Colwellia and sulfur-oxidizing bacteria: An unusual dual symbiosis in a Terua mussel (Mytilidae: Bathymodiolinae) from whale falls in the Antilles arc

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Highlights

  • Terua n. sp. is the first mussel species identified from whale fall ecosystems in the Antilles arc (Guadeloupe).

  • It derives from ancestors found in the Pacific Ocean.

  • Terua n. sp. harbors an unusual dual symbiosis involving extracellular sulfur-oxidizing and Colwellia-related bacteria.

  • This symbiosis could be an adaptation to sunken bone habitats.

Abstract

Seven individuals of a single morphotype of mussels (Bivalvia: Mytilidae) were found attached to a naturally sunken whale intervertebral disk collected in Guadeloupe (Caribbean) at 800 m depth. These specimens resemble small Idas mussels which are found worldwide at cold seeps and hydrothermal vents, and typically harbor ectosymbiotic bacteria on their gills upon which they depend for nutrition. Based on multi-locus gene sequencing, these specimens appear to belong to a new species closely related to two species now included within the genus Terua. Unexpectedly, its closest relatives are found in the Pacific, questioning how this species has reached the Antilles arc. Based on marker gene sequence analysis, electron and fluorescence microscopy, Terua n. sp. harbors two distinct and abundant extracellular bacterial symbionts located between microvilli at the apical surface of host gill epithelial cells. One is a sulfur-oxidizing bacterium similar to the symbionts previously identified in several deep-sea mussels, while the other is related to Colwellia species, a group of cold-adapted heterotrophic bacteria able to degrade organic compounds. This study provides the first evidence for the existence of a dual symbiosis in mussels from whale fall ecosystems in the Caribbean. The evolutionary history of Terua n. sp. and potential role of its Colwellia symbionts are discussed.

Introduction

Large mussels associated with deep-sea hydrothermal vents and cold seeps have been studied since their discovery in the late 1970s. Their abundance at many sites and their nutritional reliance on dense populations of gill-associated bacterial symbionts which use reduced sulfur or methane to produce their organic matter attracted interest from many researchers. They form the clade Bathymodiolinae, named after the genus Bathymodiolus in which most large mussels are included. Many papers have investigated how these symbiotic bivalves could thrive in a priori inhospitable habitats and how they had evolved (Dubilier et al., 2008, Duperron, 2010). Over the last decade smaller mussels, some described in the 19th century and overlooked since then, were shown to also belong to the Bathymodiolinae clade (Lorion et al., 2010, Lorion et al., 2013, Thubaut et al., 2013). They live in reducing habitats including vents and seeps, but also on sunken carcasses of large animals and on wood falls, the decaying of which releases reduced compounds including sulfide and methane. The classification of small mussels is challenging because genus names do not overlap with gene phylogeny-based clades, but small species are of prime importance if we are to understand the evolution of deep-sea symbiont-bearing mussels. Indeed, large mussels cluster within a limited number of terminal clades, appearing as derived specialized forms, while the rest of the Bathymodiolinae phylogeny consists of various groups of small mussels. Biologically-speaking, apart from their small size, they share important features with large mussels, including the presence of bacterial symbionts in their gills (Deming et al., 1997, Gros and Gaill, 2007, Duperron et al., 2008b, Southward, 2008). However their symbioses seem to be more diverse and flexible. Large mussels usually have one to four types of bacteria located inside their gill epithelial cells. Small mussels on the other hand can display intra- or extracellular bacteria, a greater diversity of symbionts with up to 6 distinct types co-existing in the gills of Idas modiolaeformis, not restricted to sulfur- and methane-oxidizers, and the composition of bacterial communities can vary between sites for a given species (Duperron et al., 2008a, Laming et al., 2015b). Unfortunately, because of the patchy and unpredictable occurrence of their habitats, small mussels are often collected by chance in very limited numbers. Nevertheless their study yields important information for our understanding of the diversity, biogeography and evolution of the fauna colonizing deep-sea reducing habitats.

Seven species of Bathymodiolinae mussels are currently reported from the Gulf of Mexico and Antilles arc, all from cold seep habitats. Four are large Bathymodiolus (B. keckerae, B. boomerang, B. brooksi, B. childressi and a yet unnamed species), and two are smaller species, namely Tamu fisheri usually collected from 540 to 700 m depth associated with vestimentiferan tubeworm bushes, and Idas macdonaldi from 650 m depth (Gustafson et al., 1998, Faure et al., 2015). Dell (1987) also mentions Idas dalli E.A. Smith 1885 sampled from the Culebra Island (Porto Rico, West Indies) during the Challenger expedition, but little data is available. Seven mussel individuals representing a single morphotype were recently found attached to an inter-vertebral disk of a naturally sunken whale collected near Guadeloupe (Caribbean) at 800 m depth. In this study we test whether these represent a new species, or one of the 7 previously documented from the region, using multiple marker gene sequencing and phylogeny. We also characterize associated bacterial symbionts using electron microscopy, marker gene sequencing and fluorescence in situ hybridization (FISH). Results are discussed with a special emphasis on mussel biogeography, and on the potential role of the identified symbionts. This study is the first investigation of bone-associated symbiotic mussels from the Antilles arc.

Section snippets

Sample collection and preparation

One inter-vertebral disk from a whale carcass was collected using a beam-trawl 780–820 m depth during the Karubenthos2 cruise (June 2015, chief scientist: P. Bouchet) around Guadeloupe in the Caribbean [16°23′N, 60°46′W]. Mussel individuals up to 2 cm in length were found attached to the surface of a spinal disk obtained from a unique naturally submerged whale carcass. Mussel samples were processed onboard within 1 h after collection.

Six mussel individuals were stored in 100% ethanol for molecular

Host features

The 7 small mytilids (shell length between 9 and 12 mm) displayed identical shell features, including a pink whole prodissoconch 445±13 µm in length (Figs. 1A, 2A-D) and an adult dissoconch appearing smooth under a binocular microscope. According to SEM views, prodissoconch I was small (~92±11 µm) and the prodissoconch II measured 445±13 µm with several concentric growth lines (maximum length between two adjacent lines: ~9 µm). The visceral mass of two individuals (shell lengths of 10 and 12 mm) was

Unexpected occurrence of Terua n. sp. in the Antilles arc

According to various authors, the lack of specific shell features makes the latter poor predictors of species relatedness in small deep-sea mussels (Won et al., 2008, Lorion et al., 2010). Marker gene sequence analysis has thus proven to be the most useful tool to properly assign species. Sequence-based identification has revealed that most morphology-based genera within the Bathymodiolinae were not monophyletic. Genus names are thus of little help when describing mussel evolutionary

Conclusion

Terua n. sp. Guadeloupe is the first mussel species identified from whale fall ecosystems in the Antilles arc. Its absence from previous fauna samplings at various cold seeps in the Gulf of Mexico and western Atlantic suggests that it could be a true bone specialist, derived from ancestors found in the Pacific Ocean. Its dual symbiosis is unusual because most previously described bone-associated symbioses involved either only heterotrophic bacteria such as in the vestimentiferan Osedax, or

Acknowledgments

The material from Guadeloupe was obtained during the Karubenthos2 deep-sea cruise aboard ANTHEA funded by IRD and CNRS, and we thank Dr P. Bouchet and L. Corbari, the co-principal investigators, for their invitation. We thank P-Y Pascal for his participation in collecting the samples, the captain and crew of ANTHEA, and N. Léger for excellent technical assistance. We also thank the editor and two anonymous reviewers for comments that helped improve the manuscript. Molecular work was funded by a

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