Bidle KN207-03

Ship

Cruise Party

Departure: Jun 15, 2012

Arrival: Jul 14, 2012

Operations Area

Science Objectives

The focus of this proposal is to elucidate the molecular, ecological, and biogeochemical links between vGSLs (and other polar lipids) and the global cycles of carbon and sulfur.  We propose 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, we propose to 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, we 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. Our overarching hypothesis 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.

Science Activities

A key element of the proposed research is to field test the cellular and molecular mechanisms of viral infection in natural haptophyte populations. We propose to conduct observational studies and manipulative experiments on the Northeast Atlantic (NEA) spring bloom. Satellite imagery reveals extensive coccolithophore blooms in surface waters between 50° and 63°N, as well as on the Icelandic Shelf and these blooms are likely infected and terminated by viruses, presenting an excellent venue to test our hypotheses on haptophyte-viral dynamics in surface ocean and linkages to C and S export. We propose a 30 day cruise in June 2012 aboard the R/V Knorr following a transect from Ponta Delgada, Azores to Reykjavik, Iceland. Our goal for this cruise is to transect the region of the NEA spring bloom and to extensively sample the bloom when we encounter it. Our cruise track is modeled after a recent study in this area which documented intense coccolithophore (and other haptophyte) blooms across Rockall Hatton Plateau to the Iceland Basin (55-63°N latitude) and coincided with elevated POC and TEP. For now, we envision sampling 12 water depths at 20 stations. We will also occupy three stations for several days to allow opportunities for extended experiments and sinking particulate carbon collection and flux determination. Given the timing of the bloom is difficult to predict exactly, the precise cruise track will be determined by remote sensing data (satellite and autonomous glider from Rutgers) analyzed by the PIs a few days before and during the cruise.

Our core sampling regime will focus on obtaining samples for identifying and quantifying vGSLs and linking their presence and distribution to the genetic composition of haptophytes and co-occurring viruses. vGSL samples will be analyzed by HPLC/MS as described. The presence and diversity of haptophytes and co-occurring viruses will be assessed by means of SYBR^®Green-based quantitative PCR [qPCR] and amplicon pyrosequencing using genetic markers targeting (a) haptophytes [18S rRNA genes]; (b) closely related E. huxleyi strains [genes encoding a protein with calcium-binding motifs, GPA; and cytochrome oxidase subunit I, COI] along with associated (c) coccolithovirus strains [the virus major capsid protein genes, MCP] and (d) other algal dsDNA viruses of the Phycodnaviridae [DNA polymerase gene, DNA pol (23)]. Quantitative, reverse transcriptase PCR [qRT-PCR] of viral SPT and MCP gene transcripts will be used as an additional measure of viral infection dynamics. Concomitantly, the aforementioned suite of diagnostic cellular markers will assess algal physiology and virus infection.

 We will measure core physical, chemical, biological, and biogeochemical parameters at each station and link them to the aforementioned in situ community composition, physiological state, and viral infection dynamics. These parameters will include primary productivity (via ¹⁴C-HCO₃^- fixation), calcification rates (via ¹⁴C incorporation into CaCO₃), and DMSP turnover rates. Core parameters on water column characteristics will consist of: temperature, salinity, mixed layer and euphotic zone depth, dissolved nutrients (NH₄⁺, NO₃^- + NO₂^-, TDN, PO₄^3-, silicate), bioactive sulfur compounds, DOC, particulate matter (POC, PON, PIC, BSi), pigments (chlorophyll a, 19’Hexanoyloxyfucoxanthin, HPLC accessory pigments), F_v/F_m, phytoplankton composition and abundance (via flow cytometry and microscopy). Given CaCO₃ acts as a ballast mineral and biological pump efficiency is influenced by the packaging of sinking material (e.g. in fecal pellets or as aggregates), we will also deploy drifting PIT trap arrays to determine vertical fluxes of POC, PON, and PIC. While we recognize the quantitative limitations of PIT traps on short (~24 hour) deployment times, we feel they provide an important first-order estimate on vertical C & S flux.

Given that we can’t predict in situ viral infection at our field stations, we will also conduct rigorous experimental tests by performing on-deck, manipulative bottle incubations whereby purified vGSLs, 802 lipids, myriocin, and EhV viral stocks (and combinations thereof) will be added to natural populations and the community response will be compared to unaugmented controls. This will allow for a direct assessment of whether critical determinants of viral infection indeed lubricate biogeochemical cycling of C and S. If our hypotheses are correct, then we would expect vGSLs and EhV amendments to stimulate ROS, caspase activity, DMSO production, TEP production and aggregation, which is ultimately associated with export of POC. We would also expect to see changes in the rates of primary production, calcification, and DMSP turnover. In contrast, 802 and myriocin treatments should counteract the changes in the rates of these processes. Critically, if 802 and myriocin amendments attenuate the production rates of vGSLs in bottles spiked with EhV86, this would definitively pinpoint vGSL and biomarkers for viral infection and provide strong support for Hypothesis 2c.

An important caveat in determining the influence of viral infection to ecosystem dynamics and the C & S cycles is assessing the relative contribution of grazing. As previously mentioned, culture-based experiments have shown that microzooplankton grazing is a significant pathway for DMS release, leading to ~10-fold more DMS production compared to EhV infection alone. Moreover, findings of preferential microzooplankton grazing of virally-infected cells have the potential to link viral and grazing signatures. We will perform dilution-based field experiments (24 h incubations) every 5 days to determine phytoplankton growth and microzooplankton grazing rates using a modified approach to the standard dilution technique. Apparent growth rates and grazing rates of major phytoplankton functional groups at various dilution levels (n =5) will be estimated using specific chemotaxonomic pigment markers. Pigment markers for dinoflagellates, diatoms, cryptophytes, prymnesiophytes, pelagophytes, Synechococcus and Prochlorococcus include peridinin, fucoxanthin, alloxanthin, 19’-hexanoyloxy-fucoxanthin, 19’-butanoyloxyfucoxanthin, zeaxanthin and divinyl Chl a, respectively. For comparison, growth rates of some phytoplankton classes (e.g. Synechococcus, Prochlorococcus) will also be determined directly by changes in cell numbers using flow cytometry (FCM). An advantage of using this new dilution approach is the correction in estimating phytoplankton growth rates due to photoadaptive changes in pigment concentrations (that occur during short on-deck incubations) by utilizing FCM-detected changes in red fluorescence per cell. These changes in cell fluorescence can lead to a significant underestimation in phytoplankton growth rates. Estimates of the nutrient limitation index (N_L) will also be determined by including both nutrient amended and unamended treatments. This protocol will not only allow us to determine the proportion of primary production consumed daily by microzooplankton grazing  but also to estimate the nutritional state of the phytoplankton populations. The N_L index will be compared with the physiological status of the population as estimated by the photosynthetic efficiency of PSII (i.e. F_v/F_m). Estimates of ¹⁴C -fixation utilizing standard P vs E incubations as well as Chl a synthesis rates (from dilution experiments) will allow us to estimate C/Chl ratios.  We will also estimate taxon specific C/Chl, pigment/cell and DMSO/DMSP cell^-1 ratios to reveal the effects of nutrient limitation and grazing rates.

We also acknowledge the potential influence of grazing on EhV-infected cells may have on the interpretation of our particle flux results. To assess this, we will examine the molecular composition and vGSL content of sinking fecal pellets for comparison to bulk sinking particulate matter. Sinking particles will be collected using drifting sediment net traps. These traps are distinct from PIT traps in that they are very large (1.25 m in diameter) and allow us to obtain large amounts of sinking matter for chemical analyses, including DMS/DMSO, vGSLs, 802, alkenones, TEP, pigments and viral/host genetic markers. Since net traps are not thought to yield quantitative sinking fluxes, we will measure POC, PON and pDMSO in material recovered from net traps for comparison to PIT traps, which will provide a first-order comparison between molecular composition (from net traps) and fluxes (from PIT traps). If elevated vGSLs and EhVs markers are detected in trapped particles, we want to ascertain if they were possibly delivered to depth in fecal pellets from zooplankton that had grazed on infected cells. We will address this possibility through microscopic micromanipulation of fecal pellets and subsequent analysis of vGSLs and qPCR of the aforementioned haptophyte, E. huxleyi, Phycodnaviridae, and EhV molecular markers. This will tell us if the detected vGSLs in sediment traps derived from grazing flux. 

Additional Info

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Supporting documentation:

Shipboard Equipment

Shipboard Communication

CTD/Water Sampling

Critical CTD Sensors

MET Sensors

Sample Storage

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Standard Oceanographic Cables

Portable Vans

Checklist

Checklist