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Keywords:

  • ocean acidification;
  • Cyanobacteria ;
  • Synechococcus ;
  • cyanophage

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

Increased anthropogenic CO2 emissions are expected to cause a drop in oceanic pH of c. 0.4 units within this century. According to current assessments, the consequences of this are limited for oceanic Cyanobacteria, and absent for viruses. We investigated the effect of pH on the life history of cyanophage S-PM2 and its host, Synechococcus sp. WH7803, at current pH concentrations and at predicted future concentrations. We identified significant negative effects of decreasing pH on Synechococcus growth rate, with profound negative implications for S-PM2 biogenesis and its infection cycle. The duration of the S-PM2 eclipse period increased significantly with decreasing pH. In contrast, the latent period was shorter at pH 7.6 than at pH 8, coinciding with a reduction in S-PM2 burst size from 20.1 ± 3.2 progeny phages per cell at pH 8 to 5.68 ± 4.4 progeny phages per cell at pH 7.6. At pH 7, there was no detectable progeny release. The extracellular stage of S-PM2 was insensitive to pH changes, but sensitive to light, with significant loss in infectivity (0.35–0.38 day−1) at relatively low irradiances (> 130 μmol photon m−2 s−1). Overall, the results suggest that pH has significant influence on cyanobacterial growth with important implications for the interactions between Cyanobacteria and their viruses.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

Unicellular marine Cyanobacteria are ubiquitously distributed in the world's oceans and are recognized as being a major component of the picophytoplankton (Johnson & Sieburth, 1979; Waterbury et al., 1979). Despite their small size (< 2 μm), cyanobacterial picoplankton account for c. 64% of the primary production in open oceans (Iturriaga & Mitchell, 1986; Liu et al., 1998; Uitz et al., 2010), where these chroococcoid Cyanobacteria are almost exclusively represented by the two genera Synechococcus and Prochlorococcus (Johnson & Sieburth, 1979; Waterbury et al., 1979; Partensky et al., 1999).

Viral infections are an important mortality agent of Cyanobacteria, potentially regulating global marine primary production, community structure and nutrient cycling (Proctor & Fuhrman, 1990; Suttle et al., 1990; Mühling et al., 2005; Haaber & Middelboe, 2009). Despite their importance for oceanic productivity and biogeochemical cycling, we still only have a superficial understanding of the environmental factors that control phage–host interactions in the marine environment. Thus, key parameters that determine cyanophage proliferation and survival, such as phage latent period, burst size and decay rate, are poorly characterized.

Ocean acidification occurs as a consequence of rising anthropogenic CO2 emissions and has already caused elevated dissolved inorganic carbon (DIC) levels with a concomitant drop in average pH in the surface ocean water of 0.1 pH units compared with preindustrial values (Haugan & Drange, 1996; Caldeira & Wickett, 2003). If emission levels continue to rise unabated, the pH of the ocean surface water is projected to drop c. 0.4 units by the end of this century (Haugan & Drange, 1996; Caldeira & Wickett, 2003; Raven et al., 2005). On a shorter time scale (seasonal), it has been a general assumption that pH in open ocean surface waters remains relatively stable, around 8.2 (±0.3 or less; Raven et al., 2005). Recently, the natural pH variation in coastal environments has been found to be large and highly dynamic with average diurnal pH variations around 0.24 units and annual variation of more than 1 unit (Wootton et al., 2008). Although pH oscillations are more stable in the open oceans (Joint et al., 2011), potentially, natural pH oscillations are already affecting processes in marine ecosystems, and with the ongoing anthropogenic acidification, such effects might become more obvious in the future. Although some studies have investigated the influence of increasing DIC levels and low pH on microalgal communities (Berge et al., 2010), little work has been carried out on marine picocyanobacteria, and no studies have to our knowledge focused on how a decrease in pH influences the phage–host relationship and phage decay rate.

Whilst calcareous phytoplankton (e.g. coccolithophores) have been a particular subject of concern due to their reliance on ocean chemistry to maintain their scales, the noncalcifying members of the phytoplankton have been predicted to benefit from anthropogenic acidification (Raven et al., 2005; Rost et al., 2008). Many photosynthetic microorganisms possess a carbon-concentrating mechanism (CCM), including Synechococcus sp. WH7803 (Hassidim et al., 1997), enabling them to accumulate intracellular dissolved inorganic carbon (DIC) and thereby overcome carbon limitations (Badger et al., 1980; Kaplan et al., 1980, 1991; Burns & Beardall, 1987). As CCM activity requires energy, the acidification in the oceans has been suggested to down-regulate the CCM capacity, reducing the energy cost of C transport, allocating more energy for growth (Beardall & Giordano, 2002).

Insights into the response in phage–host interactions to different pH concentrations have primarily been obtained from phages used in the dairy industry (Suárez et al., 2008), those associated with phage therapy (Smith et al., 1987; Nakai et al., 1999) and in pathogenic viruses associated with wastewater (Langlet et al., 2007). In all these studies, the pH concentrations investigated covered the low end of the pH scale, far below the pH levels anticipated from anthropogenic acidification. Consequently, little is known about the effects of pH on phage–host interactions at realistic pH ranges in the marine environment.

Although there is a significant amount of genetic and physiological diversity found within Synechococcus and Prochlorococcus species and indeed within the viruses which infect them (Kettler et al., 2007; Dufresne et al., 2008; Sullivan, 2010), no previous studies have investigated how the relationship between these globally significant genera of Cyanobacteria and their viruses will respond to ocean acidification. To redress this, we aimed to provide the first steps in an understanding of the dynamics between cyanophages and Cyanobacteria in response to decreasing pH, using the well-studied model system cyanophage S-PM2 and its host Synechococcus sp. WH7803 (Cuhel & Waterbury, 1984; Wilson et al., 1993; Bailey et al., 2004; Mann et al., 2005; Jia et al., 2010). Specifically, we investigated how pH changes on a scale (0.4–1 pH units) corresponding to the predictions of anthropogenic acidification and to natural pH variations of the oceans influence the growth of WH7803, the life cycle of S-PM2 and the dynamics of their interactions. The study demonstrated significant negative effects of pH changes on WH7803 growth, reducing phage burst size and prolonging the eclipse period whilst shortening the latent period.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

Cyanobacterial and cyanophage strains

Synechococcus sp. WH7803 was maintained in artificial sea water (ASW) medium (DIC: 0.2 mM, pH 8, unless otherwise stated; Wilson et al., 1996; Shan et al., 2008) and grown at 20.75 °C (±2.25) on a 14 : 10 h light/dark cycle. Illumination was provided from below, by cool white light with an irradiance of 35 ± 15 μmol photon m−2 s−1 (unless otherwise stated). Illumination was measured using a LI-250A© Light meter. The cyanophages used were all propagated on strain WH7803. To prevent degassing of CO2 from the cultures, which could affect pH, constant bubbling was avoided and the cultures were instead agitated at least once every 24 h by hand to prevent settling on the bottom.

Influence of pH on growth of Synechococcus sp. WH7803

Growth was measured using the optical density at wavelength 750 nm (OD750) as a proxy for total cell number. The effect of pH on the growth of WH7803 was investigated at pH values 8, 7.6 and 7. The experimental pH values were obtained by incremental adjustments of 0.05, 0.45 and 0.9 units per 24 h, with starting pH c. 8. Final pH concentrations were maintained by daily adjustments using CO2 and NaOH (0.5 M). The cultures were kept in 1800-ml conical DURAN® Fernbach culture flasks sealed with Identi-Plug® penetrated with a single silicone tube fitted with a piece of Identi-Plug® for improved air/gas diffusion. Following daily pH adjustments, each culture was briefly bubbled with atmospheric air to prevent any CO2 from lingering in the tubing system and simultaneously agitating cultures. Over the time course of the experiments (32–39 days), the average pH in the three treatments was 8.02 ± 0.21, 7.60 ± 0.35 and 7.06 ± 0.07, respectively. To ensure sufficient carbon concentration, dissolved inorganic carbon (DIC) was monitored using an infrared gas analyzer (ADC 225 MK3) during experiments.

Infection experiments – cyanophage life cycle

To determine the effect of pH on phage life cycle, the following parameters were monitored: latent period, eclipse period and burst size. pH-acclimatized WH7803 cells were used as the host in 72-h infection experiments with cyanophage S-PM2 carried out at pH 8.0, 7.6 and 7.0, under continuous light (35 ± 15 μmol photon m−2 s−1). The WH7803 cells used in the infection experiments were obtained from the growth cultures at an OD750 of 0.45 ± 0.01, subsequently readjusted to the intended pH values (8, 7.6 and 7, respectively) and then amended with phage S-PM2 at an MOI of c. 1 (pH 8 and 7.6) and 49.4 (pH 7). The S-PM2 used for all experiments originated from the same stock. The higher MOI at pH 7 was caused by lower cell concentration due to poor growth of WH7803.

The presence of mature phages inside host (eclipse period)

The end of the eclipse period is defined by the presence of the first mature virion inside the host and can be detected if the host is artificially lysed and then plated on lawns of the host cell. First, any unadsorbed free phages were inactivated with ferrous ammonium sulphate (FAS), using a modified protocol from McNerney et al. (1998) (See Supporting Information, Data S1 for details on the experimental application of the procedure for use with cyanophages). Samples were incubated with a final concentration of 10 mM FAS for 5 min. Following inactivation of free phages, samples were diluted in ASW to neutralize the inactivating effect of FAS, and host cells were lysed with 1% chloroform. The samples were screened for mature progeny phages by 5-μL spot assays on WH7803 agar plates.

Total viral and cyanobacterial counts

Samples were fixed with glutaraldehyde (2% final concentration), snap-frozen in liquid nitrogen and stored at −80 °C. Total abundance of viruses and Cyanobacteria during the infection experiments was determined using flow cytometry (BD FACSCantoTM). Samples were diluted in TE buffer and enumerated after staining with SYBR Green I (Brussard, 2004).

Decay of cyanophages

To determine how pH affects the decay rate of the cyanophage S-PM2 in the extracellular phase, phage infectivity and particle abundance were measured over time at pH 8 and 7 for up to 550 h, at three different light irradiances on a 14 : 10 h light/dark cycle: control (no light, < 1 μmol photon m−2 s−1), low light (27 ± 6 μmol photon m−2 s−1) and high light (120.5 ± 8.5 μmol photon m−2 s−1). Loss of infectivity was quantified from the decrease in number of plaque-forming units over time. By applying 5 μL of culture to lawns of WH7803 in spot assays following serial dilutions, plate assays were incubated under the same light and temperature conditions as described for the WH7803 liquid cultures, and infected phages were enumerated. Decay of S-PM2 particles was quantified by flow cytometry as described above.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

Growth of Synechococcus sp. WH7803 at different pH values

To examine the impact of ocean acidification on WH7803 and S-PM2 dynamics, the pH values used in these experiments were based on the prediction of future acidification levels of the ocean (pH 7.6), a more extreme value (pH 7) and pH 8 as a control.

Under the experimental conditions used in these experiments, WH7803 had a long lag phase before entering the exponential phase for all pH values investigated. The changes in ambient pH had strong effects on WH7803: at pH 8, WH7803 growth rate was 0.125 ± 0.005 day−1 (Fig. 1, Table 1), and the population reached a maximum density of 1.39 (OD750), whereas both the growth rate (0.094 ± 0.009 h−1, P < 0.02) and the maximum cell density (OD750 = 1.045) were significantly reduced at pH 7.6. At pH 7, growth was further inhibited with no significant increase in OD750 after 12 days (284 h). At the end of the experiment, one of three replicates at pH 7 showed a small increase in OD750 (0.102) after 37 days (c. 876 h).

Table 1. Synechococcus sp. WH7803 growth rates and cyanophage S-PM2 life history parameters and rates of infectivity loss and particle decay at experimental pH values of 8, 7.6 and 7 (n = 3)
pHGrowth rate (day−1)Eclipse period (h)Latent period (h)Burst size (phages per cell)Loss of infectivity (day−1)Particle decay rate (day−1)
  1. ND, not detected; NA, not answered; NO, no light (< 1 μmol photon m−2 s−1); LL, low light (27 ± 6 μmol photon m−2 s−1); HL, high light (120.5 ± 8.5 μmol photon m−2 s−1).

80.125 ± 0.0055.7 ± 0.412.3 ± 0.120.1 ± 3.2−0.01 ± 0.04 (NO)0.09 ± 0.09 (NO)
0.17 ± 0.05 (LL)0.05 ± 0.03 (LL)
0.35 ± 0.06 (HL)0.06 ± 0.03 (HL)
7.60.094 ± 0.0098.0 ± 0.19.9 ± 0.15.6 ± 4.4NANA
70.001 ± 0.02012.6 ± 2.0NDND−0.04 ± 0.04 (NO)0.12 ± 0.12 (NO)
0.17 ± 0.03 (LL)0.02 ± 0.05 (LL)
0.38 ± 0.03 (HL)0.22 ± 0.02 (HL)
image

Figure 1. Growth of Synechococcus sp. WH7803 at pH values of 8, 7.6 and 7 (n = 3). There was significant difference between growth rates at pH 8 and 7.6 (t-test, P < 0.02). Cell density was expressed as OD750.

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Influence of pH on the life cycle of the cyanophage S-PM2

Following the addition of S-PM2 to the cultures, the number of WH7803 cells remained constant until the end of the first infection cycle, which was evidenced by a rapid increase in phage abundance followed by a decline in WH7803. Several changes were observed in the growth parameters of S-PM2 when pH was reduced from 8 to 7.6 and 7. In the pH 8 and pH 7.6 experiments, the first infection cycle took 12.3 ± 0.1 h and 9.9 ± 0.1 h, respectively, followed by unsynchronized multiple re-infections (Fig. 2a and b, Table 1). At pH 7 (Fig. 2c), WH7803 did not show significant growth, nor was there a detectable release of phage, hence the latent period could not be determined. The eclipse period was determined by spot assays following exposure of cells to chloroform, and therefore, the measurements were independent of the flow cytometric counts used for cell and virus abundance. This meant that we were able to calculate the eclipse period for measurements at pH7 despite not being able to determine the latent period. The eclipse period is the first part of the latent period, starting when the phage enters the host cell and ending when the first mature progeny phage is ready inside the host cell. The second part of the latent period is used for further assembly and maturation of the progeny. The duration of the eclipse period increased significantly with decreasing pH, starting with 5.7 ± 0.4 h at pH 8, increasing to 8.0 ± 0.1 h at pH 7.6 and 12.6 ± 2.0 h at pH 7 (Table 1). The number of lysed cells between pH 8 and 7.6 was not significantly different (4.8 × 106 cells and 5.4 × 106 cells lysed, respectively), but the burst sizes showed notable differences: at pH 7.6, the burst size was only 5.6 ± 4 compared with 20.1 ± 3.2 at pH 8 (Table 1). Although production of phages inside the cells was verified at pH 7 during the eclipse period, no significant release of phages was detectable, and the burst size therefore was not determined.

image

Figure 2. The infection experiments with cyanophage S-PM2 (n = 3; full symbols) and the host Synechococcus sp. WH7803 (open symbols) cells observed over a c. 72-h time period with an MOI of c. 1 at pH 8 (a) and pH 7.6 (b) and 49.4 at pH 7 (c).

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Decay of S-PM2 in the extracellular phase

The decay of S-PM2 was differentiated between the loss of infectivity where the phage particles remain intact but unable to infect and decay of the phage particles, that is, destruction of the phage structure and loss of DNA. Light was the major contributor to infectivity loss in S-PM2 and showed a positive correlation between the light level and the rate of infectivity loss (Fig. 3a, Table 1), with exponential loss rates of infective units increasing from no loss in the dark to a rate of 0.35–0.38 day−1 at the highest light conditions (120.5 ± 8.5 μmol photon m−2 s−1; Fig. 3a). The pH on the other hand did not affect infectivity significantly within the tested pH range from 7 to 8 (Fig. 3a). Neither light nor pH had a significant effect on the decay rates of S-PM2 particles, which generally were much lower than infectivity loss rates (0.02–0.2 day−1, Fig. 3b, Table 1).

image

Figure 3. The decay of S-PM2 (n = 3) at pH 8 (full symbols) or 7 (empty symbols) at three different light irradiances: no light (< 1 μmol s−1 m−1, squares), low light (27 ± 6 μmol photon m−2 s−1, triangles) and high light (120.5 ± 8.5 μmol photon m−2 s−1, diamonds). (a) Loss of infectivity. (b) Decay of S-PM2 particles. Notice the difference in timeline.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

The study presented here demonstrates that decreasing pH has a significant negative effect on the growth of Synechococcus sp. WH7803 and on the intracellular development of S-PM2 during the phage life cycle. In the extracellular stage, S-PM2 was unaffected by pH, but highly sensitive to light.

Growth of Synechococcus sp. WH7803

The growth rate of Synechococcus sp. WH7803 decreased with pH decreasing from 8 to 7 (Fig. 4). Several life aspects of an organism are under influence of ambient pH, and even though marine Synechococcus is able to maintain intracellular pH (7.1–7.3) in a pH range of 6.4 to 8.3 (Kallas & Dahlquist, 1981), a lowered ambient pH can still have consequences for a cell's energy budget. Maintenance of intracellular pH is an energy-dependent process (Booth, 1985), and it is also known that the pH gradient across the cell membrane in Gram-negative bacteria facilitates protein export from the cell (Pugsley, 1993).

image

Figure 4. Synechococcus sp. WH7803 growth rate (black bars) and the S-PM2 eclipse period (grey bars) plotted against the experimental pH values.

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pH influences DIC concentration and may therefore this way indirectly affects cell growth rates and thus would negatively impact global primary production. To ensure a sufficient DIC concentration in the experiments, DIC was continuously adjusted and never decreased below 0.2 mM. In addition, WH7803 possesses a CO2-concentrating mechanism (CCM), enabling them to overcome low concentrations of extracellular DIC (Hassidim et al., 1997), and we therefore find it unlikely that DIC availability could explain the difference in growth rates at the different pH. The growth rate observed at pH 8 (0.13 day−1) was in the lower end of the range of Synechococcus growth rates obtained with natural populations during light exposure (e.g. 0.19–2.0 day−1, Landry et al., 1984; Agawin & Agustí, 1997). This could be attributed to the experimental conditions with low irradiances combined with a light/dark cycle. In addition, the fact that the WH7803 cultures in the present study were kept without continuous aeration to prevent CO2 degassing of the medium may have contributed to the relatively low growth rate.

In the present study, WH7803 cultures were exposed to incremental changes in pH which decreased by 0.05, 0.45 and 0.9 pH units per 24 h for experiments pH 8, 7.6 and 7, respectively, throughout the experimental period, and the cells were therefore acclimatized to the specific experimental pH prior to the infection experiments. For comparison, Berge et al. (2010) used stepwise pH adjustments of 0.5 units every 12 h working with marine dinoflagellates, cryptophytes, diatoms and prymnesiophytes. We therefore find it reasonable to exclude lack of acclimation as an explanation for the observed differences between pH 8 and 7.6. This is supported by the very fast acclimation (hours) to increasing CO2 concentrations observed in the coccolithophore Emiliania huxleyi (Barcelos et al. 2010). However, for the pH 7 experiment, where growth appeared to cease altogether, lack of a sufficient acclimatization period may have contributed to a long lag period.

The negative effects on WH7803 growth observed in the current study demonstrate for the first time that pH changes within the range used in the present study (0.4 units) significantly affect dominant marine phytoplankton species. More recent studies have demonstrated natural pH variations in the range used here (Middelboe & Hansen, 2007; Wootton et al., 2008; Gagliano et al., 2010) thus emphasizing that we cannot always assume a steady state pH in the ocean. Our data suggest that pH plays a key role in the regulation of Synechococcus growth not only in the context of future ocean acidification, but also during temporal pH variations at the current oceanic conditions.

Infection experiments – growth parameters of S-PM2

The effect of decreasing pH also had a significant impact on the infection cycle of phage S-PM2 causing an increase in eclipse period (Fig. 4) and a decrease in burst size (Table 1) concomitant with the decrease in host growth rate (Fig. 4) as pH decreased from 8 to 7, thus emphasizing that even natural variations in pH may have significant implications for phage–host interactions and the production of cyanophages.

A number of studies on Escherichia coli and its phages have demonstrated the impact of nutrient conditions on host growth and subsequently on virus development (Hadas et al., 1997; You et al., 2002). Studies on marine heterotrophic bacteria (Middelboe, 2000) and Cyanobacteria (Wilson et al., 1996) have found similar effects of changing nutritional conditions on virus life cycle parameters. Our findings thus supported previous studies emphasizing the importance of the physiological state of the host cell for the outcome and efficiency of phage infections. In the present study, the latent period was unexpectedly shorter at pH 7.6, and in combination with the prolonged eclipse period, this allowed less time phage maturation and accumulation. We propose that the reduced maturation period from the end of the eclipse period and until lysis contributed significantly to the decreased burst size.

Phages depend on their host for resources and energy, and mechanisms such as the host protein synthesis system (Hadas et al., 1997) and host DNA content (Brown et al., 2006) have been shown to correlate with phage assembly and burst size. Decreasing ambient pH, and thus increasing extracellular H+, changes the chemical conditions at the cell membrane, affecting the proton motive force, which drives several important cell functions. This may result in hampered cell function and additional energy being allocated for membrane-associated processes such as protein export (Pugsley, 1993).

The delay in producing the first progeny phage and the reduced latent period at pH 7.6 could therefore be a consequence of less intracellular carbon sources and energy available for phage production. This may lead to the depletion of cellular energy and thus triggering a delay in phage production and premature lysis of phages once produced, thereby increasing the eclipse time, but shortening the time between phage assembly and release, as seen at pH 7.6. This explanation is supported by studies on bacteriophages where both membrane depolarization (Wang et al., 2003) and ATP depletion (Žiedaite et al., 2005; Krupovič et al., 2007) have been demonstrated to trigger premature lysis.

Wilson et al. (1996) estimated that the S-PM2 burst size is 21.5 when propagated on WH7803, thus consistent with the burst size obtained in the present study at pH 8 (20.1 ± 3.2). The decrease in pH to 7.6 had a dramatic effect on the burst size, resulting in a 72.3% reduction (5.68 ± 4.4). A similar scenario was reported by Wilson et al. (1996), where a reduction in WH7803 growth rate caused by being grown in media without phosphate caused an apparent 80% reduction in S-PM2 burst size. In that study, it was discovered, however, that only 9.7% of infected cells produced progeny phages, whereas the rest were assumed to enter a pseudolysogenic stage, thus lowering the apparent burst size. Although exposure of WH7803 to low pH may also induce pseudolysogeny, which may be perceived as a decrease in burst size, the number of lysed WH7803 was not significantly different in the two experiments (4.8 × 106 cells and 5.4 × 106 cells lysed at pH 8 and 7.6, respectively). This suggested that infected cells were in fact lysed and that the reduced virus production at pH 7.6 reflected an actual decrease in burst size. A reduction in cyanophage production with a decreasing pH in the ocean may result in less efficient phage proliferation in terms of total number of phages produced, with a concomitant reduced role of viral lysis. However, as host Synechococcus numbers have also been reduced, there will be similar ratios of Cyanobacteria to viruses and thus viral predation will remain a significant causal factor for cyanobacterial mortality. This may be slightly lessened due to the dilution effect of there being a lower concentration of both Cyanobacteria and phages.

Effects of light and pH on S-PM2 decay rates

Several factors play a role in viral decay including sunlight, UV radiation, adhesion to nonliving particles and protozoan grazing (Suttle & Chen, 1992). Here, S-PM2 decay was investigated at relatively low light irradiances corresponding to levels occurring at the base of the photic zone (Kana & Glibert, 1987). To examine whether pH accelerates the decay rate, each pH value was combined with three different light conditions, and the decay rate was then quantified both as loss of infectivity and as the decay of the phage particles. Light was the principal factor contributing to loss of infectivity (Fig. 3a), whereas pH did not affect phage infectivity, thus supporting our suggestion that the observed effects on phage life cycle parameters were associated with pH effects on host physiology, and not related to direct effects on the phages.

The observed loss rate of infectivity in the present study (0.17–0.38 day−1) corresponds to previous reports on loss of infectivity rates of 0.19 to 0.67 day−1 in natural surface sea water (Suttle & Chen, 1992). However, the light irradiances used in the present study were > 10-fold lower (< 130 μmol photon m−2 s−1) compared with irradiances used in Suttle & Chen (1992), suggesting that S-PM2 is highly sensitive to light. The infectivity loss rates correlated positively with light intensity (t-test, P < 0.01), suggesting that light constitute an important loss factor for cyanophage infectivity, not only in the high irradiance surface waters but also at the bottom of the photic zone where the marine phycoerythrin-rich Synechococcus thrive (Suttle & Chan, 1994).

The decay of the phage particles over time occurred at a much slower rate, and neither light nor pH had a significant effect on the loss of phage particles. Previous studies (Suttle & Chen, 1992; Noble & Fuhrman, 1997) working with irradiation levels corresponding to surface waters have shown that light may contribute to virus particle destruction. The absence of light effects in the present study suggested that light irradiances below 130 μmol photon m−2 s−1 are insufficient to contribute to particle destruction of S-PM2. The loss of infectivity without a corresponding loss of the virus particles themselves is something to keep in mind when estimating natural phage titres by methods other than infectivity assays.

At present, coupled climate and ecosystem models do not include viruses and their role in relation to their host's physiology. And therefore, when predictions are made relating to how the ocean may respond to climate changes, amazingly, the viruses are left out of any models. This is primarily due to a paucity of data collected in this area. The present study demonstrates that the role of viruses in the regulation of cyanobacterial growth may be significantly altered in future climate scenarios, thus emphasizing the need for including viruses in ecosystem modelling.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

The study was supported by grants from The Danish Research Council for Independent Research, the Carlsberg Foundation to M.M. and the Leverhulme Trust Research Project Grant (ID20090588) to M.R.J.C. We wish to thank Jeanett Hansen for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors’ contribution
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
fem12199-sup-0001-DataS1.docWord document526KData S1. Data to show the impact of ferrous ammonium sulphate (FAS) on cyanophages.

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