Journal of Plankton Research Vol.21 no.5 pp.923–937, 1999
Viral lysis and grazing loss of bacteria in nutrient- and carbonmanipulated brackish water enclosures
P.Tuomi and P.Kuuppo
Finnish Environment Institute, PO Box 140, FIN-00251 Helsinki, Finland
Abstract. A 3 week enclosure experiment was carried out at the Gulf of Finland, the Baltic Sea. After
additions of inorganic nutrients [nitrogen (N) + phosphorus (P)] and a carbon source (sucrose), we
followed bacterial, viral and heterotrophic nanoflagellate (HNF) abundances, as well as bacterial
production and the frequency of bacteria visibly infected with viruses. Furthermore, the decay rate
of virus particles was measured three times during the enclosure experiment from the KCN-treated
water samples. Bacterial mortality caused by viral lysis was estimated using the decay rates and the
fraction of bacteria infected. Nutrient (N + P) additions stimulated phytoplankton growth [the chlorophyll (Chl) a concentration increased from <5 µg l–1 up to 19 µg l–1], while sucrose additions increased
bacterial production (from 4–6 3 107 cells l–1 h–1 up to 79 3 107 cells l–1 h–1). The phytoplankton
blooms affected bacterial production only slightly. Bacterial mortality that was explained by viruses
ranged from <2% to 13% when estimated from the visibly infected cells, and from 8% to 808% when
the decay rates (range 0.052–0.765 h–1) were used. Assuming a clearance rate of 5 nl flagellate–1 h–1,
the HNF community could graze 16–135% of total bacterial loss.
Introduction
In marine plankton food webs, material flux through bacteria can be large:
bacterial production can even exceed primary production (Ducklow and Carlson,
1992). In the northern Baltic Sea, annual bacterial production has been estimated
to range from 4 to 46% of net primary production (Larsson and Hagström, 1982;
Kuosa and Kivi, 1989; Lignell et al., 1993). The loss of bacterial biomass is mainly
caused by grazing and viral lysis (Fuhrman and Noble, 1995; Weinbauer and
Peduzzi, 1995) and these two processes have different consequences in carbon
fluxes and elemental cycles. If a major part of bacterial loss is due to viruses,
carbon is burned in a dissolved organic carbon (DOC)–bacteria–DOC loop
(Bratbak et al., 1992). Instead, if protists graze most of the bacterial cells
produced, the carbon will also be transferred to the higher trophic levels, and
processes other than viral lysis are important in releasing DOC and nutrients into
the water column.
Estimates of bacterial loss caused by viral lysis range from 1 to >100% (Proctor
and Fuhrman, 1990; Bratbak et al., 1992; Steward et al., 1992b; Fuhrman and
Noble, 1995; Hennes and Simon, 1995; Mathias et al., 1995). This variation can be
real, but partly due to the uncertainties in estimating the quantity of viral infection. Basically, three different approaches have been used in aquatic environments
to estimate the production rate of virus particles. First, the fraction of cells infected
can be estimated using electron microscopy to count cells that contain virus
particles (e.g. Proctor et al., 1993). With this method, it has been estimated that
5–62% of bacterial mortality may be due to viral lysis (Proctor et al., 1993;
Fuhrman and Noble, 1995; Hennes and Simon, 1995; Mathias et al., 1995). Second,
Steward et al. (1992a) measured the incorporation rate of the radioactive tracer
into the phage DNA. Using 32Pi incorporation, Steward et al. (1992b) estimated
© Oxford University Press
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P.Tuomi and P.Kuuppo
that viruses explained 1–40% of the bacterial mortality. In the third method, it is
assumed that the average decay rate of virus particles gives an estimate of the virus
production if the two processes are balanced (Heldal and Bratbak, 1991). The
decay of virus particles can be measured in the water samples where the production of new virus particles is prevented by cyanide. Using the decay rate of virus
particles, estimates of virus-induced mortality range from 16 to >100% of the total
bacterial mortality (Bratbak et al., 1992; Mathias et al., 1995).
We studied the effects of an inorganic nutrient [nitrogen (N) + phosphorus (P)]
and an organic carbon source [sucrose (S)] additions on the abundances of
bacteria, viruses and heterotrophic nanoflagellates (HNF) in a 3 week enclosure
experiment. Total mortality of bacteria was estimated from the cell numbers and
production rates. The fraction of virus-induced mortality was estimated using
measured decay rates of virus particles as well as the frequency of visibly infected
cells. Grazing loss was estimated using a clearance rate measured in the Baltic
Sea (5 nl flagellate–1 h–1) (Kuosa, 1990; Kuuppo-Leinikki, 1990; Kuuppo-Leinikki
and Kuosa, 1990) and HNF abundance.
Method
Mesocosm experiment
In July 1993, a large-scale floating enclosure experiment was carried out in an
archipelago area at the entrance to the Gulf of Finland, the Baltic Sea. Eight
enclosures, each ~50 m3 in volume (depth 14 m, diameter 2.3 m), were treated
with inorganic nutrients and sucrose additions (Table I) on days 0, 7 and 14. Using
a Ruttner type sampler, pooled water samples were taken daily, before the nutrient addition, from the upper 0–5 m with 1 m intervals.
Chlorophyll a
Subsamples for total chlorophyll (Chl a) measurements [method modified from
Marker et al. (1980)] were taken daily from all enclosures and filtered onto
Whatman GF/F filters, sonicated for 10 min and soaked in 96% ethanol for 24 h
at room temperature in darkness. The extracts were filtered through Whatman
GF/F filters and the Chl a concentrations were measured with Shimadzu RF-5001
spectrofluorometer which was standardized using pure Chl a (Sigma).
Table I. The experimental design. Treatments of different
enclosures are depicted with (–): no treatment, or (+): N + P =
15 µg PO4-P l–1 + 30 µg NH4-N l–1 + 30 µg NO3-N l–1; S is sucrose
(400 µg C l–1)
924
Enclosure
N+P
S
Control
N+P
S
N+P+S
–
+
–
+
–
–
+
+
Viral lysis and grazing loss of bacteria
Microscopy
Subsamples for microscopy were fixed every second day from all enclosures
within 1 h from the sampling. Samples for the enumeration of bacteria were
preserved with 0.2-µm-filtered 37% formalin (final concentration 2.5% formaldehyde) and counted according to Hobbie et al. (1977). Subsamples (1 ml) were
stained with acridine orange solution (final concentration 0.02%) and filtered
onto black Nuclepore filters (pore size 0.2 µm). Bacteria were counted under blue
excitation light (filter block I 2/3) using a Leiz Dialux or Diaplan epifluorescence
microscope. At least 200 bacteria from 20 fields were counted.
HNF were preserved with ice-cold 4% glutaraldehyde (final concentration 1%).
For the cell counts, 5 ml subsamples were stained with DAPI (Porter and Feig,
1980) and filtered onto black Nuclepore filters (pore size 0.2 µm). HNF were
counted under UV excitation (filter block A2) using a Leiz Dialux or Diaplan
epifluorescence microscope. From each filter, cells were counted from 100 microscope fields (yield 16–155 cells) and divided into size classes according to their
maximum dimensions (= length) with the aid of an ocular grid (Patterson globe).
Samples for the counting of viruses, bacteria visibly infected and burst size
were preserved with 25% glutaraldehyde (final concentration 1%). Subsamples
(~10 ml) were harvested by centrifugation (Beckman swing-out rotor SW 27, 3 h,
21 000 r.p.m., 78 900 g) onto duplicate 400-mesh Ni electron microscope grids
supported with carbon-coated Pioloform film (Bratbak and Heldal, 1993). Counts
were performed by transmission electron microscope (Jeol JEM-100CX, Department of Electron Microscopy, University of Helsinki, Helsinki, Finland), using
100 000 and 30 000 magnification to count viruses and bacteria infected, respectively. Virus particles were counted from one grid and from ~60 fields (in total
200–800 viruses). For the enumeration of bacteria that were visibly infected, at
least 400 cells were observed. The average burst size was counted from the
bacterial cells (in total 68 cells observed) containing virus particles.
Production measurements
Bacterial production was measured daily using the thymidine incorporation
method (Fuhrman and Azam, 1980, 1982). Duplicate 10 or 20 ml samples and a
formalin-killed control (final concentration 0.4% formaldehyde) were incubated
for 30 min in in situ temperature after the addition of [3H-methyl]thymidine (1.74
TBq mmol–1, Amersham, final concentration 10 nM). The incubation was
stopped with formalin addition. Radioactivity was measured from the coldtrichloroacetic acid (TCA)-precipitated material collected onto 0.2 µm pore size
cellulose nitrate filters (Sartorius) and counted with a 1215 Rackbeta liquid scintillation counter (LKB Wallac) using Lumagel (Lumac, The Netherlands) liquid
scintillation cocktail.
The production of virus particles was estimated from the decay rate experiments (Heldal and Bratbak, 1991), which were run on days 1, 8 and 12 using a
water sample from the control enclosure. The decay of virus particles was
observed in a cyanide (2 mM KCN) treated water sample which was incubated
925
P.Tuomi and P.Kuuppo
in glass bottles (exclude wavelengths >320 nm) at the pier in ~1 m depth. The
incubations were started in the morning (8:00–10:00 h) and lasted for 24–27 h.
During the first 4–6 h, samples for microscopy were taken at 1–2 h intervals. A
water sample without the KCN was used as a control.
Calculations
Bacterial cell production rates were calculated from the thymidine incorporation
rate using an average (2.3 3 109 cells nmol–1 thymidine) of several conversion
factors measured empirically in the study area (Kuparinen, 1988; KuuppoLeinikki, 1990; Kuuppo-Leinikki and Kuosa, 1990; Autio, 1992; Heinänen and
Kuparinen, 1992; Tuomi, 1997). The loss rate of bacteria was calculated using the
following equation: loss = (cell number on day N) + (average cell production
between days N and N + 2) – (cell number on day N + 2).
From the fraction of bacteria infected, the mortality of heterotrophic bacteria
due to virus infection was estimated assuming that virus particles can be seen
during a minimum 10% from the end of the whole lytic cycle (Proctor et al., 1993),
and that as large a fraction of lost bacteria was infected as observed for the standing stock. A decay rate-based estimate of virus production was calculated from
the virus disappearance rate and the fraction of virus particles remaining in the
decay experiments, as well as viral abundance and burst size in the enclosures
(Bratbak et al., 1992).
Grazing loss of bacteria was estimated from the bacterial and HNF abundances
assuming an average clearance rate of 5 nl flagellate–1 h–1, which has been
measured in July in the study area (Kuosa, 1990; Kuuppo-Leinikki, 1990;
Kuuppo-Leinikki and Kuosa, 1990).
Results
Temperature and phytoplankton
During the experiment, the water temperature in the surface layer of the mesocosms increased from 7 to 16°C (Figure 1), and the thermocline was between 5
and 10 m. The additions of N and P stimulated phytoplankton blooms which were
dominated by Eutreptiella gymnastica Throndsen (Chl a concentrations up to 19
µg l–l) (Figures 2 and 4). The blooms had their maximum 1–3 days after the nutrient additions, and they collapsed when the nutrients were depleted from the
water column (Olli et al., 1996). In the enclosures without nutrient additions,
Chl a concentration remained below 5 µg l–l.
Development of bacteria, HNF and viruses
At the beginning of the experiment, bacterial cell production ranged from 4 to 6
107 cells l–1 h–1 and increased up to 28 3 107 cells l–1 h–1 in the S enclosure and
up to 79 3 107 cells l–1 h–1 in the N + P + S enclosure (Figures 1–4). All manipulated enclosures differed significantly from the control unit (Sign test, P < 0.002).
We observed the increases in bacterial production 1–6 days after the nutrient
pulses or 0–4 days after the phytoplankton blooms.
3
926
Viral lysis and grazing loss of bacteria
At the beginning of the experiment, the abundance of bacteria was 2–4 3 109
cells l–1 (Figures 1–4). In the N + P + S enclosure up to 9 3 109 cells l–1 and in
other enclosures up to 4–6 3 109 cells l–1 were counted, but the differences
between the enclosures were not significant (Friedman two-way ANOVA, P =
0.70). Peaks in the bacterial cell number followed the peaks in bacterial production after 2–4 days, but the manipulations did not increase bacterial cell numbers
as much as they increased bacterial production (Table II).
Table II. The effect of the manipulations as an average percentage
increase or decrease when compared to the control unit
Chl a
Bacterial production
Bacterial abundance
HNF abundance
Virus abundance
Bacteria infected
N+P
S
N+P+S
+256%
+25%
–4%
+35%
+40%
+48%
–1%
+87%
–2%
+15%
+141%
+20%
+204%
+210%
+16%
+62%
+50%
+250%
Fig. 1. Water temperature, chlorophyll a concentration, heterotrophic nanoflagellate abundance
(HNF), bacterial cell production rate (BPROD), bacterial cell number (BAC), fraction of bacteria
visibly infected with viruses and virus total counts (VIR) in the control enclosure during the mesocosm experiment in 1993.
927
P.Tuomi and P.Kuuppo
Fig. 2. Chlorophyll a concentration, heterotrophic nanoflagellate abundance (HNF), bacterial cell
production rate (BPROD), bacterial cell number (BAC), fraction of bacteria visibly infected with
viruses and virus total counts (VIR) in the enclosure with inorganic nutrient additions (N + P) during
the mesocosm experiment in 1993.
The abundance of viruses ranged from 2 to 4 3 1010 l–1 at the beginning, and
increased towards the end of the experiment in all enclosures (Figures 1–4).
Highest viral counts (14 3 1010 l–1) were found from the S enclosure (Figure 3).
This was the only unit that differed significantly from the control unit (Sign test,
P < 0.01). Most of the viruses (73%) were in the size range 60–100 nm, the total
size range being from 10 to 300 nm. Viruses with a diameter >150 nm were present
in numbers around 108 l–1 (being ~1% of total count) and their abundance (data
not shown) followed closely the total count of viruses.
Most of the HNF (average 78%) were <5 µm in diameter. The HNF abundance
increased from the original 2–4 3 106 l–1 up to 9–12 3 106 l–1 during the second
half of the experiment (Figures 1–4). Highest counts (12 3 106 l–1) were found in
the N + P enclosures (Figure 2), where increased HNF abundances were observed
4 days after the two first phytoplankton blooms and 2 days after the last bloom.
HNF abundance was significantly higher in the N + P + S enclosure (Figure 4)
when compared to the control unit (Sign test, P < 0.02), while the rest of the
enclosures did not differ from the control (Sign test, P > 0.06).
928
Viral lysis and grazing loss of bacteria
Fig. 3. Chlorophyll a concentration, heterotrophic nanoflagellate abundance (HNF), bacterial cell
production rate (BPROD), bacterial cell number (BAC), fraction of bacteria visibly infected with
viruses and virus total counts (VIR) in the enclosure with sucrose (S) additions during the mesocosm
experiment in 1993.
Loss of bacteria
The highest average turnover time of the bacterial community was found in the
N + P + S enclosure (34 h) and the lowest in the control unit (56 h). The fraction
of bacteria that was visibly infected by viruses ranged from <0.2% to 1.3%. The
highest percentage of infected bacteria was found in the N + P + S enclosure and
the lowest in the S enclosure (Figures 1–4). Assuming that mature virus particles
can be seen only during the last 10% of the whole lytic cycle (Proctor et al., 1993),
and that lost bacteria were as frequently infected as the standing stock, virusinduced mortality explained <2–13% (average 7.5%) of total bacterial mortality
(Figure 6). Burst size, which was counted from the cells that were visibly infected,
ranged from 9 to ~200 viruses per bacterium (average 52).
Viral and bacterial abundances during the decay experiments are shown in
Figure 5. In the decay experiment on day 1, no remarkable increase in viral
numbers was observed at the start of the experiment, and the disappearance of
virus particles was slow. In the experiments on days 8 and 12, viral numbers
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P.Tuomi and P.Kuuppo
Fig. 4. Chlorophyll a concentration, heterotrophic nanoflagellate abundance (HNF), bacterial cell
production rate (BPROD), bacterial cell number (BAC), fraction of bacteria visibly infected with
viruses and virus total counts (VIR) in the enclosure with both inorganic and sucrose additions (N +
P + S) during the mesocosm experiment in 1993.
Fig. 5. Virus and bacterial counts in the decay rate experiments on days 1, 8 and 12. d, sample with
KCN (two replicates); s, control (without KCN). The dotted line shows the regression between the
sampling points when the decay was observed.
930
Viral lysis and grazing loss of bacteria
Fig. 6. Viral lysis and grazing, expressed as a percentage of calculated total bacterial mortality.
increased during the first 3–4 h, after which a decrease in the viral abundance could
be observed. For the period of the decay, a regression line was drawn (Figure 5)
from which the decay rate was estimated. On days 1, 8 and 12, virus decay rates
were 0.052, 0.300 and 0.765 h–1, respectively. Viruses decaying at this rate made up
56, 77 and 32% (on days 1, 8 and 12, respectively) of their total abundance.
Viral infection of total bacterial mortality, based on decay rates, ranged from
8 to 808% (average 239%) (Figure 6). During the first week of the mesocosm
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P.Tuomi and P.Kuuppo
experiment, viral lysis (based on the decay rates) explained 8–43% of bacterial
mortality, but during the rest of the experiment, virus-induced mortality exceeded
bacterial loss 2–10 times.
Assuming a clearance rate of 5 nl flagellate–1 h–1 (Kuosa, 1990; KuuppoLeinikki, 1990; Kuuppo-Leinikki and Kuosa, 1990), the HNF community
explained 16–135% (average 56%) of bacterial loss (Figure 6). In the control and
N + P enclosures, grazing loss increased towards the end of the experiment along
with the increasing HNF numbers.
Discussion
Bottom-up control of bacteria and HNF
The effect of different treatments on bacteria and HNF was evaluated by comparing enclosures after reducing the values from the control unit (Table II). The
availability of sucrose as carbon and energy source stimulated bacterial growth
more than inorganic nutrient additions, indicating a carbon limitation of bacteria.
In the Gulf of Finland, bacterioplankton limitation by carbon in summer has also
been suggested previously (Kivi et al., 1993; Kuuppo-Leinikki et al., 1994), when
the primary production is mainly based on regenerated nutrients and cycled
largely through the microbial food web (Uitto et al., 1997). When the carbon limitation was relieved by the sucrose addition in this experiment, bacteria were able
to compete efficiently with phytoplankton for inorganic nutrients: in the N + P +
S enclosure, the Chl a concentration did not reach as high values as in the unit
with the nutrient manipulation only (Table II). The most important loss mechanism of the phytoplankton in this experiment was mesozooplankton grazing (Olli
et al., 1996), and the carbon fixed by the blooming algae seemed not to be available for bacteria.
Nutrient and sucrose manipulations increased algal and bacterial productivity,
but the effect was much dampened on the second step of the food web. HNF
numbers did not differ significantly between the enclosures, indicating that HNF
were also top-down controlled by predators.
Estimation of virus-induced mortality of bacteria
Parameters that were used to estimate the virus-induced mortality in this study
were total viral counts, decay rate of virus particles, burst size and the fraction of
bacteria visibly infected. Some uncertainties related to these parameters are
discussed below.
Viruses infect organisms at all trophic levels of the plankton ecosystem, but
different virus strains cannot be recognized by morphology. For example, the size
range of algal (35–400 nm) and bacterial (50–128 nm) viruses is overlapping,
although on average most algal viruses (100–200 nm) are larger than bacteriophages (<100 nm) (Van Etten et al., 1991; Børsheim, 1993). Most (73%) of the
virus particles counted in this study were in the average size range of bacteriophages and only ~1% were >150 nm in size. Furthermore, the number of viruses
has been found to follow that of bacteria in aquatic ecosystems (Boehme et al.,
932
Viral lysis and grazing loss of bacteria
1993; Cochlan et al., 1993; Fuhrman and Suttle, 1993; Hennes and Simon, 1995;
Maranger and Bird, 1995; Weinbauer and Peduzzi, 1995). Similarly, on the coastal
area of the Gulf of Finland, a field study has demonstrated a coupling between
viral and bacterial abundances (Tuomi, 1997). This indicates that in marine
environments the majority of viruses are bacteriophages, which gave us grounds
to regard all viruses as bacteriophages in this study. However, if a large fraction
of virus particles were other than bacteriophages, the lysis estimates that were
based on the decay rates overestimated the virus-induced mortality. For example,
the abundance of picocyanobacteria (Synechococcus) increased towards the end
of the enclosure experiment, which may have favoured the multiplication of
cyanophages.
The virus community in the control enclosure showed decay rates (from 0.052
to 0.765 h–1) which are similar to those obtained earlier in the KCN-treated water
samples (0.01–0.64 h–1) (Heldal and Bratbak, 1991; Bratbak et al., 1992; Mathias
et al., 1995). The decay rate experiments in this study were performed in glass
bottles, excluding the solar UVB irradiation, which can cause significant loss of
virus infectivity (Noble and Fuhrman, 1997). Also the plastic cover that was used
in the enclosures excluded UV radiation (data not shown). Consequently, the
decay rates measured here may have underestimated the average disappearance
rate of viruses in the natural sea water (0.5–84 h–1; Bratbak et al., 1994), but they
can be applied to estimate the virus production rate in the enclosures.
More than a 10-fold range in decay rates has been reported for isolated bacteriophages and cyanophages (0.005–0.083 h–1) (Bratbak et al., 1994; Suttle and
Chan, 1994; Wommack et al., 1996). The use of the same decay rates for all enclosures may have caused some error to the estimates of virus-induced mortality,
because during the 3 week experiment succession of different virus strains may
have occurred.
The average number of viruses inside the infected bacteria was used to calculate the burst size (52, SD 41), which was within the values reported earlier from
the aquatic environments (21–300, average ~50) (Bratbak et al., 1990; Proctor and
Fuhrman, 1990; Heldal and Bratbak, 1991; Fuhrman and Noble, 1995; Hennes
and Simon, 1995; Mathias et al., 1995; Weinbauer and Suttle, 1996). Cultured
marine phages have higher average burst size (Børsheim, 1993), indicating that
inorganic nutrients (Wilson et al., 1996) or other environmental factors may limit
the phage particle production in natural waters. In this experiment, nutrient and
carbon pulses may have affected the burst size. In the calculation of bacterial
mortality due to viruses based on decay rates, a higher burst size than 50 results
in lower lysis estimates.
When bacterial mortality was calculated from the fraction of bacterial cells with
mature virus particles, it was considered that the period when assembled viruses
can be observed is at least 10% of the whole lytic cycle (Proctor et al., 1993). Thus,
the amount of bacteria infected was a maximum 10 times greater than the amount
of bacteria with mature virus particles. However, the period with virus particles
can be as much as 40% of the whole lytic cycle (Proctor et al., 1993), in which case
the number of bacteria actually infected would be 2.5 times the amount of
bacteria with virus particles. On the other hand, the length of the eclipse period
933
P.Tuomi and P.Kuuppo
of the lytic cycle depends on the growth rate of the host (Hadas et al., 1997). In
the starved cells after infection, the lytic cycle may not proceed before substrates
are available (Ripp and Miller, 1997). With the slow growth rate of the natural
bacterial assemblage, the eclipse period may be longer and the period with
mature virus particles relatively shorter in marine bacteria when compared to
cultured bacteria.
The fraction of cells containing virus particles was lower in this study
(0.02–1.3%) when compared to the earlier reports (0.7–40%) (Bratbak et al.,
1994). When bacteria containing virus particles are counted, a fraction of cells are
too strongly stained to be seen through. Uranyl acetate, which was used for staining, binds preferably to negatively charged DNA. Strong staining may be partly
caused by high DNA content, which could be a result of increased DNA synthesis in fast-growing cells or virus DNA replication. In addition to the possibility of
not observing all virus-containing cells, the number of cells infected can be underestimated if the cells close to lysis are disrupted during the sample preparation.
Loss of bacteria by lysis and grazing in the mesocosm experiment
Cochlan et al. (1993) and Bratbak et al. (1994) have suggested that viruses are
favoured by high energy input and biological productivity in the plankton
ecosystem. Nutritional conditions and bacterial growth rate affect the magnitude
of phage production (Heller, 1992; Hadas et al., 1997). In this experiment, the
highest fraction of bacteria infected was found from the N + P + S enclosure, and
the greatest increase in viral abundance from the S enclosure (Table II), where
the bacterial production was stimulated by carbon availability. The general
increase in viral abundances towards the end of the mesocosm experiment may
have been caused by the nutritionally favourable conditions for virus multiplication. Also, high virus densities may have resulted from the increased number
of possible host cells or the accumulation of virus particles in the water column.
The two methods measuring virus-induced mortality of bacteria gave very
different results in this study. Virus-induced mortality of bacteria was estimated
to range from <0.2 to 13% (average 7.5%) using the fraction of cells infected, and
from 8 to 808% (average 239%) using the decay rates.
Clearance rates of HNF have been found to range from 0.4 to 11.4 nl flagellate–1 h–1 (Kuosa, 1990; Kuuppo-Leinikki, 1990; Kuuppo-Leinikki and Kuosa,
1990) in the study area; using an average 5 nl flagellate–1 h–1, bacterial loss by HNF
grazing was estimated to range from 16 to 135%. During the first week of the
enclosure experiment, bacterial loss due to grazing and lysis did not explain the
whole bacterial mortality (Figure 6). During the rest of the experiment, both virus
decay rates and consequently the estimates of virus-induced mortality, as well as
HNF abundance and consequently the grazing loss, increased, exceeding the total
bacterial mortality. This demonstrates the difficulty of estimating both viral lysis
and grazing using average values within as short a time period as 3 weeks. A direct
estimate of bacterial lysis obtained from the frequency of bacteria infected gave
rather low virus-induced mortality. These lysis estimates and grazing together
could explain on average 59% of total bacterial mortality. In such a situation,
934
Viral lysis and grazing loss of bacteria
other bacterial loss mechanisms, e.g. grazing by ciliates or mixotrophic algae,
need to be considered as important as HNF grazing in the northern Baltic Sea.
Interestingly, viruses and HNF had very similar development in the N + P
enclosures, which implies that both viruses and HNF may depend on the same
organisms as host or prey. Flagellates preferably graze large bacteria (Gonzáles
et al., 1990; Kuuppo-Leinikki, 1990), which may be those growing actively (Sherr
et al., 1992). Also, viruses can only lyse active cells which are able to synthesize
new macromolecules.
In the enclosure experiment, the phytoplankton blooms, which were formed by
nutrient (N + P) manipulation of the natural planktonic community, stimulated
bacterial growth only slightly. Viral lysis and grazing controlled bacterial density
at the same time and the carbon recycled within the microbial food web was an
important substrate source for bacteria. Viral lysis causes a recycling of carbon
within the bacterial fraction (Thingstad et al., 1993). However, the magnitude of
virus-induced mortality was difficult to evaluate in this study. The methods to
measure the bacterial loss by viral infection need to be further developed and calibrated in order to get a reliable estimate of the fate of bacterial biomass.
Acknowledgements
This study was carried out under the research project PELAG at Tvärminne
Zoological Station, University of Helsinki, as part of a mesocosm experiment of
the PELAG team. We thank all participants of the mesocosm experiment, as well
as staff at Tvärminne Zoological Station, for providing background data and
facilities for this study.
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Received on September 9, 1998; accepted on December 18, 1998
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