APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2003, p. 2484–2490 Vol. 69, No. 50099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.5.2484–2490.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Virus Succession Observed during an Emiliania huxleyi BloomDeclan C. Schroeder,1 Joanne Oke,1 Matthew Hall,1 Gillian Malin,2

and William H. Wilson1,3,4*Marine Biological Association, Citadel Hill, Plymouth PL1 2PB,1 School of Environmental Sciences,

University of East Anglia, Norwich NR4 7TJ,2 Department of Biological Sciences, University ofStirling, Stirling FK9 4LA,3 and Plymouth Marine Laboratory, Prospect Place,

The Hoe, Plymouth PL1 3DH,4 United Kingdom

Received 6 November 2002/Accepted 5 February 2003

Denaturing gradient gel electrophoresis was used as a molecular tool to determine the diversity and tomonitor population dynamics of viruses that infect the globally important coccolithophorid Emiliania huxleyi.We exploited variations in the major capsid protein gene from E. huxleyi-specific viruses to monitor theirgenetic diversity during an E. huxleyi bloom in a mesocosm experiment off western Norway. We reveal that,despite the presence of several virus genotypes at the start of an E. huxleyi bloom, only a few virus genotypeseventually go on to kill the bloom.

Blooms of the unicellular marine phytoplankton Emilianiahuxleyi are known to affect the oceanic carbon pump (9) andclimate (6). Vast coastal and midocean populations of thisorganism, which are readily visualized by satellite imagery dueto their reflective calcium carbonate coccoliths, often disap-pear suddenly, causing substantial fluxes of calcite to the sea-bed (29) and cloud-forming dimethyl sulfide to the atmosphere(14). Until recently, the mechanisms of E. huxleyi bloom dis-integration were poorly understood, but it is now accepted thatviruses are intrinsically linked to these sudden crashes (13, 26).Viruses are ubiquitous in the marine environment, and theyexert significant control over bacteria and phytoplankton pop-ulations, influencing diversity, nutrient flow, and biogeochemi-cal cycling (11, 28).

Over the last decade, significant advances have been made inunderstanding the dynamics of viruses and their effects onmarine eukaryotic phytoplankton communities. The majorityof the work in this area has dealt with viruses that infectMicromonas pusilla. These viruses are widespread, geneticallydiverse, dynamic, and highly virulent (7, 8, 22). Other research-ers have looked at viruses that infect Heterosigma akashiwo.These viruses were shown to play an important role in deter-mining the clonal composition and maintaining the clonal di-versity of H. akashiwo populations (23). However, the under-standing of the dynamics and effects of viruses on E. huxleyi isstill in its infancy. Several studies have shown that virus num-bers increase dramatically following the demise of E. huxleyi-dominated blooms (1–4, 13). E. huxleyi viruses have been iso-lated from such blooms (5, 26) and have recently been assignedto a new genus, Coccolithovirus, based principally on the phy-logeny of their DNA polymerase genes (21). Coccolithovirusesbelong to the family Phycodnaviridae, a diverse family of ico-sahedral double-stranded DNA viruses that infect eukaryoticalgae (25).

Recently, we reported the cloning and sequencing of ampli-

fied segments of the major capsid protein (MCP) gene fromviruses that infect E. huxleyi (21). Significant sequence varia-tion was observed between virus strains, revealing the potentialof using this gene as a genetic tool to differentiate viral geno-types in natural communities (21). Denaturing gradient gelelectrophoresis (DGGE) is a powerful yet simple techniquethat can separate genotypes amplified from mixtures of se-quences in natural samples and is widely used in microbialecology (18). DGGE has recently been successfully used todetermine the genetic diversity within a wide range of naturallyoccurring algal viruses (22). In this study, we used PCR andDGGE to differentiate known E. huxleyi virus isolates based onthe sequence variation in the MCP gene. This technique wasused to monitor the progression of the E. huxleyi virus com-munity during an E. huxleyi-induced bloom in a mesocosmexperiment conducted in Norway during June 2000.

MATERIALS AND METHODS

Study site and flow cytometric analysis. The mesocosm experiment was car-ried out at the Marine Biological Field Station adjacent to Raunefjorden, 20 kmsouth of Bergen, Norway. The experimental design and the flow cytometricanalysis of E. huxleyi and its natural viral communities from enclosure 1 (anutrient enrichment regime with a 15:1 N/P ratio [1.5 �M NaNO3–0.1 �MKH2PO4]) are described by Jacquet et al. (13).

Collection and preparation of samples for DGGE. Samples were collecteddaily at 0900 h throughout the mesocosm experiment. Prior to concentration ofthe virus fraction, 1-liter volumes of seawater were gently filtered through 0.45-�m-pore-size Supor-450 47-mm-diameter filters (PALL Corp.). The resultingfiltrates were concentrated 1,000-fold in two steps, first by tangential-flow ultra-filtration using 50-kDa-cutoff Vivaflow 50 units (Sartorius) to a volume of 20 ml.The viruses were further concentrated in a second step by centrifugal filtrationusing Macrosep paddle filters (PALL Corp.) to a final volume of 1 ml. Theconcentrates were stored at �80°C prior to use.

PCR and DGGE. PCR of the E. huxleyi virus isolates and the concentratedvirus samples were conducted in two stages. One microliter of each of the clonalE. huxleyi virus lysates (21) and 1,000-fold virus concentrates was added to a49-�l first-stage PCR mixture containing 1 U of Taq DNA polymerase (Pro-mega), 1� PCR buffer (Promega), 0.25 mM deoxynucleoside triphosphates(dNTPs), 1 mM MgCl2, and 10 pmol of each E. huxleyi virus-specific primer,MCP-F and MCP-R (21). Viral lysates of M. pusilla (M. pusilla virus PB5;courtesy of Steven Short, University of British Columbia, Vancouver, Canada)and Phaeocystis globosa (P. globosa virus 102, a partly characterized strain iso-lated from the English Channel) and mixtures with no template added served ascontrols. The PCR was conducted with a PTC-100 cycler (MJ Research) as

* Corresponding author. Mailing address: Marine Biological Asso-ciation, Citadel Hill, Plymouth PL1 2PB, United Kingdom. Phone: 44(0)1752 633356. Fax: 44 (0)1752 633102. E-mail: whw@mba.ac.uk.

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described by Schroeder et al. (21). A 2.5-�l subsample from the first-stage PCRwas treated with ExoSAP-IT (U.S. Biochemical Corp.) to remove unutilizeddNTPs and primers.

A second-stage PCR was conducted to amplify the variable region within theMCP genes by adding each of the ExoSAP-IT-treated subsamples to a 46.5-�lPCR mixture containing 1 U of Taq DNA polymerase (Promega), 1� PCRbuffer (Promega), 0.25 mM dNTPs, 1 mM MgCl2, and 10 pmol of each E. huxleyivirus-specific primer, MCP-F2 and MCP-R2. The two oligomers, MCP-F2 (5�-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGGGTT CGC GCT CGA GTC GAT C-3�; the underlined sequence represents theGC clamp) and MCP-R2 (5�-GAC CTT TAG GCC AGG GAG-3�), were de-signed based on the alignment described by Schroeder et al. (21). The PCR wasconducted with a PTC-100 cycler (MJ Research) with an initial denaturing stepof 95°C (3 min) followed by 34 cycles of denaturing at 95°C (30 s), annealing at55°C (60 s), and extension at 74°C (90 s), and finally one cycle of denaturing at95°C (30 s), annealing at 55°C (300 s), and extension at 74°C (300 s). The PCRproducts were resolved in a 1.5% (wt/vol) agarose gel in Tris-borate-EDTAbuffer (20). The gels were stained with ethidium bromide, visualized on a UVtransilluminator, and photographed with the Gel Doc 2000 system (Bio-Rad).DGGE of second-stage PCR products was conducted using 30 to 60% lineardenaturing gradient 10% polyacrylamide gels, where 100% denaturant is a mix-ture of 7 M urea and 40% deionized formamide. Ten microliters of PCR productwas loaded into wells with 10 �l of 2� gel loading dye (70% [vol/vol] glycerol,0.05% [vol/vol] bromophenol blue, and 0.05% [vol/vol] xylene cyanol). Electro-phoresis was carried out for 3.5 h in 1� TAE (20) at 200 V and a constanttemperature of 60°C using the D-code electrophoresis system (Bio-Rad). Thegels were stained in a 0.1� SYBR Gold (Molecular Probes) solution for 20 min,and the visualized bands were photographed as described above.

Sequencing and sequence analysis. Individual bands were excised, reamplified,verified by DGGE, and sequenced directly. Twenty-seven excised gel fragmentsrepresenting 27 DGGE bands were placed in sterile microcentrifuge tubes with150 �l of 1� Tris-EDTA buffer (20) and heated to 95°C for 5 min. Two micro-liters of the resultant eluant was PCR amplified as described for the second-stagereactions. Ten microliters was loaded on a DGGE gel as described above toverify purity and confirm the correct mobility of the excised bands. In addition,7 �l was used for direct sequencing of these bands using the fluorescently labeledoligomers MCP-F2_700 (5�-TTC GCG CTC GAG TCG ATC-3�) and MCP-R2_800 (5�-GAC CTT TAG GCC AGG GAG-3�) as sequencing primers. Se-quencing was performed with the SequiTherm EXCEL II DNA SequencingKit-LC (Epicentre Technologies) with a LI-COR automated DNA sequencer(DNA Analyzer; Gene Reader 4200). The sequenced data were analyzed usinge-Seq release 1.1 software (LI-COR). Homology searches were carried out usingthe BLAST algorithm provided by the Internet service of the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

Phylogenetic analysis. DNA and protein sequences of MCP genes were ob-tained from the GenBank database. The DNA and amino acid sequences of theconserved region I (24) within the MCP genes were aligned using ClustalW (http://www.clustalw.genome.ad.jp/). Phylogenetic trees of the amino acid alignmentwere constructed using the various programs in PHYLIP (Phylogeny InferencePackage) version 3.57c (10), and the robustness of the alignments was tested withthe bootstrapping option (SeqBoot). Genetic distances, applicable for distancematrix phylogenetic inference, were calculated using the Protdist programin the PHYLIP package. Phylogenetic inferences based on the distance ma-trix (Neighbor) and parsimony (Protpars) algorithms were applied to thealignments. In both trees, the best tree or the majority-rule consensus treewas selected using the consensus program (Consense). The trees were visu-alized and drawn using TREEVIEW software version 2.1 (http://taxonomy.zoology.gla.za).

The abbreviations and GenBank accession numbers of the virus sequencesused in the phylogenetic analysis are as follows: Invertebrate iridescent virus, IIV-6(NP_149737), IIV-22 (P22166), and IIV-1 (P18162); Lymphocystis disease virus,LCDV-1 (NP_044812); Frog virus, FRGV3 (AAB01722); Paramecium bursariaChlorella virus CVT2 (AB006978), CVK2 (AB011506), G1 (AF076921), and 1(M85052); Ectocarpus siliculosus virus V-1 (NP_077601); E. huxleyi virus 84(AF453849), 86 (AF453848), 88 (AF453850), 163 (AF453851), 201 (AF453857),202 (AF453856), 203 (AF453855), 205 (AF453847), 207 (AF453853), and 208(AF453852); and E. huxleyi virus operational taxonomic unit 1 (EhVOTU1)(AY144374), EhVOTU2 (AY144375), EhVOTU3 (AY144376), EhVOTU4(AY144377), EhVOTU5 (AY144378), EhVOTU6 (AY144379), EhVOTU7(AY144380), and EhVOTU8 (AY144374).

RESULTS AND DISCUSSION

The aim of this study was to determine whether DGGEcould be successfully employed to exploit the variations previ-ously observed in the MCP gene of E. huxleyi virus isolates(21), and consequently, to genetically fingerprint E. huxleyivirus communities in natural samples.

DGGE of E. huxleyi virus isolates. MCP gene fragmentswere amplified, yielding products of 284 bp in the first-stagePCRs (data not shown) and 175 bp in the second-stage PCRs(Fig. 1A) from all 10 E. huxleyi virus isolates. DGGE analysisof the second-stage PCR fragments resulted in the differenti-ation of 7 out of the 10 E. huxleyi virus isolates (Fig. 1B).Isolates E. huxleyi virus 201, E. huxleyi virus 202, and E. huxleyivirus 84 could not be differentiated from E. huxleyi virus 205,E. huxleyi virus 207, and E. huxleyi virus 88, respectively. Thedenaturing gradient used in this analysis was too broad toseparate the individuals that differed by 1 bp (21). However, asignificant proportion of the isolates could be differentiated.Hence, we were able to demonstrate that this technique couldbe applied as a tool to genetically fingerprint E. huxleyi viruscommunities as long as a significant number of bands aresequenced to confirm their identities.

Mesocosm study. The progression of different microbialpopulations, including E. huxleyi and its associated viruses (E.huxleyi viruses), was monitored by flow cytometry during themesocosm experiment (13). Flow cytometry has been routinelyused for the analysis of different phytoplankton populations(19), bacterial communities (17), and virus communities (15,26, 27) in marine samples and is commonly accepted as areference technique in oceanography (15).

E. huxleyi numbers increased by 2 orders of magnitude, from103 to 105 cells � ml�1, between 9 and 18 June 2000 prior to theonset of the bloom crash on 19 June 2000 (Fig. 2A). The E.huxleyi numbers returned to prebloom levels of 3 � 103 cells �ml�1 at the end of the study (24 June 2000). There was anincrease of �2 orders of magnitude in E. huxleyi virus numbers,from 4.7 � 105 to 3.5 � 107 E. huxleyi viruses � ml�1, during thecollapse of the E. huxleyi bloom, suggesting it was terminatedby viral infection (Fig. 2A).

Flow cytometry was used to monitor the total abundance ofE. huxleyi viruses during the E. huxleyi bloom, and it revealeda classic lytic virus response to a susceptible host population.However, DGGE analysis of the E. huxleyi virus communityduring this bloom revealed a more dynamic sequence ofevents. In the 8 days prior to the onset of the bloom, a genet-ically diverse community of E. huxleyi viruses was observed,with a different DGGE profile each day (Fig. 2B; 6 to 13 June2000). A succession from this diverse and variable E. huxleyivirus community to a more stable E. huxleyi virus community,with the same DGGE profile each day, was observed from theonset of the E. huxleyi bloom (Fig. 2B; 14 to 24 June 2000).

The authenticity of the DGGE bands was confirmed byexcising 27 bands (Fig. 2B), verifying their purity (data notshown), and finally sequencing them. A BLAST search of theGenBank database revealed that these sequences showed highidentity to MCP gene sequences (data not shown). Phyloge-netic analysis clearly showed that they belonged to the familyPhycodnaviridae, and they clustered together with E. huxleyivirus isolates (Fig. 3). This cluster exhibited high bootstrap

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values with both the neighbor-joining and parsimony phylog-eny inferences, indicative of a strong association. The evidencecollectively confirms that the E. huxleyi virus-specific PCRprimers used in this study amplified only E. huxleyi virus se-quences.

The eight sequences obtained from the excised DGGE bandprofiles differed from the sequences obtained from the isolatescollected from the English Channel off the coast of Plymouth,United Kingdom (Fig. 4). In contrast, the DGGE bands thatmigrated like E. huxleyi virus 163 had sequences identical tothat of E. huxleyi virus 163 (Fig. 4). This was not surprising,since E. huxleyi virus 163 was isolated from this mesocosmexperiment (21). Two DGGE band profiles were observedbefore, during, and after the bloom. However, five DGGEband profiles were observed before the bloom, while one wasobserved during and after the bloom (Fig. 2B). Therefore,despite the presence of a diverse viral community prior to thedevelopment of the E. huxleyi bloom, only three dominantDGGE bands were observed during and after the bloom, sug-gesting that they were responsible for its collapse.

Similar DGGE profiles (unpublished data) were obtained insamples collected from mesocosms that were either P or Ndepleted (described by Jacquet et al. [13]). Interestingly, nu-trient availability was considered important, as it was thoughtto affect virus-host interactions (1, 13). However, contradictoryevidence exists concerning the roles of P and N limitations inthese virus-host interactions. Bratbak et al. (1) concluded thatviruses might be more sensitive to P than to N limitation,because viruses have high nucleic acid-to-protein ratios. Jac-quet et al. (13) reported that E. huxleyi virus production wasdelayed in N-depleted enclosures. However, our data suggestthat nutrient availability had no effect on the molecular suc-cession dynamics of E. huxleyi viruses, i.e., the same viruseswere responsible for the termination of the bloom irrespectiveof the growth conditions. If the nutrient conditions had af-fected host-virus interactions, then one would expect that adifferent host(s) would be selected to succeed under theseconditions. Hence, a different virus profile is expected, i.e.,either an additional band(s), the disappearance of a band(s),or a combination of both. If the same host(s) is successful

FIG. 1. Images of gel electrophoresis of PCR fragments amplified in second-stage PCR from a range of virus isolates (21). (A) Agarose gel.Lane 1, lambda DNA digested with PstI; lane 2, E. huxleyi virus 84; lane 3, E. huxleyi virus 86; lane 4, E. huxleyi virus 88; lane 5, E. huxleyi virus 163;lane 6, E. huxleyi virus 201; lane 7, E. huxleyi virus 202; lane 8, E. huxleyi virus 203; lane 9, E. huxleyi virus 205; lane 10, E. huxleyi virus 207; lane11, E. huxleyi virus 208; lane 12, M. pusilla virus PB5; lane 13, P. globosa virus 102; lane 14, no DNA. (B) DGGE gel. Lane 1, E. huxleyi virus (EhV)84; lane 2, E. huxleyi virus 86; lane 3, E. huxleyi virus 88; lane 4, E. huxleyi virus 163; lane 5, E. huxleyi virus 201; lane 6, E. huxleyi virus 202; lane7, E. huxleyi virus 203; lane 8, E. huxleyi virus 205; lane 9, E. huxleyi virus 207; lane 10, E. huxleyi virus 208; and lane 11, all E. huxleyi virus isolates.

2486 SCHROEDER ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 2. (A) Time series obtained for E. huxleyi (}) and E. huxleyi virus (EhV) concentrations (■ ) using flow cytometry between 6 and 24 June2000 in enclosure 1. B, D, and A refer to before, during, and after the bloom, respectively. (B) DGGE gel of PCR fragments amplified insecond-stage PCR from samples collected during the mesocosm experiment. The symbols indicate the 27 bands excised from the gel, and identicalsymbols indicate identical nucleotide sequences. The sequences represented by solid triangles, open circles, solid circles, solid squares, opentriangles, small open square, large open squares, and open diamond were designated E. huxleyi operational taxonomic unit numbers 1 to 8(EhVOTU1 to -8), respectively. Solid circles and solid triangles, two DGGE band profiles observed before, during, and after the bloom; opensymbols, five DGGE band profiles observed before the bloom; solid squares, one band profile observed during and after the bloom. S, standards(same as Fig. 1B, lane 11). The format for dates is day/month/year.

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under all conditions, then the same virus profile is expected.This is what was observed. Hence, the nutrient conditions didnot appear to play a role in the host-virus interactions. Inaddition, if the viral infection cycle were susceptible to theconditions tested, then either the growth rate of the host(s)would be affected or no infection would occur. Neither was thecase (13). A more likely hypothesis is that nutrient availabilitymay simply affect the growth rate of the host and consequentlythe severity of infection. A similar conclusion was reached byJacquet et al. (13).

In summary, it is clear from the DGGE profiles producedprior to the onset of the bloom that a variety of virus genotypesis present in the water column of the fjord. We hypothesizethat these viruses are remnants of previous bloom lysis events.The enrichments of the unfiltered seawater, which was col-lected from the fjord for the mesocosm experiment, resulted inthe formation of an E. huxleyi bloom. Prior to the developmentof the bloom, various virus isolates appeared and disappeareddaily. A possible explanation for this might be that as theindigenous host populations become metabolically active, theyare infected by their respective virus(es) and hence are “re-

moved” from the water column. As a consequence, the virus-(es) might reappear one or a few days later depending on thesuccess of the infection(s). EhVOTU3 is a good example ofthis. We see that this isolate is present the first 3 days of theexperiment (6 to 8 June 2000), disappears for 3 days (9 to 11June 2000), reappears for 1 day only to disappear again (12 and13 June 2000, respectively), and finally reappears throughoutthe progression and subsequent collapse of the bloom (14 to 24June 2000) (Fig. 2B). An alternative explanation for this phe-nomenon could be that it is a direct consequence of the thresh-old concentration and the relative template abundance in thewater samples. This could explain the absence of EhVOTU4prior to the bloom (Fig. 2B, 6 to 13 June 2000).

As the bloom developed, three dominant virus isolates weredetected. This observation suggests that these three isolatesinfected the bloom-forming host population. The makeup ofthis host population is unknown, i.e., we cannot be surewhether the bloom was the product of one or many E. huxleyistrains. Host range analysis revealed that certain virus isolates(separated based on differences in their MCPs) can infect anumber of host strains, while others were more host specific

FIG. 3. Phylogenetic inference based on a distance matrix algorithm between conserved domain I (24) within MCP among a few members ofthe family Phycodnaviridae and a few members of a distantly related family, Iridoviridae. The numbers at the nodes indicate bootstrap valuesretrieved from 100 replicates for both the parsimony and neighbor-joining analyses. The bar represents 1 base substitution per 10 amino acids.Abbreviations: EhV, E. huxleyi virus; PBCV, Paramecium bursaria Chlorella virus; IIV, Invertebrate iridescent virus; LCDV, Lymphocystis diseasevirus; FRGV; Frog virus; EsV, Ectocarpus siliculosus virus.

2488 SCHROEDER ET AL. APPL. ENVIRON. MICROBIOL.

(21). Therefore, the three different virus isolates representedby the three DGGE bands could infect the one E. huxleyi strainthat caused the bloom. Similarly, each virus isolate could infectits respective host strain, and thus the bloom could be theproduct of at least three different E. huxleyi strains. Ultimately,we cannot be certain unless a genetic marker is found toanswer this question. The tools presently available for theenumeration of E. huxleyi diversity are randomly amplifiedpolymorphic DNA (16) and microsatellites (12). However,both of these techniques would be extremely difficult or evenimpossible to use for evaluating community structure, as eachstrain is represented by multiple banding patterns.

The inability to determine the host composition of thebloom does not negate the significance of our observations. Weclearly demonstrated the usefulness of the MCP gene in con-junction with DGGE to distinguish between different virusisolates and hence to give an idea of the diversity of isolatespresent in a bloom dynamic. However, a note of caution iswarranted. The variability in the MCP gene as revealed byDGGE is not necessarily an absolute measure of virus func-tional diversity. However, we were able to show that a few basepair changes in the MCP gene can be correlated with differenthost range profiles and hence are significant enough to serve asmarkers for diversity (21).

In conclusion, we were able to show for the first time thatchanges occurred at the genotypic level within the E. huxleyivirus community throughout the progression of an E. huxleyibloom. This observation highlights what marine virologists hadsuspected for a long time—that we were seriously underesti-mating the level of genotypic diversity in marine viral commu-nities (11). We believe that our understanding of the extent ofvirus diversity in natural communities and their effects on theirhosts needs to be revised. These new data also raise furtherquestions regarding host susceptibility, particularly since ge-

netic diversity has been observed within E. huxleyi bloom pop-ulations (16).

ACKNOWLEDGMENTS

The mesocosm study was supported by the Access to ResearchInfrastructures scheme (Improving Human Potential Program) of theEuropean Union through contract number HPRI-CT-1999-00056. Theresearch was supported by the Marine and Freshwater MicrobialBiodiversity community program, funded by the Natural Environmen-tal Research Council of the United Kingdom (NERC). W.H.W. is aMarine Biological Association of the United Kingdom Research Fel-low. G.M. is an NERC Advanced Research Fellow.

We are grateful to Clelia Booman and Monica Martinussen, De-partment of Fisheries and Marine Biology, University of Bergen, fortheir assistance during the mesocosm study. Thanks to Steven Short(University of British Columbia) for supplying M. pusilla virus strainVPB5.

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