Conducting exploratory microbial research at deep-sea hydrothermal vents requires a number of different approaches to ensure adequate coverage of the many different micro-habitats in and around the vents. Our ultimate goal is to collect samples from as many habitats as possible for a variety of analyses and experiments including (1) protistan biomass for DNA- and RNA-based microbial diversity analysis, (2) live cells for enrichment, culturing, isolation, and identification, (3) live cells for bacterial grazing experiments at hydrostatic pressures typical of the deep-sea, and (4) samples for microscopy and digital imaging.

A primary consideration when thinking about sample collection is how to concentrate microbial biomass from an environment where it’s probable that protists of any given species are highly diluted. In the past, we’ve teamed up with the virus researchers to obtain samples from the Large Volume Water Sampler (LVWS) (see photo at top in sidebar), which pumps water from discrete locations near the seafloor that would otherwise be very difficult to sample with respect to the enormous amount of biomass that it’s able to collect.

Microbial mats and underlying sediments at Guaymas Basin are readily sampled with simple push-cores and have already yielded DNA sequences from many protists. A completely different (and new) approach to protistan sample collection in the deep sea involves deploying traps (either baited or non-baited) and fresh colonization surfaces (glass slides). Traps were deployed widely in 2007 for collection during our 2008 field season.

In addition to submersible-based sample collections carried out in close proximity to the deep-sea hydrothermal vents, we’re also interested in the distribution of protists throughout the water column above the vents, within the buoyant hydrothermal plume (a few tens of meters above the vents), and at control sites away from the immediate influence of hydrothermal vents (a few kilometers off axis). Sampling these sites using traditional oceanographic techniques (CTD/Niskin bottle casts) will help in the determination of the extent to which particular protistan species occur only in the deep-sea vent ecosystems.

Microbial mats and underlying sediments were collected with coring devices at the Guaymas Basin hydrothermal vents (top). Cores were prepared for extrusion and sectioning back on board the R/V Atlantis (left). A piston jack was used to push the sediment core up the length of the tube so that slices of the core could be recovered in 1 cm intervals starting at the top. Preliminary DNA analysis of the complex community living in the warm sediment below the orange bacterial mat indicated a diverse assemblage of ciliates.

In addition to sample collection by the submersible DSV Alvin, we use the CTD (Conductivity, Temperature, Depth) and Niskin bottles deployed from the R/V Atlantis to sample the water column in and around the hydrothermal vent ecosystem. The CTD profiling instruments can be seen below the gray Niskin water sampling bottles at right. Real-time data acquisition by the CTD and its transmission back to the ship permits the collection of discrete water samples from within the plume water above the hydrothermal vents.

A primary sampling strategy that we employed was the deployment of "Protist Traps" or "Protist Colonizers." As you can see in the images at left, the traps consisted of large sponges cut to fit inside plastic tubing with 200 µm plastic mesh fitted over the ends of the tubing to exclude metazoa (A – C).

Glass slide colonizers in conjunction with many of the sponge traps (visible in B & C attached to the side of sponge traps) were deployed at a variety of sites including detrital communities near metazoa (A), on the side of hydrothermal vent chimneys (B), and on freshly colonized basalt (C).

A previous measurement of DNA extracted from baited vs. non-baited traps deployed in an area dominated by decaying Riftia parchment tubes at a depth of 2,512 m near the Tica site indicated twice as much microbial DNA in the baited trap as the non-baited trap after a one-week deployment on the seafloor.

The colony of Riftia tubeworms most likely died off when hydrothermal venting at the location subsided, providing an example of the ephemeral nature of vent communities. The empty and decaying tubes turned out to be good source material for collection of deep-sea ciliates that were living on or within the tubes. The upper temperature limit of protists in deep-sea vent ecosystems remains unknown.