Deep-sea mussels Bathymodiolus azoricus , from Azorean hydrothermal vents, house two types of symbionts in their fleshy gills: methane-oxidizing (MOX) and sulfide-oxidizing (SOX) Gamma-proteobacteria. As soon as the mussels are collected, their symbionts are deprived from their environmental nutrient flux, and cannot rely on their usual metabolism. Recent studies have shown that the gill cells undergo high rates of apoptosis, as well as regionalized cell proliferation. This study follows the fate of the symbionts and of the hosting bacteriocytes at the ultrastructural level, during an extended starvation period. Just upon collection, we evidenced an apico-basal journey of the symbionts in the bacteriocytes, starting with (1) apical single symbiont endocytosis, (2) symbiont division, (3) symbiont storage, (4) and symbiont digestion within lysosomes, above the basal lamina. After 4-9 days starvation, endocytosis occurred with (5) empty blebbing, (6) the lysosomes increased in size, and the bacteriocytes lost their apical membrane, resulting in (7) a baso-apical return of the symbiont-containing lysosomes outside the gills, while the nucleus showed condensed chromatin, characteristic of apoptosis/necroptosis (8). Between the bacteriocytes, narrow intercalary cells appear to divide (9). Our hypothesis is that intercalary cells are stem cells that replace lost bacteriocytes. After 61 days there was no symbiont left, and the epidermis resembled those of the non-symbiotic filter-feeding mussel Mytilus edulis.
Symbiosis between Bathymodiolus and Gammaproteobacteria allows these deep-sea mussels to live in toxic environments such as hydrothermal vents and cold seeps. The quantity of endosymbionts within the gill-bacteriocytes appears to vary according to the hosts environment; however, the mechanisms of endosymbiont population size regulation remain obscure. We investigated the possibility of a control of endosymbiont density by apoptosis, a programmed cell death, in three mussel species. Fluorometric TUNEL and active Caspase-3-targeting antibodies were used to visualize and quantify apoptotic cells in mussel gills. To control for potential artefacts due to depressurization upon specimen recovery from the deep-sea, the apoptotic rates between mussels recovered unpressurised, versus mussels recovered in a pressure-maintaining device, were compared in two species from hydrothermal vents on the Mid-Atlantic Ridge: Bathymodiolus azoricus and B. puteoserpentis. Results show that pressurized recovery had no significant effect on the apoptotic rate in the gill filaments. Apoptotic levels were highest in the ciliated zone and in the circulating hemocytes, compared to the bacteriocyte zone. Apoptotic gill-cells in B. aff. boomerang from cold seeps off the Gulf of Guinea show similar distribution patterns. Deep-sea symbiotic mussels have much higher rates of apoptosis in their gills than the coastal mussel Mytilus edulis, which lacks chemolithoautotrophic symbionts. We discuss how apoptosis might be one of the mechanisms that contribute to the adaptation of deep-sea mussels to toxic environments and/or to symbiosis.
ABSTRACT Riftia pachyptila is the most conspicuous organism living at deep sea hydrothermal vents along the East Pacific Rise. To support its large size and high growth rates, this invertebrate relies exclusively upon internal chemosynthetic bacterial symbionts. The animal must supply inorganic carbon at high rates to the bacteria, which are far removed from the external medium. We found substantial differences in body fluid total inorganic carbon (ΣCO2) both within and between vent sites when comparing freshly captured worms from a variety of places. However, the primary influence on body fluid ΣCO2 was the chemical characteristics of the site from which the worms were collected. Studies on tubeworms, both freshly captured and maintained in captivity, demonstrate that the acquisition of inorganic carbon is apparently limited by the availability of CO2, as opposed to bicarbonate, and thus appears to be accomplished via diffusion of CO2 into the plume, rather than by mediated transport of bicarbonate. The greatly elevated measured at the vent sites (up to 12.6 kPa around the tubeworms), which is a result of low environmental pH (as low as 5.6 around the tubeworms), and elevated ΣCO2 (as high as 7.1 mmol l−1 around the tubes) speeds this diffusion. Moreover, despite large and variable amounts of internal ΣCO2, these worms maintain their extracellular fluid pH stable, and alkaline, in comparison with the environment. The maintenance of this alkaline pH acts to concentrate inorganic carbon into extracellular fluids. Exposure to N-ethylmaleimide, a non-specific H+-ATPase inhibitor, appeared to stop this process, resulting in a decline in extracellular pH and ΣCO2. We hypothesize that the worms maintain their extracellular pH by active proton-equivalent ion transport via high concentrations of H+-ATPases. Thus, Riftia pachyptila is able to support its symbionts’ large demand for inorganic carbon owing to the elevated in the vent environment and because of its ability to control its extracellular pH in the presence of large inward CO2 fluxes.
Deep-sea hydrothermal vents are home to a variety of invertebrate species, many of which host chemosynthetic bacteria in unusual symbiotic arrangements. The vent tubeworm Riftia pachyptila (Vestimentifera) relies upon internal chemolithoautotrophic bacterial symbionts to support its large size and high growth rates. Because of this, R. pachyptila must supply sulfide to the bacteria, which are far removed from the external medium. Internal H2S ([H2S+HS-+S2-]) can reach very high levels in R. pachyptila (2-12mmoll-1 in the vascular blood), most of which is bound to extracellular hemoglobins. The animal can potentially take up sulfide from the environment via H2S diffusion or via mediated uptake of HS-, or both. It was expected that H2S diffusion would be the primary sulfide acquisition mechanism, paralleling the previously demonstrated preferential uptake of CO2. Our data show, however, that the uptake of HS- is the primary mechanism used by R. pachyptila to obtain sulfide and that H2S diffusion into the worm apparently proceeds at a much slower rate than expected. This unusual mechanism may have evolved because HS- is less toxic than H2S and because HS- uptake decouples sulfide and inorganic carbon acquisition. The latter occurs via the diffusion of CO2 at very high rates due to the maintenance of an alkaline extracellular fluid pH. H2S accumulation is limited, however, to sulfide that can be bound by the hemoglobins, protecting the animal from sulfide toxicity and the symbionts from sulfide inhibition of carbon fixation.
