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Fig. 4 Dynamics of in vivo chlorophyll fluorescence (Fo) and photosynthetic efficiency (Fv/Fm) of Phaeocystis globosa during viral infection as assessed by fluorometry. Open symbols represent uninfected cultures, while the filled symbols represent virally infected P. globosa. Maximum fluorescence (Fm) was obtained after addition of the photosystem II inhibitor DCMU (20 pM final concentration). Fv equals Fm-Fo. Data are expressed in relative units (r.u.)

Fig. 5 Results of a mesocososm study on Phaeocystis globosa bloom dynamics and the ecological role of viruses. The measured data are represented by the symbols and the modeled results are represented by the lines. (a) measured and modeled biomass of P. globosa colonies (solid line and open symbol) and single cells (outline of grey area and filled symbol), (b) measured and modeled abundance of P. globosa viruses (PgV), and (c) modeled fraction of infective PgV without transparent exopolymer particles (TEP) present (white area) and in the presence of TEP (grey area). It was assumed that infectivity declined by 10% d_1

Fig. 5 Results of a mesocososm study on Phaeocystis globosa bloom dynamics and the ecological role of viruses. The measured data are represented by the symbols and the modeled results are represented by the lines. (a) measured and modeled biomass of P. globosa colonies (solid line and open symbol) and single cells (outline of grey area and filled symbol), (b) measured and modeled abundance of P. globosa viruses (PgV), and (c) modeled fraction of infective PgV without transparent exopolymer particles (TEP) present (white area) and in the presence of TEP (grey area). It was assumed that infectivity declined by 10% d_1

might even promote the growth of the other strain(s) indirectly as regenerated nutrients become available. There is increasing evidence accumulating that speciWc geographical populations of an algal species can be morphologically and/or genetically heterogeneous (Barker et al. 1994; Rynearson and Armbrust 2000, 2004) and viruses might actually be an important regulating factor for host heterogeneity.

Besides different PgVs infecting different P. globosa strains, some of the different PgVs infected the same range of P. globosa strains. If present at the same time in the water, these different PgV species might have to compete for these host strains. To our knowledge, this is a completely unexplored Weld of research to date. Important factors affecting the outcome of competition between viruses for the same host cell include the total standing stock of infective viruses in order to enhance the contact rate, the ability to prevent co-infection by other virus types, the burst size, and the level of resistant to loss of infectivity. Differences in burst size and sensitivity to virucidal factors have been observed (Baudoux and Brussaard 2005), but to what extent this influence the actual competition is unknown.

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 to viral infection may not be constant. A laboratory study with P. pouchetii showed that some algal host cells escaped infection with the lytic PpV, and with time the algal population increased in abundance again (Thyrhaug et al. 2003). Since PpV originating from these cultures and the original stock affected sensitive P. pouchetii cultures equally, the resistance to viral infection must have come from changes in the algal phenotype. Such phenotypic plasticity of the host's susceptibility to infection may be an important stabilizing factor in host-virus interactions as it seems to sustain long-term (one-year) coexistence of host and virus.

The research related to PgV characterization and Phaeocystis-virus interactions performed to date generally involved single cells (flagellated and non-flagellated). Phaeocystis is, however, known to form colonies and most often the embedded colonial non-flagellated cells are the predominant cell morph during bloom events. A logical question is thus if embedded colonial cells are perhaps resistant to viral infection. This question was raised already by Jacobsen et al. in 1996 upon isolation of PpV, and it was speculated at the time that the gelatinous material the colonial cells are surrounded by serves as a protection against viral infection (Jacobsen et al. 1996; Brat-bak et al. 1998). The presence of an outer thin, yet mechanically stable "skin", likely with pores <4.4 nm, has also been suggested as a defense against viral attack of the cells within the colony (Hamm et al. 1999). To test these hypotheses is nevertheless not easy as colonies will always shed at least some single cells, which become readily infected. The consequent production of viruses might thus incorrectly suggest that colonies can be infected. An attempt to infect colonial P. pouchetii cells with PpV have so far been unsuccessful (Jacobsen et al. 2005). Tests using 12 PgV isolates to infect strains of P. globosa that either formed distinct colonies or mucus aggregates were unsuccessful for all but one strain of PgV (Baudoux and Brussaard 2005). Thus, mucus formation might protect Phaeocystis cells from viral infection, but the positive infection of P. globosa strain Pg01MD-04 by PgV-01T indicates that the protection is not exclusive.

A mesocosm experiment studying the regulatory role of viruses on P. globosa population dynamics sheds more light on the topic (Brussaard et al. 2005a). Both single cells and colonies originating from the same clone were present at the start of the experiment in low concentrations. The results show that the morphology of the P. globosa cells (solitary vs. colonial) differently regulated viral control of P. globosa. Under non-limiting conditions, dense blooms of colonies and to a lesser extent single cells were formed. Viruses were found to be a significant loss factor but could not prevent bloom formation. Under conditions that restricted colony formation for the first 11 days but allowed single cells to grow, viruses were found to prevent bloom formation the moment conditions were no longer limiting colony formation. The maximum standing stock of PgV was also fivefold higher. The results suggest that the colonial form of Phaeocystis is indeed an excellent mechanism to prevent viral infection. Interestingly, a recently developed mathematical ecosystem model including a detailed virus module (Ruardij et al. 2005) suggests that the size of the colonies strongly reduces the chance of infection per cell (and not the gelatinous matrix). The physical principle of the spherical equivalent diameter determining the encounter rate was modeled earlier by Murray and Jackson (1992). With increasing diameter of the colonies, viral infection seems an insignificant loss factor (Fig. 6). The model implies that the single cells were still readily infected, which prevented a build-up of P. globosa biomass. By the time colony formation

Colony size (number of cells)

Fig. 6 Viral infection rate of P. globosa colonial cells embedded in a colonial matrix as compared to that of P. globosa single cells

Colony size (number of cells)

Fig. 6 Viral infection rate of P. globosa colonial cells embedded in a colonial matrix as compared to that of P. globosa single cells was no longer limited there was not enough standing stock of single cells to form colonies.

An additional but important indirect defense mechanism against virus infection is the formation of TEP during colony disintegration. TEP was found to be a strong stabilizing negative-feedback mechanism (Brussaard et al. 2005b; Mari et al. 2005; Ruardij et al. 2005). Without colonies and thus without TEP, fewer viruses are needed to control the population of single cells.

Environmental factors influencing virus-host interactions

Keeping in mind that algal growth is regulated by environmental factors such as irradiance, nutrients and temperature, and the fact that viral replication is dependent on the metabolism of the host, it is of interest to know the impact of these factors on virus-host interactions. Under nutrient depletion, for example, the burst size of released P. pouchetii and P. globosa viruses per infected cell is lower than under nutrient-replete conditions, especially under P-depletion (Bratbak et al. 1998; Brussaard unpubl. data). The strongly reduced burst size found for PpV upon infection of P. pouchetii in the stationary phase of growth (15 compared to 240 cell-1 for P. pouchetii in the exponential growth phase) is most likely the result of severe nutrient depletion inhibiting cell growth (Bratbak et al. 1998).

How nutrient depletion affects not only Phaeo-cystis' physiology and viral replication but also indirectly the relationship between the Phaeocys-tis host cell and the virus becomes clear when considering the different Phaeocystis morphotypes. Cells inside the colonial matrix are barely infected, but this changes when the colonies disintegrate due to nutrient depletion. Because of the presence of a diffusive boundary layer, colony formation will decrease nutrient uptake and, therefore, colonies experience nutrient depletion faster than single cells (Ploug et al. 1999). The liberation of large numbers of single cells promotes viral infection, resulting in high viral lysis rates (Ruar-dij et al. 2005). Nutrient deficiency also has another mode of controlling virus-host interactions, which is by the scavenging properties of TEP. The percentage of PgV attached to TEP was found to be higher under P- than under N-defi-ciency and consequently the number of PgV for successful infection will be lower (Brussaard et al. 2005b).

Another illustration that growth conditions can affect viral kinetics is the fourfold lower burst size for PpV-infected P. pouchetii when placed in the dark compared to those kept in the light. Interestingly, viral proliferation was not delayed or prevented in the dark, indicating that PpV was not dependent on host photosynthesis (Bratbak et al. 1998). Similar results have been found for the model system P. globosa-PgV (Brussaard, unpubl. data), which could indicate that this is a genus-wide feature. Since the level of irradiance strongly affects colony formation (Peperzak 1993), it also indirectly impacts on the level of viral control (Brussaard et al. 2005a).

UV radiation affected P. pouchetii-PpV interactions differently, depending on the type of UV radiation. UV-B radiation strongly inhibited viral infectivity, whereas UV-A radiation had no effect (Jacquet and Bratbak 2003). A fascinating additional finding was the reduced sensitivity to UV-B stress of P. pouchetii cells that previously escaped viral infection. Theoretically, these virus- and UV-resistant cells would have a huge advantage compared to sensitive cells. The fact that they are not dominating the population suggests that there must be a negative trade-off and it is tempting to speculate that these resistant cells may have lower growth rates due to inferior nutrient affinity. Anyway, it does stress the need for studies examining in more detail the effects of combined exposure to potentially regulating factors.

Virally induced mortality of Phaeocystis

Having established the presence, dynamics and diversity of Phaeocystis viruses, one may wonder what is the actual impact of those viruses on the loss rates of Phaeocystis. Earlier studies on P. globosa bloom dynamics showed that cell lysis was a relevant loss factor, with rates of up to 0.3 d"1 (Brussaard et al. 1995, 1996). Although it was not clear at that time whether viruses were causing the algal cells to undergo lysis, recent studies indicate that viruses are most likely the responsible lysis agents (Larsen et al. 2001; Brussaard et al. 2004; Baudoux and Brussaard 2005; Brussaard et al. 2005a). The use of live/dead assays indicated that viral lysis rates of infected Phaeocystis in cultures showed rates as high as 0.8 d"1 (Brussaard et al. 1999). Methods for specific and accurate determination of viral-mediated algal mortality in natural waters are however still lacking. The first attempt to estimate viral lysis rates of P. globosa cells during a bloom was performed for a set of mesocosm experiments (Brussaard et al. 2005a), and was based on the net production of PgV, a conservative viral loss rate of 0.07 d"1 used to correct viral production, and an assumed viral burst size of 300 (Baudoux and Brussaard 2005). The estimated viral lysis rates were around 0.2 d"1 during the bloom, and largely accounted for most of the total cell lysis, which was either obtained using the dissolved esterase activity assay or by subtracting the net algal growth rate (equals the change in net abundance) and the microzooplankton grazing rate (using the dilution method) from the gross algal growth rate (derived from DNA cell cycle analysis).

The only field study specifically estimating virally mediated mortality of P. globosa cells to date (Baudoux et al. 2006) was executed using a newly developed method, being an adaptation of the classical dilution method to determine microzooplankton grazing rates (Landry and Hassett

1982; Evans et al. 2003). Besides dilution of the natural sample in 0.2 ^m pore-size filtered seawa-ter at different ratios, parallel samples were diluted in seawater that was made virus-free by ultrafiltration through 30 kDa cartridges. As dilution results in a reduction in viral infection and/or grazing pressure, the subsequent increase in algal cell abundance after 24 h incubation is used to the determine the actual impact of grazing and viral lysis on the algal population. The usefulness of this adapted method for Phaeocystis was validated by testing a culture of P. globosa cells in the presence of viruses and the absence of grazers. During two consecutive years, natural viral lysis and microzooplankton rates were determined for P. globosa during the annual spring bloom events. Whereas during bloom development microzooplankton grazing seemed to be the dominant loss factor for P. globosa cells, viral lysis became increasingly important over the course of the bloom, with rates comparable to the grazing rates (max. 0.35 d_1). At times, viral lysis made up more than 50% of the total losses of the single-cell population (Baudoux et al. 2006).

Despite the dependence on certain assumptions, the discussed studies clearly imply that viral lysis is an ecologically significant mortality factor for Phaeocystis cells. Modeling also backs up the evidence that viral lysis can be an important cause of P. globosa cell mortality, especially under conditions where single cells dominate (Ruardij et al. 2005). Comparing model situations that lack and contain colonies, grazing as well as viral lysis are strongly enhanced with sixfold higher maximum the outline of the black area, microzooplankton grazing by grazing rates and 40-fold higher maximum lysis rates (Fig. 7). Colonies thus seem to have the advantage of being largely protected against grazing and viral infection, explaining their build-up of biomass (bloom). The only significant loss factor for cells inside colonies seems to be automor-tality, which becomes important during nutrient deficiency as a result of strongly limited nutrient diffusion (Ploug et al. 1999; Ruardij et al. 2005).

Note that the model suggests that grazing indirectly affects the impact of viral infection on the population dynamics of P. globosa (assuming equal grazing on both uninfected and virally infected cells). Without grazing single-cell biomass would increase faster, but viral infection would take place earlier, resulting in an overall lower 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 of Phaeocystis primary production is of importance for the distribution of energy and biogeochemical cycling within the pelagic and benthic ecosystems (Schoemann et al. 2005). Substantial viral lysis of Phaeocystis will provide a sizable source of dissolved organic matter, thereby promoting a retentive system that oxidizes organic matter and regenerates inorganic nutrients in the euphotic zone (Brussaard et al. 1996; Gobler et al. 1997; Wilhelm and Suttle 1999; Ruardij etal. 2005).

line represents the total biomass of P. globosa cells

Fig. 7 Modeled abundance of Phaeocystis globosa cells and 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 by the grey area, and automortality by the white area. Auto-mortality affects cells with a net growth rate of <0.002 d_1, which was only found to be of importance for cells embedded in the colonies under nutrient depletion. The dotted

Fig. 7 Modeled abundance of Phaeocystis globosa cells and 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 by the grey area, and automortality by the white area. Auto-mortality affects cells with a net growth rate of <0.002 d_1, which was only found to be of importance for cells embedded in the colonies under nutrient depletion. The dotted

Bratbak et al. (1998) showed that viral infection of P. pouchetii resulted in a conversion of the entire algal biomass to dissolved organic carbon (DOC) within three days. In contrast, this was only a maximum of 20% in the uninfected cultures. In response to viral lysis of a Phaeocystis blooms, bacterial production has been shown to increase rapidly (Brussaard et al. 2005b). Assuming a bacterial conversion factor of 0.35 (intermediate of values reported for phytoplankton cell debris and lysis products; Biddanda 1988; Van Wambeke 1994), most to all of the bacterial C demand could be accounted for by P. globosa cellular C release upon viral lysis. Concomitantly, a shift in bacterial community composition was observed, which was most likely the result of the difference in the DOC pathway. Instead of the normally slow and steady release of small amounts of photosynthetic release of DOC (favoring K-strategists or equilibrium populations), there is the sudden virally induced release of large amounts of readily degradable and organic nutrient-rich DOC (favoring r-strategists or opportunistic populations).

Most of the field data that are available to date relate to Phaeocystis blooms. Although the virally mediated cell lysis of Phaeocystis during blooms is notably substantial, there is no proof of infection of single cells beyond the bloom period. This does not mean that there could not be signiWcant viral lysis of Phaeocystis outside the blooming period. Production of single cells can also take place at high rates when colony formation is not possible, e.g., low irradiances and nutrient concentrations. This provides not only the potential of grazing by microzooplankton, but also of viral infection of the single cells. The relatively small fraction of dissolved organic matter (DOM) obtained in this way may constitute a signiWcant portion of the cycling of rapidly degradable carbon in the pelagic zone.

The theoretical models considering the inXu-ence of (algal) viruses on the carbon cycle that exist to date are steady-state models assuming a fixed percentage of the algal population dying due to viral lysis. A bloom of Phaeocystis in, for example, temperate eutrophic coastal waters is, however, clearly not a steady-state situation. Based on the ecosystem model by Ruardij et al. (2005), we established 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 primary production only when colonies were present (68% of total), but was still one-third of the total primary production under conditions dominated by single cells. The averaged daily flux of viral lysis of P. globosa was tenfold higher for the model situation with only single cells present compared to the situation including colonies (115 vs. 11 |ig C L_1). Without colonies, viral lysis made up for 53% of the modeled daily P. globosa primary production (only 2% when colonies were present).

The reason that the averaged daily uptake of C by bacteria is not higher under the conditions that single cells dominate (compared to colony dominated conditions) and bacterial secondary production is stimulated due to P. globosa viral lysis, is due to the absence of TEP production upon disintegration of colonies. Because of the large influence colonies have on the C cycling as a result of their high content of intracellular carbon, the C budget for a model run without viruses resembles largely that of the run with viruses present (data not shown). When excluding both colonies and

Fig. 8 Simplified representation of a pelagic food web used to calculate the C budget during a Phaeocystis globosa bloom as presented in Table 1. The release of cellular carbon due to viral lysis as sources of DOC, as well as auto-mortality was only modeled for P. globosa. Excretion of DOC due to photosynthetic release was taken into account for all phytoplankton groups (Phytopl.: Phaeocystis and other algae). Micrograzers (Micropl.) include heterotro-phic nanoflagellates (HNF) and ciliates. Export was considered a negligible loss

Fig. 8 Simplified representation of a pelagic food web used to calculate the C budget during a Phaeocystis globosa bloom as presented in Table 1. The release of cellular carbon due to viral lysis as sources of DOC, as well as auto-mortality was only modeled for P. globosa. Excretion of DOC due to photosynthetic release was taken into account for all phytoplankton groups (Phytopl.: Phaeocystis and other algae). Micrograzers (Micropl.) include heterotro-phic nanoflagellates (HNF) and ciliates. Export was considered a negligible loss

Table 1 Modeled C budget of the wax and wane of a Phae-ocystis globosa bloom (Brussaard et al. 2005a)

With colonies No colonies (standard run)

Phytoplankton

0 0

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