Table of Contents

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

Note: The detailed protocols are described in Thomy et al., 2024

For eukaryote isolation, seawater sampling was carried out on March 02, 2011 from the oligotrophic open-ocean site at Station ALOHA, in the North Pacific Subtropical Gyre at a depth of 45 meters. Seawater samples were enriched with Keller (K) medium and unialgal cultures were then isolated by serial dilution to extinction. Florenciella sp. strain UHM3020 was further identified by small subunit ribosomal RNA gene (18S rRNA gene) sequencing. DNA was extracted from the pellets using the MasterPure Complete DNA and RNA Purification Kit (Epicentre). Florenciella 18S rRNA was amplified by PCR then cloned and extracted using the Zyppy Plasmid Miniprep Kit (Zymo Research). Near-full-length 18S rRNA gene for Florenciella was sequenced using Sanger method.

For virus isolation, seawater sampling was carried out on September 15, 2014 from the same site as described previously for the host isolation at a depth of 25 meters. Forty liters of seawater was filtered through 0.8 μm pore size filters to remove larger cells while minimizing losses of large viruses. Viral particles in the filtrate were concentrated by tangential flow filtration (TFF; 30 kDA molecular weight cut-off). The concentrate was amended with nutrients to match K medium and then used to challenge a culture of a healthy Florenciella culture isolated previously from the same water. Viruses in the filtrate were concentrated by TFF (30 kDa) to 300 mL volume, further concentrated to 0.5 mL by centrifugal ultrafiltration (30 kDa) and then purified in a CsCl buoyant density gradient. DNA was extracted from the virus peak in the gradient using Masterpure Complete DNA and RNA Purification Kit (LGC Biosearch Technologies). DNA was sequenced using Illumina (NextSeq System) and PacBio methods.

The FloV-SA2 complete genome sequence was deposited in GenBank with accession number PP542043 (see related datasets) as well as the ubiquitin-60S ribosomal protein eL40 gene sequence encoded in the Florenciella sp. host genome PP665604 (see related datasets). The gene annotations were published as Supplementary Table 1 in Thomy et al., 2024.

The FloV-SA2 genome was assembled from PacBio sequencing reads using Canu v1.0 and polished using a combination of pbalign v0.2.0.141024 and Quiver v2.0.0.  

Initial gene prediction was conducted with Prokka v1.14.5. Functional annotations  were performed using a BLASTp search using Diamond (v2.1.4) against: 

*NCBI Refseq databases (O’Leary et al., 2015)

*InterProScan (Jones et al., 2014)

-imported "Data-samples-info-V2-2024-10-15.xlsx" into the BCO-DMO data system
-split column containing lat and lon
-converted lon to negative to represent decimal degrees
-converted date to YYYY-mm-dd
-renamed fields to conform with BCO-DMO naming conventions
-exported file as "949101_v1_eukaryotic_viruses_flov-sa2.csv"

NSF Award Abstract:
Viruses can infect all forms of life. Viruses are highly diverse, and one aspect of diversity is size: genomes of viruses vary more than a thousandfold in length, and the size of viral particles varies nearly a millionfold. The discovery of “giant” viruses was astounding because they can be physically larger and code for more genes than many free-living microorganisms. There is growing evidence that giant viruses are widespread and diverse in the ocean, but much about their ecology remains unknown. What critical ecological tradeoffs vary with virus size, allowing small and large viruses to coexist? Do these tradeoffs cause the distribution of virus sizes to vary across habitats? This project aims to answer these questions for viruses that infect phytoplankton, the microscopic plants that are the foundation of ocean productivity. This research can also influence a diverse array of scientific fields because virus size varies greatly in other ecosystems and host-associated microbiomes. The fundamental constraints on size may be broadly similar across systems, but the processes driving virus size have not been thoroughly investigated in any of them. This project supports the training of a postdoctoral researcher, two graduate students, and undergraduate students in integrative science that includes field, laboratory, and modeling components. National Science Foundation-supported Research Experience for Undergraduates and Tribal Colleges and Universities programs at UH Manoa that serve Pacific Islanders and other underrepresented groups are used for recruiting students. In addition, science outreach at public events in Hawai’i includes an interactive game to communicate ideas about giant viruses and their role in the ocean.

Large viruses may have four advantages over smaller viruses: i) ability to infect a greater diversity of host genotypes, ii) better control of host metabolism, iii) large enough size to enter host cells by ingestion, and iv) greater persistence in the extracellular environment. These advantages may compensate for the advantages held by smaller viruses: higher contact rates with their hosts and greater offspring number per infection. The advantages of large size may be more consequential in oligotrophic habitats, where the microbial eukaryote community is primarily small phagotrophic flagellates (mixotrophs and heterotrophs), at low population densities, with resource-limited growth. The project goals are: (1) To test whether giant viruses indeed dominate in the oligotrophic ocean compared to a productive coastal location, as suggested by initial observations of this research team; (2) To test the above four hypotheses about the advantages of large size by conducting laboratory experiments with diverse viral isolates, and (3) To use an eco-evolutionary model of eukaryotic microbes and their viruses to explain observed size patterns.

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.