DOI: 10.5061/dryad.q573n5tfr
title: AQUACOSM VIMS-Ehux – Core data

description: The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular alga
 that forms massive oceanic blooms covering thousands of square kilometers
 (Tyrrell & Merico 2004). The intricate calcite exoskeleton of E.
 huxleyi accounts for ~1/3 of total marine CaCO3 production (Monteiro et
 al. 2016). E. huxleyi blooms are an important source of DMS, which is, by
 far, the most abundant volatile sulfur compound in the surface ocean and
 the best studied aerosol precursor (Simó 2001) with a significant
 climate-regulating role that enhances cloud formation (Alcolombri et al.
 2015; Simó 2001). Biotic interactions that regulate the fate of these
 blooms play a profound role in determining carbon and nutrient cycling in
 the ocean and feedback to the atmosphere. Annual E. huxleyi spring blooms
 are frequently terminated following infection by a specific large dsDNA
 virus (EhV) that belongs to the Coccolithovirus group (Schroeder et al.
 2002). Despite the huge ecological importance of host-virus interactions,
 the ability to assess their ecological impact is limited to questions that
 focus mainly on quantification of viral abundance and diversity in a
 reductionist manner. The project in which this dataset was collected is a
 holistic approach to untangle the complexity in alga-virus-bacterium
 interactions during an E. huxleyi bloom, their effect on the metabolome of
 the phycosphere, and their possible implications to C and S cycles. The
 project took place for 24 days, including daily sampling for various
 biological and physiochemical parameters. Flow cytometry was used to
 monitor different populations of phytoplankton, bacteria and virus-like
 particles (VLP). Additionally, physiochemical properties of the water such
 as salinity, temperature and nutrient concentrations were acquired, as
 well as viral abundances estimated by qPCR. These data compose the
 contextual data for various scientific papers.
description: <strong><em>Mesocosm
 setup</em></strong> The mesocosm experiment
 AQUACOSM VIMS-Ehux was carried out for 24 days between 24th May (day 0)
 and 16th June (day 23) 2018 in Raunefjorden at the University of Bergen’s
 Marine Biological Station Espegrend, Norway (60°16′11N; 5°13′07E). The
 experiment consisted of seven enclosure bags made of transparent
 polyethylene (11 m<sup>3</sup>, 4 m deep and 2 m wide, 90%
 photosynthetically active radiation) mounted on floating frames and moored
 to a raft in the middle of the fjord. The bags were filled with
 surrounding fjord water (day -1; pumped from 5 m depth) and continuously
 mixed by aeration (from day 0 onwards). Each bag was supplemented with
 nutrients at a nitrogen to phosphorous ratio of 16:1 (1.6 µM
 NaNO<sub>3</sub> and 0.1 µM
 KH<sub>2</sub>PO<sub>4 </sub>final concentration)
 on days 0-5 and 14-17, whereas on days 6, 7 and 13 only nitrogen was
 added. <em><strong>Sampling for flow
 cytometry analysis</strong></em> Samples
 for flow cytometric counts were collected twice a day, in the morning
 (07:00 AM) and evening (08:00-09:00 PM) from each bag and the surrounding
 fjord, which served as an environmental reference. Water samples were
 collected in 50 mL centrifugal tubes from 1 m depth, pre-filtered using 40
 µm cell strainers, and immediately analysed with an Eclipse iCyt (Sony
 Biotechology, Champaign, IL, USA) flow cytometer. A total volume of 300 µL
 with a flow rate of 150 µL/min was analyzed. A threshold was applied based
 on the forward scatter signal to reduce the background noise.
 <em><strong>Enumeration of phytoplankton cells by
 flow cytometry</strong></em> Phytoplankton
 populations were identified by plotting the autofluorescence of
 chlorophyll versus phycoerythrin and side scatter: calcified <em>E.
 huxleyi</em> (high side scatter), <em>Synechococcus</em>
 (high phycoerythrin), nano- and picophytoplankton (high and low
 chlorophyll, respectively). 
 PHYTOPLANKTON GATES Criteria 1 Criteria 2 Group Low Chlorophyll (Low FL4) High phycorerythrin (High FL3 Low FSC) Syn = Synechococcus Low phycorerythrin (Low FL3) Pico-Euks = Pico-eukaryotes High Chlorophyll (High FL4) High Side Scatter Neuks_HighSS = Calcified Ehux Low Side Scatter Neuks_LowSS = Diatoms, Dinoflagellates, or uncalcified Ehux Chlorophyll fluorescence was detected by FL4 (excitation (ex): 488nm and emission (em): 663-737 nm). Phycoerythrin was detected by FL3 (excitation (ex): 488 nm and emission (em): 570-620 nm). Raw .fcs files were extracted and analyzed in R using ‘flowCore’ and ‘ggcyto’ packages. <em><strong>Enumeration of large virus-like particles and bacteria by flow cytometry</strong></em> For extracellular VLP counts, 200 µL of sample were fixed with 4 µL glutaraldehyde 20% (final concentration of 0.5%) for one hour at 4°C and flash frozen. They were thawed and stained with SYBR gold (Invitrogen) that was diluted 1:10,000 in Tris-EDTA buffer, incubated for 20 min at 80°C and cooled to room temperature. Bacteria, VLP and larger VLP were counted and analysed using a Cytoflex and identified based on the Violet SSC-A versus FITC-A by comparing to reference samples containing fixed EhV201 and bacteria from lab cultures. A total volume of 60 µL with a flow rate of 10 µL/min was analyzed. A threshold was applied based on the forward scatter signal to reduce the background noise. <em><strong>Measurement of dissolved inorganic nutrients</strong></em> Unfiltered seawater aliquots (10 mL) were collected from each bag and the surrounding fjord water in 12 mL polypropylene tubes and stored frozen at −20 °C. Dissolved inorganic nutrients were measured with standard segmented flow analysis with colorimetric detection (Hansen & Grasshoff 1983), using a Bran & Luebe autoanalyser. <em><strong>Measurement of water temperature and salinity </strong></em> Water temperature and salinity were measured in each bag and the surrounding fjord water using a SD204 CTD/STD (SAIV A/S, Laksevag, Norway). Data points were averaged for 1-3 m depth (descending only). When this depth was not available, the available data points were taken. Data is missing for the fjord in days 0-1. Outliers were removed for the following samples: bag 1 at days 0, 4, 15; bag 7 at day 15. <em><strong>Enumeration of extracellular EhV abundance by qPCR</strong></em>.<em> </em> Each filter from the core microbiome was diluted 100 times, and 1 µL was then used for qPCR analysis. EhV abundance was determined by qPCR for the major capsid protein (<em>mcp</em>) gene: 5′-acgcaccctcaatgtatggaagg-3′ (mcp1F<sup>5</sup>) and 5′-rtscrgccaactcagcagtcgt -3′ (mcp94Rv). All reactions were carried out in technical triplicates. For all reactions, Platinum SYBER Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA, USA) was used as described by the manufacturer. Reactions were performed on a QuantStudio 5 Real-Time PCR System equipped with the QuantStudio Design and Analysis Software version 1.5.1 (Applied Biosystems, Foster City, CA, USA) as follows: 50°C for 2 min, 95°C for 5 min, 40 cycles of 95°C for 15 s, and 60° C for 30 s. Results were calibrated against serial dilutions of EhV201 DNA at known concentrations, enabling exact enumeration of viruses. Samples showing multiple peaks in melting curve analysis or peaks that were not corresponding to the standard curves were omitted.
description: FCM data (containing
 phytoplankton, VLP, and bacteria counts), dissolved inorganic nutrients
 data, water temperature, salinity data and viral qPCR abundances are
 available in the Excel files
 "AQUACOSM_VIMS-Ehux_CoreData.xlsx". All .pdf documents containing the
 suffix "_Gates" represent the different phytoplankton population
 gates per bag. NOTE: Version published 28
 February 2024 was published to correct units of qPCR analysis of mcp gene
 from µL to mL.