Viral lysis and bacterivory during a phytoplankton bloom in a coastal water microcosm

Abstract

The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated. A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation. Virus-like particles (VLP) were determined by using three methods of counting (DAPI [4',6-diamidino-2-phenylindole] staining, YOPRO staining, and transmission electron microscopy). Virus counts increased from the beginning until the end of the experiment. However, different methods produced significantly different results. DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers. Bacteria reached the maximal abundance at 122 h (3 x 10(7) bacteria ml-1), after the peak of chlorophyll a (80 μg liter-1). Phototrophic nanoflagellates followed the same pattern as for chlorophyll a. Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment. The specific bacterial growth rate increased until 168 h (2.6 day-1). The bacterivory rate reached the maximal value at 96 hours (0.9 day-1). Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide. The %VIB was always lower than 1% during the experiment. VDR were used to estimate viral production. Viral production increased 1 order of magnitude during the experiment (from 10(6) to 10(7) VLP ml-1 h-1). The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10%. The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment. Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9. A balance between bacterial losses and bacterial production was calculated for each sampling interval. At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses. At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured. At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality. In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions.

FIG. 1

VLP abundance during the experiment as determined by the different counting methods.

FIG. 2

(A) Chlorophyll a (Chla) and bacterial abundance (BN) throughout the experiment. (B) HNF and PNF abundance throughout the experiment.

FIG. 3

(A) BHP during the experiment. Bars indicate the standard errors based on two replicates for each culture. When no bars are visible, the errors were smaller than the marker points. (B) Specific growth rate (μ) of the bacteria during the experiment. (C) Bacterivory rate during the experiment. d, day.

FIG. 4

Viral-decay experiments performed after 0 (A), 2 (B), 4 (C), 7 (D), and 9 (E) days. At day 0, the viral-decay experiment was performed with the natural sample. Bars indicate the standard errors based on two replicates for each experiment. When no bars are visible, the errors were smaller than the marker points.

FIG. 5

(A) VDR calculated as the slope of the log-linear part of each decay experiment. Bars indicate the standard error for each slope. Significance and r² values for these slopes and the interval of hours used to calculate them are shown in Table 2. (B) VLP produced per milliliter per hour, calculated according to the VDR and according to the percentage of infected cells (%VIB). In the latter case we have assumed a burst size of between 100 to 300 VLP per cell. Error bars correspond to the standard error of the estimated values with this range of burst sizes. (C) Bacterial cells lost per hour and per milliliter due to viral lysis and due to bacterivory. The bacterial mortality due to viral lysis corresponds to the values calculated from the VDR. Error bars correspond to the standard error of the estimated values with a range of burst sizes of between 100 and 300 VLP per cell.

FIG. 6

Visibly infected bacteria at day 7 of the experiment.

FIG. 7

(A) Bacteria ingested by bacterivores or lysed by viruses as percentages of bacterial abundance (% BN) per hour at different days of the experiment. The values of viral lysis were calculated from the VDR. N.S., natural sample. Error bars indicate the lowest and the highest numbers of bacteria lysed by viruses calculated by assuming a range of burst sizes of between 100 and 300 VLP released per cell. (B) Bacteria ingested by bacterivores or lysed by viruses as percentages of BHP at different days of the experiment for both cultures. The values of viral lysis were calculated from the VDR. N.S., natural sample. Error bars indicate the lowest and highest values of bacteria lysed by viruses calculated by assuming a range of burst sizes of between 100 and 300 VLP released per cell. (C) Bacteria ingested by bacterivores or lysed by viruses as percentages of BHP and abundance (BN) per hour at different days of the experiment. Numbers of bacteria lysed by viruses have been calculated from the percentage of infected cells. The two arrowheads indicate samples where infected cells could not be detected.

FIG. 8

Bacterial abundance (BN ml⁻¹) as determined by epifluorescence microscopy at the end of each time interval BN_{di + 2} (A) and bacterial abundance calculated as follows: BN_di + average HBP_{di, di + 2} + average VL_{di, di + 2} + BV_di (B), where i is 0, 2, 4, and 7 days. Values were calculated for each culture separately.