Collaborative Lubrication of oceanic carbon and sulfur biogeochemistry by viral glycosphingolipids and a host-virus chemical arms race

Ship

R/V Knorr

Vehicles

Cruise Party

Kay Bidle: Principal Investigator, Chief Scientist
Rutgers University, Institute of Marine and Costal Sciences 71 Dudley Road New Brunswick, NJ United States of America 08901
+1 848 932 3467
bidle@marine.rutgers.edu

Marco Coolen: Principal Investigator
Woods Hole Oceanographic Institution Fye 120A, MS#04 Woods Hole, Ma. USA 02543
+1 508 289 2931
mcoolen@whoi.edu

Giacomo DiTullio: Principal Investigator
College of Charleston 31 Fort Johnson Charleston, SC USA 29412
+1 843 953 9196
DitullioJ@cofc.edu

Benjamin Van Mooy: Principal Investigator, Chief Scientist
Woods Hole Oceanographic Institution Fye 119, MS#04 Woods Hole, Ma. USA 02543
+1 508 289 2322
bvanmooy@whoi.edu

Assaf Vardi: Principal Investigator
Weizman Institute of Science Rehovot, Israel 76100
+1 9728 934 2914
assaf.vardi@weizmann.ac.il

Departure: Ponta Delgada, Azores on Jun 15, 2012

Arrival: Reykjavik, Iceland on Jul 14, 2012

Mobilization Date: Jun 13, 2012

Demobilization Date: Jul 16, 2012

Supporting documentation:

»2012NorthAtlantic_ProposedTransectStations.pdf

Operations Area: North Atlantic (transect from Ponta Delgada, Azores to Reykjavik, Iceland

Lat/Lon: 37° 0.0′ N / 26° 0.0′ W

Depth Range: surface / 200 m

Will the vessel be operating within 200 NM of a foreign country? Azores (Portugal), Iceland, possibly Greenland (Denmark)
Are visas or special travel documents required? no

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. 

Pre-cruise planning meeting: Teleconference/Visit WHOI

I think that both a teleconference and visit to WHOI would be useful for the cruise planning.
Media personnel on board: Video
A key facet of our fieldwork in the Northeast Atlantic (NEA) will be to post a web/video blog as part of IMCS/Rutgers' successful program (http://marine.rutgers.edu/main/blog/) to bring scientists at sea to classroom students and the general public. Given the R/V Knorr is fully networked, we will plan to post blog content directly from the ship. WHOI has budgeted resources to bring a freelance videographer on the NEA cruise to gather multimedia content including video, photos, and audio, which will be used to create post-cruise deliverables, such as podcasts, an audio slideshow, images of the day, and photo essays. The WHOI Communications group will provide editorial guidance and production expertise at its own cost. Targeted outlets will be the WHOI and IMCS websites, Oceanus magazine, NSF 360, and Google Earth. We have also budgeted resources to work with Dr. Ari Daniel Shapiro in the production of 'Ocean Gazing'€™ podcasts through COSEE NOW http://coseenow.net/category/ocean/) in association with our NEA cruise work. Shapiro received his PhD in biological oceanography from the MIT/WHOI Joint Program in 2008 and is currently an independent science radio and multimedia producer. His pieces have appeared on NPR, Radio Lab and The World. The ‘Ocean Gazing’ podcasts allow the public to look at, listen to and touch the ocean by presenting oceanographic research and interviewing oceanographers

Stations:

Funding Agency: NSF #OCE-1061883

- added NSF #OCE-1061883 on Dec 13, 2011 11:57 PM by Dr. Kay D Bidle

R/V Knorr

Shipboard Equipment

Deionized Water System
Science Underway Seawater System
Fume Hood

Shipboard Communication

Is there a need to receive data from shore on a regular basis?
Is there an expectation to use Skype or any other real-time video conference program?
Basic Internet access via HiSeasNet

CTD/Water Sampling

Wet Labs ECO-AFL fluorometer
Wet Labs C*Star transmissometer (660nm wavelength)
Seapoint STM turbidity sensor
SBE43 oxygen sensor
911+ Rosette 24-position, 10-liter bottle Rosette with dual T/C sensors
Biospherical underwater PAR (1000m depth limit) with reference Surface PAR

Critical CTD Sensors: 

MET Sensors

Barometric Pressure
Air temperature
Precipitation
Relative Humidity
Wind speed and direction
Short Wave Solar Radiation

Sample Storage

Freezer -70°C 25 cu. ft.
Freezer -20°C
Freezer -70°C 3.2 cu. ft. ea.
Refrigerator 8.6 cu. ft.


Storage Notes:

Navigation

Will you be using Long Base Line (LBL) navigation? no

Will you be using Doppler/GPS navigation? no

Navigation

GPS

Navigation Notes:


Winch Notes:



Wire Notes:

Slip ring required? no	Number of conductors:
Non-standard wire required? no	Type:
Traction winch required? no	Describe:

Portable Vans

Isotope Van
Hydrographic Van
Chemical Storage Van

Other Science Vans:

Other Science Vans:

Science Van 1
Type/size: standard/20 ft		Location: main deck
Water: no		Power:no
Science Van 2
Type/size: Standard/20 ft		Location: main deck
Water: yes		Power:yes
Science Van 3
Type/size: Standard/20 ft		Location: main deck
Water: yes		Power:yes

Specialized Deck Equipment

Mooring Deployment/Recovery Equipment Required: no	Type:
Cruise Specific Science Winch Required: no	Type:
Nets Required: no	Type:

Over the Side Equipment

Will you be bringing any equipment (winches, blocks, etc.) that lowers instruments over the side? yes

Details: We intend to deploy a glider and a couple of profiling floats to better characterize the in situ physical and optical conditions of blooms when they are encountered

Special Requirements

Elecrical Power: no	Identify:
Equipment Handling: yes	Identify: glider deployment, profiling float deployment
Inter/intraship Communications: no	Identify:
Science Stowage: no	Identify:
Water: yes	Identify: MilliQ or Distilled

Additional Cruise Items/Activities

Explosive Devices: no Portable Air Compressors: no Flammable Gases: no

Small Boat Operations: no SCUBA Diving Operations: no

Hazardous Material

Will hazardous material be utilized? yes

Describe deployment method and quantity:

Radioactive Material

Radioiosotopes: yes

Additional Information

Is night time work anticipated on this cruise? yes

Specialized tech support (Seabeam, coring, other): We will be deploying a glider and a couple of profiling floats with optical sensors on them. It will be good to have some tech support for these.

Other required equipment and special needs: 
Date Submitted: Dec 16, 2011 12:47 PM by Dr. Kay D Bidle