Virally Induced Mortality of Phaeocystis Globosa During Two Spring Blooms in Temperate Coastal Waters

AQUATIC MICROBIAL ECOLOGY Vol. 44: 207–217, 2006 Published October 10 Aquat Microb Ecol Virally induced mortality of Phaeocystis globosa during two spring blooms in temperate coastal waters Anne-Claire Baudoux, Anna A. M. Noordeloos, Marcel J. W. Veldhuis, Corina P. D. Brussaard* Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, The Netherlands ABSTRACT: This study reports virally mediated mortality rates of Phaeocystis globosa single cells in the southern North Sea during 2 consecutive spring blooms (2003 and 2004). An adapted dilution method was used to estimate viral lysis and microzooplankton grazing simultaneously. Parallel dilution experiments were performed with 30 kDa ultrafiltrate (virus and grazer-free diluent) and 0.2 µm filtered seawater (grazer-free, but virus-containing diluent). Specific viral lysis rates were calculated from the difference in P. globosa growth rates between the 2 dilution series after 24 h incubation under natural conditions. The validity of this method was tested using a culture of P. globosa infected with a known P. globosa virus (PgV). The field data show that virally induced mortality can be a substantial loss factor for P. globosa single cells (up to 0.35 d–1), comparable to that due to microzooplankton grazing (up to 0.4 d–1). Viral lysis was the major cause of total P. globosa cell lysis. Assum- ing no loss due to sinking, viral lysis accounted for 5 to 66% of the total mortality of P. globosa single cells. Viral lysis and total putative PgV abundance increased concomitantly with abundance of P. globosa single cells whilst the increase in infective PgV was delayed. This delay may be caused by the formation of transparent exopolymeric particles that are generated when P. globosa colonies disrupt and are known to passively adsorb viruses. Viruses and microzooplankton were shown to be major controlling agents of P. globosa single cells, although their relative significance varied over the course of the bloom and between years. KEY WORDS: Algae · Virally induced mortality · Cell lysis · Dilution assay · Microzooplankton grazing · Phaeocystis globosa · North Sea Resale or republication not permitted without written consent of the publisher INTRODUCTION encounter between the virus and the host, which is directly affected by their abundance. During algal With phytoplankton forming the basis of the pelagic bloom events, which are defined by high cell abun- marine food web, their dynamics critically influence dance, virally induced mortality has indeed been the functioning of marine ecosystems. Traditionally, reported to be a substantial loss factor (Bratbak et al. grazing and sinking are considered important causes 1993, Brussaard et al. 1996b, Tomaru et al. 2004). of phytoplankton mortality but over the last decade, Phaeocystis globosa (Prymnesiophyte) is a bloom- cell lysis has also been recognized as a significant loss forming phytoplankter with a world-wide distribution. factor for phytoplankton (Van Boekel et al. 1992, Brus- This marine microphytoplankter is well known for its saard et al. 1995, Agusti et al. 1998). Viral infection is a complex polymorphic life cycle, including flagellated major cause of phytoplankton cell lysis, which affects cells (5–7 µm diameter) and colonies (up to 1–2 cm), population dynamics and diversity (for review see which consist of colonial cells embedded in a poly- Brussaard 2004b). Successful infection depends on the saccharide (mucus) matrix. Typically, in the southern *Corresponding author. Email: [email protected] © Inter-Research 2006 · www.int-res.com 208 Aquat Microb Ecol 44: 207–217, 2006 North Sea, P. globosa develops into high biomass spring Helder & De Vries 1979, Grasshoff 1983), and reactive blooms, which contribute the bulk of the local primary silicate (Strickland & Parsons 1968). The limit of de- production (Lancelot & Billen 1984). These blooms also tection was 0.03 µM for phosphate, 0.1 µM for ammo- affect microbial food web dynamics and biogeochemi- nium, 0.01 µM for nitrite, 0.15 µM for nitrate, cal processes (Stefels & Van Boekel 1993, Brussaard and 0.05 µM for silicate. et al. 1995, 1996a, 2005b). The concentration of transparent exopolymeric par- Viruses infecting Phaeocystis globosa (PgVs) have ticles (TEP) in µg equiv. gum xanthan (equiv. GX) l–1 been isolated and characterized (Brussaard et al. 2004, was measured according to Passow & Alldredge Baudoux & Brussaard 2005). A recent mesocosm ex- (1995). Replicate samples (30 to 75 ml) were filtered periment demonstrated that virally mediated mortality through 0.4 µm pore-size polycarbonate filters (Poret- of P. globosa accounted for 30 to 100% of the total lysis ics). The particles retained on the filter were stained of P. globosa single cells. In contrast, cells embedded in with 500 µl of a 0.02% solution of Alcian blue prepared a colonial matrix tend to escape viral infection (Brus- in 0.06% acetic acid (pH 2.5). After staining (<2 s), saard et al. 2005a, Ruardij et al. 2005). To our knowl- the filters were rinsed 3 times with Milli-Q (Millipore) edge, estimates of virally mediated mortality under to remove excess dye. The filters were immediately natural P. globosa bloom conditions do not exist. The transferred into 20 ml glass tubes and soaked for 3 h in dynamics of viral abundance, the virus to P. globosa a solution of 80% H2SO4 with gentle agitation every ratio and total cell lysis rates of P. globosa during 30 min. The samples were analyzed spectrophoto- bloom events, however, suggest that viruses are an metrically at 727 nm (U-3010 Hitachi). important source of mortality for this alga (Brussaard et Microbial abundances. Samples collected for phyto- al. 2004, 2005a). plankton pigments (150 to 700 ml) were filtered through Direct methods for estimating viral lysis of phyto- GF/F glass fiber filters (Whatman) and stored at –50°C. plankton are rare, and, to date, most viral lysis rates The extract from the filters was analyzed by high pres- recorded in the literature rely on theoretical cal- sure liquid chromatography (HPLC) after extraction in culations or are based on indirect measurements (for 4 ml of 100% methanol buffered with 0.5 mol l–1 am- review see Brussaard 2004b). Recently, an adaptation monium acetate and homogenized for 15 s. The rela- of the classical dilution approach (Landry & Hassett tive abundance of the taxonomic group Prymnesio- 1982) provided estimates of the viral lysis of the pico- phyceae (specifically Phaeocystis globosa during our phytoplankter Micromonas pusilla in mesocosms (Evans study) was determined using CHEMTAX (Mackey et et al. 2003). The current study has applied this method al. 1996, Riegman & Kraay 2001). to Phaeocystis globosa under natural conditions. Our Phaeocystis globosa single cells were enumerated work aimed at elucidating the relative significance of in 50 µm-sieved and unfixed samples using a Beck- viral lysis compared to microzooplankton grazing man Coulter XL-MCL flow cytometer equipped with a and total cell lysis of P. globosa single cells during 2 488 nm air-cooled laser. Special care was taken to consecutive spring blooms. avoid rupture of P. globosa colonies during sieving using a small volume of sample. Fixation of the sample resulted in the disintegration of the colonial matrix; MATERIALS AND METHODS therefore the total abundance of P. globosa cells (including both single and colonial cells) could be Study site and sampling. Sampling of the coastal obtained from unfiltered samples that were fixed to a southern North Sea was performed twice a week 1% final concentration with formaldehyde:hexamine between March (Day 60) and June (Day 180) in 2003 solution (18% v/v:10% w/v). These fixed samples were and 2004 from the jetty of the Royal Netherlands Insti- frozen in liquid nitrogen and stored at –80°C until flow tute for Sea Research (NIOZ). Because the jetty is cytometry analysis. Fixation and freezing did not located at the outer border of a major tidal inlet, sam- affect the P. globosa cell counts. P. globosa cells were ples were collected on the incoming high tide. Samples discriminated on the basis of their natural red chloro- containing freshwater run-off (salinity <27‰) were not phyll autofluorescence and forward scatter signal. taken into account (1 out of 46 samples). The abundance of virus-like particles resembling Chemical parameters. Nutrient samples (ca. 5 ml) PgV was determined on glutaraldehyde fixed samples were gently filtered through 0.2 µm pore-size polysul- (final concentration 0.5% glutaraldehyde, frozen in fone filters (Acrodisc, Gelman Sciences) and stored at liquid nitrogen and stored at –80°C prior analysis) –50°C (or 4°C for the reactive silicate) until analysis. using a Beckton-Dickinson FACSCalibur flow cytome- Analyses were performed using a TrAAcs 800 auto- ter, with a 15 mW 488 nm air-cooled argon-ion laser analyzer for dissolved orthophosphate (Murphy & according to Brussaard (2004a). Thawed samples were Riley 1962), nitrogen (nitrate, nitrite and ammonium; diluted (dilution factor >25) in 0.2 µm filtered sterile Baudoux et al.: Virally induced mortality of Phaeocystis globosa 209 TE-buffer (pH 8) and stained with the nucleic acid- and compared to noninfected controls. Those dilutions specific dye SYBR Green I at a final concentration of that showed signs of cell lysis were scored positive 0.5 × 10–4 of the commercial stock (Molecular Probes). when PgV proliferation could be confirmed (using flow Putative PgV could be discriminated on the basis of the cytometry as described above). The positive scores green fluorescence and side scatter signature (Fig. 1), were converted to abundance of infective PgV using which was identical to that of PgV isolates from the an MPN assay computer program (Hurley & Roscoe same geographical location, and kept in culture at the 1983). Royal NIOZ (Brussaard et al. 2004). Loss parameters of Phaeocystis globosa.

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