Relative abundances of methane- and sulfur-oxidizing symbionts in gills of the deep-sea hydrothermal vent mussel Bathymodiolus azoricus under pressure

Highlights

• We investigate the dynamics of bacterial symbionts of a deep-sea vent mussel using FISH.

• We compare isobaric and non-isobaric sampling procedures.

• Live specimens were exposed to several treatments in IPOCAMP pressurized vessels.

• Sampling procedures did not significantly impact symbiont relative abundances.

• Exposure to reduced sulfur resulted in dominance of sulfur-oxidizing symbionts in gills.

Abstract

The deep-sea mussel Bathymodiolus azoricus dominates hydrothermal vent fauna in the Azores region. The gills of this species house methane- and sulfur-oxidizing bacteria that fulfill most of the mussel’s nutritional requirements. Previous studies suggested that the ratio between methane- and sulfur-oxidizers could vary in response to the availability of electron donors in their environment, and this flexibility is considered a key factor in explaining the ecological success of the species. However, previous studies were based on non-isobaric recovery of specimens, with experiments at atmospheric pressure which may have induced artifacts. This study investigates the effect of pressure-related stress during recovery and experimentation on the relative abundances of bacterial symbionts. Mussel specimens were recovered for the first time using the pressure-maintaining device PERISCOP. Specimens were subsequently transferred into pressurized vessels and exposed to various chemical conditions. Using optimized fluorescence in situ hybridization-based approaches, relative abundance of symbionts were measured. Our results show that the recovery method (isobaric versus non-isobaric) does not influence the abundances of bacterial symbionts. Significant differences occur among specimens sampled from two contrasting sites. Exposure of mussels from the deeper site to sulfide and bicarbonate, and to bicarbonate alone, both resulted in a rapid and significant increase in the relative abundance of sulfur-oxidizers. Results reported herein are congruent with those from previous reports investigating mussels originating from shallow sites and kept at ambient pressure. Isobaric recovery and maintenance allowed us to perform in vivo experiments in specimens from a deeper site that could not be maintained alive at ambient pressure, and will greatly improve the chances of identifying the molecular mechanisms underlying the dialogue between bathymodioline hosts and symbionts.

Introduction

Bathymodiolinae mussels (family Mytilidae) are part of the remarkable fauna colonizing ecosystems such as hydrothermal vents and cold seeps in the deep-sea (Desbruyères et al., 2000, Duperron, 2010, Duperron et al., 2009, Von Cosel et al., 1999, Von Cosel et al., 2001). These mussels rely upon sulfur- or methane-oxidizing (SOX and MOX) bacteria occurring in their gill epithelial cells for all or part of their nutrition (Cavanaugh et al., 1981, Felbeck, 1981). The symbionts of mussels exploit compounds present in vent or seep fluids for their metabolism (Van Dover, 2000, Van Dover et al., 2002). Although sulfide is toxic to animals, symbiotic sulfur-oxidizers use hydrogen sulfide from the fluids as the source of energy for their metabolism and to fix inorganic carbon (Cavanaugh et al., 1988). Methane-oxidizing bacteria use methane both as a carbon and an energy source (Cavanaugh et al., 1992, Childress et al., 1986). Organic carbon compounds are subsequently transferred to their animal host and ultimately contribute to ecosystem productivity in habitats where only a small fraction of the photosynthetic primary production from upper layers of the oceans is brought in by sedimentation or advective transport (Cavanaugh, 1983, Corliss et al., 1979, Karl et al., 1980).

Bathymodiolus azoricus and its sister species Bathymodiolus puteoserpentis dominate several vent sites on the Mid-Atlantic Ridge (MAR). They possess both sulfur- and methane-oxidizing symbionts in their gill bacteriocytes, as demonstrated through ultrastructural studies, 16S rRNA-encoding gene sequence analyses, and enzyme assays (Cavanaugh et al., 1992, Distel et al., 1995, Duperron et al., 2006, Fiala-Medioni et al., 2002, Fisher et al., 1993). Dual symbiosis is thought to increase the environmental tolerance of hosts because the distinct metabolism of the sulfur- and methane-oxidizing symbionts may help the holobiont adapt to varying availability of reduced sulfur and methane (Distel et al., 1995, Fiala-Medioni et al., 2002). Several studies point to a high flexibility of the symbiont populations (Kádár et al., 2005, Riou et al., 2008). Bacteria indeed disappear from B. azoricus gill bacteriocytes when subjected to starvation in sulfide- and methane-free sea-water, but can be recovered when mussels return to sulfide-enriched aquaria (Kádár et al., 2005). The relative volume occupied by each symbiont type in bacteriocytes of B. azoricus varies within vent sites, and between sites displaying different chemical signatures (Halary et al., 2008). Experiments using mussels maintained in controlled conditions at atmospheric pressure with one, both or none of the electron donors necessary for endosymbiont metabolism confirm that symbiont relative abundances can change rapidly in response to changes in the availability of their respective substrates (Halary et al., 2008, Riou et al., 2010, Riou et al., 2008).

However, the previously mentioned results suffer numerous potential biases. First, specimen recovery from the MAR vent sites, which are located at depths between 800 m (Menez Gwen) and 3500 m (Logatchev), involved rapid (usually a few hours) and large de-pressurization of specimens (8 to 35 MPa). This results in high levels of stress, ultimately resulting in the death of specimens from the deepest sites (Halary et al., 2008). Second, specimens used in in vivo experiments are usually from shallower vent sites (Menez Gwen) and maintained in the laboratory at atmospheric pressure, i.e. ~80-fold lower than in situ conditions (Kádár et al., 2005, Riou et al., 2008). Results from these studies are thus potentially affected by artifacts associated with depressurization, and it remains to be confirmed whether observed symbiont dynamics were the consequence of these stresses or true biological responses. Another issue is with the quantification of symbionts itself. Several studies are based on a 3D fluorescence in situ hybridization (FISH) approach, which measures the fraction of the total volume occupied by each type of symbiont within bacteriocytes (Halary et al., 2008, Riou et al., 2008, Duperron et al., 2011). Although reliable, this approach is time consuming because it involves the acquisition of 3D images of gill sections, manual cropping of individual bacteriocytes, and computing volumes using a dedicated ImageJ plugin (Halary et al., 2008). This has limited the number of specimens that could be analyzed, reducing the statistical power of comparisons (Prosser, 2010).

Several pressurized vessels for live maintenance and pressurized recovery that prevent or reduce pressure-related stress have become available in recent years (Boutet et al., 2009). The first aim of the present study is to investigate relative symbiont abundances in B. azoricus mussels recovered from the Menez Gwen (800 m depth) and Rainbow (2300 m depth) vent sites using the pressure-maintaining PERISCOP sampling cell (Shillito et al., 2008). The second aim is to investigate these abundances in Rainbow specimens exposed to substrates used by the sulfide-oxidizing bacteria at their native pressure in the IPOCAMP vessel (Shillito et al., 2014). The percentage of total bacterial volume corresponding to methane- plus sulfur-oxidizing symbionts is measured by means of FISH and image analysis. In order to optimize the method, we compare results from three FISH-based approaches, and images acquired from the anterior and posterior regions of the gills. We then compare isobaric vs. non-isobaric recovery, Menez Gwen and Rainbow specimens, and the effect of treatments applied to live specimens from Rainbow. Results are discussed in relation to improvements of the methods used to investigate symbiont dynamics in animal tissue in light of previous reports, based on specimens from non-isobaric recoveries and exposed to various experimental conditions at atmospheric pressure.