Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Abstract

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.

Figure 1

Bayesian tree obtained from the analysis of the COI mtDNA dataset. The substrate from which each specimen was collected is given. Substrate labels within brackets correspond to previous records without molecular support. Labels of newly sequenced specimens are in bold and those used to study symbiotic associations are highlighted in grey. Substitution model selected from MrAIC: GTR+[Γ]+I. PP and bootstraps values obtained from ML analysis are given above and below nodes, respectively. PP and bootstraps values lower than 0.90 and 50%, respectively, are not shown. Scale bar represents 10% estimated base substitution. Filled square, vent; open square, seep; grey-filled circle, turtle bone; open circle, whale bone; black-filled circle, wood.

Figure 2

TEM images of gill filaments and epithelial cells of A. crypta colonizing various substrates. (a,b) Gill filaments of the specimen from whale bones. Bacteriocyte (BC) cytoplasm is filled with intracellular bacterial symbionts and secondary lysosome-like particles (Ly). (a) Very few bacteria can be observed at the periphery of the cell, located between microvilli. Higher magnification of intracellular bacteria (b) shows that symbionts are typical Gram-negative bacteria. Their DNA occupies most of the volume of the bacterial cytoplasm. The periplasmic space does not contain sulphur granules (usually appearing as electron-lucent vesicles), but there are numerous electron-dense granules (curved arrows) in the cytoplasm representing β-polyhydroxybutyrate storage granules. Small arrows indicate the microvilli from the host cell contained inside the large phagocytosis vacuole. m: mitochondria (c,d) TEM view of gill filaments from A. crypta collected on sunken woods. Bacteria are mostly located at the apical pole of the bacteriocytes; the basal part of the host cell contains mostly secondary lysosome-like structures (Ly). Bacterial symbionts are less numerous per phagocytosis vacuole (stars) with more bacteria located outside the host cell. Such extracellular bacteria probably became enclosed in the vacuoles by phagocytosis (arrows). Bacteria do not possess the dark β-polyhydroxybutyrate granules. BL: blood lacuna; N: nucleus.

Figure 3

TEM images of gill filaments and epithelial cells of lineage C colonizing various substrates. (a) Gill filaments from an individual collected on whale bone (Vanu 32) at 441 m depth. The thickness of the bacterial layer is greater than the bacteriocyte's thickness. Bacteria, which are located extracellularly between microvilli of the host cells, are mostly ovoid: bacteriocyte cytoplasm possesses few secondary lysosomes (Ly), characterized by their heterogeneous aspect. No bacteria are found inside phagosomes. BL: blood lacuna. N: nucleus. (b) Gill filament of the lateral zone from a wood-inhabiting specimen (Vanu 47) collected at 802 m depth. Bacteriocytes (BC) harbour few layers of extracellular bacteria. In the bacteriocyte cytoplasm, numerous small lysosomes are seen. Extracellular symbionts (arrows) are located on the apical surface of the bacteriocytes in contact with microvilli. (c) Lateral zone of a gill filament from a wood-inhabiting specimen (Vanu 44) collected at 290 m depth. Bacteriocyte cytoplasm contains mostly mitochondria. (d) Lateral zone of a gill filament from a wood-inhabiting specimen (Vanu 46) collected at 802 m depth. The thickness of the extracellular bacteria above the apical pole of the bacteriocytes reaches 15 μm. Similar bacteria can be observed either in the thick layer of extracellular bacteria or inside the phagosomes (stars) without apparent degradation. The bacteriocytes contain various lysosome-like structures in their cytoplasm.

Figure 4

Bayesian tree obtained from the analysis of the 16S rRNA dataset. Sequences obtained in this study are in bold. Substitution model selected from MrAIC: GTR+[Γ]+I. Posterior probabilities (PP) and bootstraps values obtained from ML analysis are given above and below nodes, respectively. PP and bootstraps values lower than 0.90 and 50%, respectively, are not shown. Scale bar represents 2% estimated base substitution. The broken branch represents 13% estimated base substitution.