Identity of epibiotic bacteria on symbiontid euglenozoans in O2-depleted marine sediments: evidence for symbiont and host co-evolution

Abstract

A distinct subgroup of euglenozoans, referred to as the 'Symbiontida,' has been described from oxygen-depleted and sulfidic marine environments. By definition, all members of this group carry epibionts that are intimately associated with underlying mitochondrion-derived organelles beneath the surface of the hosts. We have used molecular phylogenetic and ultrastructural evidence to identify the rod-shaped epibionts of the two members of this group, Calkinsia aureus and B.bacati, hand-picked from the sediments of two separate oxygen-depleted, sulfidic environments. We identify their epibionts as closely related sulfur or sulfide-oxidizing members of the epsilon proteobacteria. The epsilon proteobacteria generally have a significant role in deep-sea habitats as primary colonizers, primary producers and/or in symbiotic associations. The epibionts likely fulfill a role in detoxifying the immediate surrounding environment for these two different hosts. The nearly identical rod-shaped epibionts on these two symbiontid hosts provides evidence for a co-evolutionary history between these two sets of partners. This hypothesis is supported by congruent tree topologies inferred from 18S and 16S rDNA from the hosts and bacterial epibionts, respectively. The eukaryotic hosts likely serve as a motile substrate that delivers the epibionts to the ideal locations with respect to the oxic/anoxic interface, whereby their growth rates can be maximized, perhaps also allowing the host to cultivate a food source. Because symbiontid isolates and additional small subunit rDNA gene sequences from this clade have now been recovered from many locations worldwide, the Symbiontida are likely more widespread and diverse than presently known.

Figure 1

Differential interference contrast (DIC) light microscope images of B. bacati and Calkinsia aureus. (a–d) C. aureus showing the epibionts that disassociate with the host almost immediately upon stress. Cells can be seen floating free of host cell in (b) and (c). (e and f) B. bacati showing black inclusions within the anterior part of the cell. Scale bar=20 μm except panel (c), which is 10 μm.

Figure 2

Electron micrographs of the epibionts on Calkinsia aureus and B.bacati. (a) SEM of B. bacati showing rod-shaped epibiotic bacteria and spherical-shaped, extrusive epibiotic bacteria (arrow). Note that this image is the same magnification as C. (b) TEM of the peripheral area of B. bacati showing epibionts connected to the plasma membrane of B. bacati by a glycocalyx. Hydrogenosome-like organelles (H) were located under the plasma membrane. Note that this image is the same magnification as D. (c) SEM of Calkinsia aureus showing the rod-shaped epibionts on the cell surface. (d). TEM of the peripheral area of C. aureus showing epibionts connected to a robust extracellular matrix (Ex) through a glycocalyx. Arrow indicates conduits in the extracellular matrix. Hydrogenosome-like organelles (H) were located under the plasma membrane (arrow). (e) A longitudinal section of the epibionts of C. aureus showing fibrous material (arrowhead). (f) The transverse section of the epibionts of C. aureus showing fibrous material (arrowhead). Scale bar=2 μm in (a and c), 300 nm in (b, d and f) and 500 nm in (e).

Figure 3

Phylogenetic analysis of 16S rDNA genes from the epibionts of B.bacati and Calkinsia aureus. Tree is based on an alignment of 1389 nucleotides. Bootstrapping and determination of the test estimate of the ML tree topology for these data sets were conducted with the Rapid Bootstrapping algorithm of RAxML version 7.0 under the GTR+I model running on the CIPRES portal (Stamatakis, 2006; Stamatakis et al., 2008) (www.phylo.org).

Figure 4

General comparison of tree topologies and branch lengths in phylogenetic analyses of host and epibiont small subunit rDNA sequences.

Figure 5

CARD-FISH. (a) Non-probe DAPI, (b) non-probe Alexa488, (c) EUB338-probe DAPI, (d) EUB338-probe Alexa488, (e) epsilon-proteobacterial probe EPS549 DAPI, (f) epsilon-proteobacterial probe EPS549 Alexa488, (g) Arcobacter-specific probe ARC94 Alexa488, (h) epsilon-Proteobacterial probe EPS549 confocal fluorescein isothiocyanate (FITC). Scale bars=10 μm.