Phaeocystis and its interaction with viruses

Corina P. D. Brussaard • Gunnar Bratbak • Anne-Claire Baudoux • Piet Ruardij

Received: 10 November 2005 / Accepted: 14 February 2006 / Published online: 7 April 2007 © Springer Science+Business Media B.V. 2007

Abstract Over the years, viruses have been shown to be mortality agents for a wide range of phytoplankton species, including species within the genus Phaeocystis (Prymnesiophyceae). With its polymorphic life cycle, its worldwide distribution, and the capacity of several of the Phaeocystis species to form dense blooms, this genus is a key player for our understanding of biogeochemical cycling of elements. This paper provides an overview of what is know to date about the ecological role of viruses in regulating Phaeocystis population dynamics. It explores which variables affect the algal host-virus interactions, and examines the impact of virally induced cell lysis of Phaeo-cystis on the function and structure of the pelagic food web as well as on the flow of organic carbon and nutrients.

Keywords Characteristics • Mortality • Phaeocystis • Phycodnaviridae • PgV • Viruses

C. P. D. Brussaard (&) • A.-C. Baudoux • P. Ruardij Department Biological Oceanography, Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790AB Den Burg, The Netherlands e-mail: [email protected]

G. Bratbak

Department of Biology, University of Bergen, Jahnebakken 5, 5020 Bergen, Norway

Abbreviations

PgV Phaeocystis globosa virus

PpV Phaeocysis pouchetii virus

TEP Transparent exopolymeric particles

MPN Most probable number

TEM Transmission electron microscopy DsDNA Double-stranded DNA

HAB Harmful algal bloom species

PFGE Pulsed-field gel electrophoresis

DMS Dimethyl sulfide

DMSP Dimethylsulfoniopropionate

DOC Dissolved organic carbon

DOM Dissolved organic matter

Introduction

The presence of viruses in marine environments has been acknowledged for many years, and it is now well established that viruses are dynamic and important members of the microbial food web (Bergh etal. 1989; Proctor and Fuhrman 1990; Gobler et al. 1997; Fuhrman 1999; Wommack and Colwell 2000; Weinbauer 2004). Viruses infect not only the numerically dominant bacteria but also prokaryotic and eukaryotic primary producers. Unicellular photosynthetic organisms are a major group of organisms in natural aquatic communities and viruses have been recognized as mortality agents for phytoplankton (Van Etten et al. 1991; Reisser 1993; Brussaard 2004). Viruses or virus-like particles have been reported in many different taxa of eukaryotic algae, 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, whether through sinking, grazing or cell lysis, has major implications for carbon and energy cycles in marine ecosystems. Lysis-mediated release of the cellular content can greatly enhance bacterial activity and subsequently force the food web towards a more-regenerative system. Energy and nutrients released by cell lysis and excretion is transferred to higher trophic levels via the microbial loop (Azam et al. 1983). Lytic viral infection of phytoplankton causes rapid cell lysis, which may affect not only the energy and nutrient flow, but also the phyto-plankton community composition and succession. Recognizing viral lysis of phytoplankton as a major process has emphasized the importance of the microbial loop including a viral shunt.

Theoretical models suggest that a 2-10% loss of phytoplankton due to viral infection increases the flow of organic carbon, bacterial production and respiration by more than 25% (Fuhrman 1999; Wilhelm and Suttle 1999). Especially during algal blooms, when high algal cell abundances enhance the virus-host encounter rates, virally mediated lysis can have a profound effect on population dynamics, community diversity and transfer of energy and matter within the pelagic food web.

The Phaeocystis genus, with its cosmopolitan distribution, includes several high-biomass-form-ing species (Cadée and Hegeman 2002; Verity and Medlin 2003; Schoemann et al. 2005). Phaeo-cystis has a life cycle dominated by single cells (with and without flagellae) and embedded colonial non-flagellated cells. Several species of Phaeocystis, e.g., P. pouchetii and P. globosa, regularly dominate the phytoplankton community and sequester huge amounts of nutrient resources, predominantly in the form of colonies. These blooms occur mostly in colder and temperate waters, such as the coastal zone of the North Atlantic and the North Sea. Because of the importance of these blooms for the pelagic ecosystem and the socioeconomic interest in these HAB species, substantial research has been conducted on factors controlling the wax and wane of these blooms. With light and nutrients as impor tant factors initiating Phaeocystis blooms, grazing and viruses are considered the relevant loss factors. Field studies indicate that these viruses are a dynamic component, notably involved in the decline of the blooms. Laboratory and seminatu-ral studies provided insight into host-virus interactions and revealed how environmental factors may influence viral infection. The scope of the present paper is to provide a summary and synthesis of available information and some unpublished data related to Phaeocystis and the viruses infecting it.

Isolation and characterization of viruses infecting Phaeocystis

Viruses that infect species of Phaeocystis have been isolated during and directly after natural blooms (Jacobsen et al. 1996; Brussaard et al. 2004; Baudoux and Brussaard 2005). Phaeocystis pouchetii viruses (PpV) were successfully isolated after 100-fold concentration by continuous centri-fugation and exposure to ultraviolet (UV) for 15 and 30 s (Jacobsen et al. 1996). Exposure to UV light was intended to cause induction of virus production in algal cells containing lysogenic viruses, but as the virus isolated was lytic this treatment was most likely not necessary. So far, all viruses infecting eukaryotic microalgae are lytic and none have been reported to enter a lysogenic relationship with the host. Phaeocystis globosa viruses (PgV) were isolated from filtered (GF/F Whatman glass fiber filters) natural water that was added to exponentially growing P. globosa host cultures (Baudoux and Brussaard 2005). Incubation of the natural seawater with the addition of nutrients for a week at in situ temperature and irradiance (excluding UV) before adding a sub-sample to cultures of P. globosa occasionally advanced successful isolation of PgV. At the decline of the bloom, when most free viruses can be expected to occur, nutrients regularly become depleted. By adding nutrients more algal biomass was generated and the encounter rate between algal host and virus enhanced.

The Phaeocystis viruses isolated so far are species specific, i.e., they only infect one of the Phaeocystis species (Jacobsen et al. 1996; Bau-

doux and Brussaard 2005). Not all P. globosa or P. pouchetii host strains are infected by the same virus isolates, and not all viruses isolated infect the same algal host strains. There can be a high degree of specificity for these algal viruses.

All intracellular Phaeocystis virus particles observed microscopically to date have been located in the cytoplasm of the algal host cell, are hexagonal in their profile, are non-enveloped, and lack a tail. The particles are between 100 and 160 nm in diameter based on transmission electron microscopy (TEM) thin sectioning micrographs (particles tend to shrink when subjected to fixation and dehydration during preparation for thin sectioning). The hexagonal profiles of the viruses suggest that virions may contain an icosahedral capsid (Fig. 1). A recent investigation of the native morphology of PpV at a resolution of 3 nm using electron cryomicroscopy and three-dimensional image reconstruction methods revealed that the capsid had a maximum diameter of 220 nm between opposite vertices, and was composed of 2,192 capsomers that were organized in large triangular and pentagonal aggregates (Yan et al. 2005).

A common ancestor for P. globosa viruses and viruses infecting another prymnesiophyte (Chrys-ochromulina brevifilum) has also been suggested based on the phylogenetic analysis using the inferred amino acid sequences of a DNA polymerase gene fragment (Brussaard et al. 2004). This study also showed that seven PgV isolates formed a distinct monophyletic group with other eukaryotic algal viruses, despite differences in

Fig. 1 Transmission electron micrographs of thin sections of infected cells of Phaeocystis pouchetii. (a) The virus-like particles (indicated by arrow) are found in the cytoplasm of the cells. (b) Detail of virus-like particles showing the hexagonal outline of the viruses their genome size and other phenotypical characteristics. Wilson et al. (2006) recently isolated a virus infecting P. globosa from surface water in the English Channel, UK that did not cluster with the other PgVs. Instead, it was more closely related to C. brevifilum.

The fact that the DNA polymerase gene could be amplified using the algal virus-specific primers AVS1 and AVS2 (Chen and Suttle 1996) allows assignment of these viruses to the family Phy-codnaviridae (Van Etten 1995). Many of the characterized phytoplankton viruses are indeed assigned to this family of large double-stranded (ds) DNA viruses that infect eukaryotic algae. The use of the highly conserved DNA polymerase gene turned out to be a good genetic marker for classification 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 staining with a sensitive nucleic-acid-specific dye such as SYBR Green I, these large genome sized Phaeo-cystis viruses could be readily detected using epi-fluorescent microscopy or flow cytometry (Marie et al. 1999). Furthermore, the use of flow cytome-try allowed the discrimination of these viruses from other viruses such as many other algal viruses (Brussaard et al. 2000) or bacteriophages in natural samples (Larsen etal. 2001; Baudoux and Brussaard 2005). The ability to detect, discriminate and enumerate samples containing Phaeocystis viruses in a rapid and objective manner promotes laboratory research on virus-host interactions, and ecological studies in the field.

Detailed laboratory studies showed that the total length of the lytic growth cycles of the Phae-ocystis viruses infecting exponentially growing host cells ranged between 25 and 50 h (Jacobsen et al. 1996; Baudoux and Brussaard 2005). For PpV the latent period, the time period from infection until the first increase in the abundance of extracellular free viruses, was around 12-18 h (Jacobsen et al. 1996). The study by Baudoux and Brussaard (2005) showed three different latent periods for the various PgV isolates in culture, i.e., 10, 12 and 16 h (Fig. 3). These periods match the range of latent periods for all characterized phytoplankton viruses so far, and are somewhat

Fig. 1 Transmission electron micrographs of thin sections of infected cells of Phaeocystis pouchetii. (a) The virus-like particles (indicated by arrow) are found in the cytoplasm of the cells. (b) Detail of virus-like particles showing the hexagonal outline of the viruses

Fig. 2 Viral genome sizes of different Phaeocystis globosa virus isolates (PgV) determined by pulsed-field gel electrophoresis (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 of 465 kb). The small-sized band (approximately 45 kb) as seen in lanes 1-3 correspond to bacteriophages since algal cultures were not axenic

Fig. 2 Viral genome sizes of different Phaeocystis globosa virus isolates (PgV) determined by pulsed-field gel electrophoresis (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 of 465 kb). The small-sized band (approximately 45 kb) as seen in lanes 1-3 correspond to bacteriophages since algal cultures were not axenic shorter or comparable to the maximum growth rates of their host (Schoemann et al. 2005; Vel-dhuis et al. 2005). Based on the decline in the algal host population and the increase in extracellular virus particles, a conservative estimate of the burst size (number of viruses released per host cell that underwent lysis) can be estimated. For both Phaeocystis species, burst sizes ranged on average between 250 and 500, with the higher burst sizes for P. pouchetii. The variation in burst sizes between different PgV isolates was considerable, with values down to around 100 despite the exponential growth of the algal host cells (Bau-doux and Brussaard 2005). Although the burst size strongly affects the chances for infection of the remaining cells in the host population, not all viruses are infectious. In exponentially growing cultures, the percentage of infective Phaeocystis viruses produced as determined by the most probable number (MPN) method is generally relatively high, ranging from 60 to about 100% (Bratbak et al. 1998; Brussaard, unpubl. data).

The devastating eVect on Phaeocystis cells of an infection by lytic viruses is well illustrated by the morphological, physiological and viability status of the host population during infection (Jacobsen et al. 1996; Bratbak et al. 1998, Brussaard et al. 1999, 2001). Even though the photo-synthetic apparatus of the infected algal cells seem to be active during the Wrst hours after infection, sudden and sharp declines in the photo-synthetic efficiency of the cells were observed at the end of the latent period (Fig. 4). In contrast to the above-mentioned assays, which reflect the status of the entire population, the use of flow cytometry allows the analysis of individual cells. Changes in the cell characteristics of the virally infected cells are dynamic in time, with the proportion of cells with increased cellular DNA increasing in the Wrst hours after infection, followed by a decline in cellular scatter signals when virions are formed. Prior to cell lysis, the red autofluorescence declined concomitantly with the disruption of the organelles (as observed by transmission electron microscopy). By the time the Wrst viruses are released from the host cells the portion of dead cells increased (Brussaard et al. 2001). Finally, during the period of cell lysis a subpopulation of cells showing reduced concentration of cellular DNA developed and increased with time.

Occurrence and dynamics of Phaeocystis viruses

Although the observations of Phaeocystis cells containing virus-like particles and the isolation of viruses infecting Phaeocystis species suggest that viruses may be potentially important, it is the succession of Phaeocystis algal cells and free viruses under seminatural conditions that implies a direct causative relationship and ecological signiWcance. At present, two mesocosm studies and two Weld studies have been performed, all showing highly dynamic Phaeocystis virus abundances with time

0 10 20 30 40 50 Time (h)

Fig. 3 Abundance of Phaeocystis globosa (a, c, e) and PgV (b, d, f) according to Baudoux and Brussaard (2005). Open square symbols represent uninfected cultures, while the filled 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 viral growth cycles of (b) PgV-07T, (d), PgV-05T, and (f) and PgV-04T. The length of the latent period (indicated by the dotted line) was 10 h for PgV-07T, 12 h for PgV-05T, and 16 h for PgV-04T

Fig. 3 Abundance of Phaeocystis globosa (a, c, e) and PgV (b, d, f) according to Baudoux and Brussaard (2005). Open square symbols represent uninfected cultures, while the filled circles represent virally infected P. globosa. (a) P. globosa infected with PgV-07T, (c) with PgV-05T, and (e)

and closely linked to the abundance of their host (Larsen et al. 2001; Brussaard et al. 2004; Brussaard et al. 2005a; Baudoux et al. 2006). For all these studies the abundance of Phaeocystis viruses was 30- to 100-fold higher during bloom maxima than the abundance of their host, suggesting that viruses should indeed be regarded as important mortality agents for P. globosa and P. pouchetii. During the periods of study, the Phaeocystis viruses generally made up between 0 and 5% of the total virus population, independent of whether Phaeocystis was dominating the phytoplankton community or not. As bacteria are the numerically most abundant, the low share of Phaeocystis viruses is to be expected. However, under specific conditions favoring the single-cell morph as compared to the colonial form, the portion of PgV increased up to 30% of total virus abundance (Brussaard et al. 2005a). Under such conditions viruses were actually able to prevent a build-up of standing stock (i.e., bloom) of P. globosa.

A critical note here is that successful infection of Phaeocystis does not depend on the total abundance of Phaeocystis viruses, but on the number of infectious viruses. A recently published ecosystem model that was calibrated with a large data set from P. globosa mesocosm experiments (Ruardij et al. 2005) suggested that the fraction of

with PgV-04T. Filled diamond symbols represent the viral growth cycles of (b) PgV-07T, (d), PgV-05T, and (f) and PgV-04T. The length of the latent period (indicated by the dotted line) was 10 h for PgV-07T, 12 h for PgV-05T, and 16 h for PgV-04T

infective PgV successfully infecting P. globosa cells increased steeply over the course of the bloom to a maximum of 0.035 (Fig. 5). The fraction of infective PgV that successfully infect P. globosa was highest when single cells dominate. The absorption of PgV to transparent exo-polymer particles (TEP) that are formed upon disintegration of colonies reduces the available infective PgV and subsequently the fraction of PgV that successfully infects P. globosa (Fig. 5). The very low value prior to bloom formation (0.0005%) represents the situation at the start of the growing season when PgV standing stock was subjected for a long time (autumn till spring) to loss of infectivity and decline in actual virus particles. At the same time new virus production was insigniWcant for P. globosa when host cells were present in very low numbers and barely growing (due to light and temperature limitation during winter). A Weld study in the turbid coastal waters of the southern North Sea showed that the fraction of infectious PgV was around 0.04 (Bau-doux etal., 2006), matching very well with the model situation. It is noteworthy that for P. pouchetii also a low host-virus adsorption efficiency was needed to produce reasonable good Wt between simulation and experimental observations (Bratbak et al. 1998).

0 0

Post a comment