INTRODUCTION

Autotrophic picoplankton (solitary cyanobacteria and algae less than 2 μm in size) are present in all types of freshwater ecosystems (Stockner, 1991). In the majority of these ecosystems, the concentrations of picocyanobacteria are an order higher than of picoalgae and the former are the main component of the autotrophic picoplankton (Mikheeva, 1998).

Picocyanobacteria (PC) are an important component of the planktonic food webs, and heterotrophic nanoflagellates, infusoria, and multicellular fine filter-feeders actively consume them. In the Upper Volga reservoirs, PC play an important role for plankton. In summer, a considerable part of them dies out due to viral infection and lysis (Kopylov et al., 2010). Although the Boreal waterbodies are covered by ice for half the year, these processes during the freezing period have not yet been studied.

The goal of the present paper is to determine the level of quantitative development of virioplankton and planktonic picocyanobacteria, as well as to assess the intensity of viral infection in picocyanobacteria during the freezing period at low water temperatures.

MATERIALS AND METHODS

The studies were carried out at the Volzhskii reach of Rybinsk Reservoir at one deep-water (site 1, 58°05.61′ N, 38°18.04′ E) and two shallow-water (site 2, 58°05.77′ N, 38°17.53′ E and site 3, 58°08.83′ N, 38°22.75′ E) sampling sites. At each site the water was sampled on February 2, 15, and 26 and March 11 and 27, 2008, using a 0.5 L Plexiglas Ruttner bathometer. The water was sampled at 2–3 layers of the water column including the surface (about 20 cm under ice) and near-bottom (about 50 cm above the bottom) layers.

Planktonic virus particles were analyzed by the epifluorescence microscopy technique using SYBR Green I stain and Anodisc (Wathman, United States) aluminum oxide filter with a 0.02 µm pore size (Noble and Fuhrman, 1998). The number of PC was determined using epifluorescence microscopy by the autofluorescence of their cell pigments (McIsaac and Stockner, 1993) on a black nuclear filter with 0.2 μm pores (JINR, Dubna, Russia). The viruses and PC were counted using Olympus BZ51 (Olympus, Japan) epifluorescence microscope equipped with the system for analysis of the images.

To determine the frequency of clearly visible cells of picoautotrophs infected with the viruses (FVIC, % of the total number of PC) and the mean number of mature phages in the infected cells (Burst size (BS), particles/cell), we used transmission electron microscopy. The viruses and PC were precipitated on nickel Pioloform/carbon coated grids for electron microscopy (400 mesh density) with by centrifugation at 100 000 g (35 000 rpm) for 1 h at 4°С on an OPTIMA L-90k ultracentrifuge (Beckman Coulter, USA) equipped with a 45 Ti rotor. The grids were analyzed at 50 000–150 000 magnification on a JEM 1011 electron microscope (Jeol, Japan). The cells of picoautotrophs were considered infected if they contained four or more mature phages.

Two stages in the cycle of virus lytic infection of bacterial cells are defined: latent period and lysis. The latent period is the time from the beginning of cell infection to the beginning of lysis. Mature phages appear in the cell directly before the lysis. This is why the viruses are invisible in the cells during most of the latent period. The share (%) of infected cells containing clearly visible mature cyanophage viruses in the total number of PC (FVIC) is an important parameter in the studies of viruses and cyanobacteria. The time from the beginning of infection to the first appearance of visible virus particles is called the “eclipse-period.” Such particles are present in the infected bacterial cells from the end of the eclipse-period to the lysis. Thus, the share of all infected cells (the frequency of infected cells, FIC) in the PC population is much higher than FVIC. It was proposed to assess FIC as a product of FVIC and the ratio of the duration of the eclipse-period to the duration of the latent period (ε) (Proctor and Fuhrman, 1990). In our calculations the ε value was taken as 0.75 (Suttle, 2000; Mann, 2003).

RESULTS AND DISCUSSION

At the deep-water sites of the reservoir, the depth varied from 9.5 to 10.6 m; at the shallow-water sites, 3.5–4.5 m (Table 1). Water transparency was 2.8–3.5 m; the thickness of the ice was 40–60 cm; the thickness of the snow cover above the ice reached up to 10 cm. The values of pH were weakly alkaline: 7.31–7.88 (7.60, on average); electric conductivity was 374–400 (385, on average) μSm/cm. The water temperature fluctuated from 0.3 to 0.9°C and was higher at the deep-water site. In March the water temperature was higher (0.67°C, on average) than in February (0.54°C, on average). The concentration of water borne oxygen varied from 7.3 to 11.3 (9.1, on average) mg/L. No clear stratification of the water column during the freeze-up period was observed at the sites studied.

Table 1.   Characteristics of the sampling sites in Rybinsk Reservoir in February–March 2008
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During the study period, the number of virioplankton (NV) was high (Table 2). At the deep-water sites in the water column, the minimal and maximal values of NV differed by 1.1–2.0 times. In February the mean for the number of viruses ((57.2 ± 3.6) × 106 particles/mL) in the water column differed slightly from the number in March ((66.8 ± 2.4) × 106 particles/mL). At the shallow-water sites 2 and 3, the mean for the water column values of NV in February ((56.1 ± 2.2) × 106 and (50.5 ± 6.5) × 106 particles/mL, respectively) differed from the March values insignificantly (56.5 ± 2.3) × 106 and (53.9 ± 4.9) × 106 particles/mL, respectively).

Table 2. Total number of planktonic viruses (NV, 106 particles/mL), number of picocyanobacteria (NPC, 103 cells/mL), number of picocyanobacteria with viruses attached to their cells (NAPC, % NPC) and number of viruses and number of viruses attached to picocyanobacteria (NVPC, particles/ml) during the freezing period in Rybinsk Reservoir (February–March 2008)
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The number of PC (NPC) at the studied sites of the reservoir was low, being three orders of magnitude lower than the number of planktonic viruses (Table 2). In February the values of NPC at all sites at various depths differed by a factor of not more than 1.6 while in March the minimal values of this parameter in the near-bottom water layer were 1.5–4.8 times lower than the maximal values. In February at the deep-water site 1, the mean for the whole water column NPC value (50728 ± 3430 cells/mL) was lower than in March (34 883 ± 9488 cells/mL). At sites 2 and 3, the mean for the water column values of NPC in February (69 450 ± 1974 and 61 900 ± 2654 cells/mL, respectively) were also considerably higher than in March (37 500 ± 10 818 and 39 781 ± 10 109 cells/mL, respectively). Virus particles were attached to the surfaces of a considerable part of PC (Table 2). At the deep-water site 1, the mean for the whole span of the observation period share of PC with attached viruses in NPC (12.7 ± 1.5%) was slightly higher than for the shallow-water sites (site 2, 10.2 ± 1.1%; site 3, 10.0 ± 1.7%).

The number of viruses attached to one picocyanobacterium reached 11, and on average for the sample ranged from 1.2 to 3.0 viruses. The number of cyanophage viruses attached to cyanobacterial cells varied from 3831 to 34742 (on average, 9426 ± 1189), which accounted for 0.01–0.06 (on average, 0.02 ± 0.10)% NV.

Table 3.   Frequency of visible infected picocyanobacteria cells (FVIC, % NPC), frequency of infected picocyanobacteria (FIC, % NPC) and number of viral particles inside the picocyanobacteria cells (BS, particles/cell)
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At the deep-water site, the FVIC values changed from 1.0 to 7.6 NPC, averaging 2.9 ± 0.5% (Table 3). The FIC values varied from 3.0 to 22.8% (8.8 ± 1.4%, on average) of NPC. In February the values of FVIC and FIC (4.0 ± 0.5 and 12.0 ± 1.6% of NPC, respectively) were higher than in March (1.4 ± 0.2 and 4.2 ± 0.3% of NPC), respectively.

At the shallow-water sites 1 and 2, the values of FVIC and FIC fluctuated from 0 to 4.7 (1.7 ± 0.3%, on average) and from 0 to 1.41% (5.1 ± 0.8%, on average) NPC, respectively. In March the intensity of infection by viruses (FVIC = 0.86 ± 0.20% and FIC = 2.58 ± 0.61% of NPC) was also lower than in February ((FVIC = 2.31 ± 0.37% and FIC = 6.93 ± 1.10% of NPC). A positive correlation (R = 0.55; p = 0.05) between the number of visibly infected PC and the number of viruses attached to the cells of cyanobacteria was observed.

The study revealed that the intensity of infection of PC by viruses decreased during the observation period from the beginning of February to the end of March. It is likely that the high intensity of viral infection observed in PC in February was the reason for the high mortality of the latter during this period. As a result, and presumably owing to the lack of gain in the PC number in March, their number decreased considerably and the intensity of viral infection dropped sharply. At the same time, lysis of cyanobacteria and a relatively high number of mature phages inside their cells lead to the release of free viruses into the environment. The latter fact may be one of the reasons for the high number of free viruses under the ice.

In Rybinsk Reservoir the numbers of virioplankton in February, March, and July differed inconsiderably but the abundance of PC during the freezing period was, on average, 3.2 times lower than in summer (Table 4). The share of visibly infected cells in NPC and the number of mature viruses inside the cells in February were considerably higher than in summer; in March, they were lower.

Table 4. Number of virioplankton (NV, 106 particles/mL) and picocyanobacteria (NPC, 103 cells/mL), frequency of visible infected picocyanobacteria cells (FVIC, % NPC), and number of mature cyanophages inside infected picocyanobacteria cells (BS, particles/cell) in July 2007 and February–March 2008
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CONCLUSIONS

In Rybinsk Reservoir during the freezing period, the number of virioplankton was high, being comparable to its number during the vegetation period. From February to March, the share of the infected picocyanobacteria cells decreased considerably, but, on average, for the observation period it was approximately the same as in summer.