Intellectual Merit: Despite the critical importance of viruses in shaping marine microbial ecosystems, very little is known about the molecular mechanisms mediating phytoplankton-virus interactions. As a consequence, we currently lack biomarkers to quantify active viral infection in the oceans, significantly hindering our understanding of its ecological and biogeochemical impacts. The coccolithophore Emiliania huxleyi (Prymnesiophyceae, Haptophyte) is a cosmopolitan unicellular photoautotroph whose calcite skeletons account for about a third of the total marine CaCO3 production. E. huxleyi forms massive annual spring blooms in the North Atlantic that are infected and terminated by lytic, giant double-stranded DNA containing coccolithoviruses. Findings that lytic viral infection of E. huxleyi recruits the hosts programmed cell death (PCD) machinery demonstrate that viruses employ a sophisticated, co-evolutionary "arms race" in mediating host-virus interactions. The investigators recently demonstrated that viral glycosphingolipids (vGSLs), derived from unexpected cluster of sphingolipid biosynthetic genes, a pathway never before described in a viral genome, play a crucial functional role in facilitating infection of E. huxleyi. The observations of vGSLs in the North Atlantic and Norwegian fjords further suggest that they may be novel, diagnostic biomarkers for viral infection of coccolithophore populations. At the same time, the discovery of vGSLs and a distinct, protective 802 lipid argues that a host-virus, co-evolutionary chemical arms race plays a pivotal role in regulating viral infection and in lubricating upper ocean biogeochemical fluxes of Carbon and Sulfur.
The focus of this project is to elucidate the molecular, ecological, and biogeochemical links between vGSLs (and other polar lipids) and the global cycles of carbon and sulfur. The team of investigators proposes a multi-pronged approach combing a suite of lab-based, mechanistic studies using several haptophyte-virus model systems along with observational studies and manipulative field-based experiments the Northeast Atlantic. Using these diagnostic markers, they will document active viral infection of natural coccolithophore populations and couple it with a suite of oceanographic measurements in order to quantify how viral infection (via vGSLs) influences cell fate, the dissolved organic carbon (DOC) pool, vertical export of particular organic (POC) and inorganic carbon (PIC; as calcium carbonate, CaCO3) (along with associated alkenone lipid biomarkers and genetic signatures of viruses and their hosts) and the upper ocean sulfur cycle (via the cycling of dimethylsulfide [DMS] and other biogenic sulfur compounds). Furthermore, given they are unique to viruses, the investigators propose that vGSLs can be used to trace the flow of virally-derived carbon and provide quantitative insights into a "viral shunt" that diverts fixed carbon from higher trophic levels and the deep sea. The overarching hypothesis for this study is that vGSLs are cornerstone molecules in the upper ocean, which facilitate viral infection on massive scales and thereby mechanistically "lubricate" the biogeochemical fluxes of C and S in the ocean.
Broader Impact: This research blends concepts in physiology, molecular biology, biochemistry, viriology and lipid chemistry, with oceanography and biogeochemistry, thereby providing an opportunity whereby researchers with different educational backgrounds can interact and develop. This project provides excellent hands-on training for development of postdocs, graduate students and undergraduate students. The research provides resources and opportunities for inter-institutional exchange Rutgers-WHOI-College of Charleston and builds both on established national and international collaborations and will foster new ones. The PIs will work with COSEE NOW and Networked Ocean World to increase ocean literacy by integrating scientific research with K-12 educators and public audiences. An important component of this project is to bring scientists at sea in touch with classroom students and the general public. As such, the project incorporates several concrete strategies, including: (1) posting web/video blogs from sea; (2) incorporating a freelance videographer to collect multimedia content on a cruise to the Northeast Atlantic, which will be used in diverse post-cruise deliverables; (3) producing "Ocean Gazing" podcasts so the general public can look at, listen to and touch the ocean and unpack some of its secrets by presenting ongoing oceanographic research and interviewing oceanographers; and (4) integrating the research activities with ongoing K-12 teacher workshops as part of the Marine Activities, Resources and Education program and through interactions with Laura Dunbar, a Science/Technology teacher at Sea Girt Elementary School (Sea Girt, NJ).
Intellectual Merit. The coccolithophore Emiliania huxleyi is a unicellular phytoplankton that forms massive blooms in the global ocean that span up to 100,000 km2 and can be observed with Earth-observing satellites. As a photosynthetic organism that uses sunlight to convert CO2 into biomass, it plays a key role in Earth’s carbon cycle. This impact on the carbon cycle is two fold given its ability to also biomineralize calcium carbonate into calcite cell walls (coccoliths). In the oceans, E. huxleyi cells also face a challenge, an ‘arms race’ of sorts, in that they are routinely infected, lysed, and terminated by specific, double-stranded DNA containing viruses called Coccolithoviruses or EhVs (Figure 1). This arms race is a natural aspect of the ecology of all marine microbes, as viruses are the most abundance entities in the oceans (averaging 10 million viruses per milliliter of seawater) and have an ancient evolutionary history. As members of the Phycodnaviridae, EhVs are giant microalgal viruses (~180 nm in diameter) with an extensive genetic capability (~407 kb genomes) to manipulate host metabolic pathways for their replication. Owing to the collective insight gained from genomics (both host and virus genomes have been sequenced) and the array of genetically diverse host (sensitive and resistant) and virus strains in culture, the E. huxleyi–EhV host-virus is one of the best model systems for investigating algal host-virus interactions and the cellular processes that mediate infection dynamics. This project uncovered that coccolithoviruses employ a sophisticated, coevolutionary arms race to rewire and manipulate host lipid metabolism, altering a specific class of lipids known as glycosphingolipids (GSL), and in turn, regulate cell fate by inducing reactive oxygen species and key proteolytic enzymes (caspases and metacaspases) that trigger host programmed cell death (Figures 1-2). These unique GSL lipid molecules are central to successful infection E. huxleyi and its EhV viruses, and given their unique chemical signature and bioactivity, can be used as novel diagnostic biomarkers to detect the extent of virus infection in the oceans—an ability that microbial oceanographers have lacked until now. This project elucidated the molecular, ecological, and biogeochemical links between GSLs, EhV infection of E. huxleyi, and the global cycles of carbon and sulfur (E. huxleyi also produces dimethyl sulfide, an important sulfur-based gas that can moderate Earth’s climate). Our work combined a suite of lab-based, mechanistic studies using E. huxleyi-virus model systems along with observational studies and manipulative field-based experiments in Norwegian fjords and the Northeast Atlantic. Using a suite of different GSLs and other cellular targets as diagnostic markers, we documented active viral infection of different natural E. huxleyi populations and coupled it with a suite of oceanographic measurements in order to quantify how viral infection (via GSLs) influences cell fate, the dissolved organic carbon (DOC) pool, sinking and vertical export of particular organic (POC) and inorganic carbon (PIC; as calcium carbonate, CaCO3) and the upper ocean sulfur cycle (via the cycling of DMS and other biogenic sulfur compounds. Our work showed that vGSLs are cornerstone molecules in the upper ocean, which facilitate viral infection on massive scales and mechanistically ‘lubricate’ the biogeochemical fluxes of C and S in the ocean (Figure 3). A key finding from our work was that active EhV infection (and the cellular pathways induced by it) play key role in the formation of aggregates and the sinking flux of organic matter into the deep ocean (Figure 4). This is counter to the traditional paradigm invoking cell lysis and the retention of this carbon in the upper ocean, fueling the ‘microbial respiration’. This has important implications for the sequestration of carbon into the deep ocean. Broader Impact: Research blended concepts in physiology, molecular biology, biochemistry, virology and lipid chemistry, with oceanography and biogeochemistry, thereby providing an opportunity whereby researchers with different educational backgrounds can interact and develop. This project provided unique hands-on training for development of postdocs, graduate students and undergraduate students. Research provided resources and opportunities for inter-institutional exchange between Rutgers-WHOI-College of Charleston and built both on established national and international collaborations and fostered new ones with several different universities. The PIs worked closely with the education and outreach teams at Rutgers and WHOI to increase ocean literacy by integrating scientific research with K-12 educators and public audiences. Activities brought scientists at sea in touch with classroom students and the general public using several concrete strategies, including: (1) posting web/video blogs from sea; (2) integrating research activities with ongoing K-12 teacher workshops; and (3) the development of a novel video series on how science works, specifically how scientists use ‘Collaborations’ and ‘Sampling & Proxies’, to highlight scientific practices through real research investigations during the NA-VICE research expedition in the North Atlantic in 2012. The videos will be open access and are designed for incorporation into middle and high schools, as well as undergraduate classes.
