Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

We sequenced the 16S V4 hypervariable region of deep-water samples collected with a Suspended Particulate Rosette Sampler (SuPR) (Mclane Research Laboratories, Inc. Falmouth, MA USA) or Universal Fluid Obtainer (UFO) (National Deep Submergence Facility, Falmouth, MA, USA) using the ROV Jason. At each vent field 15.71–47.51 L of water were pumped through 2–4 0.22 µM Express Plus Membrane filters (MilliporeSigma, MA, USA) both within close proximity to hydrothermal vent animal communities (~10-20 cm above animal assemblages) and away from hydrothermal activity (~10s to 100s of meters distance). A 10 µM custom nylon mesh pre-filter (Sefar Inc., NY, USA) was assembled in front of each sample filter to prevent collection of microbes associated with shed animal cells. Because the SuPR device failed during deployment at Tu’i Malila and during one dive at each ABE and Kilo Moana, 10.5 L deep water samples were additionally taken with the UFO at these localities. To account for cross-contamination using this device, empty filters were deployed but not pumped and used as negative controls. On board ship, filters were preserved in RNALater™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and frozen at –80°C. DNA from filter samples was extracted with a phenol:chloroform protocol. The 3 Strain Tagged Genomic DNA Even Mix (ATCC, VA, USA) was added as spike-in control and dilutions of the spike-in without DNA extract were prepared to assess cross-contamination during sample processing in the laboratory. 

For the collection of microbial biofilms on various mineral and other substrates, we used weighted PVC bait cages containing vertically held open-ended 2-mL polypropylene tubes packed with crushed mineral substrates (andesite, basalt, glass beads or Alviniconcha shells) and contained by window screen mesh on either end for microbial colonization. Three of these cages were deployed during the first dive at each vent field and recovered approximately two weeks later using the ROV Jason. On board ship, water and biofilm samples were preserved in RNALater™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and frozen at –80°C. DNA from substrate biofilms was extracted with the E.Z.N.A. Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA), including an additional 45 second bead beating step prior to proteinase K digestion.

Samples were sequenced at the Argonne National Laboratory (Lemont, IL, USA) using amplicon library preparation with the 515F/806R primer pairs (Apprill et al. 2015; Parada et al. 2016). All libraries were 2x150 bp paired-end sequenced on a Illumina NextSeq2000 instrument.

- Imported "TN401_16S_Sequencing_NCBI.xlsx" into BCO-DMO system
- Split lat and lon into two fields, with negative and positive decimal degrees
- Removed period from parameter name
- Exported file as "964227_v1_tn401_16s_sequencing_ncbi.csv"

NSF Abstract:

Symbiosis with microbes is ubiquitous and critical to fundamental biological functions such as development and nutrition. Thus, the success of a host animal may depend on its ability to find and associate with its microbial partner(s). While some hosts directly transmit their symbionts from parent to offspring in order to guarantee this, acquisition of microbial symbionts from the environment is vital for the survival of many obligately-symbiotic animals. An understanding of the free-living symbiont population and how the host acquires those symbionts is fundamental to our comprehension of ecological processes in all ecosystems, yet almost nothing is known about either. Hydrothermal vent ecosystems provide important opportunities to investigate the role of microbial symbionts in host-, community-, and ecosystem-level ecology, since these ecosystems are dominated by animals whose survival is clearly linked to the acquisition of one or a few specific symbionts. This project begins to fill a gap in our understanding of the factors driving community structure at hydrothermal vents by addressing the potential for free-living symbiont populations to affect host animal establishment, while also expanding our general knowledge regarding the impact of host-associated microbes on fundamental ecological processes that apply across ecosystems. The results of this project will be shared via educational videos and live-broadcasts to the Smithsonian Institution's National Museum of Natural History and University-run museums. The investigators will also design and implement an educational program about symbiosis and hydrothermal vent biology suitable for middle and high school classes. Finally, the investigators will train a diverse group of undergraduate and graduate students in both research and the development of science educational programs.

This project will focus on two sister genera of snails, Alviniconcha and Ifremeria, which predominate at vents in the southwestern Pacific. At vents in the Lau Basin (Tonga), three species of Alviniconcha and one species of Ifremeria coexist. These four species all host distinct lineages of chemoautotrophic proteobacteria in their gill tissue as adults that provide the bulk of their nutrition. Previous work in this region showed a structured snail species distribution that corresponds to the concentrations of key chemical substrates for symbiont chemoautotrophic metabolism, suggesting that snail species are sorting into geochemical habitats based on symbiont physiology. It is not clear if this sorting is occurring among established snail-bacteria symbioses, or whether environmental effects on the availability of specific symbionts are influencing the recruitment of host species, since arriving and developing snail larvae must obtain their symbionts from the environment. This study aims to 1) assess the larval supply and population structure of symbiotic vent snails via collections of larval, juvenile, and adult snails, 2) investigate the developmental timing of symbiont acquisition through microscopy and marker gene sequencing of gametes, larvae, and juveniles, and 3) use metagenomic sequencing to quantify the availability of free-living symbionts in the environment to arriving larvae. Altogether, this series of interlinked efforts will allow for an improved understanding of free-living bacterial symbiont populations, the timing of symbiont acquisition, and host snail life history, as well as how these things interact to shape vent communities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.