Fig 1.
Decay of fresh and incubated trophosome over time under cold and warm incubation conditions.
a, f) Fresh trophosome, b, g) one day cold incubated trophosome, c, h) six day cold incubated trophosome, d, i) one day warm incubated trophosome, e, j) six day warm incubated trophosome. a-e) Fluorescence in situ hybridization (FISH) on LR-White sections of fresh and incubated trophosome shows a perfect overlap of signals for the symbiont specific and general bacterial probe mix. Red: EUB I, II, III mix; green: symbiont probe RifTO445; blue: DAPI; scale bars: 20 μm. f-j) Transmission electron micrographs (TEM) of fresh and incubated trophosome. A star indicates symbiont presence in the trophosome; black arrow: symbiont outer and cell membrane; white arrow: host symbiosome membrane; scale bars: 500 nm.
Fig 2.
Agar diffusion test for the inhibition of bacterial growth by freshly fixed and gradually degraded trophosome and freshly fixed skin.
Inhibition of growth of a-f) Bacillus subtilis and g-l) Listeria welshimeri by a, g) freshly fixed trophosome, b, h) one day cold incubated trophosome, c, i) six days cold incubated trophosome, d, j) one day warm incubated trophosome and e, k) six days warm incubated trophosome. f, l) No growth inhibition of Bacillus subtilis and Listeria welshimeri by freshly fixed Riftia skin. The inhibition of both strains by the trophosome correlates significantly with the peak areas of the free fatty acids from the HPLC-CAD analysis. Star indicated zone of inhibition, tr: trophosome, sk: skin, scale bars: 1 mm.
Fig 3.
a) Measured inhibition zones in mm induced by freshly fixed and incubated (cold and warm) trophosome samples and freshly fixed skin on Bacillus subtilis, Listeria welshimeri, Flavobacterium johnsoniae, Mycobacterium smegmatis and Staphylococcus aureus. b) Absolute peak areas of the lipids 2-palmitoleyl-1-lyso-PE (1-LPE), 1-palmitoleyl-2-lyso-PE (2-LPE), palmitoleic acid (C16:1), palmitic acid (C16:0), and oleic acid (C18:1) obtained by HPLC-CAD analysis of the corresponding ethanol supernatants. The correlation of the inhibition zone of Bacillus subtilis and Listeria welshimeri with the peak areas of the three free fatty acids is highlighted with the colours. sk: skin, tr: trophosome (S3 Table).
Fig 4.
HPLC-CAD chromatograms of the ethanol supernatant after fixation of a) freshly fixed trophosome, b) trophosome after one day warm incubation, c) trophosome after six days warm incubation, and d) freshly fixed skin of Riftia pachyptila.
Peaks 1–5 were tentatively identified by HPLC-MS as 2-palmitoleyl-1-lyso-PE (1-LPE), 1-palmitoleyl-2-lyso-PE (2-LPE), palmitoleic acid, palmitic acid, and oleic acid, respectively.
Table 1.
Spearman correlation coefficients between the peak areas of 2-palmitoleyl-1-lyso-PE (1-LPE), 1-palmitoleyl-2-lyso-PE (2-LPE), palmitoleic acid (C16:1), palmitic acid (C16:0), and oleic acid (C18:1) and the growth inhibitory effect of freshly fixed and incubated trophosome and skin on Bacillus subtilis, Listeria welshimeri, Flavobacterium johnsoniae, and Mycobacterium smegmatis.
Correlations are between 1-LPE and 2-LPE, between 2-LPE, palmitoleic acid and oleic acid, and between all three fatty acids. The peak areas of all three fatty acids correlate with the inhibition of Bacillus subtilis and Listeria welshimeri.
Fig 5.
Symbiont viability in the course of host degradation.
In fresh specimens, the host and symbiont are alive and the trophosome and skin samples represent the lipid composition of the holobiont. Upon host death, i.e., after trophosome incubation for one day of cold and warm incubations and after six days of cold incubations in our experiments, symbiont digestion ceases. The in situ membrane degradation of symbionts quantitatively exceeds the degradation of host membranes upon host death, and therefore LPEs and fatty acids decrease. Symbiont and remaining host autolysis between day one and day six of warm incubations results in an increase of the concentration of free fatty acids. Red: host alive; pink: symbiont alive; dark green: host dead; light green: symbiont dead.