DOI: 10.5061/dryad.h44j0zpjc
title: Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise

description: Marine viruses are the most abundant biological entity in the ocean and
 are considered as major evolutionary drivers of microbial life (Suttle,
 2007). Yet, we lack quantitative approaches to assess their impact on the
 marine ecosystem. Here, we provide quantification of active viral
 infection in the bloom forming single celled phytoplankton Emiliania
 huxleyi infected by the large virus EhV, using high-throughput single
 molecule mRNA in situ hybridization of both virus and host transcripts. In
 natural samples, viral infection reached only 25% of the population
 despite synchronized bloom demise exposing co-existence of infected and
 non-infected subpopulations. We prove that photosynthetically active cells
 chronically release viral particles through non-lytic infection, and that
 viral-induced cell lysis can occur without viral release, thus challenging
 major assumptions regarding the life cycle of giant viruses. We could also
 assess active infection in cell aggregates, linking viral infection and
 carbon export to the deep ocean (Laber et al., 2018) and suggest a
 potential host defense strategy by enrichment of infected cells in sinking
 aggregates. Our approach can be applied to diverse marine microbial
 systems, opening a mechanistic dimension to the study of biotic
 interactions in the ocean.
description: Culture growth and viral infection
 The non-calcifying <i>E. huxleyi</i> strain CCMP 2090
 was used for this study. Cells were cultured in K/2 medium with
 antibiotics (Ampicillin and Kanamycin) and incubated at 18°C with a 16:8h
 light-dark illumination cycle. A light intensity of 100 μmol photons
 m<sup>-2</sup> s<sup>-1</sup> was provided by cool
 white light-emitting diode lights. All experiments started with
 exponential phase cultures (5*10^5 cells mL<sup>-1</sup>). The
 virus used for this study is EhV201 propagated on CCMP2090 with
 antibiotics. Five day before infection, Most Probable Number assays were
 performed to assess the fraction of infectious viruses in the stock (48).
 <i>E. huxleyi</i> was infected with 5:1 multiplicity of
 infection (MOI) ratio of infectious virus per cell, hence guaranteeing
 that all cells encountered an infectious particle 30 minutes post
 infection. Time course of infected and non-infected cultures were done
 simultaneously, in triplicates. A second MPN experiment was performed on
 the day of the experiment with the same virus and same algae to assess the
 exact MOI at the beginning of the experiment (Table S1).
 <i>mcp</i>, and
 <i>psbA</i> probe design and conjugation for
 smFISH The smFISH
 technique identifies a single mRNA based on the binding of multiple small
 probes targeted to different locations to the mRNA of interest. Fasta
 sequences of the target genes <i>mcp </i>and
 <i>psbA</i> were first submitted to Stellaris Probe Designer
 to obtain potential probes. We designed 48 probes per gene with a probe
 length of 20 nucleotides. Probes with more than 70% GC content were
 discarded. To discard off-targets and decrease nonspecific signal, each
 probe sequence was blasted against the <i>E. huxleyi</i>
 transcriptome and EhV201 genome. Probes with an off-target gene matching
 over 17 nucleotides were discarded. A minimum of 20 probes per gene are
 necessary to detect a single molecule. Validated probes were ordered with
 3’ amine groups through the Custom Oligo Service of BioSearch Technologies
 (see Probe_Sequences.xlsx). Fluorophores with succinimidyl ester group
 were ordered from Click Chemistry Tools: Tetramethylrhodamine (TMR) and
 sulfo-Cyanine5 (Cy5). Probes were coupled to fluorophores and purified
 according to (23). All the conjugated probes of a given gene are used as a
 mixture each time we target the expression of that gene. Therefore, the
 <i>mcp</i> mRNA was detected using 47 probes of 20 nucleotides
 each, and <i>psbA</i> mRNA was detected using 48 probes of 20
 nucleotides each. The position of the probes on the reference gene are
 shown in Figure S9A and S9B, and their respective positions and sequences
 indicated in the file Probe_Sequences.xlsx
 Sample fixation and hybridization for
 smFISH in laboratory samples
 At each time point, 50 mL of each flask were fixed in cold 1%
 paraformaldehyde final concentration, and incubated for 1h at 4°C with
 gentle agitation. Each sample was then centrifuged for 2 min at 4°C and
 3000g. The supernatant was discarded, the pellet resuspended in 1 mL of
 cryopreservant solution (prepared in 1X PBS containing 4% paraformaldehyde
 and 30% sucrose), transferred to a 1.7 mL Eppendorf and incubated 1h at
 4°C with agitation. Tubes were then centrifuged for 2 min at 4°C and
 3000g, the supernatant removed, and stored at 80°C until
 hybridization. A day before
 ImageStreamX acquisition, selected samples were thawed and chlorophyll
 extracted by a first wash using 900 μL of 70% ethanol applied for 3 min,
 followed by centrifugation 3 min at 3000g and removal of supernatant. A
 second wash was performed with 900 μL of 100% ethanol applied for 3 min,
 followed by centrifugation of 3 min at 3000g and removal of supernatant.
 Samples were treated with 500 μL ProteinaseK at 10 μg/mL final
 concentration (Ambion #AM2546) for 10 min at room temperature and washed
 with 3 minutes at 3000g centrifugation. Samples were then resuspended in
 50 mL of hybridization buffer (17.5% formamide concentration) containing
 equal concentrations of the different target genes at 0.1ng/mL final
 concentration. <i>Mcp</i> probes were conjugated to TMR, and
 <i>psbA</i> probes were coupled to Cy5. Hybridization was
 performed overnight in 30°C shakers. The day of
 ImageStreamX acquisition, hybridization buffer was washed away by 3 min
 centrifugation at 3000g. Samples were stained for dsDNA with 500 μL of
 GLOX buffer (prepared in nuclease free water with 0.4% final concentration
 of glucose, 2X final concentration SSC Ambion #AM9765, and 10 mM final
 concentration of Tris pH 8.0) with DAPI in 10μg/mL final concentration
 (except the single stains). DAPI staining was done for 30 min in 30°C,
 followed by centrifugation 3 min at 3000g, removal of supernatant and
 resuspension in 40μL of GLOX buffer before being acquired in the
 ImageStreamX. DAPI was used for several reasons. The first one is
 technical: DAPI is required for the fluorescent microscope to localize
 cells when Brightfield channel is not available. We used DAPI to draw the
 contour of the nucleus using Cell Profiler in Figure 1 and Figure S12.
 DAPI is also important for gating of the cells in the imaging
 flow-cytometer. Gating on brightfield signal and on the DAPI achieves
 better detection of the cells and thus better downstream statistics. The
 second major reason to use DAPI is that it provides important information
 regarding the general DNA content of the cells, its cell cycle phase, and
 is a nucleus marker that serves as a reference point in our
 cells. The following
 day, samples were resuspended in 300 μL of GLOX with the
 3,3'-Dihexyloxacarbocyanine Iodide membrane stain at final
 concentration of 1μM (ThermoFisher Catalog number D273). Excessive dye was
 removed by 3 min centrifugation at 3000g and removal of supernatant,
 followed by resuspension in 4 μL GLOX. 2 μL were deposited on a microscope
 slide with 2 μL of antibleach solution, and the sample was imaged with the
 epifluorescent microscope. Imaging Flow
 Cytometry acquisition, compensation and analysis for laboratory
 samples Samples were
 acquired using the ImageStreamX MarkII machine (ISX, Amnis, Luminex).
 Three excitation wavelengths were used: 405 nm (DAPI - Channel 7- 50 mW),
 561 nm (TMR - Channel 3 – 200 mW) and 642 nm (Cy5 - Channel 11 – 120 mW).
 For each sample, at least 50,000 cells were acquired and imaged with 60X
 magnification. Single stained samples were acquired using the compensation
 wizard of ISX. On average, less than 10 μL of
 2x10<sup>^6</sup> cell stock concentration were necessary to
 collect the optimal amount of cells, except towards the end of the
 experiment where cells were scarce. Data was
 analyzed using IDEAS6.2 (Amnis, Luminex). The compensation matrix was
 built using the IDEAS wizard and manually checked, before being applied to
 all the acquired files. Based on the Area (the number of microns squared
 in a mask) and Circularity (the degree of the mask’s deviation from a
 circle) of DAPI three populations are identified as single cells (mainly
 DAPI area &lt; 60 a.u), doublets and aggregates (mainly DAPI area
 &gt; 60 a.u). Single cells were additionally selected in the same
 focal plane using the BF gradient and contrast (both gradient and contrast
 measure the sharpness quality of an image by detecting large changes of
 pixel values in the image). All gates were defined on a single file before
 being applied to the total data set (See Fig. S10). Each file was then
 manually inspected to check the accuracy of single cell and aggregates
 gating. All the data (fluorescent intensities, morphological features,
 populations) was then exported for each cell of each file for analysis in
 R.  Similarity
 calculation. As defined in the IDEAs User Manual (Imagestream software)
 "The Similarity feature is the log transformed Pearson’s Correlation
 Coefficient and is a measure of the degree to which two images are
 linearly correlated within a masked region.". In Fig S4D, we
 correlate DAPI and <i>mcp</i> masks for each cell of two
 subpopulations (<i>mcp</i>+/<i>psbA</i>+,
 <i>mcp</i>+/<i>psbA</i>-) and plot its
 distribution. The threshold used to declare co-localisation was Similarity
 &gt; 1.2
description: <b>Software</b> The files can be open with the software IDEAS 6.5.   <b>Files types:</b> Files with .cif extension contain the compensation matrix used to analyze the data based on single stain acquisition. Files with .daf extension contain the actual data for each single cell of that time point, as well as the gates used to define the different populations. IDEAS requires both .cif and .daf files for a given sample.   <b>Naming scheme:</b> Files are named with the following scheme: Treatment_Replicate_TimePoint If Treament = Ctrl: the samples are from non-infected <i>E. huxleyi</i> cultures If Treament = EhV201: the samples are from <i>E. huxleyi</i> cultures infected with EhV201 Replicate = R1-R2-R3 The Control R1 was kept for microscopy controls. TimePoint = 0, 8, 24, 48, 72 hours post infection (hpi).