Research:

Project OViD: The sampling details

Viral sampling methods:

At each of the 550 sampling sites, between 1 and 9 depths are sampled (varies per expedition and per ocean features). Duplicate large-volume viral fractions (<0.22µm filtrate, paired with the microbial fraction) are concentrated using our new highly efficient viral concentration method [1], and archived according to standard procedures [2-4]. A portion of each viral concentrate will be used to extract DNA, while the remainder will remain archived for future use. Additionally, replicate non-concentrated filtrates are collected for isolations [5], viral counts [6], and electron microscopy morphological analysis [7, 8].

A wealth of context: environmental metadata and exceptional parallel scientific characterization

Environmental metadata from both cruises: Each sample on both the Tara Oceans and Malaspina cruises has extensive metadata characterizing its physics, chemistry and biology. Most notable for our work are three substantive biological characterizations done by community members:

  1. community viral genetic diversity (by RAPD-PCR),
  2. microbial community characterization (microbial diversity by 16S pyrotags; flow cytometry counts for microbes, Prochlorococcus, Synechococcus, and picoeukaryotes and nanoflagellates), and
  3. community gene expression on select samples (based on diversity data) will be investigated for protistan (Tara Oceans) and microbial (both expeditions) gene expression, through metatranscriptomic sequencing (sensu [9]).

In addition, chemical and physical metadata will be collected at each station (0 - 4,000m depth profiles for Malaspina vs select depths on Tara Oceans as detailed). From 0-2000m, Tara will sample temperature, salinity, O2, particulate organic and inorganic carbon, dissolved inorganic carbon, chlorophyll a (vertical profile), photosynthetic pigments (by HPLC), NO2, NO3 (chemically), high-resolution NO3 (by ISUS sensor), PO4, and SiO2. From 0-500m, Tara will also measure and additional properties of organic matter, including particulate matter spectral absorption coefficient, retrodiffusion coefficient, colored dissolved organic matter (CDOM, by UV attenuation coefficient), as well as chlorophyll a (by fluorescence). Autonomous PROBIO profiler floats will also remain deployed at each station after Tara moves on, and will measure temperature, turbidity, salinity, pH, pCO2, luminescence and fluorescence, as well as vertical particle flux and size class distribution using sediment traps.

Tara Oceans specific:

Community biodiversity: Beyond microbial diversity, the Tara consortium will document genetic diversity for eukaryotes (4 size fractions, Colomban de Vargas), and “giruses” (Giant viruses, Hiroyuki Ogata), and the overall viral community (RAPD-PCR, Weinbauer). These genetic data are coupled to exceptional documentation of the morphological diversity of microbes, protists, and plankton, through a series of cutting-edge imaging approaches (details below) to produce a catalog of morphotype images. Teams of experts will help identify and curate the imaging database, which will then be used for sampling image auto-annotation, and be made publicly available. Relevant to our proposed work, these genetic and morphological diversity characterizations provide rich information about higher trophic-level structure, which may be related back to viral diversity.

At each sampling location, plankton, protists, microbes and viruses will be collected from the surface and deep chlorophyll maximum, and examined using a variety of methods. Plankton identification will be accomplished by standard microscopy methods complemented by an Underwater Video Profiler (1mm- to 10cm-scale quantitative measurements), an on-board Cytopeia Influx flow-cytometer (underway monitoring and sorting of microbes and phytoplankton), a binocular fluorescent microscope (for high-throughput screening), and EMBL’s Single Plane Illumination Microscope (for µm- to mm-scale 3D and fluorescence imaging of specimens). Identifications will be performed by a variety of experts and in collaboration with the Scientific Committee on Ocean Research (SCOR, http://www.scor-int.org/) and the Census of Marine Life (COML, http://www.coml.org/).

Malaspina specific:

Expanded depth sampling and process measurements: Malaspina will provide greater coverage of the deep oceans, while also conducting process measurements including bacterial production, nitrogen fixation and grazing rates, and enzyme activities.

Expanded Nutrient measurements: Urea: FIA on board. C, N, O, and S isotopic ratios: mass spectrometry connected to either a TOC or an elemental analyzer, upon return. DMS: gas chromatography on board.

Vertical diffusive fluxes of nutrients: calculated from concentration gradients and ADCP and CTD data; isoprene and CH3I:GC and mass spectrometry on board. Deep ocean DOM and POM sampling: aboard ultrafiltration and in situ pump filtration, respectively.

References cited:

  1. John, S.G., et al., A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environmental Microbiology, 2010. in press.
  2. Suttle, C.A. and A.M. Chan, Marine cyanophages infecting oceanic and coastal strains of Synechococcus: abundance, morphology, cross-infectivity and growth characteristics. Marine Ecological Progress Series, 1993. 92: p. 99-109.
  3. Waterbury, J.B. and F.W. Valois, Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophage abundant in seawater. Applied and Environmental Microbiology, 1993. 59(10): p. 3393-3399.
  4. Wilson, W.H., et al., Isolation and molecular characterization of five marine cyanophages propogated on Synechococcus sp. strain WH 7803. Applied Environmental Microbiology, 1993. 59: p. 3736-3743.
  5. DeLong, E.F., Archaea in coastal marine environments. Proceedings of the National Academy of Sciences, USA, 1992. 89(12): p. 5685-9.
  6. Noble, R.T. and J.A. Fuhrman, Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquatic Microbiology and Ecology, 1998. 14: p. 113-118.
  7. Suttle, C.A., Marine viruses - major players in the global ecosystem. Nature Reviews Microbiology, 2007. 5: p. 801-812.
  8. Laybourn-Parry J., H.J.S., Sommaruga R. , Viruses in the plankton of freshwater and saline Antarctic lakes. Freshwater Biology, 2001. 46: p. 1279-1287.
  9. Frias-Lopez, J., et al., Microbial community gene expression in ocean surface waters. Proceedings of the National Academy of Sciences, USA, 2008. 105(10): p. 3805-10.