Dynamics and genotypic composition of Emiliania huxleyi and their co-occurring viruses during a coccolithophore bloom in the North Sea

We studied the temporal succession of vertical proﬁles of Emiliania huxleyi and their speciﬁc viruses (EhVs) during the progression of a natural phytoplankton bloom in the North Sea in June 1999. Genotypic richness was assessed by exploiting the variations in a gene encoding a protein with cal-cium-binding motifs (GPA) for E. huxleyi and in the viral major capsid protein gene for EhVs. Using denaturing gradient gel electrophoresis and sequencing analysis, we showed at least three different E. huxleyi and EhV genotypic proﬁles during the period of study, revealing a complex, and changing assemblage at the molecular level. Our results also indicate that the dynamics of EhV genotypes reﬂect ﬂuctuations in abundance of potential E. huxleyi host cells. The presence and concentration of speciﬁc EhVs in the area prior to the bloom, or EhVs transported into the area by different water masses, are signiﬁcant factors affecting the structure and intraspeciﬁc succession of E. huxleyi during the phytoplankton bloom.


Introduction
Emiliania huxleyi is considered the most abundant coccolithophore in the ocean (Green & Leadbeater, 1994), has a wide distribution, forms intense blooms (Egge & Heimdal, 1994;Tyrrell & Taylor, 1996) and plays an important role in the biogeochemistry of the ocean by significantly influencing the sulphur and carbon cycles Malin et al., 1993;Simó , 2001). Emiliania huxleyi is a major contributor to the oceanic carbonate budget (Balch et al., 1992;Beaufort et al., 2007) which renders it a key species in ocean acidification studies, yet there exist contradictory claims on their physiological responses to increased CO 2 (Iglesias-Rodriguez et al., 2008;Beaufort et al., 2011). Vast E. huxleyi blooms occur during spring and summer in offshore, coastal and oceanic waters at mid-latitudes (45-55°) (Ackleson et al., 1988). Indeed, coc-colithophore blooms are seasonally predictable in certain areas including the North Sea (Holligan et al., 1983). These blooms are not formed by a cosmopolitan E. huxleyi population with a common gene pool; instead, there is evidence of gene pools fragmentation and adaptation of local populations to their environment, where morphological and calcifying differentiation occurs (Beaufort et al., 2011). It is therefore crucial to properly differentiate morphotypes and ecotypes to avoid extrapolating findings from studies using a limited number of strains and ecosystems.
Previous studies using mesocosm systems have investigated the role viruses have in structuring different microbial components (e.g. (Bratbak et al., 1993;Wilson et al., 1998;Martínez Martínez et al., 2007;Sorensen et al., 2009). It is clear from these studies that viruses are instrumental in the collapse of E. huxleyi blooms and allow succession of different microalgae following rapid bacterial remineralization of organic matter Larsen et al., 2001). Emiliania huxleyi-specific viruses (EhVs) are known to be diverse at the genotypic level (Wilson et al., 2002b;Schroeder et al., 2003;Martínez Martínez et al., 2007). Furthermore, they have been reported to show location-specific distinctions (Rowe et al., 2011). The study of natural E. huxleyi blooms in different oceanic regions is indispensable for determining intraspecific diversity, clarifying the importance of viruses as mortality agents in the ocean and determining community spatial dynamics.
An opportunity to investigate the aspects mentioned earlier, among others, was given during a multidisciplinary cruise that followed the progression of a developing E. huxleyi-rich phytoplankton bloom in a programme called 'DImethyl Sulphide biogeochemistry within a COccolithophore bloom (DISCO)', in the northern North Sea in June 1999. The study comprised analyses of the biological, optical and physical properties of the patch of water containing the bloom as well as studies of sulphur compounds, nutrients, halocarbons, methylamines, carbon monoxide, dissolved organic carbon and total dissolved nitrogen. In addition, the role of viruses, bacteria, phytoplankton and zooplankton, the dynamics of primary production, plankton respiration, grazing and sedimentation were investigated in relation to the biogeochemical cycling of dimethyl sulphide (DMS). The results were published elsewhere, for an overview see Burkill et al.(2002). As part of the DISCO cruise, Wilson et al. (2002a) investigated the E. huxleyi-and E. huxleyi-specific virus (EhV) dynamics by examining their concentrations through vertical profiles by analytical flow cytometry. Their aim was to obtain high-intensity sampling data of E. huxleyi and EhVs to gain information on their temporal and spatial dynamics in an open-water site.
In the current study, we have gone a step further in the investigation of the dynamics of E. huxleyi and their co-occurring virus by assessing changes in their genotypic composition during the bloom progression using specific primers. We have exploited the variations found in a gene encoding a protein with calcium-binding motifs (GPA) in E. huxleyi (Schroeder et al., 2005) and in the major capsid protein gene (MCP) of the E. huxleyi-specific viruses (Schroeder et al., 2002(Schroeder et al., , 2003 to analyse samples taken during the cruise using denaturing gradient gel electrophoresis (DGGE) and sequencing analysis.

Study site and sampling
The samples were collected during a research cruise aboard, the RRS Discovery, between the 5 and 29 of June 1999, that followed a coccolithophore-rich phytoplankton bloom originally located at 59°N 01°E in the North Sea. The experimental design and the flow cytometry (FCM) analysis are described by Wilson et al. (2002a). Briefly, the cruise was split into three parts: (1) An initial survey to identify the bloom combining satellite imagery and measurements of E. huxleyi concentrations by FCM. (2) A lagrangian time series study was then conducted between the 18 and 23 of June, when the selected patch of water was traced with sulphur hexafluoride (SF 6 ) using methods described previously (Law et al., 1998); during this period, the water column could be divided into three layers (surface, subsurface and bottom) based on the 10.5 and 8.5°C isotherms. (3) A final survey of the bloom (June 24-29) after the entrance of a patch of warmer, lower salinity water in the sampling area, which formed a new surface layer above the 11.5°C isotherm. Further details of the physical structure of the study site were described by Burkill et al. (2002).
Seawater was collected twice daily from a depth profile, down to approximately 100 m, typically just after midnight and midday, using a stainless steel CTD sampler system equipped with 12 Niskin bottles (30 L). From each depth sample, 1 L of seawater was filtered onto 0.45-lm-pore size Supor-450 47-mm-diameter filters (PALL Corp). The filters were transferred to 2-mL cryotubes, snap frozen in liquid nitrogen and stored at À20°C until further processing for total genomic DNA preparations. For virus and host enumeration using FCM, subsamples were also collected from each depth as described by Wilson et al. (2002a).

DNA isolation
Genomic DNA was isolated from the particulate matter retained on the Supor filters using an adapted phenol/ chloroform method. The filters were cut into small easily dissolvable pieces (approximately 0.5 cm 2 ) and placed in a 2-mL Eppendorff tube. Following the addition of 800 lL GTE buffer (50 mM glucose, 25 mM Tris-Cl pH 8.0 and 10 mM EDTA), 10 lL Proteinase K (5 mg mL À1 ), 100 lL 0.5 M filter sterilized EDTA and 200 lL 10% SDS, samples were incubated at 65°C for 1-2 h. DNA was then purified by phenol extraction as described by Schroeder et al.(2002).

Polymerase chain reaction (PCR) amplification and DGGE
Emiliania huxleyi genotypic richness was studied by nested PCRs on the total genomic DNA preparations using three oligomers designed to the GPA gene of  Table S1). Two-stage PCRs (firstly with primers GPA-F1/GPA-R1 and secondly with GPA-F2/GPA-R1) were conducted to amplify the variable region within the GPA gene that allows separation of the alleles into genotypes and coccolith morphology motif groups (CMM) (Schroeder et al., 2005). The PCRs were performed using 100 ng of total genomic DNA for the first reaction, then a 2-lL sub-sample from the first-stage PCR for the second reaction. Cycling conditions consisted of an initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 58/60°C (first-/second-stage reaction) for 45 s and 72°C for 60 s and a final extension for 5 min.
Viral diversity studies were also conducted by two-stage PCRs using oligomers designed for the MCP gene (Schroeder et al., 2002(Schroeder et al., , 2003 (Table S1). PCRs using these primers coupled as MCP-F1/MCP-R1 and MCP-F2/MCP-R2 were conducted as described by Schroeder et al. (2002Schroeder et al. ( , 2003, respectively. Host and viral second-stage PCR products were treated with Mung Bean nuclease (Promega) according to the manufacturer's recommendations to degrade single-stranded DNA ends. DGGE analysis of those PCR products was conducted using 30-50% linear denaturing gradient 8% polyacrylamide gels, where 100% denaturant is a mixture of 7 M urea and 40% deionized formamide. Ten microlitres of PCR products (it was estimated that all samples had approximately the same DNA concentration based on band intensity on an agarose gel) were loaded into wells with 5 lL of 29 gel loading dye [70% (v/v) glycerol, 0.05% (v/v) bromophenol blue and 0.05% (v/v) xylene cyanol]. Electrophoresis conditions and visualization of bands were as previously described by Schroeder et al. (2003).

DNA sequencing and sequence analysis
Selected representative single bands were excised from the DGGE gels for both E. huxleyi and viruses. Unfortunately, E. huxleyi DGGE gels for days 18-26 were lost before bands could be excised; therefore, limiting the GPA sequence database. The excised bands were incubated in 50 lL of molecular water, re-amplified and verified by DGGE. PCR products were subsequently sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, UK) on an ABI 3100 capillary sequencer (Applied Biosystems) according to the manufacturer's recommendations. The data for each fragment were aligned using CLUSTALW (http://www.ebi.ac.uk/clustalw/). The GPA sequences from this study (see Table 1 for Gen-Bank accession numbers) were aligned with the GPA sequences from 15 E. huxleyi isolates (Schroeder et al., 2005) Table S2 for GenBank accession numbers). The virus MCP sequences from this study (see Table 1 for GenBank accession numbers) were aligned with the MCP sequences of 10 clonal viruses isolated between 1999 and 2001 from the English Channel (Schroeder et al., 2002;Wilson et al., 2002b) and also with the MCP sequences from samples collected during the 2 mesocosm experiments in the Norwegian fjord (see Table S2 for GenBank accession numbers).

EhV diversity
Here, we present key parts of gels that help illustrate and pinpoint the timing of significant changes in temporal and spatial (depth) structure of the EhV assemblage during the cruise. The full series of gels are presented in Supporting Information, Fig. S1. DGGE analysis of virus MCP fragments revealed a diverse and dynamic EhV assemblage throughout the period of study (Fig. 1, Fig.   S1). DGGE MCP bands represent different genotypes (Schroeder et al., 2002(Schroeder et al., , 2003Martínez Martínez et al., 2007), and sequencing of representative selected bands corroborates that bands that migrated at the same rate (even among different gels) have the same sequence (Fig. 1). The symbols, abbreviations and GenBank accession numbers of the MCP bands and their sequences are summarized in Table 1. DGGE gels showed a relatively stable EhV assemblage composition at all depths between June 18 and 23 (midday) ( Fig. 1 and Fig. S1). For clarity, the DGGE gel images were sorted into two layers, from surface to 38 m depth (Fig. 1a) and from 38 to 100 m depth (Fig. 1b). The 38m-depth threshold was just above the 8.5°C isotherm (Wilson et al., 2002a). During the period of June 18-23, two more intense bands (a and b) were present in samples from all depths. Low-intensity bands during the same period had a more variable pattern and were not always easily visualized on the gels. An example of this is band (d).
From midnight of June 23, we observed a clear change in the genotypic composition of the EhV assemblage,  Table 1). Standards (S), bands of known EhV isolates. The full temporal and spatial series collected during the cruise is presented in Fig. S1. concurrent with the influx of a patch of warmer water into the sampling area (Wilson et al., 2002a). At this point, a new intense band, (e), became present in the upper 60 m of the water column ( Fig. 1; 23 June 00:00 hours), and at some depths, this was actually the only distinguishable band by DGGE. Band (e) prevailed almost to the end of the study, but it was not detectable between 5 and 24 m on the last sampling day -29 June (Fig. 1a). Notably, based on their relative intensity, the previously most intense bands, (a) and (b), no longer predominated in the surface 20 m from midnight of the 23 through to the 24 June, but reappeared as intense bands in the surface from the 25 June (Fig. S1).
From the 25 June, at least six bands had high intensity: (a) and (b) which were detected from surface to deep layers; (e) that was present after the 23 June; (g) (not sequenced) that was only detected in samples collected below 50 m; and (c) and (f) which followed a more irregular distribution pattern ( Fig. 1 and Fig. S1).
The alignment of MCP virus sequences from DGGE bands (Fig. S2) showed that two of the six EhV genotypes detected during the bloom were identical in the amplified region to virus isolates from the English Channel [eh-vOTU21 (e) was identical to EhV-84; ehvOTU22 (f) was identical to EhV-86], one more EhV genotype, ehvOTU1 (b), was the same as the virus isolate EhV-163 (isolated from a Norwegian fjord) and two other EhV genotypes, ehvOTU3 (a) and ehvOTU5 (c), were detected in this study and in the samples from the Norwegian mesocosm studies. Genotype ehvOTU20 (d) did not match with any genotype in GenBank.

E. huxleyi diversity
DGGE analysis of PCR products amplified with the specific primers for the GPA gene revealed a broad range of E. huxleyi bands (Fig. 2). Changes in presence/absence of bands were observed both temporally and spatially.  Table 1). The ovals mark a distinctive band only detectable below 40 m depth on 18 and 19 June and at surface on 24 June. Virus-host dynamics in a coccolithophore bloom As some E. huxleyi strains contain a single GPA allele while other strains contain two alleles (Schroeder et al., 2005), the bands revealed in this study indicate the different alleles present instead of quantitative richness of E. huxleyi strains. Table 1 summarizes the symbols, abbreviations and GenBank accession numbers given to each of the GPA bands excised from the DGGE gels. The time/depth DGGE profile for E. huxleyi allelic richness was partial as we were not able to amplify the GPA gene from all the samples. As in the EhV analysis, we divided the samples into surface and deep water layers to facilitate the interpretation of the DGGE gel images (Fig. 2). The 38-m-depth threshold corresponding to the 8.5°C isotherm also marks the depth at which E. huxleyi numbers are just starting to reach the limit of detection by FCM analysis (Wilson et al., 2002a), indicative of very low cell concentrations in the deep layer.
In general, a similar profile of E. huxleyi bands was observed throughout the water column from June 18 to 26, with a few bands changing intensity in certain samples (Fig. 2). Interestingly, on 18 and 19 June, the DGGE profiles in the deep layer were most similar to those observed in the surface layer on the 24 June, specifically the noticeable common encircled bands in Fig. 2. The E. huxleyi assemblage composition changed significantly from 27 June. A more irregular band profile was observed during the 27-29 June period (Fig. 2), during which the combination of band migration rate in the DGGE gel (Fig. 2) and sequencing analysis (Fig. S3) of excised bands showed the presence of at least 12 different E. huxleyi alleles. Sequencing information alone from these short fragments was not enough to determine allelic richness as it did not allow differentiation of all the bands at the genotype level. However, the sequence data from excised bands revealed the presence of five different CMM groups of the A and B E. huxleyi morphotypes (Fig. S3). Four of those genotypes (CMM I to IV) were previously characterized by Schroeder et al. (2005). We were not able to determine to which morphotype CMM V belonged.

Discussion
In their original report on virus-host dynamics of this study site in the North Sea, Wilson et al. (2002a) suggested that large viruses (EhVs) were actively infecting hosts. However, EhV concentrations were lower than expected, grazing rates were relatively high  and viruses showed no evidence of influencing Dimethyl sulphide/Dimethylsulphoniopropionate (DMS/ DMSP) production (Wilson et al., 2002a). The implication was, therefore, that viruses played a minor role in the dynamics of this coccolithophore-dominated phyto-plankton bloom. However, the molecular data presented here, from samples collected during the same bloom, reveal a dynamic virus-host system, concealed by what appears to be relatively uninteresting numerical population data. The sensitivity of PCR has allowed us to explore these dynamics in much greater detail, essentially revealing changing assemblages of viruses and their hosts during the course of this naturally occurring bloom in the North Sea (Figs 1 and 2, Fig. S1) beyond the limits of detection for FCM. While the FCM analysis only detected E. huxleyi cells up to 45 m deep, PCR and DGGE revealed the presence of different E. huxleyi alleles as deep as 100 m. It is worth noting that despite their wide use for describing microbial community structure based on extracted DNA (Petersen & Dahllöf, 2005) PCR and DGGE only provide presence/absence data and not abundance. This is mainly owing to the qualitative nature of PCR and limitations in DGGE resolution. To our knowledge, this is the first time such a comprehensive temporal and spatial analysis of E. huxleyi and their corresponding viruses has been presented at the molecular level during the progression of a natural coccolithophore bloom.
Our results showed at least three significantly different E. huxleyi genotypic profiles during the period of study: (1) The combination of DGGE and FCM (Wilson et al., 2002a) data showed the progression and termination of a diverse genotypic E. huxleyi assemblage in the surface layer between June 18 and 23 (Fig. 2). The concurrent decrease followed by an increase in EhV concentrations in the surface during the same period suggests an active infection process of E. huxleyi followed by the release of EhV progeny (Fig. 1). DGGE analysis showed a stable EhV assemblage (Fig. 1, Fig. S1) until the entrance of a warm patch surface water (23 June), indicating that those EhV genotypes (Fig. 1, Fig. S2) were linked to the initial E. huxleyi assemblage (Fig. 2) and were likely involved to a certain extent in control of the bloom through infection following 'kill the winner' dynamics (Thingstad, 2000).
(2) The influx of warm water probably caused mixing as well as the entrance of new hosts and viruses. In turn, the new E. huxleyi assemblage progressively disappeared (Wilson et al., 2002a;Fig. 2). This disappearance could be partly attributed to grazing  and viral infection. The concomitant detection of new EhV genotypes (c and f, Fig. 1) suggests specific active infection of the decreasing E. huxleyi assemblage and the subsequent release of those EhVs into the water in enough numbers to allow detection by PCR and DGGE. In addition, the persistence in the water column of the EhV genotypes (a) and (b) until the last day of study (Fig. 1) hints that the remaining viruses from previous lysis events were also propagated by infection of the incoming E. huxleyi assemblage.
(3) As the surface E. huxleyi assemblage diminished in numbers, a new assemblage formed at 30-40 m depth between 26 and 29 June (Wilson et al., 2002a;Fig. 2). It is likely that E. huxleyi cells that bloom in surface layers sink out with time and can be later detected in deeper water. This was especially evident between 26 and 29 June owing to the available sequence data collected for this period. For example, E. huxleyi alleles (L) and (C) were first detected at 30-35 m depth on the 27 and 28 June, respectively, and at 60-80 m depth on the 28 and 29 June (Fig. 2). Following this reasoning, we hypothesize that the E. huxleyi genotype on 18 -19 June at depths below 40 m (marked by an oval in Fig. 2) revealed the presence of a remnant E. huxleyi genotype, from a previous bloom event, that sank to deeper water as the bloom declined. The presence of the same band at surface on 24 June (Fig. 2) can be explained by either mixing caused by the warm surface water influx or entry of similar E. huxleyi strains with that water patch.
Low impact of E. huxleyi and EhVs in DMSP production was recorded during the DISCO study . However, Steinke et al. (2002) measured maximum DMSP lyase activity at approximately 50 m depth on 22 June, 40 m on 23 June and 35 m on 24 June, concurrently with the lowest cell numbers recorded for the E. huxleyi assemblage, suggesting the production of DMSP by dying E. huxleyi cells. Yet, the low impact of E. huxleyi and EhVs in DMSP production could be explained first by the fact that coccolithophores accounted for less than 30% of the phytoplankton biomass . Secondly, different E. huxleyi strains are known to have different DMS production rates (Steinke et al., 1998). It is possible that the dominant strains during the main E. huxleyi bloom were not high DMS producers. It may be that the E. huxleyi contribution to the standing stocks of DMSP and the importance of EhVs as agents of DMS production were higher after the influx of warm surface water (24-29 June) when new E. huxleyi and EhV communities developed. However, DMSP and DMS measurements are lacking for this period. Further investigation would aid in establishing links between the results presented here and sulphur biogeochemical cycles in the sea. The use of well-established molecular tools, as the ones employed in this study, may be the key to answer unknown questions regarding the implications of the E. huxleyi virus system in local ecology, climate and biogeochemistry cycling and production of compounds such as DMS, calcite and carbon.
In summary, DGGE and sequencing analysis of E. huxleyi and EhV groups provided additional information about the dynamics of E. huxleyi blooms in open waters. Depth profiles showed 'past, present and future' of the progression and structuring within a natural coccolitho-phore-dominated bloom. The findings in this study are of great value because despite being one of the best-studied eukaryotic phytoplankton virus systems, we still know little about E. huxleyi natural bloom dynamics, diversity and evolution. Our results revealed highly dynamic and diverse E. huxleyi genotypic assemblages perhaps driven by a response to infection by equally diverse EhV genotypes seemingly following the 'Red Queen' effect (van Valen, 1973), in which viral pressure leads to increased host diversity. Another possibility is a 'Cheshire Cat' scenario (Frada et al., 2008) in which viral infection induces the transformation or succession from diploid to virusresistant haploid E. huxleyi cells, which may explain reoccurrence of certain diploid genotypes when specific virus pressure disappear. Previous studies have reported that the same limited number of E. huxleyi and associated EhV genotypes can reoccur over a 3-year period in a Norwegian fjord (Martínez Martínez et al., 2007) and can even persist for centuries as shown in the Black Sea (Coolen, 2011). A combination of both scenarios, 'Red Queen' and 'Cheshire Cat', is more plausible. Wilson

Supporting Information
Additional Supporting Information may be found in the online version of this article: Fig. S1. DGGE gels of PCR fragments amplified with MCP primers for analysis of EhV diversity. Fig. S2. Multiple sequence alignment of the EhV-MCP fragments produced in this study (ehvOTUs). Fig. S3. Clustal alignment of fragments within the partial E. huxleyi GPA-sequences obtained from excised DGGE bands from this study ('ehuxOTUs') (Table 1), from two mesocosm studies ('ehuxOTUs') (Table S2) and from isolates in culture (Schroeder et al., 2005). Table S1. Primers used to assess host (GPA prefix) and viral (MCP prefix) diversity. Table S2. List of E. huxleyi and EhV genotypes from mesocosm studies in a Norwegian fjord and GenBank references for their sequence data (Martínez Martínez et al., 2007).
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Virus-host dynamics in a coccolithophore bloom