Abstract Over the years, viruses have beenshown to be mortality agents for a wide range ofphytoplankton species, including species withinthe genus Phaeocystis (Prymnesiophyceae). Withits polymorphic life cycle, its worldwide distribu-tion, and the capacity of several of the Phaeocystisspecies to form dense blooms, this genus is a keyplayer for our understanding of biogeochemicalcycling of elements. This paper provides an over-view of what is know to date about the ecologicalrole of viruses in regulating Phaeocystis popula-tion dynamics. It explores which variables aVectthe algal host–virus interactions, and examinesthe impact of virally induced cell lysis of Phaeo-cystis on the function and structure of the pelagicfood web as well as on the Xow of organic carbonand nutrients.
The presence of viruses in marine environments hasbeen acknowledged for many years, and it is nowwell established that viruses are dynamic andimportant members of the microbial food web(Bergh et al. 1989; Proctor and Fuhrman 1990;Gobler et al. 1997; Fuhrman 1999; Wommack andColwell 2000; Weinbauer 2004). Viruses infect notonly the numerically dominant bacteria but alsoprokaryotic and eukaryotic primary producers. Uni-cellular photosynthetic organisms are a major groupof organisms in natural aquatic communities andviruses have been recognized as mortality agents forphytoplankton (Van Etten et al. 1991; Reisser 1993;Brussaard 2004). Viruses or virus-like particles have
C. P. D. Brussaard (&) · A.-C. Baudoux · P. RuardijDepartment Biological Oceanography, Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790AB Den Burg, The Netherlandse-mail: [email protected]
G. BratbakDepartment of Biology, University of Bergen, Jahnebakken 5, 5020 Bergen, Norway
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been reported in many diVerent taxa of eukaryoticalgae, including harmful algal bloom (HAB) species(see review by Brussaard 2004; Nagasaki et al. 2004;Tomaru et al. 2004; Baudoux and Brussaard 2005).
The fate of phytoplankton biomass, whetherthrough sinking, grazing or cell lysis, has majorimplications for carbon and energy cycles inmarine ecosystems. Lysis-mediated release of thecellular content can greatly enhance bacterial activ-ity and subsequently force the food web towards amore-regenerative system. Energy and nutrientsreleased by cell lysis and excretion is transferred tohigher trophic levels via the microbial loop (Azamet al. 1983). Lytic viral infection of phytoplanktoncauses rapid cell lysis, which may aVect not onlythe energy and nutrient Xow, but also the phyto-plankton community composition and succession.Recognizing viral lysis of phytoplankton as a majorprocess has emphasized the importance of themicrobial loop including a viral shunt.
Theoretical models suggest that a 2–10% loss ofphytoplankton due to viral infection increases theXow of organic carbon, bacterial production andrespiration by more than 25% (Fuhrman 1999;Wilhelm and Suttle 1999). Especially during algalblooms, when high algal cell abundances enhancethe virus-host encounter rates, virally mediatedlysis can have a profound eVect on populationdynamics, community diversity and transfer ofenergy and matter within the pelagic food web.
The Phaeocystis genus, with its cosmopolitandistribution, includes several high-biomass-form-ing species (Cadée and Hegeman 2002; Verityand Medlin 2003; Schoemann et al. 2005). Phaeo-cystis has a life cycle dominated by single cells(with and without Xagellae) and embedded colo-nial non-Xagellated cells. Several species of Phae-ocystis, e.g., P. pouchetii and P. globosa, regularlydominate the phytoplankton community andsequester huge amounts of nutrient resources,predominantly in the form of colonies. Theseblooms occur mostly in colder and temperatewaters, such as the coastal zone of the NorthAtlantic and the North Sea. Because of theimportance of these blooms for the pelagic eco-system and the socioeconomic interest in theseHAB species, substantial research has been con-ducted on factors controlling the wax and wane ofthese blooms. With light and nutrients as impor-
tant factors initiating Phaeocystis blooms, grazingand viruses are considered the relevant lossfactors. Field studies indicate that these virusesare a dynamic component, notably involved in thedecline of the blooms. Laboratory and seminatu-ral studies provided insight into host–virus inter-actions and revealed how environmental factorsmay inXuence viral infection. The scope of thepresent paper is to provide a summary and syn-thesis of available information and some unpub-lished data related to Phaeocystis and the virusesinfecting it.
Isolation and characterization of viruses infecting Phaeocystis
Viruses that infect species of Phaeocystis havebeen isolated during and directly after naturalblooms (Jacobsen et al. 1996; Brussaard et al.2004; Baudoux and Brussaard 2005). Phaeocystispouchetii viruses (PpV) were successfully isolatedafter 100-fold concentration by continuous centri-fugation and exposure to ultraviolet (UV) for 15and 30 s (Jacobsen et al. 1996). Exposure to UVlight was intended to cause induction of virus pro-duction in algal cells containing lysogenic viruses,but as the virus isolated was lytic this treatmentwas most likely not necessary. So far, all virusesinfecting eukaryotic microalgae are lytic and nonehave been reported to enter a lysogenic relation-ship with the host. Phaeocystis globosa viruses(PgV) were isolated from Wltered (GF/F What-man glass Wber Wlters) natural water that wasadded to exponentially growing P. globosa hostcultures (Baudoux and Brussaard 2005). Incuba-tion of the natural seawater with the addition ofnutrients for a week at in situ temperature andirradiance (excluding UV) before adding a sub-sample to cultures of P. globosa occasionallyadvanced successful isolation of PgV. At thedecline of the bloom, when most free viruses canbe expected to occur, nutrients regularly becomedepleted. By adding nutrients more algal biomasswas generated and the encounter rate betweenalgal host and virus enhanced.
The Phaeocystis viruses isolated so far arespecies speciWc, i.e., they only infect one of thePhaeocystis species (Jacobsen et al. 1996; Bau-
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doux and Brussaard 2005). Not all P. globosa or P.pouchetii host strains are infected by the samevirus isolates, and not all viruses isolated infectthe same algal host strains. There can be a highdegree of speciWcity for these algal viruses.
All intracellular Phaeocystis virus particlesobserved microscopically to date have beenlocated in the cytoplasm of the algal host cell, arehexagonal in their proWle, are non-enveloped, andlack a tail. The particles are between 100 and160 nm in diameter based on transmission electronmicroscopy (TEM) thin sectioning micrographs(particles tend to shrink when subjected to Wxationand dehydration during preparation for thinsectioning). The hexagonal proWles of the virusessuggest that virions may contain an icosahedralcapsid (Fig. 1). A recent investigation of the nativemorphology of PpV at a resolution of 3 nm usingelectron cryomicroscopy and three-dimensionalimage reconstruction methods revealed that thecapsid had a maximum diameter of 220 nmbetween opposite vertices, and was composed of2,192 capsomers that were organized in large trian-gular and pentagonal aggregates (Yan et al. 2005).
A common ancestor for P. globosa viruses andviruses infecting another prymnesiophyte (Chrys-ochromulina breviWlum) has also been suggestedbased on the phylogenetic analysis using theinferred amino acid sequences of a DNA poly-merase gene fragment (Brussaard et al. 2004).This study also showed that seven PgV isolatesformed a distinct monophyletic group with othereukaryotic algal viruses, despite diVerences in
their genome size and other phenotypical charac-teristics. Wilson et al. (2006) recently isolated avirus infecting P. globosa from surface water inthe English Channel, UK that did not cluster withthe other PgVs. Instead, it was more closelyrelated to C. breviWlum.
The fact that the DNA polymerase gene couldbe ampliWed using the algal virus-speciWc primersAVS1 and AVS2 (Chen and Suttle 1996) allowsassignment of these viruses to the family Phy-codnaviridae (Van Etten 1995). Many of the char-acterized phytoplankton viruses are indeedassigned to this family of large double-stranded(ds) DNA viruses that infect eukaryotic algae.The use of the highly conserved DNA polymerasegene turned out to be a good genetic marker forclassiWcation of dsDNA algal viruses. The Phaeo-cystis viruses have indeed large dsDNA genomes,about 485 kb in size for PpV (Castberg et al.2002), and either 177 or 466 kb for PgV (Fig. 2;Baudoux and Brussaard 2005). After stainingwith a sensitive nucleic-acid-speciWc dye such asSYBR Green I, these large genome sized Phaeo-cystis viruses could be readily detected using epi-Xuorescent microscopy or Xow cytometry (Marieet al. 1999). Furthermore, the use of Xow cytome-try allowed the discrimination of these virusesfrom other viruses such as many other algalviruses (Brussaard et al. 2000) or bacteriophagesin natural samples (Larsen et al. 2001; Baudouxand Brussaard 2005). The ability to detect, dis-criminate and enumerate samples containingPhaeocystis viruses in a rapid and objective man-ner promotes laboratory research on virus-hostinteractions, and ecological studies in the Weld.
Detailed laboratory studies showed that thetotal length of the lytic growth cycles of the Phae-ocystis viruses infecting exponentially growinghost cells ranged between 25 and 50 h (Jacobsenet al. 1996; Baudoux and Brussaard 2005). ForPpV the latent period, the time period from infec-tion until the Wrst increase in the abundance ofextracellular free viruses, was around 12–18 h(Jacobsen et al. 1996). The study by Baudoux andBrussaard (2005) showed three diVerent latentperiods for the various PgV isolates in culture,i.e., 10, 12 and 16 h (Fig. 3). These periods matchthe range of latent periods for all characterizedphytoplankton viruses so far, and are somewhat
Fig. 1 Transmission electron micrographs of thin sectionsof infected cells of Phaeocystis pouchetii. (a) The virus-likeparticles (indicated by arrow) are found in the cytoplasm ofthe cells. (b) Detail of virus-like particles showing the hex-agonal outline of the viruses
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shorter or comparable to the maximum growthrates of their host (Schoemann et al. 2005; Vel-dhuis et al. 2005). Based on the decline in thealgal host population and the increase in extracel-lular virus particles, a conservative estimate of theburst size (number of viruses released per hostcell that underwent lysis) can be estimated. Forboth Phaeocystis species, burst sizes ranged onaverage between 250 and 500, with the higherburst sizes for P. pouchetii. The variation in burstsizes between diVerent PgV isolates was consider-able, with values down to around 100 despite theexponential growth of the algal host cells (Bau-doux and Brussaard 2005). Although the burstsize strongly aVects the chances for infection of
the remaining cells in the host population, not allviruses are infectious. In exponentially growingcultures, the percentage of infective Phaeocystisviruses produced as determined by the most prob-able number (MPN) method is generally rela-tively high, ranging from 60 to about 100%(Bratbak et al. 1998; Brussaard, unpubl. data).
The devastating eVect on Phaeocystis cells ofan infection by lytic viruses is well illustrated bythe morphological, physiological and viabilitystatus of the host population during infection(Jacobsen et al. 1996; Bratbak et al. 1998, Bruss-aard et al. 1999, 2001). Even though the photo-synthetic apparatus of the infected algal cellsseem to be active during the Wrst hours afterinfection, sudden and sharp declines in the photo-synthetic eYciency of the cells were observed atthe end of the latent period (Fig. 4). In contrast tothe above-mentioned assays, which reXect the sta-tus of the entire population, the use of Xowcytometry allows the analysis of individual cells.Changes in the cell characteristics of the virallyinfected cells are dynamic in time, with the pro-portion of cells with increased cellular DNAincreasing in the Wrst hours after infection, fol-lowed by a decline in cellular scatter signals whenvirions are formed. Prior to cell lysis, the redautoXuorescence declined concomitantly with thedisruption of the organelles (as observed by trans-mission electron microscopy). By the time theWrst viruses are released from the host cells theportion of dead cells increased (Brussaard et al.2001). Finally, during the period of cell lysis a sub-population of cells showing reduced concentrationof cellular DNA developed and increased with time.
Occurrence and dynamics of Phaeocystis viruses
Although the observations of Phaeocystis cellscontaining virus-like particles and the isolation ofviruses infecting Phaeocystis species suggest thatviruses may be potentially important, it is the suc-cession of Phaeocystis algal cells and free virusesunder seminatural conditions that implies a directcausative relationship and ecological signiWcance.At present, two mesocosm studies and two Weldstudies have been performed, all showing highlydynamic Phaeocystis virus abundances with time
Fig. 2 Viral genome sizes of diVerent Phaeocystis globosavirus isolates (PgV) determined by pulsed-Weld gel electro-phoresis (PFGE). Lane M: Lambda concatamers ladder,Lane 1: uninfected culture of P. globosa, Lane 2: PgV-04(genome size of 175 kb), Lane 3: PgV-12 T (genome size of465 kb). The small-sized band (approximately 45 kb) asseen in lanes 1–3 correspond to bacteriophages since algalcultures were not axenic
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and closely linked to the abundance of their host(Larsen et al. 2001; Brussaard et al. 2004; Bruss-aard et al. 2005a; Baudoux et al. 2006). For allthese studies the abundance of Phaeocystis viruseswas 30- to 100-fold higher during bloom maximathan the abundance of their host, suggesting thatviruses should indeed be regarded as importantmortality agents for P. globosa and P. pouchetii.During the periods of study, the Phaeocystisviruses generally made up between 0 and 5% ofthe total virus population, independent of whetherPhaeocystis was dominating the phytoplanktoncommunity or not. As bacteria are the numericallymost abundant, the low share of Phaeocystisviruses is to be expected. However, under speciWcconditions favoring the single-cell morph as com-pared to the colonial form, the portion of PgVincreased up to 30% of total virus abundance(Brussaard et al. 2005a). Under such conditionsviruses were actually able to prevent a build-up ofstanding stock (i.e., bloom) of P. globosa.
A critical note here is that successful infectionof Phaeocystis does not depend on the total abun-dance of Phaeocystis viruses, but on the numberof infectious viruses. A recently published ecosys-tem model that was calibrated with a large dataset from P. globosa mesocosm experiments(Ruardij et al. 2005) suggested that the fraction of
infective PgV successfully infecting P. globosacells increased steeply over the course of thebloom to a maximum of 0.035 (Fig. 5). The frac-tion of infective PgV that successfully infectP. globosa was highest when single cells domi-nate. The absorption of PgV to transparent exo-polymer particles (TEP) that are formed upondisintegration of colonies reduces the availableinfective PgV and subsequently the fraction ofPgV that successfully infects P. globosa (Fig. 5).The very low value prior to bloom formation(0.0005%) represents the situation at the start ofthe growing season when PgV standing stock wassubjected for a long time (autumn till spring) toloss of infectivity and decline in actual virus parti-cles. At the same time new virus production wasinsigniWcant for P. globosa when host cells werepresent in very low numbers and barely growing(due to light and temperature limitation duringwinter). A Weld study in the turbid coastal watersof the southern North Sea showed that thefraction of infectious PgV was around 0.04 (Bau-doux et al., 2006), matching very well with themodel situation. It is noteworthy that forP. pouchetii also a low host–virus adsorptioneYciency was needed to produce reasonable goodWt between simulation and experimental observa-tions (Bratbak et al. 1998).
Fig. 3 Abundance of Phaeocystis globosa (a, c, e) and PgV(b, d, f) according to Baudoux and Brussaard (2005). Opensquare symbols represent uninfected cultures, while theWlled circles represent virally infected P. globosa. (a) P.globosa infected with PgV-07T, (c) with PgV-05T, and (e)
with PgV-04T. Filled diamond symbols represent the viralgrowth cycles of (b) PgV-07T, (d), PgV-05T, and (f) andPgV-04T. The length of the latent period (indicated by thedotted line) was 10 h for PgV-07T, 12 h for PgV-05T, and16 h for PgV-04T
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The strong dynamics of the Phaeocystis virusesindicate that, besides substantial production, theviral particles are also lost. Ways of removal caninclude passive adsorption of viruses onto theabundant organic aggregates (TEP) that aregrazed upon, or onto inorganic colloids (clay,sand) that are removed from the euphotic zone bysinking (Kapuscinski and Mitchell 1980; Bruss-aard et al. 2005b). Other factors aVecting the lossof the virus particles or infectivity are grazing byprotozoa, enzymatic hydrolysis, and UV radiationas it damages the viral nucleic acids (Kapuscinskiand Mitchell 1980; Suttle and Chen 1992; Gon-zález and Suttle 1993; Noble and Fuhrman 1997;Jacquet and Bratbak 2003). Despite the dynamicnature and the substantial losses of PgV, pheno-typic characterization and molecular analysis ofPgV isolates collected one year apart from thesame area revealed identical sequences, indicatingconsiderable stability of these PgV populations
(Brussaard et al. 2004; Baudoux and Brussaard2005). Isolation of PgV during periods with verylow to undetectable P. globosa host abundance,furthermore, suggests this robust group of viruseshas a constant presence in the water column inthese coastal areas where Phaeocystis occurs.
Diversity of Phaeocystis viruses and its ecological role
To achieve successful infection a virus depends onthe encounter rate and thus on the abundance ofits host species. The Phaeocystis virus isolateshave, however, not only a species-restricted hostranges but most often also a strain-speciWc spec-trum of infection (Jacobsen et al. 1996; Baudouxand Brussaard 2005). Thus, not all strains of aPhaeocystis species (e.g., P. globosa) will becomeinfected, even when coexisting in the same watermass. Factors inXuencing this are the ability of thevirus to bind optimally to a proper host cell, aswell as the sensitivity of the host to infection.
Based on the structural capsid protein compo-sition, 12 PgV isolates that originate from thesouthern North Sea could be divided in twogroups (Baudoux and Brussaard 2005). The pro-teins on the surface of free viruses serve as ameans of virus-to-cells attachment and allowtransfer of viral genomes into the host cells. Anychanges in the composition of these structuralproteins between viruses may aVect the binding tothe host cell’s receptors, selecting for host rangerestriction.
Considering that some of these diVerent PgVisolates and their algal host strains originatefrom the same water sample, the ecologicalimpact of such virus–host diversity is intriguing.Infection by a speciWc PgV does not act merelyat the total host species population level, butrather on the subpopulation level. Essentialadvantages of such virus diversity could be thepromotion of coexisting P. globosa strains toguarantee the availability of algal hosts. If one(dominating) strain of P. globosa gets infectedand undergoes lysis, another resistant P. globosastrain that otherwise might be less Wt for compe-tition for nutrients, for example, can Wll theniche. The lysis of the infected P. globosa strain
Fig. 4 Dynamics of in vivo chlorophyll Xuorescence (Fo)and photosynthetic eYciency (Fv/Fm) of Phaeocystis glob-osa during viral infection as assessed by Xuorometry. Opensymbols represent uninfected cultures, while the Wlled sym-bols represent virally infected P. globosa. Maximum Xuo-rescence (Fm) was obtained after addition of thephotosystem II inhibitor DCMU (20 �M Wnal concentra-tion). Fv equals Fm-Fo. Data are expressed in relative units(r.u.)
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might even promote the growth of the otherstrain(s) indirectly as regenerated nutrientsbecome available. There is increasing evidenceaccumulating that speciWc geographical popula-tions of an algal species can be morphologicallyand/or genetically heterogeneous (Barker et al.1994; Rynearson and Armbrust 2000, 2004) andviruses might actually be an important regulat-ing factor for host heterogeneity.
Besides diVerent PgVs infecting diVerentP. globosa strains, some of the diVerent PgVsinfected the same range of P. globosa strains. Ifpresent at the same time in the water, these diVer-ent PgV species might have to compete for thesehost strains. To our knowledge, this is a com-pletely unexplored Weld of research to date.Important factors aVecting the outcome of com-petition between viruses for the same host cellinclude the total standing stock of infectiveviruses in order to enhance the contact rate, theability to prevent co-infection by other virustypes, the burst size, and the level of resistant toloss of infectivity. DiVerences in burst size andsensitivity to virucidal factors have been observed(Baudoux and Brussaard 2005), but to whatextent this inXuence the actual competition isunknown.
Resistance to viral infection
As discussed above, some strains of the Phaeocys-tis species are resistant to viral infection. But even
for one Phaeocystis strain the susceptibility toviral infection may not be constant. A laboratorystudy with P. pouchetii showed that some algalhost cells escaped infection with the lytic PpV,and with time the algal population increased inabundance again (Thyrhaug et al. 2003). SincePpV originating from these cultures and the origi-nal stock aVected sensitive P. pouchetii culturesequally, the resistance to viral infection must havecome from changes in the algal phenotype. Suchphenotypic plasticity of the host’s susceptibility toinfection may be an important stabilizing factor inhost–virus interactions as it seems to sustain long-term (one-year) coexistence of host and virus.
The research related to PgV characterizationand Phaeocystis–virus interactions performed todate generally involved single cells (Xagellatedand non-Xagellated). Phaeocystis is, however,known to form colonies and most often theembedded colonial non-Xagellated cells are thepredominant cell morph during bloom events. Alogical question is thus if embedded colonial cellsare perhaps resistant to viral infection. This ques-tion was raised already by Jacobsen et al. in 1996upon isolation of PpV, and it was speculated atthe time that the gelatinous material the colonialcells are surrounded by serves as a protectionagainst viral infection (Jacobsen et al. 1996; Brat-bak et al. 1998). The presence of an outer thin, yetmechanically stable “skin”, likely with pores<4.4 nm, has also been suggested as a defenseagainst viral attack of the cells within the colony(Hamm et al. 1999). To test these hypotheses is
Fig. 5 Results of a mesocososm study on Phaeocystis glob-osa bloom dynamics and the ecological role of viruses. Themeasured data are represented by the symbols and themodeled results are represented by the lines. (a) measuredand modeled biomass of P. globosa colonies (solid line andopen symbol) and single cells (outline of grey area and
Wlled symbol), (b) measured and modeled abundance ofP. globosa viruses (PgV), and (c) modeled fraction of infec-tive PgV without transparent exopolymer particles (TEP)present (white area) and in the presence of TEP (greyarea). It was assumed that infectivity declined by 10% d¡1
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nevertheless not easy as colonies will always shedat least some single cells, which become readilyinfected. The consequent production of virusesmight thus incorrectly suggest that colonies canbe infected. An attempt to infect colonialP. pouchetii cells with PpV have so far beenunsuccessful (Jacobsen et al. 2005). Tests using12 PgV isolates to infect strains of P. globosa thateither formed distinct colonies or mucus aggre-gates were unsuccessful for all but one strain ofPgV (Baudoux and Brussaard 2005). Thus, mucusformation might protect Phaeocystis cells fromviral infection, but the positive infection of P.globosa strain Pg01MD-04 by PgV-01T indicatesthat the protection is not exclusive.
A mesocosm experiment studying the regula-tory role of viruses on P. globosa populationdynamics sheds more light on the topic (Brussaardet al. 2005a). Both single cells and colonies origi-nating from the same clone were present at thestart of the experiment in low concentrations. Theresults show that the morphology of the P. globosacells (solitary vs. colonial) diVerently regulatedviral control of P. globosa. Under non-limitingconditions, dense blooms of colonies and to alesser extent single cells were formed. Viruseswere found to be a signiWcant loss factor but couldnot prevent bloom formation. Under conditionsthat restricted colony formation for the Wrst11 days but allowed single cells to grow, viruseswere found to prevent bloom formation themoment conditions were no longer limiting colonyformation. The maximum standing stock of PgVwas also Wvefold higher. The results suggest thatthe colonial form of Phaeocystis is indeed anexcellent mechanism to prevent viral infection.Interestingly, a recently developed mathematicalecosystem model including a detailed virus mod-ule (Ruardij et al. 2005) suggests that the size ofthe colonies strongly reduces the chance of infec-tion per cell (and not the gelatinous matrix). Thephysical principle of the spherical equivalentdiameter determining the encounter rate wasmodeled earlier by Murray and Jackson (1992).With increasing diameter of the colonies, viralinfection seems an insigniWcant loss factor (Fig. 6).The model implies that the single cells were stillreadily infected, which prevented a build-up ofP. globosa biomass. By the time colony formation
was no longer limited there was not enough stand-ing stock of single cells to form colonies.
An additional but important indirect defensemechanism against virus infection is the forma-tion of TEP during colony disintegration. TEPwas found to be a strong stabilizing negative-feed-back mechanism (Brussaard et al. 2005b; Mariet al. 2005; Ruardij et al. 2005). Without coloniesand thus without TEP, fewer viruses are neededto control the population of single cells.
Keeping in mind that algal growth is regulated byenvironmental factors such as irradiance, nutri-ents and temperature, and the fact that viral repli-cation is dependent on the metabolism of thehost, it is of interest to know the impact of thesefactors on virus–host interactions. Under nutrientdepletion, for example, the burst size of releasedP. pouchetii and P. globosa viruses per infectedcell is lower than under nutrient-replete condi-tions, especially under P-depletion (Bratbak et al.1998; Brussaard unpubl. data). The stronglyreduced burst size found for PpV upon infectionof P. pouchetii in the stationary phase of growth(15 compared to 240 cell¡1 for P. pouchetii in theexponential growth phase) is most likely theresult of severe nutrient depletion inhibiting cellgrowth (Bratbak et al. 1998).
Fig. 6 Viral infection rate of P. globosa colonial cellsembedded in a colonial matrix as compared to that ofP. globosa single cells
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How nutrient depletion aVects not only Phaeo-cystis’ physiology and viral replication but alsoindirectly the relationship between the Phaeocys-tis host cell and the virus becomes clear when con-sidering the diVerent Phaeocystis morphotypes.Cells inside the colonial matrix are barelyinfected, but this changes when the colonies disin-tegrate due to nutrient depletion. Because of thepresence of a diVusive boundary layer, colony for-mation will decrease nutrient uptake and, there-fore, colonies experience nutrient depletion fasterthan single cells (Ploug et al. 1999). The liberationof large numbers of single cells promotes viralinfection, resulting in high viral lysis rates (Ruar-dij et al. 2005). Nutrient deWciency also hasanother mode of controlling virus–host interac-tions, which is by the scavenging properties ofTEP. The percentage of PgV attached to TEP wasfound to be higher under P- than under N-deW-ciency and consequently the number of PgV forsuccessful infection will be lower (Brussaard et al.2005b).
Another illustration that growth conditions canaVect viral kinetics is the fourfold lower burst sizefor PpV-infected P. pouchetii when placed in thedark compared to those kept in the light. Interest-ingly, viral proliferation was not delayed or pre-vented in the dark, indicating that PpV was notdependent on host photosynthesis (Bratbak et al.1998). Similar results have been found for themodel system P. globosa–PgV (Brussaard,unpubl. data), which could indicate that this is agenus-wide feature. Since the level of irradiancestrongly aVects colony formation (Peperzak1993), it also indirectly impacts on the level ofviral control (Brussaard et al. 2005a).
UV radiation aVected P. pouchetii–PpV inter-actions diVerently, depending on the type of UVradiation. UV-B radiation strongly inhibited viralinfectivity, whereas UV-A radiation had no eVect(Jacquet and Bratbak 2003). A fascinating addi-tional Wnding was the reduced sensitivity to UV-Bstress of P. pouchetii cells that previously escapedviral infection. Theoretically, these virus- andUV-resistant cells would have a huge advantagecompared to sensitive cells. The fact that they arenot dominating the population suggests that theremust be a negative trade-oV and it is tempting tospeculate that these resistant cells may have lower
growth rates due to inferior nutrient aYnity. Any-way, it does stress the need for studies examiningin more detail the eVects of combined exposure topotentially regulating factors.
Virally induced mortality of Phaeocystis
Having established the presence, dynamics anddiversity of Phaeocystis viruses, one may wonderwhat is the actual impact of those viruses on theloss rates of Phaeocystis. Earlier studies onP. globosa bloom dynamics showed that cell lysiswas a relevant loss factor, with rates of up to0.3 d¡1 (Brussaard et al. 1995, 1996). Although itwas not clear at that time whether viruses werecausing the algal cells to undergo lysis, recentstudies indicate that viruses are most likely theresponsible lysis agents (Larsen et al. 2001; Bruss-aard et al. 2004; Baudoux and Brussaard 2005;Brussaard et al. 2005a). The use of live/deadassays indicated that viral lysis rates of infectedPhaeocystis in cultures showed rates as high as0.8 d¡1 (Brussaard et al. 1999). Methods for spe-ciWc and accurate determination of viral-mediatedalgal mortality in natural waters are however stilllacking. The Wrst attempt to estimate viral lysisrates of P. globosa cells during a bloom was per-formed for a set of mesocosm experiments(Brussaard et al. 2005a), and was based on the netproduction of PgV, a conservative viral loss rateof 0.07 d¡1 used to correct viral production, andan assumed viral burst size of 300 (Baudoux andBrussaard 2005). The estimated viral lysis rateswere around 0.2 d¡1 during the bloom, andlargely accounted for most of the total cell lysis,which was either obtained using the dissolvedesterase activity assay or by subtracting thenet algal growth rate (equals the change in netabundance) and the microzooplankton grazingrate (using the dilution method) from the grossalgal growth rate (derived from DNA cell cycleanalysis).
The only Weld study speciWcally estimatingvirally mediated mortality of P. globosa cells todate (Baudoux et al. 2006) was executed using anewly developed method, being an adaptation ofthe classical dilution method to determine micro-zooplankton grazing rates (Landry and Hassett
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1982; Evans et al. 2003). Besides dilution of thenatural sample in 0.2 �m pore-size Wltered seawa-ter at diVerent ratios, parallel samples werediluted in seawater that was made virus-free byultraWltration through 30 kDa cartridges. As dilu-tion results in a reduction in viral infection and/orgrazing pressure, the subsequent increase in algalcell abundance after 24 h incubation is used to thedetermine the actual impact of grazing and virallysis on the algal population. The usefulness ofthis adapted method for Phaeocystis was vali-dated by testing a culture of P. globosa cells in thepresence of viruses and the absence of grazers.During two consecutive years, natural viral lysisand microzooplankton rates were determined forP. globosa during the annual spring bloom events.Whereas during bloom development microzoo-plankton grazing seemed to be the dominant lossfactor for P. globosa cells, viral lysis becameincreasingly important over the course of thebloom, with rates comparable to the grazing rates(max. 0.35 d¡1). At times, viral lysis made upmore than 50% of the total losses of the single-cell population (Baudoux et al. 2006).
Despite the dependence on certain assump-tions, the discussed studies clearly imply that virallysis is an ecologically signiWcant mortality factorfor Phaeocystis cells. Modeling also backs up theevidence that viral lysis can be an important causeof P. globosa cell mortality, especially under con-ditions where single cells dominate (Ruardij et al.2005). Comparing model situations that lack andcontain colonies, grazing as well as viral lysis arestrongly enhanced with sixfold higher maximum
grazing rates and 40-fold higher maximum lysisrates (Fig. 7). Colonies thus seem to have theadvantage of being largely protected against graz-ing and viral infection, explaining their build-upof biomass (bloom). The only signiWcant loss fac-tor for cells inside colonies seems to be automor-tality, which becomes important during nutrientdeWciency as a result of strongly limited nutrientdiVusion (Ploug et al. 1999; Ruardij et al. 2005).
Note that the model suggests that grazing indi-rectly aVects the impact of viral infection on thepopulation dynamics of P. globosa (assumingequal grazing on both uninfected and virallyinfected cells). Without grazing single-cell bio-mass would increase faster, but viral infectionwould take place earlier, resulting in an overalllower standing stock of single cells (Ruardij et al.2005).
Impact of viral lysis of Phaeocystis on the microbial food web and element cycling
From earlier studies we know that the fate ofPhaeocystis primary production is of importancefor the distribution of energy and biogeochemicalcycling within the pelagic and benthic ecosystems(Schoemann et al. 2005). Substantial viral lysis ofPhaeocystis will provide a sizable source of dis-solved organic matter, thereby promoting a reten-tive system that oxidizes organic matter andregenerates inorganic nutrients in the euphoticzone (Brussaard et al. 1996; Gobler et al. 1997;Wilhelm and Suttle 1999; Ruardij et al. 2005).
Fig. 7 Modeled abundance of Phaeocystis globosa cellsand mortality rates of P. globosa during a mesocosm study(Ruardij et al. 2005). (a) A model run with colonies, and (b)a run without colonies present. Viral lysis is represented bythe outline of the black area, microzooplankton grazing by
the grey area, and automortality by the white area. Auto-mortality aVects cells with a net growth rate of <0.002 d¡1,which was only found to be of importance for cells embed-ded in the colonies under nutrient depletion. The dottedline represents the total biomass of P. globosa cells
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Bratbak et al. (1998) showed that viral infectionof P. pouchetii resulted in a conversion of theentire algal biomass to dissolved organic carbon(DOC) within three days. In contrast, this wasonly a maximum of 20% in the uninfected cul-tures. In response to viral lysis of a Phaeocystisblooms, bacterial production has been shown toincrease rapidly (Brussaard et al. 2005b). Assum-ing a bacterial conversion factor of 0.35 (interme-diate of values reported for phytoplankton celldebris and lysis products; Biddanda 1988; VanWambeke 1994), most to all of the bacterial Cdemand could be accounted for by P. globosacellular C release upon viral lysis. Concomitantly,a shift in bacterial community composition wasobserved, which was most likely the result of thediVerence in the DOC pathway. Instead of thenormally slow and steady release of smallamounts of photosynthetic release of DOC(favoring K-strategists or equilibrium popula-tions), there is the sudden virally induced releaseof large amounts of readily degradable andorganic nutrient-rich DOC (favoring r-strategistsor opportunistic populations).
Most of the Weld data that are available to daterelate to Phaeocystis blooms. Although the virallymediated cell lysis of Phaeocystis during blooms isnotably substantial, there is no proof of infectionof single cells beyond the bloom period. This doesnot mean that there could not be signiWcant virallysis of Phaeocystis outside the blooming period.Production of single cells can also take place athigh rates when colony formation is not possible,e.g., low irradiances and nutrient concentrations.This provides not only the potential of grazing bymicrozooplankton, but also of viral infection ofthe single cells. The relatively small fraction of dis-solved organic matter (DOM) obtained in this waymay constitute a signiWcant portion of the cyclingof rapidly degradable carbon in the pelagic zone.
The theoretical models considering the inXu-ence of (algal) viruses on the carbon cycle thatexist to date are steady-state models assuming aWxed percentage of the algal population dying dueto viral lysis. A bloom of Phaeocystis in, for exam-ple, temperate eutrophic coastal waters is, how-ever, clearly not a steady-state situation. Based onthe ecosystem model by Ruardij et al. (2005), weestablished a carbon budget for the main players
during the wax and wane of a P. globosa bloom(Fig. 8 and Table 1). P. globosa dominated pri-mary production only when colonies were present(68% of total), but was still one-third of the totalprimary production under conditions dominatedby single cells. The averaged daily Xux of viral lysisof P. globosa was tenfold higher for the model sit-uation with only single cells present compared tothe situation including colonies (115 vs. 11 �g CL¡1). Without colonies, viral lysis made up for53% of the modeled daily P. globosa primary pro-duction (only 2% when colonies were present).
The reason that the averaged daily uptake of Cby bacteria is not higher under the conditions thatsingle cells dominate (compared to colony domi-nated conditions) and bacterial secondary pro-duction is stimulated due to P. globosa viral lysis,is due to the absence of TEP production upon dis-integration of colonies. Because of the large inXu-ence colonies have on the C cycling as a result oftheir high content of intracellular carbon, the Cbudget for a model run without viruses resembleslargely that of the run with viruses present (datanot shown). When excluding both colonies and
Fig. 8 SimpliWed representation of a pelagic food web usedto calculate the C budget during a Phaeocystis globosabloom as presented in Table 1. The release of cellular car-bon due to viral lysis as sources of DOC, as well as auto-mortality was only modeled for P. globosa. Excretion ofDOC due to photosynthetic release was taken into accountfor all phytoplankton groups (Phytopl.: Phaeocystis andother algae). Micrograzers (Micropl.) include heterotro-phic nanoXagellates (HNF) and ciliates. Export was consid-ered a negligible loss
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viruses, grazing on P. globosa becomes logicallymore important (data not shown). This modelingexercise suggests that Phaeocystis viruses have asigniWcant impact on organic C cycling, but theunderlying processes and their interactions arecomplex.
The lysis of cells does not only release organiccarbon into the surrounding waters, but also othergeochemical important elements such as, N, P, S,Fe, Mn and Zn become available for biologicalcycling (Gobler et al. 1997; Ruardij et al. 2005).Nitrogen and phosphorus are well-known bot-tom-up controllers of phytoplankton productivity.Especially during periods that heterotrophic bac-teria are carbon- instead of nutrient-deWcient intheir growth (common during Phaeocystisblooms) the shunting of lysis products through
the viral shunt can result in a net remineralizationof N and P. Since the available inorganic nutrientscan nourish algal growth again, viral infection ofPhaeocystis leads to death on one side and sus-tained primary production of uninfected Phaeo-cystis strains or diVerent phytoplankton specieson the other.
Iron is a nutritive trace element whose role as alimiting agent for algal growth has been demon-strated in areas where certain Phaeocystis species(e.g., P. antarctica) are also commonly found.Viral lysis will aVect the absolute concentration ofiron that is potentially available for biologicalrequirement, but may also directly aVect the spe-ciation and bioavailability as iron is mostly com-plexed with organic ligands and colloids.
As a major dimethyl sulWde (DMS) and dim-ethylsulfoniopropionate (DMSP) producer, virallysis of Phaeocystis cells has been reported animportant mechanism for the release of theseorganic sulphur compounds (Malin et al. 1998).Viral infection of P. pouchetii resulted in an eight-fold increase of DMS concentration acomparedto an uninfected culture. The high productivityassociated with Phaeocystis blooms in combina-tion with its world-wide distribution (Schoemannet al. 2005) makes the genus not only an impor-tant contributor to the marine carbon Xux butalso to the global sulphur cycle. It was estimatedthat the contribution of Phaeocystis to the globalXux of DMS is 5–10% (Schoemann et al. 2005).As the atmospheric trace gas DMS aVects cloudcover, viral lysis of Phaeocystis cells is a majorlink in the biogeochemical cycling of this climate-relevant element.
Future perspectives
To date viruses have been isolated and broughtinto culture for the bloom-forming species P.pouchetii and P. globosa. The genus includes,however, more species of which only one other isknown to form a colony bloom (P. antarctica).Given the dependence of viral induced algal mor-tality on virus and host cell abundance, it wouldbe of special interest to be able to bring into cul-ture virus–host model systems of species that donot form such dense blooms and compare with
Table 1 Modeled C budget of the wax and wane of a Phae-ocystis globosa bloom (Brussaard et al. 2005a)
The daily C Xuxes (�g C L¡1 d¡1 ) originate from an eco-system model by Ruardij et al. (2005) and are averaged val-ues over a period of 36 days. The standard run representsthe situation as was observed during the mesocosm experi-ment, with both P. globosa single cells and colonies present.Viral lysis of phytoplankton is speciWc for P. globosa. Virallysis of bacteria is a second order density-dependent mor-tality. Respiration and high refractory DOC were modeledbut are not included in the table
C-Xux (�g C L¡1)
With colonies (standard run)
No colonies
PhytoplanktonPrimary production
P. globosa605 215
Primary production other algae
290 435
Viral lysis P. globosa 11 115Automortality
P. globosa4.2 0
Excretion P. globosa 189 35Excretion other algae 104 117Bacteria
ZooplanktonGrazing on bacteria 64 53Grazing on P. globosa 158 62Grazing on other algae 54 143Grazing on
microzooplankton143 115
Zooplankton excretion 125 113
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those that do. Furthermore, the geographical dis-tribution of the diVerent Phaeocystis species isdiverse which would provide an excellent oppor-tunity to investigate the role of Phaeocystisviruses in the various ecosystems. A start hasbeen by comparing the two diVerent virus–hostmodel systems that are available in culture, basedon not only the more traditional basic virologicalcharacteristics but also on their genetic material.Full genome sequencing of PpV and PgV iscurrently in progress (University of Bergen,Norway and DOE-JGI). Once the sequences areavailable, exciting possibilities such as the devel-opment of methods for speciWc gene detection,function and activity assessment will be withinreach.
As there are diVerent virus types infecting thesame Phaeocystis species, primer/probe develop-ment for the speciWc viruses will enhance ourknowledge on virus diversity related issues. Thistype of research could also be of great help tostudy the ecological role of viruses for Phaeocystisduring periods or in regions of low Phaeocystisabundance. Comparative genomics of the diVer-ent virus types as planned for PgV is a challengethat is expected to provide insight into how theseviruses coexist for the same host.
The newly developed data-based ecosystemmodel including a virus module by Ruardij et al.(2005) has proven to be very useful providinginsight towards a comprehensive understandingof the role of viruses for P. globosa populationdynamics and C cycling. It is, however, still farfrom complete. Aspects such as processes inXu-encing PgV infectivity and losses are essentiallyunstudied. For example, is grazing of Phaeocystisviruses by protozoans an ecologically importantloss factor? More detailed mechanistic-orientatedexperiments will help solve these unknowns.
Acknowledgements We acknowledge Runar Thyrhaugfor providing TEM images of PpV viruses and PpV infect-ed Phaeocystis pouchetii cells.
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