Affiliations between bacteria and marine fish leeches (Piscicolidae), with emphasis on a deep-sea species from Monterey Canyon, CA.

Leeches within the Piscicolidae are of great numerical and taxonomic importance, yet little is known about bacteria that associate with this diverse group of blood-feeding marine parasites of fish and elasmobranchs. We focused primarily on the bacteria from a deep-sea leech species of unknown identity, collected at ∼ 600 m depth in Monterey Canyon, CA, along with two shallow-living leech genera, Austrobdella and Branchellion, from Los Angeles Harbor, CA. Molecular analysis of all five leech species revealed a dominance of gammaproteobacteria, which were distinct from each other and from previously reported freshwater leech symbionts. Bacteria related to members of the genus Psychromonas (99% similarity in 16S rRNA) were dominant in the deep-sea leech species (80-94% of recovered ribotypes) collected over 19 months from two different locations. Psychromonas was not detected in cocoons or 2-16 week-old juveniles, suggesting that acquisition is via the environment at a later stage. Transmission electron microscopy did, however, reveal abundant bacteria-like cells near areas of thinning of the juvenile epithelial surface, as well as Psychromonas sparsely distributed internally. Electron and fluorescence in situ microscopy of adults also showed Psychromonas-like bacteria concentrated within the crop. Despite the apparent non-transient nature of the association between Psychromonas and the deep-sea leech, their functional role, if any, is not known. The prevalence, however, of an abundant bacterial genus in one piscicolid leech species, as well as the presence of a dominant bacterial species in singular observations of four additional marine species, suggests that members of the Piscicolidae, possibly basal within the class Hirudinea, form specific alliances with microbes.

Osedax: bone-eating marine worms with dwarf males.

We describe a new genus, Osedax, and two new species of annelids with females that consume the bones of dead whales via ramifying roots. Molecular and morphological evidence revealed that Osedax belongs to the Siboglinidae, which includes pogonophoran and vestimentiferan worms from deep-sea vents, seeps, and anoxic basins. Osedax has skewed sex ratios with numerous dwarf (paedomorphic) males that live in the tubes of females. DNA sequences reveal that the two Osedax species diverged about 42 million years ago and currently maintain large populations ranging from 10(5) to 10(6) adult females.

Biogeography and ecological setting of Indian Ocean hydrothermal vents.

Within the endemic invertebrate faunas of hydrothermal vents, five biogeographic provinces are recognized. Invertebrates at two Indian Ocean vent fields (Kairei and Edmond) belong to a sixth province, despite ecological settings and invertebrate-bacterial symbioses similar to those of both western Pacific and Atlantic vents. Most organisms found at these Indian Ocean vent fields have evolutionary affinities with western Pacific vent faunas, but a shrimp that ecologically dominates Indian Ocean vents closely resembles its Mid-Atlantic counterpart. These findings contribute to a global assessment of the biogeography of chemosynthetic faunas and indicate that the Indian Ocean vent community follows asymmetric assembly rules biased toward Pacific evolutionary alliances.

The ionic composition of the hydrothermal vent tube worm Riftia pachyptila: evidence for the elimination of SO2-4SO and H+ and for a Cl-/HCO-3HCO shift.

Riftia pachyptila is one of the most specialized invertebrate hosts of chemoautotrophic symbionts. Crucial to the functioning of this symbiosis is how these worms cope with fluctuating ion concentrations. Internal sulfate levels in R. pachyptila appear comparable with other benthic marine invertebrates, despite the production of sulfate internally by means of the bacterial oxidation of hydrogen sulfide, suggesting that these worms are able to eliminate sulfate effectively. Internal chloride levels appear comparable; however, coelomic fluid chloride levels decrease significantly as the amount of coelomic fluid bicarbonate increases, demonstrating a 1:1 stoichiometry. We believe this shift in chloride, out of the body fluids, is needed to compensate for changes in electrochemical balance caused by the large increase (up to and greater than 60 mmol L-1) in negatively charged bicarbonate. Riftia pachyptila fits the general pattern of monovalent ion concentrations that is seen in other benthic marine invertebrates, with a high [Na+] : [K+] ratio extracellularly and low [Na+] : [K+] ratio intracellularly. Extracellular pH values of 7.38+/-0.03 and 7.37+/-0. 04 for coelomic fluid and vascular blood, respectively, as well as intracellular pH values of 7.37+/-0.04 and 7.04+/-0.05 for plume and trophosome tissue, respectively, were measured. On the basis of significant decreases in extracellular pH and, in some cases, Na+ and K+, in worms exposed to carbonyl cyanide m-chlorophenylhydrazone, sodium vanadate, and N-ethylmaleimide, we suggest that high concentrations of H+-ATPases, perhaps Na+/H+- or K+/H+-ATPases, are involved in H+ elimination in these animals.

Physiological Functioning of Carbonic Anhydrase in the Hydrothermal Vent Tubeworm Riftia Pachyptila.

On the basis of our experiments, it is clear that carbonic anhydrase (CA) plays an important role in the CO2-concentrating mechanisms in Riftia pachyptila. Plume tissue from freshly collected animals had the highest CA activity, 253.7 +/- 36.0 {mu}mol CO2 min-1 g-1 wet wt, and trophosome activity averaged 109.4 +/- 17.9 {mu}mol CO2 min-1 g-1 wet wt. Exposure of living worms to ethoxyzolamide, a carbonic anhydrase inhibitor, resulted in a 99% decrease in CA activity (from 103.9 +/- 38.6 to 0.7 +/- 0.2 {mu}mol CO2 min-1 g-1 wet wt in the plume tissue and 57.6 +/- 17.9 to 0.04 +/- 0.11 {mu}mol CO2 min-1 g-1 wet wt in the trophosome) and essentially a complete cessation of {Sigma}CO2 uptake. High concentrations of CA appear to facilitate the equilibration between inorganic carbon (Ci) in the external and internal environments, greatly enhancing the diffusion of CO2 into the animal. In summary, R. pachyptila demonstrates very effective acquisition of inorganic carbon from the environment, thereby providing the symbionts with large amounts of CO2. This effective acquisition is made possible by three factors: extremely effective pH regulation, a large external pool of CO2, and, described in this paper, high levels of carbonic anhydrase.

Sulfide acquisition by the vent worm Riftia pachyptila appears to be via uptake of HS-, rather than H2S.

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.

Inorganic carbon acquisition by the hydrothermal vent tubeworm Riftia pachyptila depends upon high external PCO2 and upon proton-equivalent ion transport by the worm

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 (CO2) both within and between vent sites when comparing freshly captured worms from a variety of places. However, the primary influence on body fluid CO2 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 PCO2 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 CO2 (as high as 7.1 mmol l-1 around the tubes) speeds this diffusion. Moreover, despite large and variable amounts of internal CO2, 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 CO2. 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 PCO2 in the vent environment and because of its ability to control its extracellular pH in the presence of large inward CO2 fluxes.