Extracellular and mixotrophic symbiosis in the whale-fall mussel Adipicola pacifica: a trend in evolution from extra- to intracellular symbiosis

Abstract

Background: Deep-sea mussels harboring chemoautotrophic symbionts from hydrothermal vents and seeps are assumed to have evolved from shallow-water asymbiotic relatives by way of biogenic reducing environments such as sunken wood and whale falls. Such symbiotic associations have been well characterized in mussels collected from vents, seeps and sunken wood but in only a few from whale falls.

Methodology/principal finding: Here we report symbioses in the gill tissues of two mussels, Adipicola crypta and Adipicola pacifica, collected from whale-falls on the continental shelf in the northwestern Pacific. The molecular, morphological and stable isotopic characteristics of bacterial symbionts were analyzed. A single phylotype of thioautotrophic bacteria was found in A. crypta gill tissue and two distinct phylotypes of bacteria (referred to as Symbiont A and Symbiont C) in A. pacifica. Symbiont A and the A. crypta symbiont were affiliated with thioautotrophic symbionts of bathymodiolin mussels from deep-sea reducing environments, while Symbiont C was closely related to free-living heterotrophic bacteria. The symbionts in A. crypta were intracellular within epithelial cells of the apical region of the gills and were extracellular in A. pacifica. No spatial partitioning was observed between the two phylotypes in A. pacifica in fluorescence in situ hybridization experiments. Stable isotopic analyses of carbon and sulfur indicated the chemoautotrophic nature of A. crypta and mixotrophic nature of A. pacifica. Molecular phylogenetic analyses of the host mussels showed that A. crypta constituted a monophyletic clade with other intracellular symbiotic (endosymbiotic) mussels and that A. pacifica was the sister group of all endosymbiotic mussels.

Conclusions/significance: These results strongly suggest that the symbiosis in A. pacifica is at an earlier stage in evolution than other endosymbiotic mussels. Whale falls and other modern biogenic reducing environments may act as refugia for primal chemoautotrophic symbioses between eukaryotes and prokaryotes since the extinction of ancient large marine vertebrates.

Figure 1. Adipicola mussels.

Transmission electron micrographs of transverse sections of ctenidial filaments. (A)–(C) Adipicola pacifica. (A) Epithelial cells of the ctenidial filament. Gram-negative bacterial symbionts (arrows) are visible on the surface of the cells. Arrowheads indicate pseudopodium-like structures. (B) Bacterial symbionts (arrows) contained in vacuoles accompanied by microvilli (arrowheads). (C) Intracellular degradation of symbionts. Relics of decomposed bacteria (arrows) located in vacuoles of host cells and accompanying host microvilli (arrowheads). (D) Adipicola crypta. Intracellular gram-negative symbiotic bacteria within epithelial cells of the ctenidial filament. Arrowheads indicate the symbionts in vacuoles and arrows indicate digested bacteria in lysosomes.

Figure 2. Adipicola mussels.

Scanning electron micrographs of gill surfaces. (A)–(C) Adipicola pacifica. (A) & (B) Well-developed microvilli and numerous bacterial symbionts on the gill surface. Arrowheads indicate the symbionts and arrows indicate microvilli. (C) Higher magnification of the bacterial symbionts. Well-developed filamentous networks are visible. Arrowheads indicate the symbionts. (D) Adipicola crypta. The gill surface was flat and smooth and few bacteria are visible.

Figure 3. Phylogeny of bacterial symbionts from whale-fall Adipicola mussels based on 16S rRNA gene sequences.

Bayesian (BA) tree of the γ-Proteobacteria are shown. Scale bar represents 0.05 nucleotide substitution per sequence position. A BA posterior probability greater than 0.5 and bootstrap values greater than 50% are shown for each branch, with left, center and middle values representing posterior probability in BA and bootstrap values in the maximum-likelihood (ML) and neighbor-joining (NJ) methods, respectively. Symbionts of the mussels examined in this study are highlighted. The accession numbers used for this study are shown in parentheses following the operational taxonomic unit names.

Figure 4. Adipicola mussels.

Images of Fluorescence in situ hybridization (FISH) microscopy of bacterial symbionts in transverse sections of gill filaments of A. pacifica (A, B) and A. crypta (C, D) are shown. Hybridizations with the Symbiont A-specific probe SymA labeled with Alexa 647 (shown in red) and the Symbiont C-specific probe SymCx labeled with Alexa 555 (shown in green) are shown in A and B. Hybridizations with the A. crypta symbiont-specific probe SymAc labeled with Alexa 647 (shown in pink) are shown in C and D. All images are embedded sections (4-µm thickness) that were also stained with DAPI after hybridization (shown in blue). CZ: ciliated zone, LZ: lateral zone.

Figure 5. Phylogeny of whale-fall Adipicola mussels based on sequences of three eukaryotic genes: 18S rRNA, cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4).

BA tree of mytilid mussels is shown. Scale bar represents 0.02 nucleotide substitution per sequence position. BA posterior probability greater than 0.5 and bootstrap values greater than 50% are shown for each branch, with left, center and middle values representing posterior probability in BA and bootstrap values in ML and NJ, respectively. Adipicola mussels examined in this study are highlighted. The accession numbers used for this study are shown in Table 2.

Figure 6. Stable isotopic compositions of soft tissues of whale-fall Adipicola mussels and whale tissues.

(A) The δ¹³C and δ¹⁵N. (B) The δ¹³C and δ³⁴S. Open circle: A. crypta, solid circle: A. pacifica, open square: whale tissue. Each error bar indicates standard deviation among specimens.

Figure 7. Hypothetical schemes for the evolution of symbiont-harboring mytilids.

Mussel habitats and representative symbiotic forms in mussels from each habitat are shown. Open ellipse: mussel habitat, solid arrow: emigration of mussel, n: nucleus.