Fig 1.
FISH and TUNEL labelling on gill filaments of B. azoricus.
A: Transverse section of gill filaments with FISH labelling. Nuclei from host tissue in blue (DAPI); sulfur-oxidizing symbionts in pink, and methane-oxidizers in green. B, C and D TUNEL-labelled nuclei in green, DAPI staining in red. Arrows point to apoptotic nuclei. B: Ciliated zones often contain many labelled nuclei. C: Bacteriocyte zone that displays many apoptotic nuclei (arrows), apoptotic hemocytes (arrowheads) and an apoptotic cell detached from the basal lamina (circle). D: Abfrontal zone of the gill filament showing apoptotic nuclei in cells with no symbiont (arrows), and apoptotic cells detached from the basal lamina (circles). Abbreviations: BZ: Bacteriocyte zone; FCZ: Frontal Ciliated Zone; H: Hemolymph zone; IFCJ Inter-Filamentary Ciliated Junction. Scale bars: 50μm.
Fig 2.
TUNEL labelling on gill filaments of B. puteoserpentis.
TUNEL-labelled nuclei in green, DAPI staining in red. A: Frontal ciliated zones often contain many labelled nuclei. B: Bacteriocyte zone that displays only a few apoptotic nuclei. C: Inter Filamentary ciliated Junction often contain many labelled nuclei. D: Abfrontal zone of the gill filament with apoptotic nuclei. Abbreviations: BZ: Bacteriocyte zone; FCZ: Frontal Ciliated Zone; H: Hemolymph zone; IFCJ Inter-Filamentary Ciliated Junction. Scale bars: 50μm.
Fig 3.
Active Caspase-3 labelling in the gill of B. azoricus.
Typical labelling in green surrounds the nucleus (arrows). DAPI signal in blue. Scale bar: 50μm.
Fig 4.
Percentages of apoptotic nuclei in the bacteriocyte zone of B. azoricus (Ba) and B. puteoserpentis (Bp) from the three sites.
White-colored boxplots indicate specimens collected non-isobarically, and grey boxplots indicate those collected isobarically, respectively. Percentages were not significantly different (a, see materials and methods). The letter n indicates the number of individuals analyzed for each species. Boxplot whiskers correspond to minimal and maximal values on a single image, the line inside the box is the median, and the upper and lower frames of the boxes represent the first and third quartile respectively.
Fig 5.
Percentages of apoptotic nuclei in the hemolymph, ciliated and bacteriocyte zone of B. azoricus and B. puteoserpentis.
Different letters (a,b) indicate the plots in which median values are statistically different (Wilcoxon test) and n indicates the number of specimens. Boxplot whiskers indicate minimal and maximal values on a single image, the line inside the box is the median, and the upper and lower frames of the boxes represent the first and third quartile respectively.
Fig 6.
TUNEL labelling on gill filaments of Bathymodiolus aff. boomerang (A) and Mytilus edulis (B-C). TUNEL-labelling in green or yellow, DAPI staining in red. A: Bathymodiolus aff. boomerang displays many labelled nuclei mostly in the frontal ciliated zone. B-C: Mytilus edulis gills show very few TUNEL-labelled nuclei, two are visible in the insert (C). Same abbreviations as in Fig 1. Scale bar: 50μm.
Fig 7.
Percentages of apoptotic nuclei compared between vent and seep species in the ciliated, hemolymph and bacteriocyte zones of B. azoricus, B. puteoserpentis and B. aff. boomerang (Ba, Bp and Bb, respectively).
The letter n indicates the number of specimens. Boxplot whiskers indicate minimal and maximal values on a single image, line inside the box is the median, and the upper and lower frames of the boxes represent the first and third quartile respectively.