ELSEVIER
FEMS Microbiology
Ecology
19 (1996) 263-269
Dynamics of virus abundance in coastal seawater
G. Bratbak a**, M. Heldal a, T.F.Thingstad
’
Department
of Microbiology,
Uniaersip
b hiirminne
Received 28 September
of Bergen.
Zoological
Jahnebakken
a, P. Tuomi b
5, N-5020
Bergen,
Nornq
Station, 10900 Hat&o, Finland
1995; revised 1 March 1996; accepted 8 March 1996
Abstract
The short term dynamics of virus abundance in coastal sea water was investigated by frequent sampling of open
ecosystems and of water incubated in bottles in situ. Sampling intervals were 6-10 min. The viral abundance showed
significant temporal fluctuations both in situ and in the bottles and it changed in some cases by a factor of 2-4 within lo-20
min. Laboratory incubations showed that production and release of viruses were not induced or stimulated by nutrient
addition, high light intensities or transient increase in temperatures ca. 10°C. Our interpretation of these results is that they
result from synchronous lysis and release of virus particles from bacterial hosts and a rapid disintegration of these particles
when released in sea water.
Keyvords:
Virus; Phage; Viral-like
particle (VLP); Bacteria;
Population
1. Introduction
The abundance of viral-like particles (VLP) in sea
water ranges from < lo4 ml-’ to > lo8 ml- ’ [l].
Their distribution is correlated with chl a concentration, particulate DNA, bacterial abundance and other
biological parameters [2,3]. Thus, the highest abundances are generally found in coastal surface waters
during the productive part of the year while the
lowest are found in the open ocean, in deep waters
and during the unproductive part of the year [Il.
The rate of production of virus particles in aquatic
environments
has been assessed from observed net
increase in viral abundance over time [4], from viral
decay rates [5,6], and from incorporation of radioactive orthophosphate [7,8].
* Corresponding
author. E-mail: [email protected]
016%6496/96/$15.00
0 1996 Federation
PII SOl68-6496(96)00023-2
of European
Microbiological
dynamics
Estimation of virus production from the rate of
viral decay in samples where the host community
has been poisoned by the addition of metabolic
inhibitors such as cyanide [5] is based on a number
of underlying assumptions. The rate of viral decay
must be independent of the metabolic activity of the
microbial community in the sample and the stability
of the viral particles must be unaffected by the
metabolic inhibitor added. Suttle and Chen [9] found
that cyanide, the poison used as metabolic inhibitor
in decay experiments, caused decay rates to decrease
and they concluded that biological processes were
involved in the decay process. The rate of inactivation of experimentally
added tracer virus is usually
found to be higher in untreated sea water than in
autoclaved or filtered sea water [lo]. Decay experiments where the metabolic activity has been inhibited may thus underestimate the in situ decay rate. If
the daily virus production
Societies.
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is to be estimated
from the
264
G. Bratbali
et al. / FEMS Microhiolop
rate of viral decay measured in bottle incubation
experiments lasting only a few hours, it must also be
assumed that the rates of the processes taking place
inside the bottles are representative for the average
in situ rates for an extended period of time. This may
not be the case if the virus production in the bacterial
community is synchronized or if the rate of production or the rate of decay shows large daily fluctuations.
In the radiotracer method developed by Steward
et al. [7,8] the rate of virus production is estimated
from the amount of tracer incorporated
into the
nucleic acids of released viruses. This approach is
based on a model where the tracer is taken up by the
host, incorporated
into the viral nucleic acid and
released as free labeled viral particles. The method
depends on filtration, precipitation and chemical or
enzymatic hydrolysis for quantitative
separation of
labeled virus nucleic acid from the much larger pools
of unused tracer, labeled bacteria and labeled free
nucleic acids. The degree of isotope dilution may be
large as many phages rely on the use of the host
genome as a source of nucleotides rather than de
novo synthesis [l 11. The reliability of both experimentally determined and theoretically derived conversion factors for estimating virus production from
the amount of label incorporated into viral nucleic
acids, is uncertain. The method is new and may be
questioned, but only further application testing and
development can reveal the overall reliability of the
approach and the results obtained.
The method based on observation of net change in
viral abundance
over time assumes that the virus
production and the virus decay are not closely coupled and that this leads to temporal changes in viral
abundance.
To observe these changes and to use
them in rate estimates the sampling frequency must
be short compared to the frequencies in the fluctuations in abundance over time (c.f. Shannons sampling theorem). Nevertheless, the method yields minimum estimates of virus production as the rate of the
simultaneous
reverse process, viral decay, is unknown.
In die1 experiments where the sampling intervals
have been 2-4 h, serrated graphs with one-data-point
peaks and valleys in viral abundance are often obtained [5,6.12]. These changes suggests that the rate
of change in viral abundance for short periods may
Ecology
I9 (1996) 263-269
be on the order of 1- 10 h- ’ or higher. The reliability of these estimates are. however, questionable as
they, in many cases. are based on two consecutive
data points with no intermediate values. The purpose
of the present study is to test the hypothesis that such
rates of change in viral abundance are real and not
caused by bad counting statistics or experimental
errors. The approach we have used is simply to
sample at high frequency and observe net change in
viral abundance over time. We have also attempted
to sort out factors that may affect virus production in
natural waters.
2. Materials and methods
2. I. Samphg
sites
Sea water samples were collected in Raunefjorden
about 20 km south of Bergen, western Norway
(60”16’N, 5”12’E) and in Osterfjorden about 20 km
north of Bergen (60”34’N, 5”24’E). The field work in
Raunefjorden was carried out on June 29 1993 using
a small rowboat while the work in Osterfjorden was
carried on September
16 1993 using a zodiac
launched from R/V Haakon Mosby.
2.2. Labnruto~
experiments
The effect of increased nutrient supply, increased
irradiance and transient increase in temperature (temperature shock) on virus production in a natural
bacterial assemblage was investigated using a surface water sample collected in Raunefjorden on January 15 199 1. The water was collected in 10-l highdensity-polyethylene
barrels, brought back to the
laboratory within 2 h, transferred to 5-l glass carboys
and treated as follows: (al the control carboy was
incubated in the dark at 10°C (in situ temperature
was 8°C); (b) the nutrient carboy was incubated as
the control but received an addition of 70 mg I -’
yeast extract (Difco); (c) the light carboy was incubated as the control but with constant illumination
from an Osram Powerstar 0 dysprosium lamp (400
WI which has a relatively flat spectral composition
in the visible range. The irradiance at the surface of
the bottle was 40 nE s-‘cm-‘;
and (d) in the last
carboy the temperature was increased from 8 to 18°C
G. Bratbak et al. / FEMS Microbiology
with the aid of warm (ca. 60°C) water while vigorously agitating the carboy and then cooled to 10°C
with cold tap water. The whole operation took about
15 min. Samples (100 ml) from the carboys were
withdrawn at intervals, preserved and harvested as
described below.
2.3. Field experiments
For the bottle incubations carried out in Raunefjorden we used glass flasks of different sizes in
order to detect any volume dependent effects of
confinement.
One 20-l flask, one l-l flask and 14
30-ml flasks with screw caps were filled with water
from ca. 10 cm below the surface. The larger flasks
had silicone stoppers penetrated by two silicone tubings, one for subsampling and one for gas exchange
during sampling. The flasks were incubated in situ at
the surface tied to a drifting buoy. Subsamples from
the larger flasks (one at each timepoint) were collected by the aid of 50 ml syringes, while the entire
content of one small flask were sampled at each
timepoint. To trace in situ changes in viral abundance we collected water samples with a syringe
from 10 cm below the surface (one sample at each
timepoint). In this experiment the bottles were filled
at 10.30 a.m. and the first samples were collected
after 10 min. The experiment carried out in Osterfjorden was similar to the Raunefjord experiment,
but in this case we used two parallel l-l flasks filled
with water from 50 cm below the surface, and to
trace in situ changes we collected two independent
parallel samples from 50 cm below the surface at
each timepoint. The bottles were filled at 12.15 p.m.
and the first samples were collected after 4 min. In
both field experiments the samples were transferred
to plastic scintillation vials, preserved and harvested
as described below.
2.4. Counting
of Lit-us and bacteria
Ecology I9 (19961263-269
265
3. Results
3.1. Laboratory
experiments
The results from the laboratory incubations (Fig.
1) shows that there was a rapid fluctuation in viral
abundance occurring in the control bottle during the
first 1.5 h of the incubation.
Similar fluctuations
occurred in the experimental bottles but none of the
treatments caused a significant
stimulation
of the
virus production compared to the control. The bacterial abundance in the water used in this experiment
was 1.8 * 0.3 X lo6 ml- ’ (mean f S.D.) and there
was no significant change during the incubation.
3.2. Field experiments
3.2. I. Raunejjorden
The variation in viral abundance in the bottles
incubated in situ in Raunefjorden was characterized
by two notable peaks, one at 30 min and one at 100
min after the start of the incubation (Fig. 2a). There
were no significant differences among the three bottle sizes used for the incubation (the l-l bottle accidentally broke after 60 min). The viral abundance in
situ initially decreased and then showed one major
peak at 50-60 min. (Fig. 2b). The bacterial abundance was 1.3 f 0.2 X lo6 ml- ’ (mean f S.D.) and
showed no significant variation over time either in
the bottles or in situ.
10
0
0
20
40
60
80
Temperature
100
120
Minutes
All water samples were preserved with 2.5% glutaraldehyde (final concentration).
Particles were harvested onto electron microscope grids by centrifugation and prepared for counting of virus and bacteria
in the transmission electron microscope as described
earlier [ 131.
Fig. 1. Fluctuations
in total counts of virus WLP. virus-like
particles) in sea water samples incubated under different conditions, 0: Control incubated in the dark at 10°C; V: Incubated
with an addition of 70 mg 1-l yeast extract; 0 : Incubated with a
constant illumination 40 nE s- ’ cm-’ d): 0: Incubated after a
transient (15 min) temperature increase from 8 to 18 to 10°C.
Error bars are standard error of mean, 3-4 parallel counts
266
G. Bratbak et al. / FEM.7 Microbiology
1
6-a
5432‘:
2
’
4
0
6
6-b
-
54321 0
‘.‘,l,l,I,I,’
0
20 40
60
80
100
120
Minutes
Fig. 2. Fluctuations
in total counts of virus (VLP, virus-like
particles) in Raunefjorden,
June 1993. (a) Bottles of different
volume (0 : 20 1; v : 1 1; and 0: 30 ml) incubated in situ. The l-l
bottle accidentally broke after 60 min. Line is drawn through the
mean. (b) Samples collected in situ 10 cm below the surface.
Error bars indicate counting error (1 /Jn).
a
*t
0
0
20
40
60
60
Minutes
Fig. 3. Fluctuations
in total counts of virus (VLP, virus-like
particles) in Osterfjorden.
September 1993. (a) Two parallel (0
and v ) 1-l bottles incubated in situ. (b) Samples collected in situ
50 cm below the surface. Two independent samples were collected each time.
Ecology 19 (1996) 263-269
3.2.2. Ostefjorden
The variation in viral abundance in the two parallel bottles incubated in situ in Osterfjorden is characterized by lag period of 20-30 min followed by a
gradual increase for the next 30-40 min and then a
sudden decrease (Fig. 3a). The increase in virus
abundance from 1.9 * 0.4 X 10’ ml-’ (mean + S.D.)
in the lag period (O-24 min, n = 8) to 6.5 & 0.8 X
lo7 ml-’ (mean i S.D.) at the maximum (54-64
min, iz = 3) is statistically significant (t-test, P <
0.001). The in situ viral abundance in Osterfjorden
showed no significant changes the first hour but it
then decreased by a factor of about 2 within lo- 1.5
min, whereafter it increased by a factor of 6 within
8-10 min (Fig. 3b). The bacterial abundance was
2.1 + 0.6 X lo6 ml-’ (mean + S.D.) and showed no
significant variation during the experiment either in
the bottles or in situ.
4. Discussion
The maximum rates of increase in virus abundance that may be estimated from these experiments
are 13 h- ’ for the bottle experiments and 6 h- ’ for
the observations made on in situ changes. The maximum rates of decrease were 5 h- ’ and 11 hh' .
respectively. The steady increase in viral abundance
observed between 30 and 54 min in the bottles in
Osterfjorden (Fig. 3a) had a rate of 2.5 hh ‘. These
rates are minimum estimates as the simultaneous
opposite rates are unknown. They are nevertheless
1-2 orders of magnitude higher than the rate of most
other biological production and consumption
processes that may be observed in natural marine systems. We will first argue that the observations made
are real and not due to some experimental artifact
and then discuss the nature of the biological processes behind the observed rates.
The rapid changes in viral abundance were observed in water samples incubated in duplicate and
triplicate, and in parallel in situ samples. The samples from the different time series were harvested
onto electron microscope grids, prepared for counting and counted independently to ensure that systematic errors should not affect the results. This supports
the view that the observed changes are real and not
due to some artifact in our procedures.
G. Bratbak et al. / FEMS Microbiology Ecology 19 (1996) 263-269
The fact that the variations were observed in
bottles with a IO-fold range in surface to volume
ratio (e.g. from 0.2 to 2.3 cm-‘) suggests that the
variations were not due to confinement or some kind
of ‘bottle effect’ [ 14,151, which might be expected to
be more severe in small than in large bottles. In any
case, if handling and containment
of a water have
such immediate and dramatic effects on viral abundance, it seems reasonable to assume that events in
natural waters, such as the breaking of waves and
turbulence caused by wind and marine animals may
cause similar changes in viral abundance. The variations observed in the bottles may thus mirror what
happens in situ.
The water samples for the in situ time series were
taken close to a drifting buoy and at the same depth
all the time to ensure that we sampled from the same
water mass. For the bottle incubations, spatial variations or patches must have been on the scale of
centimeters or less, as we observed the same variations in a 20-l carboy, in l-l flasks and by total
sampling of 30-ml tubes. The fact that these experiments were carried out in duplicate or triplicate,
excludes the possibility that the observed variations
can be explained by spatial variations in the bottles.
For the in situ samplings spatial variations may have
been more important. The time scale of variations
observed in the bottles suggest to us that we must be
dealing with relatively small patches as it is difficult
to imagine what factors or processes could dictate
such variations for large areas or water masses. The
variations in the 30-ml tubes and the 20-l carboy
were similar so the patches must be larger than that,
but it is difficult to say how large. On the other hand,
it seems reasonable
to assume the existence of
short-lived micro-scale patches caused by the lysis
and release of virus from infected single cells. Temporal variations can explain the variations observed
in the bottles and in this case spatial variations can
be excluded. The in situ observations
can be explained by temporal variations
alone, but spatial
variations cannot be excluded. Our conclusion is thus
that the variations observed both in the bottles and in
situ are real. The rapid variations may also explain
why there were, in some cases, differences in viral
abundance between bottles and in situ values at the
start of the experiments.
To approach a biological
interpretation
of the
267
variations we may ask the question: What frequency
and amplitude
would one expect for oscillations
caused by infective cycles? In a simple predator-prey
system with constant prey growth rate p and constant predator death rate 6, the theoretical period of
oscillations for small amplitudes is of the order of
21r@
as can be shown by lineariziation
of the
traditional Lotka-Volterra
equations. Exploring numerical solutions for these equations (using the Stella
II V3.0 for Windows programming
system, results
not shown) we found this approximation to be fairly
robust also for larger amplitudes. Assuming bacterial
growth rates and viral ‘death’ rates to be in the order
of 1 dd’ and 100 d-‘, respectively, the host-phage
systems would be expected to oscillate with a period
of about 15 h. In a diverse bacterial community with
many host-phage systems, one would not necessarily
be able to detect oscillations in individual phage-host
systems as oscillations
in total bacterial and viral
numbers. The number of simultaneously
coexisting
host-phage systems is presently unknown but theoretical calculations
suggest that there is room for
100-300 systems [ 16,171 of equal magnitude. If this
is the case, oscillations in one system would only be
expected to make changes on the order of 1% in total
numbers, and, since lowered population size in one
type of host would make room for another, it seems
easy to imagine mechanisms producing oscillations
of opposite phase blurring total number variations. In
a diverse community
the observed oscillations
in
total numbers would thus only be possible if the
virus production in the different host-phage systems
is synchronized.
However, the alternative explanation, which assumes that the diversity of the system
is low, i.e. dominated by a few different host phage
systems or phages with a broad host range, cannot be
ruled out, although it disputes our present conception
of a high diversity in natural aquatic bacterial communities [ 18-201. The third hypothesis accounting
for the variations in viral abundance is based on the
assumption that the bacteria are lysogenic. The virus
production
may then be explained as a result of
induction and the oscillations in the system may be
considered event driven.
Our interpretation
of the observed variations is
thus that the increases in virus abundance are due to
a more or less synchronous lysis and release of viral
particles from host cells that at some time point have
268
G. Bmtbak et al. / FEMS Microbiology
been infected or induced. The rate of increase in
viral particles does not represent the rate of biosynthesis of virus particles, but the rate at which the
particles are released from the lysing host cells. For
the bottle incubations it is possible that the sampling
and incubation
procedures affected cell lysis and
release of viral particles and made the release process more, or less, synchronous than it would be in
the undisturbed natural environment.
This may explain why the development in the bottles were different from the development observed in situ.
Disintegration
of the viral particles is the most
reasonable explanation for their disappearance as the
rates are too high to be explained by other processes
such as adsorption to larger particles. Assuming that
every collision between virus and larger particles
(say bacterial-sized,
i.e. 0.1 pm31 results in adsorption (i.e. the adsorption rate constant k =: 1 X 10e9
cm3 min- ‘1, the concentration of the other particles
must have been in the order of 10’ ml-’ if adsorption was to explain the observed rates of decrease in
viral abundance. Thus, the viruses must be assumed
to have adsorbed non-specifically
to all bacteria as
well as to any other type of particles. In view of the
generally high host specificity of most viruses, this is
rather unreasonable.
The numerical increases in viral abundance observed in this study were in the order of 3-7 X 10’
VLP ml-‘.
Assuming
an average burst size for
bacterial hosts between 50 and 100 [5,21] this increase would require the lysis of 0.3-1.4 X 10’ bacteria per ml. Our estimates of the bacterial abundance were not done with an accuracy high enough
to resolve this expected decrease. For data on rates
of virus production to make sense in an ecological
context with respect to carbon and nutrient flow they
must be expressed as mean rates on a time scale of at
least a day. The changes we have observed occurred
on a time scale of minutes and the sampling program
in the field experiments
lasted for less than two
hours. This data set does not allow us to estimate the
per day virus production. The viral community
is
presumably made up of populations that have very
different turnover times. The size of these populations are unknown,
but those with the shortest
turnover times may have varied between zero and
3-7 X 10’ VLP ml-’ and accounted for most of the
observed variations, while populations with longer
Ecology I9 llYY61263-269
turnover times accounted for a concentration
between 2 and 4 X 10’ VLP ml -I. The reason for the
wide range in turnover time is open for speculation,
but it is known that many temperate phages are less
stable than lytic phages [22]. Another possibility is
that synchronized
release of premature viral particles, which may be structurally unstable, will result
in short lived peaks in the VLP abundance.
Although we interpret our results to indicate that
cell lysis and release of viral particles is a synchronous process, we do not know if the foregoing
processes, i.e. induction, infection and virus biosynthesis, in the bacterial community are synchronous as
well. The latter is possible, but then we might also
expect that bacterial growth as such is also synchronized. We have earlier argued that induction of
lysogenic bacteria seems to be the most reasonable
process initiating virus production in natural bacterial communities
[ 1,231. Others have, however, obtained results interpreted to indicate that infection
may be the most important process [24]. If the virus
production in the bacterial community is synchronized it may be difficult to discriminate between the
different processes.
Acknowledgements
This work was supported by funding from The
Research Council of Norway to the MAST-II MEICE, contract
number
MAS2-CT92-003 1 (G.B..
T.F.T. and M.H.), by funding from the Academy of
Finland to the PELAG project (P.T.), and by funding
from Nordic Academy for Advanced Study (P.T.).
Anne Naess Myhrvold and Tori11 Roeggen are thanked
for technical assistance and for providing data on the
laboratory experiments. The EM work was done at
the Laboratory for Electron Microscopy, University
of Bergen.
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