VIRAL INFECTION OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) LEADSTO ELEVATED PRODUCTION OF REACTIVE OXYGEN SPECIES1

Claire Evans,2 Gillian Malin, Graham P. Mills

Laboratory for Global Marine and Atmospheric Chemistry, School of Environmental Sciences, University of East Anglia,

Norwich NR4 7TJ, UK

and

William H. Wilson

Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UK

The effect of viral infection of Emiliania huxleyi(Lohman) Hay and Mohler on the concentration ofintracellular reactive oxygen species (ROS), hydro-gen peroxide (H2O2) excretion and cell photosyn-thetic capacity (CPC) was examined. During thecrash of an E. huxleyi culture induced by virusesintracellular ROS concentrations were generally el-evated and reached levels of approximately doublethose observed in non-infected control cultures.H2O2 concentrations also increased in the mediaof the infected cultures from background levels ofaround 130 nM to approximately 580 nM while lev-els in the controls decreased. These data suggestthat oxidative stress is elevated in infected cells. Al-though the precise mechanism for ROS productionwas not identified, a traditional defense related oxi-dative burst was ruled out, as no evidence of a rapidintracellular accumulation of ROS following addi-tion of the virus was found. CPC declined substan-tially in the infected culture from a healthy 0.6–0arbitrary units. Clearly infection disrupted normalphotosynthetic processes, which could lead to theproduction of ROS via interruption of the electrontransport chain at the PSII level. Alternatively, ROSmay also be a necessary requirement for viral rep-lication in E. huxleyi, possibly due to a link with vi-ral-induced cell death or associated with generaldeath processes.

Key index words: Emiliania huxleyi; Fv/Fm; hydro-gen peroxide; oxidative stress; photosynthesis;reactive oxygen species; virus

Abbreviations: CM-H2DCFDA, 5-(and-6)-chlorome-thyl-20,70-dichlorodihydrofluorescein diacetate;CPC, cell photosynthetic capacity; DCF, dichloro-fluorescein; DCFH, 2070-dichlorohydrofluorescin;DCMU, 3(3,4-dichlorophenyl)-1,1-dimethyl urea;EhV, Emiliania huxleyi virus; HIV, human immuno-deficiency virus; MOI, multiplicity of infection;PCD, programmed cell death; POPHA, (p-hydro-

xyphenyl) acetic acid; ROS, reactive oxygen spe-cies; TE, Tris EDTA buffer

Viruses are active components of the marine microbialcommunity and potentially significant mediators of algalmortality (Brussaard 2004). In particular, viruses can dec-imate microalgal blooms (Tarutani et al. 2000, Wilsonet al. 2002) and have been linked to low-level, continuouscropping of phytoplankton populations (Cottrell andSuttle 1995). As the extent of virus involvement in themortality of eukaryotic photosynthetic organisms has be-come apparent, the impact of viral infection on physio-logical parameters such as membrane integrity, enzymeactivity and photosynthesis in phytoplankton has beeninvestigated (Seaton et al. 1995, Brussaard et al. 1999,2001). However, to our knowledge no study has deter-mined the effect of viral infection on the production ofreactive oxygen species (ROS) in algal cells.

ROS are potent oxidants and can be radicals or re-active, nonradical compounds derived from oxygenincluding the superoxide anion (O2 � � ), the hydroxylradical (HO � ), and hydrogen peroxide (H2O2). All liv-ing cells produce ROS as a consequence of normalmetabolism and possess a broad range of mechanismsincluding enzymes and antioxidants compounds to re-move them (Ledford and Niyogi 2005). Oxidativestress is the result of damage to cellular constituentscaused by the presence of detrimental levels of ROSthat occurs when ROS production exceeds the cell’selimination capacity. This is a common phenomenonduring viral infection of both plant and animal cells asa result of a number of mechanisms (Schwarz 1996).Following pathogen recognition, some cells exhibit de-fense related oxidative bursts, which are characterizedby the intense accumulation of ROS within hours andsometimes minutes of infection (Doke 1985). Oxidativebursts serve a number of protective functions includ-ing direct damage to the pathogen (De Gara et al.2003) and ultimately the induction of programmed celldeath (PCD), which is thought to limit the spread ofthe invading organism (Jones and Dangl 1996). ROSproduction during viral infection may also result from

1Received 26 July 2005. Accepted 26 June 2006.2Author for correspondence: e-mail ceva@pml.ac.uk.

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J. Phycol. 42, 1040–1047 (2006)r 2006 by the Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2006.00256.x

disruption to metabolic pathways by the alteration ofelectron transport (Balachandran et al. 1997), which inplants has been linked to the interruption of photo-synthesis (Montalbini and Lupattelli 1989). Finally, insome viral systems of both plants and animals, elevatedlevels of ROS have been implicated in the viral repli-cation process (Schwarz 1996).

ROS are important with regards to both the ecologyand biogeochemistry of the oceans. Certain phyto-plankton species produce ROS under non-stressedconditions without triggers or stimulants (Oda et al.1998), although most reports of ROS production fromalgae are in response to chemical or environmentalfactors such as metal toxicity and UV radiation(Rijstenbil 2001, Pinto et al. 2003). Studies to assessthe distribution and concentration of ROS in seawaterhave focused on the relatively stable compound H2O2.Typically, H2O2 is found at concentrations in the tens ofnanometer although much higher concentrations havebeen recorded, e.g. 220 nM in the Atlantic Ocean(Yuan and Shiller 2001). In addition to biological for-mation, H2O2 is formed by chemical and predomi-nantly photochemical means and can be introduced bywet and dry deposition (Zika et al. 1982, Cooper andZika 1983). At the upper concentrations observed inseawater, H2O2 is inhibitory to bacteria (Xenopoulosand Bird 1997) and some phytoplankton species areassociated with ROS-mediated toxic effects on fish(Yang et al. 1985). ROS can modify the speciation oftrace elements in seawater, which may affect the toxic-ity and/or bioavailability of a number of biogeochem-ically and ecologically relevant compounds includingiron, copper, and arsenic (Moffett and Zika 1987).

The aim of the present study was to determinewhether virally infected algae exhibited elevated levelsof ROS production and whether photosynthetic proc-esses were affected and therefore might be implicatedin the generation of ROS in infected cells. The Em-iliania huxleyi host-virus system was selected as thereare several reports of viral-induced demise of E. huxleyiblooms (Bratbak et al. 1995, Brussaard et al. 1996,Wilson et al. 2002), indicating that viral infection ofthis alga may be a common phenomenon, and anumber of well characterized E. huxleyi virus (EhV)isolates are available in culture (Castberg et al. 2002,Schroeder et al. 2002). Oxidative stress in the E. huxleyicells was assessed by measuring intracellular concent-rations of ROS and H2O2 excretion into the culturemedium. Photosynthetic processes were monitored bymeasuring the photosynthetic capacity of the cultures.We revealed for the first time that virally infectedE. huxleyi exhibit oxidative stress and decreased cellphotosynthetic capacity (CPC) indicating that, in com-bination with other pathways, disruption of photosyn-thesis may be linked to ROS generation.

MATERIALS AND METHODS

Culturing. Axenic cultures of E. huxleyi strain CCMP 1516were obtained from Scripps Institution of Oceanography,

San Diego, CA, USA. Cultures were maintained in f/2 medi-um (Guillard 1975) at 151 C under a light:dark cycle of14:10 h and at an illumination of 250 mmoL pho-tons �m� 2 � s�1. The virus pathogen EhV strain EhV86 wasused (Schroeder et al. 2002) and virus lysates were generatedby the addition of 1–10 mL of viral stock to exponentiallygrowing, 2 L cultures of E. huxleyi resulting in a multiplicity ofinfection (MOI) of 0.1–0.01. Cultures were monitored for theoccurrence of lysis, which was indicated by a change in theculture appearance from a green color to a chalky white.Lysates were then filtered through 0.22 mm pore size sterile1 L filter units (Corning, New York, NY, USA) and stored inthe dark at 41 C until required.

Intracellular ROS concentration; reagents, protocol, and vali-dation. Algal intracellular ROS were measured by thefluorescent probe 5-(and-6)-chloromethyl-2 0,7 0-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA; excitation488 nm, emission 522 nm). The CM-H2DCFDA probe is non-fluorescent and membrane permeable, allowing it to diffuseinto the cells where it is hydrolyzed by esterases to 20,7 0-di-chlorohydrofluorescin (DCFH). As DCFH is polar, it becomestrapped inside the cell where it can then be oxidized to thefluorescent compound dichlorofluorescein (DCF) (Haugland2002). Intracellular DCF fluorescence can then be measuredby flow cytometry. The CM-H2DCFDA probe was purchasedfrom Molecular Probes Inc. (Eugene, OR, USA) in 50 mg al-iquots and stored at � 201 C until required. Fresh 1 mMstock solutions were made up before use by dilution in et-hanol. According to the method of (Malanga and Puntarulo1995) a final CM-H2DCFDA concentration of 5 mM was used.Intracellular fluorescence was measured on a FACSCalibur(Becton Dickinson, San Jose, CA, USA) flow cytometer withthe standard filter set up and Milli Q as the sheath fluid.Stained E. huxleyi cells were discriminated on the basis oftheir autofluorescence (610 nm) versus the green fluores-cence of the CM-H2DCFDA stain at 522 nm. The average percell green fluorescence of the E. huxleyi population was takenas a relative measure of ROS concentration when comparedwith the control population. In order to determine the ef-fectiveness of this stain concentration and the optimum stain-ing period, two experiments were conducted on E. huxleyiCCMP 1516. In the initial experiment, H2O2 was used to ar-tificially elevate the intracellular ROS concentration. H2O2 isthe most stable of the ROS and is capable of rapid diffusionacross cell membranes (Salin 1988). E. huxleyi cells were in-cubated either alone, in the presence of 100 mM H2O2, in thepresence of 5 mM CM-H2DCFDA or with both H2O2 and CM-H2DCFDA. Duplicate analyses were made of all treatmentsover a 2 h period. This experiment was repeated using 1 mMmethyl viologen (MV) (Sigma-Aldrich Gillingham, Dorset,UK), a compound known to induce ROS formation in cells(Halliwell and Gutteridge 1989), instead of H2O2. It was de-termined that 5 mM was a suitable stain concentration andthat 60 min was an effective staining period.

H2O2 production; reagents, protocol, and validation. H2O2 ex-creted into the culture medium was measured by an enzyma-tic assay, based on catalytic reduction of H2O2 by horseradishperoxidase in the presence of the hydrogen donor molecule(p-hydroxyphenyl) acetic acid (POPHA). For every H2O2

molecule, one molecule of the fluorescent dimer 6,6 0-di-hydroxy-3,3 0-biphenyldiacetic acid (excitation 320 nm, emis-sion 400 nm) is produced and therefore the fluorescenceintensity is directly proportional to the H2O2 concentration(Guilbaul et al. 1968). A working stock of fluorescent reagentsolution was made by dissolving 255 mM POHPA (Sigma-Ald-rich), 0.25 M Tris and 50 units/mL of horseradish peroxidase(Sigma-Aldrich), in distilled water and adjusting the pH to8.8 with HCl. This solution was stored in the dark at 71 C. Forthe experimental assay, 200 mL of the solution was mixed

REACTIVE OXYGEN AND VIRAL INFECTION 1041

with 3.8 mL of sample and allowed to react for 15 min. Flu-orescence was measured using a fluorometer consisting of aphosphor-coated mercury lamp (Jelight Company Inc., Ir-vine, CA, USA) as the excitation source and a HamamatsuR268 photomultiplier tube (Hamamatsu Photonics, WelwynGarden City, Hertfordshire, UK) for fluorescence detection.The assay was calibrated using H2O2 standards made up in f/2 medium. Background concentrations of H2O2 in the me-dium were determined by measuring fluorescence with andwithout the addition of catalase (Sigma-Aldrich). A workingstock of catalase was made up to 500 units mL�1 in distilledwater. For catalase additions, 100 mL of the working stock wasadded to 3.7 mL of sample and allowed to react for 10 minbefore the addition of fluorescent reagent solution. Fluores-cence signals were corrected for the presence of E. huxleyicells by determining per cell fluorescence in the absence ofexperimental reagents. This was performed by measuringthe fluorescence of a serial dilution of an uninfected cultureof known cell concentration in experimental medium. Theeffectiveness of the assay was validated by assessing H2O2

production from E. huxleyi in the presence of 1 mM MV overa 3 h period.

ROS and cell photochemical capacity (CPC) during viral infec-tion. Four duplicate, 500 mL E. huxleyi cultures were set up inseparate 1 L Erlenmeyer flasks. Fresh filtered lysate of virusstrain EhV86 was added to two of the cultures during theexponential stage of growth at an MOI of approximately 1.Daily measurements were made of all parameters except forat 24 h where intracellular ROS concentration was not deter-mined. In addition, from approximately 48 h onwards, thefrequency of sampling for ROS was increased in order toexamine the crash period in more detail. On the third daysamples were taken for catalase addition to confirm thatH2O2 was the peroxide being detected by the horseradishperoxidase-POPHA assay. In a second experiment with thesame set up, daily measurements of CPC were made duringthe decline of a virally infected culture from in vivo fluores-cence measurements made using the photosynthetic inhibi-tor 3(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU; Sigma-Aldrich) according to the method describe by Parkhill et al.(2001). Before measurement samples were dark adapted byincubation at 151 C for 30 min. Fluorescence was measuredon a 10-AU Turner Designs fluorometer (Fresno, CA, USA)before and 1 min after the addition of 50 mL of 3 mM DCMUin ethanol. CPC expressed as Fv/Fm was calculated accordingto the following equation:

Fv=Fm ¼ ðFm � FoÞ=Fm ð1Þ

where Fo is the minimum fluorescence, obtained after theperiod of dark adaptation, Fm is the maximum fluorescence,obtained after the addition of DCMU, which closes all thereaction centres in PSII and reduces the probability of pho-tochemistry to 0, and Fv is the difference between Fm and Fo.

E. huxleyi and virus enumeration. E. huxleyi counts wereperformed using a Multisizert 3 coulter counter (BeckmanCoulter, High Wycombe, UK) with a 100 mm orifice. Auto-claved, 0.2 mm filtered seawater was used as the electrolyte.

For virus enumeration, 1 mL samples were fixed in 0.5%final concentration glutaraldehyde for 4 h at 71 C. Sampleswere then fast frozen in liquid nitrogen and stored at � 801 Cuntil analysis. Analysis was performed using SYBR Green Inucleic acid gel stain (Molecular Probes Inc.) according tomethod of Marie et al. (1999). Fixed frozen samples were de-frosted and diluted with TE buffer (10 mM Tris, 1 mM EDTAadjusted to pH 7.5) which had been pre-filtered through a Vi-vaFlow 200 (Sartorius, Goettingen, Germany) equipped with apolyether sulfone 30 kDa module and autoclaved. The sampleswere then mixed with SYBR Green I at a final concentration of10�4 and maintained at 801 C for a 10 min staining period.

Flow cytometric analysis was carried out for 2 min on high flowrate and data acquisition was triggered on green fluorescenceversus side scatter.

RESULTS

Intracellular oxidative stress assay validation. In theabsence of CM-H2DCFDA, per cell fluorescence re-mained low and constant in the E. huxleyi cells in thecontrol, H2O2 or MV incubations (Fig. 1). When sub-jected to CM-H2DCFDA per cell fluorescence in-creased in a linear fashion with time, probably as aresult of ROS production due to normal cellularmetabolism. With the addition of H2O2 andCM-H2DCFDA per cell fluorescence in the cultureincreased during the first 20 min and then remainedconstant for the following 100 min, suggesting that all

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CLAIRE EVANS ET AL.1042

DCF formation occurred within the first 20 min (Fig.1A). With the addition of MV, fluorescence began toincrease after 30 min and continued on for the du-ration of the experiment to levels well in excess of themaximum observed in the presence of H2O2. Thissuggests that the plateau in fluorescence in responseto H2O2 addition was not a result of insufficient avail-ability of DCFH but rather that all the H2O2 waseliminated by the cells within the first 20 min.

H2O2 assay validation. In the absence of fluorescentreagent (POPHA, Tris and horseradish peroxidase)low levels of H2O2, approximately 25 nM, were indi-cated by the assay for the E. huxleyi cultures with andwithout MV (Fig. 2). At the initial sampling point, theconcentration of H2O2 in the E. huxleyi cultures wasapproximately 80 nM. In the control, increaseswere observed over the first hour and then levelsstabilized at 120 nM, indicating relatively low back-ground levels. In the culture containing MV, H2O2

was produced throughout the experiment andreached approximately 390 nM after 3 h.

ROS and CPC during viral infection. In the exper-iment to determine ROS production during viralinfection, virus lysis caused E. huxleyi cell numbersto decline steadily in the infected cultures concomi-tant with the accumulation of viral particles, whereascontrol cultures grew normally (Fig. 3A). Stainingwith CM-H2DCFDA indicated that initial levels ofintracellular ROS were the same in all the cultures(Fig. 3B). H2O2 concentrations were slightly higherin infected cultures at time 0 immediately after theaddition of virus particles (Fig. 3C), suggesting thatlysates contained H2O2.

During the intensive sampling period from 48 to62 h, levels of intracellular ROS were higher in the in-fected cultures compared with the controls, and by thefinal sampling point at 76 h were more than double

those exhibited by the controls (Fig. 3B). Levels ofintracellular ROS increased in the control cultureover the light period reaching a maximum just beforethe lights were turned off. Levels then began to de-crease, and by the start of the next light period weresimilar to what was seen at the same time on the pre-vious day, indicating a diel cycle of ROS production inE. huxleyi. In the infected cultures, this pattern was re-

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REACTIVE OXYGEN AND VIRAL INFECTION 1043

versed with levels of intracellular ROS decreasing overthe light period and increasing during the dark.

Over the first 24 h, extracellular H2O2 concentra-tions decreased in all cultures, however by 48 h theconcentrations in the infected cultures had increasedto almost double those exhibited by the controls. Fromthis point onwards H2O2 accumulated rapidly in themedium of the infected cultures and by 76 h hadreached a concentration of 578 nM, more than eighttimes the control value. Addition of catalase at 53.5 hreduced the levels of peroxide detected by the assay by97%, confirming that the majority of the peroxide de-tected by the HP POPHA assay was H2O2.

In a separate viral infection experiment to assess theeffect of viral infection on CPC, the control culturesgrew while viral lysis caused cell numbers to declineand virus particles to accumulate (data not shown).Levels of CPC increased slightly in the control culturesover the course of the experiment, but remained char-acteristic of those for healthy cells at 40.5. However inthe infected cultures, levels fell to zero indicatingthat photosynthesis was disrupted in infected cells(Fig. 4).

DISCUSSION

Phytoplankton exhibit oxidative stress in responseto a variety of factors including temperature, ultravio-let radiation, high light intensity, CO2, and nutrientdeficiency (Rijstenbil 2001) however, the influence ofviral infection had not been previously investigated.Our results show that the concentration of intracellularROS and excretion of H2O2 was increased in virallyinfected cultures of E. huxleyi indicating that levels ofoxidative stress are elevated in infected cells. The in-creases in ROS were generally comparable with thoseobserved in response to other stressors including ele-vated temperature and UV levels (Malanga and Punt-arulo 1995, Lesser 1996, Rijstenbil 2002). In plants

and animals increased ROS production may be associ-ated with pathogen-induced oxidative bursts. Thereare no previous reports of pathogen-induced oxidativebursts in microalgae, however studies of macroalgaehave indicated they can be produced in response tooligosaccharide elicitors or pathogen extracts (Bouar-ab et al. 2001). Such oxidative bursts have been shownto eliminate seaweed pathogens and are examples ofincompatible (not-disease causing) host–pathogen in-teractions whereby the ROS produced serve a numberof protective functions and ultimately result in the in-duction of PCD of infected cells (Jones and Dangl1996).

In infected E. huxleyi cultures only a small initial in-crease in intracellular ROS was detected immediatelyafter addition of viral particles (data not shown) andthis could have derived from H2O2 in the virus lysate,indicating that a defense-related oxidative burst didnot occur. This suggests that the interaction ofE. huxleyi with its virus is a compatible relationship(disease-causing), which is supported by the fact thatmost of the E. huxleyi cells are lost during infection ofthe culture. Therefore, the large elevation in ROS pro-duction that follows viral infection of E. huxleyi wasprobably associated with later stages of the infectionprocess. Indeed ROS production within the cultureonly appeared to become significant towards the latterpart of the culture decline, although this was probablyexaggerated to some extent by the fact that a highproportion of the cells present in the culture at thestart of the experiment would have been uninfected.Because intracellular ROS concentrations were as-sessed by taking an average for the whole population,healthy cells may have masked the increases occurringin the infected cells. In addition, algal cells are able todecompose H2O2 (Wong et al. 2003) and these unin-fected cells may have therefore reduced levels of H2O2.

Elevated H2O2 production by infected cells couldoccur as a result of specific biochemical alterations im-posed on the cell during viral replication, for exampleROS generation may occur due to the alteration ofelectron transport (Asada 1994). If the electron trans-port chain is interrupted, the likelihood of electrontransfer to molecular oxygen is increased, leading tothe production of superoxide that is converted toH2O2 by superoxide dismutase. The interruption ofphotosynthesis is of particular relevance to photosyn-thetic organisms and several plant-virus systems havebeen used to demonstrate lower photosynthetic elec-tron transport rates in infected plants at the PSII level(Takahashi and Ehara 1992). During viral infection ofE. huxleyi, CPC was significantly reduced in infectedcultures. Reduction of photosynthesis has been ob-served in aquatic communities following viral enrich-ment (Suttle et al. 1990, Suttle 1992, Hewson et al.2001), and viral addition to marine microalgal cultureshas also been shown to decrease photosynthesis(Waters and Chan 1982). In a detailed study of pho-tosynthesis during infection of Chlorella NC64A withthe virus PBCV-1 a large decrease in photochemical

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CLAIRE EVANS ET AL.1044

quenching was observed that correlated with an in-crease in the reduction of the primary electron accept-ing plastoquinone of PSII (Seaton et al. 1995). Theysuggested that this resulted from photosynthetic limi-tations involving electron transport, and found rates ofphotochemical quenching increased with the additionof MV, an artificial PSI electron acceptor indicatingthat viral infection indirectly interfered with electronflow. Similar interference by EhV infection with thephotosynthetic apparatus of E. huxleyi could accountfor both the reduction of CPC and the increase inH2O2 production in the infected culture. However, itshould be noted that ROS levels increased in the in-fected culture during the dark period, indicating that,if interruption of electron transport from PSII was re-sponsible, this was in conjunction with other ROS pro-duction pathways. Furthermore, during the lightperiod from approximately 50 to 58 h intracellularROS levels decreased slightly in the infected culture,although it should be noted they were consistentlyhigher then those observed in the control. Indeed, thisapparent decrease was potentially another artifact ofthe ratio of infected to healthy cells. If cells began tolyse during the light period this ratio would decrease,therefore reducing the average intracellular ROS con-centration in the infected culture.

An alternative explanation for the rise in H2O2

could be that it is a necessary requirement of EhV86replication, as studies have implicated ROS as neces-sary for the replication of some viruses (Schwarz 1996).The evidence for this theory comes principally fromstudies of the human immunodeficiency virus (HIV)because HIV-seropositive humans have decreasedantioxidants levels (Staal et al. 1992) and increased in-dicators of oxidative stress (Roederer et al. 1991). Fur-thermore, cell culture studies have shown that H2O2

promotes the replication of HIV whereas antioxidantsinhibit it (Roederer et al. 1990, Staal et al. 1990)and so ROS have been implicated in viral gene ex-pression and protein synthesis (Harakeh et al. 1990,Kslebic et al. 1991, Treitinger et al. 2000). Plant stud-ies have also indicated that a reduction in free radicalscavenging capacity may be required before a rapidincrease in virus replication can take place (Clarkeet al. 2002).

ROS are widely implicated in apoptosis or PCD andhigh levels of oxidative stress have been observed dur-ing the autocatalysed decline of a number of phyto-plankton species including Trichodesmium spp and thedinoflagellate Peridinium gatunense (Vardi et al. 1999,Berman-Frank et al. 2004). A possible theory to ac-count for the requirement of ROS production duringreplication of the EhVs is that these viruses may induceapoptosis within host cells as part of the replicationprocess. Wilson et al. (2005) identified a pathway forthe production of ceramide, an intracellular signal forapoptosis (Obeid et al. 1993) in the EhV86 genome.PCD is thought to occur in E. huxleyi and markers ofapoptosis have been identified in ageing E. huxleyi cells(Bidle and Falkowski 2004).

Further research will be needed to determine theprecise mechanism for ROS production during viralinfection of E. huxleyi and to elucidate how ROS areinvolved in the interactions of phytoplankton and theirviral pathogens. In particular it would be pertinent toinvestigate the possible link between photosynthesisand ROS production and the effect of antioxidantson EhV86 replication in order to asses whether oxida-tive stress is a required element of replication. How-ever, this study has shown that virally infectedphytoplankton should be considered as a potentiallyimportant source of H2O2 to the marine environment.As E. huxleyi is capable of the production of massiveblooms that may be destroyed by viral infection(Wilson et al. 2002) leading to the production of highconcentrations of ROS, this raises the profile ofE. huxleyi as an ecologically and biogeochemicallysignificant species.

The research was supported by the Marine and FreshwaterMicrobial Biodiversity (M&FMB) community programme,funded by the Natural Environmental Research Council ofthe United Kingdom (NERC) under grant reference NER/T/S/2000/00638. C. E. was supported by a studentship funded bythe Isle of Man education authority and the Marine BiologicalAssociation of the UK. G. M. is a NERC Advanced ResearchFellow under grant reference NERC NE/B501039/1.W. H. W.is supported through the NERC-funded core strategic re-search programme of Plymouth Marine Laboratory.

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