Phage Bacteriolysis, Protistan Bacterivory Potential, and Bacterial

Production in a Freshwater Reservoir: Coupling with Temperature

A.S. Pradeep Ram, D. Boucher, T. Sime-Ngando, D. Debroas and J.C. Romagoux
Laboratoire de Biologie des Protistes, Universite Blaise Pascal (Clermont-Ferrand II), UMR CNRS 6023, F-63177, Aubie`re Cedex, France

Received: 26 May 2004 / Accepted: 4 October 2004 / Online publication: 29 July 2005

Introduction
Abstract
Over the last 15 years, our view of aquatic microbial food
Phage abundance and infection of bacterioplankton
webs has changed with the discovery of the abundance
were studied from March to November 2003 in the Sep
and activities of viruses. Viruses are among the most
Reservoir (Massif Central, France), together with tem-
abundant biological entities in natural waters [17]. They
perature, chlorophyll, bacteria (abundance and pro-
play an important role in the mortality of microbial
duction), and heterotrophic nanoflagellates (abundance
communities [36] and in mediating processes such as
and potential bacterivory). Virus abundance (VA) ran-
transduction, lysogenic conversion, and species succes-
ged from 0.6 to 13 1010 viruses l)1, exceeding bacterial
sions [48]. On average, 10%40% of bacterial production
abundance (BA) approximately sixfold on average. In
is lysed by viruses in both marine and freshwaters. This is
terms of carbon, viruses corresponded to up to 25% of
a significant departure from the traditional view that
bacterial biomass. A multiple regression model indicated
predation and resource availability are the main factors
that BA was the best predictor for VA (R2 = 0.75). The
controlling bacterial abundance and production in pela-
frequency of infected bacteria (estimated from the per-
gic systems [13, 30, 43, 54, 55].
centage of visibly infected cells) varied from 1% to 32%
The abundance and activities of the virioplankton
and was best explained by a combination of tempera-
have been shown to vary according to various factors,
ture (R2 = 0.20) and bacterial production (R2 = 0.25).
including water depth and oxygen concentration [11, 46],
Viruses and flagellates contributed about equally to
trophic state of the system [9, 21, 42, 45], temperature
bacterial mortality. Both factors destroyed 55% of bac-
[17, 18], light, especially ultraviolet (UV) light level [53],
terial production, with a shift from phage bacteriolysis
suspended particulate matter [16], bacterial diversity
in early spring to protistan bacterivory in late summer.
[10], and bacterivorous activity of protists [29]. Results
The vertical differences in most of the biological vari-
from a few investigations conducted over a short time
ables were not significant, contrasting with the seasonal
period have shown substantial shifts in abundances and
differences (i.e., spring vs. summer-autumn). All bio-
activities but few consistent temporal patterns or rela-
logical variables under study were indeed significantly
tionships with environmental and microbial factors [3,
coupled to temperature. We regarded this to be the
18, 35, 47]. However, very few seasonal studies on virus
consequence of the enhanced discharge of the reservoir
distribution have been reported in freshwater systems,
in 2003 (compared to previous years). This substantially
and most of those observations come from studies made
weakened the stability and the thermal inertia of the
in natural lakes [4, 5, 30, and references therein]. Studies
water column, thereby establishing temperature as a
pertaining to reservoirs are rare, and lack of opportunity
stronger forcing factor in setting the conditions for
to document viral ecology in such systems has resulted in
optimal metabolic activity of microbial communities.
a paucity of knowledge about these environments. To the
best of our knowledge, viruses have so far been examined
only in two reservoir systems, the Rimov Reservoir
(Czech Republic) by Simek et al. [29] and Weinbauer
et al. [44], and the Sau Reservoir (Spain) by Sommaruga
Correspondence to: T. Sime-Ngando; E-mail: Telesphore.SIME- et al. [32]. Both studies in the Rimov Reservoir were
NGANDO@univ-bpclermont.fr based on a summertime spot surface sampling followed

64 DOI: 10.1007/s00248-004-0110-y d Volume 50, 6472 (2005) d  Springer Science+Business Media, Inc. 2005
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON
by size-fractionation, incubation in situ for few days, and
examination of microbial communities for the effects of
enhanced grazing and resource enrichment (i.e., river
inflow vs dam transplanted incubations) on bacterial
composition and viral production. In the Sau Reservoir,
the size distribution of viruses was analyzed, also from a
summertime spot surface sampling.
Disturbance frequencies are much higher in reser-
voirs than in lakes, with rapid, often irregular and large,
changes in flushing rates, water level, and the stability of
the water column. In shallow reservoirs, the enhanced
changes in the physical environment can overrule the
importance of other regulating factors for biotic com-
munities [50]. This study aimed to document the seasonal
abundance and activity of viruses in a small reservoir (Sep
Reservoir, France), characterized by enhanced flushing
rates in summer to meet agricultural irrigation needs. The
specific objectives were to (1) determine the variability
(vertical and temporal) in virus abundance and phage
infection of the bacterioplankton, (2) determine the rel-
ative importance of viruses in bacterial mortality com-
pared to the potential bacterivory from nanoflagellates,
and (3) determine the significance of relationships be-
tween viruses and abiotic and biotic variables.
Materials and Methods
Study Site and Sampling. The Sep Reservoir was cre-
ated from a dam built in 1994 across the Sep Stream for
the summertime irrigation of an agricultural region, the
Haute Morge, Massif Central, France (ca. 46N, 3E). At
its full supply level, the reservoir contains 4.7 106 m3 of
water and has a surface area of 33 ha, with a maximum
depth of 37 m. The outlet is located near the bottom of
the dam. The Sep reservoir is generally classified as an
oligotrophic to mesotrophic lake, based on the annual
pigment and nutrient concentrations, following Organi-
zation for Economic Co-operation and Development
(OECD) recommendations [25]. Fluctuations of the
water column height during the present study are shown
in Figure 1A. For more details on the characteristics of
the Sep Reservoir, see Jugnia et al. [20] and Tadonle ke
et al. [40].
Sampling was carried out from March to November
(2003) in the deepest central point of the reservoir.
Samples were collected biweekly from April to September
and monthly in March, October, and November, using a
horizontal Van Dorn bottle. The spring period corre-
sponds here to the period lasting from the beginning of
the study to June 19, and the summer-autumn period
refers to the period from June 26 to the end of the study,
when the reservoir was progressively emptied for agri-
cultural needs. The sampling depths were located in the
epilimnion (1 m below the surface), metalimnion (7 m
below the surface), and hypolimnion (1 m above the
IN RESERVOIRS 65
sediments), and are hereafter referred to as representative
of these layers. As the depth of the reservoir decreased
from the start of the summer period, the 7 m samples
could not be taken from mid-August onward (Fig. 1A).
Physicochemical Variables. Water temperature
and dissolved oxygen were measured in situ with an
oxycal-SL 197 multiparameter probe (WWT, Limonest,
France). Secchi depth (Zs) measurements were used to
estimate the euphotic depth (Zeu) according to the rela-
tionship Zeu = 2.42 Zs, assuming that 15% of the incident
light is transmitted to the Zs depth [51]. Samples for
nutrients, namely dissolved ammonium (NH4-N), ni-
trate (NO3-N) and nitrite (NO2-N) nitrogen, and soluble
orthophosphate (PO4-P) and total phosphorus (TP) were
analyzed spectrophotometrically with standard methods
[2, 51]. Dissolved nutrients were obtained from samples
previously filtered through Whatman GF/C glass fiber
filters. Chlorophyll a concentrations (Chl) were deter-
mined spectrophotometrically from samples (500 ml)
collected on Whatman GF/F filters. Pigments were ex-
tracted in 90% acetone overnight in the dark at 4C, and
concentrations were calculated from SCOR-UNESCO
[28] equations.
Bacterial and Viral Abundances. For the enu-
meration of virus-like particles and bacteria, water sam-
ples were fixed with 0.02-lm filtered buffered alkaline
formalin (final concentration 2% v/v, from a 37% w/v
solution of commercial formaldehyde). Within 1 h after
sampling, subsamples (12 ml) were filtered (<15 kPa
vacuum) through 0.02-lm pore size Anodisc filters
(Whatman, Maidstone, England), with 1.2-lm pore size
cellulose acetate backing filters. After they were stained
with SYBR Green I fluorochrome (Molecular Probes
Europe, Leiden, Netherlands) (final dilution, 2.5 10)3
fold) as described by Noble and Fuhrman [24], filters
were air dried on absorbent paper and mounted between
a slide and a glass coverslip with a special antifading
mountant. The mounting medium Citifluor (Citifluor,
London, UK) was amended with ca. 20% (v/v) of Vecta
Shield (Vector Laboratories Inc., Burlingame, CA). This
modification significantly reduced fading of the fluoro-
chrome and gave highly stable fluorescence. When not
analyzed immediately, slides were stored at )20C until
counting under an epifluorescent microscope (LEICA
DC 300F model).
Bacterial Production. Bacterial production (BP)
was determined by the incorporation of labeled thymi-
dine (3H-TdR) into bacterial DNA [14]. First, 10 ml of
freshly collected water samples (in triplicates) and one 0.5
N NaOH killed control were inoculated with labeled
thymidine (specific activity = 87 Ci mmol)1, Amersham
Biosciences, UK) at a final concentration of 20 nM in
66
Pyrex tubes. These samples were then incubated in situ
and in the dark for 5060 min. Thymidine incorporation
was stopped by adding 0.5 N NaOH. Radioactive samples
were filtered though 0.22-lm cellulose acetate filters
(Sartorius, Goettingen, Germany), and rinsed twice with
3 ml ice-cold 5% trichloroacetic acid. Filters were placed
in vials, allowed to dry, and solubilized with 0.5 ml of
ethyl acetate. After the addition of 5 ml of scintillation
cocktail, radioactivity was counted with a liquid scintil-
lation counter (Beckman, LS 6500). Bacterial production,
calculated in moles of TdR incorporated into DNA, was
converted into the number of bacterial cells produced by
applying a conversion factor (1.67 1018 cells mol)1)
previously determined for the Sep Reservoir bacterial
communities using the derivative method [19].
Phage Bacteriolysis. In formalin-fixed samples,
bacteria contained within 8-ml subsamples were har-
vested by ultracentrifugation onto 400 mesh copper
electron microscope grids with carbon-coated Formvar
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON IN RESERVOIRS
Figure 1. Seasonal variations in the maximum and
euphotic depths (A), the water temperature (B), and
chlorophyll a concentration (C) in the Sep Reservoir,
MarchNovember 2003. Black points included in A
represent the sampling depths. Note that the x-axis in A is
not spaced linearly according to the sampling dates.
film [31], using a Centrikon TST 41.14 swing-out rotor
at 70,000 g for 20 min at 4C [cf. 35]. Each grid was
then stained for 30 s with uranyl acetate (2% w/v) and
examined in a JEOL 1200EX transmission electron
microscope (TEM) operated at 80 kV at a magnification
of 20,00040,000. At least 300500 cells per sample
were examined to determine the frequency of visibly
infected bacterial cells (FVIC). Cells were scored as in-
fected if they contained five or more intracellular viru-
ses. Because mature phages are visible only late in the
infection cycle (i.e., ratio eclipse to latent period), FVIC
were converted to the frequency of infected bacteria
(FIC) using the equation: FIC = 9.524FVIC ) 3.256
(with data given as percentages) derived from a viral
dilution approach [49]. Assuming a steady state, that
the infected and uninfected bacteria were grazed at the
same rate, and that the latent period equaled the bac-
terial generation time, the FIC were converted to the
frequency of bacterial mortality due to viral lysis (FMVL
as a percentage per generation, i.e., assuming that
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON IN RESERVOIRS 67

Table 1. Mean (coefficient of variation) and range in the chemical characteristics of the water column of the Sep Reservoir, March to
November 2003
Parameters Epilimnion Metalimnionb Hypolimnion
)1 a
Dissolved oxygen (mg L ) 8.03 (13) 4.01 (47) 4.72 (46)
min-max 6.449.12 1.666.02 1.426.83
NH4-N (mg L)1) 0.08 (39) 0.08 (43) 0.13 (97)
min-max 0.020.12 0.020.11 0.020.26
NO2-N (mg L)1) 0.02 (28) 0.02 (24) 0.01 (28)
min-max 0.010.03 0.010.03 0.010.02
NO3-N (mg L)1) 0.75 (59) 0.94 (48) 0.89 (68)
min-max 0.251.96 0.511.81 0.282.32
PO4-P (mg L)1) 0.02 (141) 0.02 (108) 0.02 (89)
min-max und0.11 und0.06 und0.06
Total phosphorus (mg L)1) 0.14 (53) 0.09 (12) 0.13 (44)
min-max 0.090.34 0.070.10 0.080.27
und: undetectable. For seasonal differences, see the main text.
a
Epilimnion differed significantly from metalimnion (p < 0.003) and hypolimnion (p < 0.002).
b
Correspond to the period MarchJuly only (cf. Fig. 1A).

FMVL equals the losses of BP) using the equation:
FMVL = (FIC + 0.6FIC2)/(1 ) 1.2FIC) [6].
Protist Abundance and Grazing Potential. Samples
for the measurements of heterotrophic nanoflagellate
(HNF) abundance were fixed immediately after sampling
with alkaline Lugol solution (final concentration, 0.5%)
and decolorized with borate-buffered formalin (final
concentration, 2%). Primulin-stained HNF collected on
0.8-lm polycarbonate black filters (25 mm diameter)
were counted under UV excitation in a LEICA epifluo-
rescent microscope [8]. Loss of bacteria to HNF grazing
was estimated from in situ bacterial abundance, total
HNF abundance, and an assumed mean clearance rate
(CR) of 0.7 nl HNF)1 h)1 (range: 03.3 nl HNF)1 h)1),
reported by Thouvenot et al. [41] during a seasonal study
(AprilAugust 1997) in the Sep Reservoir.
Statistical Analysis. Differences in physicochemi-
cal and biological variables between sampled depths and
seasons were tested by one-way analysis of variance
(ANOVA). Potential relationships among variables were
tested by linear pair-wise correlations (i.e., Pearson cor-
relation analysis) and stepwise multiple regressions. Data
were log transformed to satisfy the requirements of
normality and homogeneity of variance necessary for
parametric statistics. All statistical analyses were per-
formed with Minitab software for Windows (Release 12,
Minitab).
than in summer-autumn (Fig. 1A). Water temperatures
ranged from 5C in mid-March in the bottom waters to
25C in July-August in the epilimnion (Fig. 1B). The
water column was markedly warm during the summer,
with a pronounced stratification. However, the stratifi-
cation was artificially removed during the second half of
the summer, because of the increasing discharge from the
reservoir (Fig. 1B). The epilimnion was well oxygenated,
with oxygen contents ranging from 6.4 to 9.1 mg l)1. This
differed significantly from the metalimnion (p < 0.003)
and hypolimnion (p < 0.002), where dissolved oxygen
levels were lower. However, no anoxic conditions were
observed in the bottom waters (Table 1).
The nitrate concentration was higher than that of
other nitrogen species (i.e., ammonia and nitrite), rang-
ing from 0.3 to 2.3 mg l)1 in the water column. Inorganic
phosphate ranged from undetectable to 0.11 mg l)1 and
accounted for 0 to 36% of total phosphorus. There was
no significant variation with depth and seasons in the
nutrient concentrations (Table 1).
Chlorophyll a concentrations ranged from 1.2 to 42.6
lg l)1 in the euphotic depths (epilimnion and metalim-
nion) and from 0.5 to 38.5 lg l)1 in the hypolimnion.
The highest values in the epilimnion and hypolimnion
were observed in the late summer period, i.e., coinciding
with the artificial mixing of the reservoir. No significant
depth-related difference was noted, while concentrations
in summer-autumn (>10 lg l)1) were significantly higher
(AVOVA, p < 0.001) than in spring (<5 lg l)1) (Fig. 1C).

Standing Stocks. The abundances of viruses (VA)

Results
Physicochemical Environment. During the present
study, significant water discharge from the reservoir (i.e.,
for agricultural needs) started from mid-June when the
maximum depth of the water column changed from >30
m to <10 m in August and onward. The euphotic depth
fluctuated from 1.9 to 14.5 m, with higher values in spring
and bacteria (BA) varied from 0.6 to 13.0 1010 L)1
(mean SD = M = 2.3 2.2 1010 L)1), and from 0.7
to 12.2 109 cells L)1 (M = 4.1 2.6 109 cells L)1),
respectively. Both variables peaked in the August 27
sample, the values recorded in summer-autumn being
significantly higher than in spring (ANOVA, p < 0.001).
No significant vertical difference was noted (Fig. 2A,B).
68
Figure 2. Spatial and temporal variability in the abundances of
viruses (A) bacteria (B) and heterotrophic nanoflagellates (HNF,
C) in the Sep Reservoir, MarchNovember 2003.
The virus to bacteria ratio (VBR) ranged from 2.1 to 51.7
(M = 5.5 2.7), and no significant difference with depth
or season was noted.
The abundance of HNF ranged from 0.5 to
11.9 106 cells L)1, the highest value being recorded
on August 27 in the epilimnion (Fig. 2C). Like the
values for Chl, BA, and VA, HNF abundance was
significantly higher in summer-autumn than in spring
(ANOVA, p < 0.001), but no vertical difference was
observed.
Bacterial Production. Bacterial production (BP)
varied between 0.7 and 18.7 107 cells L)1 h)1 and
was significantly higher in the epilimnion than in the
hypolimnion (ANOVA, p < 0.001). Temporally, BP
peaked in mid-April in the epilimnion and in mid-July
and mid-August at the three depths sampled, prior to
the late-August peaks noted in the abundances of
viruses, bacteria, and flagellates (Figs. 2, 3A). Bacterial
production increased between early June and mid-Au-
gust at all depths, although low values were recorded
on July 30. However, there was no significant differ-
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON IN RESERVOIRS
Figure 3. Spatial and temporal variability in the bacterial pro-
duction (BP, A), the frequency of infected bacterial cells (FIC, B)
and the ratio viral bacteriolysis: flagellate bacterivory in the Sep
Reservoir, MarchNovember 2003. Standard errors of the mean,
i.e., variability between triplicates, are shown for bacterial pro-
duction (BP).
ence between the spring and summerautumn periods
(ANOVA, p > 0.05), because of a marked spring peak
in BP in the surface waters (Fig. 3A).
Viral Lysis and Protistan Grazing Potential. Fre-
quency of visibly infected bacterial cells ranged from 0.5
to 3.7% (M = 1.6 0.7), and FIC from 1.0 to 31.5%
(M = 12.3 7.1%). The seasonal fluctuations of these
variables roughly increased in spring, but the highest
values were recorded at the end of the summer (Fig. 3B).
There was no significant difference between spring and
summer-autumn. The proportion of total bacterial
mortality from viral lysis (FMVL) varied broadly from
1% to 60% (M = 20.1 10.9%).
The potential grazing rates of HNF on bacteria ran-
ged from 0.02 to 5.6 107 cells L)1 h)1, corresponding to
0.1% to 96.7% of bacterial production. The mean ratio of
viral lysis to flagellate grazing decreased with depth, from
1.2 in the epilimnion to 0.8 and 0.4 in the metalimnion
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON IN RESERVOIRS 69

Table 2. Pearson product-moment correlation coefficient (r) between different parameters during MarchNovember 2003 in the Sep
Reservoir, n = 38
Temperature C TP Chl VA BA BP FIC
TP 0.55***
Chl 0.43** 0.48**
VA 0.52*** 0.38* 0.68***
BA 0.69*** 0.44** 0.64*** 0.86***
BP 0.61*** 0.43** NS NS NS
FIC 0.43** NS NS NS NS 0.49***
HNF 0.43** NS 0.45** 0.56*** 0.63*** 0.41** NS
BP:BA NS NS NS NS )0.31* 0.66*** 0.44**
Levels of significance: *p < 0.05, **p < 0.01, ***p < 0.001. NS = not significant. TP: total phosphorus; Chl: chlorophyll a concentration; VA: viral abundance;
BA: bacterial abundance; BP: bacterial production; FIC: frequency of infected cells; HNF: abundance of heterotrophic nanoflagellates.

and hypolimnion, respectively. This decrease was related
to the shift noted in the dominant source of bacterial
mortality throughout the study, from phage bacteriolysis
in early spring in the surface waters to protistan
bacterivory in late-summer-autumn in the bottom waters
(Fig. 3C). For the study period, 11% to 116%
(M = 55 21%) of bacterial production was removed by
viral lysis and HNF grazing.
Correlations. Temperature was correlated with all
the biological variables under study, namely Chl, BA, VA,
BP, FVIC, FIC, FMVL, and HNF abundances and grazing
rates. Among biological variables, the variability in VA
was better explained by BA than by Chl, TP, or the HNF
abundance. The sole biological variable explaining the
variance in FIC was BP. In addition, the bacterial cell-
specific production (i.e., the ratio BP: BA) was correlated
with FIC (Table 2). A multiple regression analysis was
carried out to determine the relative importance of the
main correlates for VA and FIC. The models for which
the highest R2 were obtained clearly highlight the
importance of BA for VA (i.e., VA= )0.780.06 Temp +
0.04 Chl + 9 BA, R2 = 0.82, n = 38), and of the combi-
nation of temperature and BP for FIC (FIC = 2.24 + 0.38
Temp + 0.29 BP, R2 = 0.31, n = 38).
Discussion
The Seasonal Abundance of Viruses. This study
provides the first documentation of the seasonal standing
stock of viruses and phage infection in relation to biotic
and abiotic variables in a freshwater reservoir, adding to
the growing knowledge of the fate of bacterial production
in aquatic systems. The viral abundances in the Sep
Reservoir were within the range of those reported in
other freshwater systems [30]. Among various correlates
(i.e., temperature, TP, Chl, BA, and HNF) the dominant
one for VA was BA, suggesting that bacteria were the
major hosts for viruses. Comparable correlations between
VA, BA, and Chl have been reported in temperate lakes
[4, 21, 46], as well as in coastal and oceanic waters, where
BA generally is the best predictor for VA [1, 9, 33].
Our correlation coefficient between VA and BA (i.e.,
0.86) is, however, higher than those (<0.6) reported in
the other studies. The reasons for this likely include the
strong covariations of biological variables with respect to
the temperature during our study, but also a tight cou-
pling between bacteria and viruses. The variations in the
virus-to-bacterium ratio indeed were lower (CV = 49%)
compared to those in VA (CV = 96%) and BA
(CV = 63%), indicating the tight coupling between
viruses and their major hosts, and a relatively constant
level of virus production and loss [26]. The VBR levels in
the Sep Reservoir were close to those reported in lakes
located in the same geographical region [4], and they
were well within the typical range (i.e., 520) reported in
other pelagic environments [30, 43, 55]. However, our
mean VBR (i.e., about 6) was lower than the predicted
value of 20 for freshwater systems [21, 55]. Finally, no
significant vertical difference was noted for VA, BA, and
VBR, contrasting with some studies where the highest
values of these variables occurred in the thermocline [3,
4, 11, 45]. We consider that the general absence of ver-
tical differences in biological variables in the Sep Reser-
voir is the result of the artificial mixing generated by the
drawdown [21].
Phage Infection and Its Ecological Implica-
tions. The observed dynamics in VA indicate that a
minimum host cell concentration is necessary for viral
replication. From a linear regression analysis, we found
that our recorded BA were well above the minimum (i.e.,
7 108 bacteria L)1) necessary for a measurable level of
viral infection. This is close to the typical threshold host
concentration of 5 108 bacteria L)1 reported by Stew-
ard et al. [34] in the sea. Data from northern Adriatic
seawater samples also indicated that there was no infec-
tion when the number of bacterial hosts fell below
2 108 cells L)1 [47]. The requirement of a minimum
host density for efficient viral propagation has also been
demonstrated for cyanophages in coastal seawaters [38].
In our samples, the product of viral and bacterial abun-
dances was always larger than 1015 L)1, suggesting that
bacteriavirus interactions could be a significant source
70
of bacterial mortality [52]. The presence of infected cells
in our samples and the correlation between these cells
and bacterial production (i.e., total and cell specific) in-
deed imply that viruses were produced in situ.
Our data on visibly infected cells were within the
typical range (i.e., <5%) reported in pelagic systems
studied with the same methodological approach, i.e., the
whole cell method [30, 55]. Although this approach
generally yields estimates with large errors; e.g., for our
samples, 300500 cells were examined for 0.5%3.7% of
visibly infected cells. Peaks in FIC were noted during
spring and autumn, similar to the results reported re-
cently in two temperate lakes [5]. However, in contrast to
the latter study, FIC in the Sep Reservoir generally in-
creased during the spring period, and was correlated with
temperature and total and cell-specific bacterial produc-
tion. This suggests that the maintenance of a high
number of viruses in the water column is dependent on
the active fraction of the bacterioplankton [33].
The viral-mediated bacterial mortality (FMVL) ran-
ged from 1% 60% during the study period. These
values are similar to those reported in freshwater envi-
ronments [30]. The average FMVL in the Sep Reservoir
was found to be 21%, which is close to the value of 17.1%
reported in the Rimov Reservoir [29], and the 20% re-
ported in aquatic systems in general (reviewed in [30, 43,
55]). Assuming a carbon content of 0.2 and 20 fgC
particle)1 for viruses and bacteria respectively [53], the
viral biomass in this study (range = 1.6 26 lg Cl)1,
M = 4.6 4.4 lg Cl)1) corresponded up to 25% of
bacterial biomass (14244 lg Cl)1, 82 52 lg Cl)1),
suggesting that their role in recycling prokaryotic and
eukaryotic biomass through cell lysis may be more pro-
found than expected [7, 15]. To what extent viruses
contribute to carbon cycling and to the dynamics of
bacterial diversity remains unanswered.
Comparison with the Nanoflagellate Grazing Po-
tential. In order to compare bacterial losses resulting
from viral lysis with those resulting from protistan pre-
dation, the potential grazing by heterotrophic nanofla-
gellates was estimated using an assumed clearance rate
(CR) known from the literature. A similar approach has
been previously used in virioplankton studies [3, 33, 46].
In our study, we minimized errors related to this ap-
proach by applying a mean CR of 0.7 nL ind)1 h)1
(range = 03.3 nL ind)1 h)1) previously estimated in the
Sep Reservoir [41]. These values fell within the range of
values published for freshwater systems [27]. Results
indicated that viruses and HNF contributed almost
equally to bacterial mortality in the water column. The
above finding did not significantly vary when the grazing
rate was calculated with the maximum CR as indicated
above. On average, viruses and HNF together were
responsible for 55% of bacterial mortality, implying that
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON IN RESERVOIRS
the remaining 45% of bacterial production may be di-
rectly available for microzooplankton. This is in accor-
dance with earlier studies in the Sep Reservoir showing
that cladocerans such as Daphnia longispina and
Ceriodaphnia quandrangula are important bacterivores in
this reservoir, where they can remove more than 50% of
the bacterial production [41]. There was a shift in the
dominant source of bacterial mortality over the study
period, from phage bacteriolysis in the early spring to
protistan bacterivory in the late summer-autumn period.
The dominance of viruses over HNF in causing bacterial
mortality was thus noted earlier in the spring, i.e., during
the clear water phase, as indicated by the patterns in
euphotic depth and chlorophyll. This suggests that in
such conditions, viral activity could result in a greater
release of labile organic matter, which can be beneficial to
non-infected bacteria. Our observations thus agree with
the model reported by Murray and Elridge [23] where the
greatest impact of viral activity on food webs occurs
under clear water conditions. This was recently con-
firmed in a comparative lake study [5].
Coupling with Temperature. An important finding
is the coupling between the biological variables under
study and the temperature. Temperature explained about
20%50% of the variance in biological variables. Al-
though the abundance and activity of microheterotrophs
in the Sep Reservoir generally covary with temperature
and resource availability (cf. [20]), such extensive corre-
lations between temperature and biological communities
have not been observed previously in the Sep Reservoir
(cf [20, 39, 40]). Concerning the virioplankton standing
stock and activity, few correlations with temperature have
been reported in previous studies. In Tampa Bay (USA),
Jiang and Paul [17] reported a significant positive cor-
relation between the abundance of viruses and tempera-
ture. The same was found for the viral decay rates in a
backwater system of the River Danube [22]. There are
also data showing a positive correlation between infec-
tious cyanophages and temperature in the Gulf of Mexico
[37]. More recently, Frederickson et al. [12] provided
evidence that the physical structure of the water column
(i.e., temperature, salinity) influenced the composition of
cyanophages and the relatedness of communities found
in British Columbian inlets.
In the Sep Reservoir, we suspected that the general
coupling between biological variables and temperature
was a consequence of the increased summer time dis-
charge from the reservoir for agricultural needs. The
discharge from the reservoir indeed resulted in a signif-
icant decrease in the depth, stability, and thermal inertia
of the water column; thereby naturally establishing the
temperature as a major environmental forcing factor for
biological activities. This was certainly more pronounced
in 2003, a year characterized by a net precipitation deficit
A.S. PRADEEP RAM ET AL.: PHAGE INFECTION OF BACTERIOPLANKTON
from February to September and a significant increase in
the summer temperature (http://www.meteo.fr/meteon-
et/actu/eve.htm). During this study, we recorded a sig-
nificant temperature increase (by 12C) in the water
column during the summer period, compared to 1996
2002. In contrast, the depth of the water column was
decreased by up to 10 m more than during the previous
years [20, 39, 40, unpublished data]. We thus argue that
the unusually warm summer and the enhanced discharge
activity established temperature as a stronger driving
factor in setting the conditions for optimal metabolic
activity of microorganisms. This study thus highlights the
importance of temperature on the seasonal dynamics of
viral abundance and activity and the microbial interac-
tions in pelagic systems, in relation to the hydrographic
conditions. We speculate that any environmental changes
causing a shift to a reduced thermal inertia or to high
temperatures could increase the impact of this parameter
on the variability within biological processes in aquatic
systems.
Acknowledgments
A.S.P. and D.B. were supported by postdoctoral and
doctoral fellowships from the French Government. We
gratefully acknowledge financial support from the Euro-
pean Community and from the following French
national and regional organizations: Ministe`re de lEn-
vironnement, Agence de leau Loire-Bretagne, Conseil
Re gional dAuvergne, Conseil Ge ne ral du Puy-de-Dome,
and Syndicat des Agriculteurs Irrigants de la Haute
Morge. We thank Mr Denis Sargos and Ms Agne`s Vellet
for their timely help in the enumeration of heterotrophic
nanoflagellates and data analysis.
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