixenosomes has two main stages. In stage I, cells with typical prokaryotic structure divide by binary fission. Stage II cells show complex organization with different cytoplasmic compartments where an extrusive apparatus within a proteinaceous matrix, although not membrane-bounded, differs from the remaining cytoplasm. The ejection process is involved in defense; extrusive apparatus is surrounded by a basket consisting of bundles of tubules. These tubules, 22 +/- 3 nm in diameter, delimited by a wall made up of globular structures, are sensitive to inhibitor of tubulin polymerization (nocodazole/4 degrees C temperature) and react positively with different antitubulin antibodies, two of which are monoclonal. The prokaryotic vs. eukaryotic nature of epixenosomes was resolved by comparative sequence analysis of amplified small subunit rRNA genes and in situ hybridization with fluorescently labeled rRNA-targeted polynucleotide probes. These unique ectosymbionts are phylogenetically related to Verrucomicrobia. Epixenosomes represent marine symbionts in this recently discovered division of the Bacteria.

Figure 1

Epixenosomes and their host. (a) Dorsal view of E. itoi at scanning electron microscope. Arrows indicate epixenosomes in the cortical band. (Bar = 100 μm.) (b) Sectioned stage I epixenosomes. Arrow indicates the dividing one. (c) Stage II epixenosomes sectioned at different levels. DZ, Apical (dome-shaped) chromatin-like zone. EA, Extrusive apparatus. BT, Microtubule-like elements forming a “basket” around EA. (d) Section through the head of an ejected epixenosome. (Bars = 1 μm.) (e) The tube at the end of ejection. ER, Epixenosome remnant. H, Head. (Bar = 10 μm.)

Figure 2

Phylogenetic tree based on 16S rRNA sequences showing the position of epixenosomes within the major lineages of Bacteria. The shown tree is derived from an optimized global tree reconstructed from more than 10,000 aligned SSU rRNA sequences belonging to all three domains using various treeing programs included in the arb package.

Figure 3

Detection of epixenosomes on E. arenarium. (a) Detail on ejected extrusive processes in phase contrast superimposed with the green signal of epixenosome specific probe. The heads of ejected epixenosomes are clearly labeled. (b and c) Double hybridization with the Cy3-labeled oligonucleotide probe Eare-832-Cy3 specific for Euplotidium (red signal), and with the fluorescein-labeled polynucleotide probe specific for epixenosomes (green signal). Both organisms are specifically labeled. The green fluorescence is visible only at the level of the epixenosomal band or on the heads of ejected tubes. (d and e) Corresponding phase contrast of b and c. (Bars = 10 μm.)