Limnol. Oceanogr.

, 55(2), 2010, 885898

2010, by the American Society of Limnology and Oceanography, Inc.

Effect of viruses and protists on bacteria in eddies of the Canary Current region
(subtropical northeast Atlantic)
Julia A. Boras,a,* M. Montserrat Sala,a Federico Baltar,b Javier Arstegui,b Carlos M. Duarte,c and
Dolors Vaquea
a Institut

de Cie`ncies del Mar (Consejo Superior de Investigaciones Cientficas CSIC), Barcelona, Spain
de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
c Institut Mediterrani dEstudis Avanc
ats (Consejo Superior de Investigaciones Cientficas CSICUniversidad de las Islas Baleares),
Mallorca, Spain
b Facultad

Abstract
The effect of oceanic eddies on microbial processes, with emphasis on bacterial losses due to protists and
phages, was examined in the Canary Current region (subtropical northeast Atlantic) through the water column
(down to 1000 m) during August 2006. Sampling stations were located in cyclonic and anticyclonic eddies, as well
as in regions situated outside the influence of the eddy field (far-field stations). In the euphotic zone, in cyclonic
eddies losses of bacteria due to viruses and protists were from 25.6% to 69.8%, and from not detected to 46.8% of
bacterial production (BP) d21, respectively. In anticyclonic eddies, these values ranged from 20.6% to 90.2% of
BP d21 for viruses, and from 8.0% to 79.4% of BP d21 for protists. At far-field stations, losses of bacteria ranged
from 48.7% to 66.9% for viruses, and from not detected to 44.8% for protists. In addition, covering all stations
and depths (from the epipelagic to the bathypelagic layer), bacterial losses due to viruses were significantly higher
than losses by protists, and did not differ significantly among depths except for the stations situated in
anticyclonic eddies, where they were significantly higher in the epipelagic layer. Lysogenic infection was more
frequent at anticyclonic stations, where the highest pressure of protists on bacteria was observed. Because of the
importance of viral activity, we suggest that lysis products from bacteria may be a source of regenerated nutrients
in the surface of the oligotrophic ocean, in addition to the input of nutrients upwelled by eddies.

Mesoscale eddies are common features in the ocean.

Cyclonic eddies raise deep, nutrient-rich water to the
surface, and anticyclonic eddies deepen the warm, nutrient-poor surface water. Eddies are thus a key mode of
vertical nutrient transport in the ocean. Because of the
overall nutrient limitation of primary producers in the
ocean, eddies can regulate oceanic primary production.
Some studies have shown that nutrient input by cyclonic
eddies can significantly enhance the production of autotrophs in the euphotic zone (Falkowski et al. 1991).
Because of the tight coupling between marine prokaryotes
and primary producers, eddies should also affect processes
within the microbial heterotrophic community. Indeed,
higher bacterioplankton abundances inside cyclonic eddies
relative to surrounding waters have been reported (Lochte
and Pfannkuche 1987). However, the evidence of the effect
of eddies on the functioning of microbial food webs is still
scarce (Olaizola et al. 1993). For instance, virioplankton
are recognized to be an essential component of the
microbial loop, but their distribution and activity (production and lysis of prokaryotes) in oceanic eddies has not yet
been described. Moreover, the effect of eddies on microbial
processes should not be restricted to surface waters.
Enhanced primary production at the surface of cyclonic
eddies may lead to an increased flux of particulate organic
matter (POM) to their meso- and bathypelagic waters. The
interpretation of the effect of these mechanisms in mesoand bathypelagic layers is difficult, because there is still

lack of knowledge of microbial food web processes therein

(Magagnini et al. 2007).
Viral infection and subsequent cell lysis recycles organic
carbon (C) and nutrients contained in bacterial cells, which
can be particularly important in nutrient-limited systems
(Fuhrman 1999), whereas protistan grazing on bacteria
transfers the POM up into the food web. Models predict
that viral infections of bacterioplankton will be more
prevalent in eutrophic than in oligotrophic systems,
because of higher host density (Murray and Jackson
1992), as supported by some empirical observations
(Weinbauer and Suttle 1999). In contrast, protistan grazing
is considered to be the principal cause of bacterial mortality
in oligotrophic waters (Guixa-Boixereu et al. 1996). Trophy
of the system may also affect the life strategy of viruses, and
thus a higher percentage of lysogens was found in
oligotrophic than in eutrophic waters (Weinbauer et al.
2003). Because bacteria are the major node in C cycling in
oligotrophic systems (del Giorgio et al. 1997), the nature of
bacterial mortality, cell lysis or protistan grazing, shapes
the C flow in oligotrophic oceanic ecosystems.
Here we compare the bacterial mortality mediated by
viruses and protists in contrasting mesoscale eddies in the
Canary Current region, where a recurrent eddy field is
located south of the Canarian Archipelago. We hypothesize
that the relaxation of oligotrophic conditions in cyclonic
eddies would lead to a higher control of viruses over
bacteria along the euphotic zone, whereas protists would be
the dominant source of bacterial mortality outside these
eddies (far-field stations). In the euphotic zones of

* Corresponding author: boras@cmima.csic.es

885

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Boras et al.

Fig. 1. Map of the area studied during the RODA1 cruise. FF, far-field stations; CE,
cyclonic eddy stations; AE, anticyclonic eddy stations. Black lines correspond to XBT sections
across eddies, represented in Fig. 2.

anticyclonic eddies, we expected to find no changes in

microorganism growth and activity compared with those in
the far-field stations, as no modification in water trophy
was assumed to result from the deepening of surface water
masses. Lysogeny was expected to be the dominant type of
infection in anticyclonic and far-field stations where
oligotrophic conditions prevail, and should be less frequent
in waters under the influence of cyclonic eddies. Also, a
higher percentage of lysogeny was expected in deeper
waters than in the euphotic zone, because of the lower
abundance of host.

Methods
Study area and samplingThe study was carried out at
stations located at the subtropical northeast Atlantic, in the
Canary Current region (26.530uN, 1523.1uW; Fig. 1), on
board the Buque de Investigacion OceanograficaHesperides
during the Remolinos Oceanicos y Deposicion Atmosferica
(RODA1) cruise from 11 August to 05 September 2006. Six
stations were sampled: two stations in cyclonic eddies
(CE1, CE2), two stations in anticyclonic eddies (AE1,
AE2), and two presumably undisturbed far-field stations,
located in regions where no eddies were detected (FF1,
FF2; Fig. 1). At each station, temperature, salinity and
fluorescence were recorded down to 2000-m depth using a
SeaBird 911 Plus conductivitytemperaturedepth (CTD)
system, mounted on a General Oceanics rosette sampler,
equipped with 12-liter Niskin bottles. Samples for microbial abundances and processes were taken at intervals of
25 m up to 100 m, at 150 m, and at 200 m, including the
deep-fluorescence maximum (DFM) layer, and from 200 m
at intervals of 100 m down to 1000 m. Furthermore,
samples for viral abundance (VA) and bacterial abundance
(BA) were taken also at 2000 m. At FF2, CE2, and AE1,
samples for nutrient analysis (phosphate, nitrate + nitrite,
and ammonium) were collected down to 500 m. Samples
for the determination of the dissolved inorganic phosphate
concentrations and the nitrate + nitrite concentrations were
kept frozen until analyzed in a Bran + Luebbe AA3

autoanalyzer following standard spectrophotometric methods (Hansen and Koroleff 1999), and ammonium concentrations were measured spectrofluorometrically within 1 h
of collection.
Microorganism abundancesIn situ VA and BA were
determined by flow cytometry. Subsamples (2 mL), taken
at 12 selected depths, were fixed with glutaraldehyde for
viruses (0.5% final concentration) or paraformaldehyde for
bacteria (1% final concentration). Samples of viruses were
fixed at 4uC during 1530 min, then quick-frozen in liquid
nitrogen (N) and stored at 280uC as described in Brussaard
(2004). Samples of bacteria were analyzed on board
immediately after fixation. Counts were made on a
FACSCalibur (Becton and Dickinson) flow cytometer.
Samples of viruses were stained with SYBR Green I and
run at a medium flow speed (Brussaard 2004), and samples
of bacteria were stained with dimethyl sulfatediluted
SYTO13 and run at a low speed using 50 mL of 0.92-mm
yellow-green latex beads as an internal standard (del
Giorgio et al. 1996).
In situ nanoflagellate abundance was determined by
epifluorescence microscopy (Olympus BX40-102/E at
10003). Subsamples (100 mL) were taken at 6 selected
depths, fixed with glutaraldehyde (1% final concentration),
filtered through 0.6-mm black polycarbonate filters, and
stained with 4,6-diamidino-2-phenylindole (DAPI) at a
final concentration of 5 mg mL21 (Sieracki et al. 1985).
Heterotrophic nanoflagellates (HNF) and phototrophic
nanoflagellates were distinguished under ultraviolet (UV)
and blue light (B2 filter). At least 20100 HNF were
counted per sample. The HNF were grouped into four size
classes: # 2 mm, 25 mm, 510 mm, and 1020 mm. To
determine ciliate abundance and community composition,
1 liter of water from the same depths as for nanoflagellate
counts was immediately fixed with acidic Lugol (2% final
concentration). The fixed samples were allowed to settle for
48 h in the same sampling bottles, and the supernatant was
gently removed, leaving , 200 mL. One hundred milliliters
of this concentrate was further sedimented in 100 mL

Bacterial mortality in ocean eddies

887

chambers for at least 48 h before enumeration at 4003

magnification. Ciliates were counted in an inverted
microscope (Zeiss AXIOVERT35), and identified to genus
level when possible. No ciliate samples were taken at Sta.
CE1.

Net BP (BPN) in the incubation bottles was obtained:

Bacterial production and mortalityExperiments to

determine bacterial production (BP), viral production
(VP), and bacterial losses due to protists (protist-mediated
mortality [PMM]) and viruses (virus-mediated mortality [VMM]), were run using water samples from four
selected depths: DFM, 200 m, 700 m (800 m for FF2
and CE2), and 1000 m. Those depths were chosen in
order to select the most active ocean layer (DFM and
200 m, which is a boundary of the euphotic zone), and
potentially low-productive deeper layers (7001000 m).
Depths of 700800 m correspond to the minimum oxygen
concentration layer, selected as an example of mesopelagic
waters, and 1000 m was chosen as an example of deep-sea
waters.
Bacterial mortality due to protists was evaluated
following the fluorescent-labeled bacteria (FLB) disappearance method (Sherr et al. 1987). For each grazing
experiment, duplicates (1 liter of seawater each) and one
control (1 liter of virus-free water) were prepared in 2-liter
polycarbonate bottles. All bottles (control and duplicates)
were inoculated with FLB at 20% of the natural bacterial
concentration. The FLB were prepared with a culture of
Brevundimonas diminuta (strain obtained from the Spanish
Type Culture Collection, http://www.cect.org/index2.html).
B. diminuta was heat-killed and stained with 5-([4,6
dichlorotriazin-2yl) amino]-fluorescein. Bottles were incubated in a thermostatic chamber, simulating in situ
temperature and in the dark. Samples from the DFM were
incubated during 48 h, and samples from the rest of the
depths during 72 h. Samples for evaluation of BA and FLB
abundance were taken at the beginning and at the end of
the experiments. BA and FLB abundance were assessed by
epifluorescence microscopy (Olympus BX40-102/E, 10003
magnification). To this end, aliquots of 20 mL sample were
filtered through 0.2-mm black polycarbonate filters and
stained with DAPI at a final concentration of 5 mg mL21
(Sieracki et al. 1985). Natural bacteria were identified by
their blue fluorescence when excited with UV radiation,
whereas FLB were identified by their yellow-green fluorescence when excited with blue light. Control bottles showed
no decrease of FLB during the experiments.
Grazing rates of bacteria were obtained following the
equations of Salat and Marrase (1994), based on the
specific grazing rate (g) and the specific net growth rate (a),
and calculated as follows:

G~(g=a)|BPN cells mL{1 d{1 

g~{(1=t) ln (Ft =F0 ) FLB mL{1 

a~(1=t) ln (BAt =BA0 ) cells mL{1 

where t is the incubation time, Ft is the abundance of FLB

at the final time, F0 is the abundance of FLB at the initial
time, and BAt and BA0 are BAs at the end and at the
beginning of the experiment, respectively.

BPN ~BA0 |(eat {1) cells mL{1 d{1 

Then, grazing rate (G) was calculated:

4

Finally, PMM was calculated as the percentage of

bacterial standing stock (BSS) and BP:
PMMBSS ~(G|100)=BA0 % d{1 

PMMBP ~(G|100)=BP % d{1 

To determine VP and bacterial losses due to phages, we

followed the virus-reduction approach (VRA; Wilhelm et
al. 2002). Briefly, 2 liters of seawater was prefiltered
through an 0.8-mmpore size cellulose filter (Whatman;
except 1000-m-depth samples), and then concentrated by a
spiral-wound cartridge (0.22-mm pore size, VIVAFlow
200), obtaining 40 mL of bacterial concentrate. Virus-free
water was collected by filtering 1 liter of seawater using a
cartridge of 100 kDa molecular mass cutoff (VIVAFlow
200). A mixture of virus-free water (160 mL) and bacterial
concentrate (40 mL) was prepared and distributed into 4
sterile Falcon plastic tubes. Two of the tubes were
maintained without any manipulations as controls, whereas
in the other two, mitomycin C (Sigma) was added (1 mg mL21
final concentration) as inducing agent of the lytic cycle in
prophages. All Falcon tubes were incubated in a thermostatic chamber simulating in situ temperature and in the dark
during 12 h. Samples for VA and BA were collected at time
zero and every 4 h of the experiment, fixed with glutaraldehyde, and stored as described before for viruses. Viruses
and bacteria from VP experiments were counted by flow
cytometry. The number of viruses released by bacterial cell
(burst size [BS]) was estimated from VP experiments, as in
Middelboe and Lyck (2002), Wells and Deming (2006) and
Boras et al. (2009). Increase of VA during short time
intervals (4 h) in VP experiments was divided by a decrease
of BA in the same period of time. We assumed that the BP
and viral decay in this time interval were negligible. The
estimated BS ranged from 5 to 299 viruses per cell.
VMM was determined as previously described in Weinbauer et al. (2002) and Winter et al. (2004). Briefly, viral
increase in the control tubes represents lytic VP (VPL), and
the difference between viral increase in the mitomycin C
treatments and VPL gives the lysogenic production (VPLG).
To compare the values of VPL and VPLG from different
experiments, we multiplied them by the bacterial loss factor
(0.48.9), as the loss of part of the in situ BSS during
tangential flow filtration was observed (Winget et al. 2005).
Then, we calculated the rate of lysed cells (RLC) dividing
VPL by BS, following the method of Guixa-Boixereu (1997):
RLC~VPL =BS cells mL{1 d{1 

RLC was used to calculate VMM as a percentage of BSS

(VMMBSS):

888

Boras et al.
VMMBSS ~(RLC|100)=BA0 % d{1 

where BA0 is the initial BA in the VP experiment.

Assuming that percentage of losses of BSS due to viruses
is the same in Falcon tubes and in the grazing bottles, we
used VMMBSS to calculate RLC in the grazing experiment
(RLCGR):
RLCGR ~(VMMBSS |BAGR )=100 cells mL{1 d{1 

where BAGR is BA in the grazing bottles at time 0.

Finally, using RLCGR, VMM as a percentage of BP
(VMMBP) could be calculated:
VMMBP ~(RLCGR |100)=BP) % d{1 

10

BP was calculated as the sum of BPN, grazing rate (G)

and rate of lysed cells (RLCGR):
BP~BPN zGzRLCGR cells mL{1 d{1 

11

Nutrient fluxesNutrient fluxes from bacteria to the

dissolved organic matter (DOM) pool, or to higher trophic
levels as POM, were determined transforming lysed or
consumed bacteria to C, N, and phosphorus (P) units,
using the following cell factors: 12 fg C cell21 (Simon and
Azam 1989), 5.6 fg N cell21 (Simon and Azam 1989), and
0.5 fg P cell21 (Fagerbakke et al. 1996).
Statistical analysesThe Shapiro-Wilk W-test was used
to check normal distribution of data, and data were
logarithmically transformed prior to analyses if necessary.
One-way ANOVA for normal distributions and the
Wilcoxon test for non-normal distributions were used to
evaluate the differences between the three types of stations
or between water layers. Pearson correlation and regression
analyses were used to determine the relationships between
the various properties examined. These statistical analyses
were performed using the JMP program. Canonical
correspondence analysis (CCA) was performed to evaluate
multivariate patterns in the data, using XLSTAT-ADA
software. Depth, fluorescence, salinity, and temperature
were used as predictor (abiotic) variables, and VA, BA,
HNF abundance, VP, BP, and losses of BP due to phages
and grazers as response variables (biotic). The permutation
test confirmed the significance of both canonical correspondence axes.

Results
Oceanographic conditionsCyclonic and anticyclonic
eddies were identified during the cruise by satellite seasurface temperature (Advanced Very High Resolution
Radiometer [AVHRR]). Once the approximate location
was obtained, high-resolution expendable bathythermograph (XBT) transects were carried out to determine the
thermal gradients of the mesoscale eddy field (Fig. 2). Four
eddies were selected for this study: two cyclonic, CE1 and
CE2, and two anticyclonic, AE1 and AE2 (see Fig. 1 for
locations and Fig. 2 for vertical potential temperature

Fig. 2. Vertical sections of potential temperature (uC) across

the different eddies studied (from west to east; see Fig. 1). Arrows
on the top axis indicate XBT stations. Vertical dotted lines
indicate positions at the eddy centers where CTD casts were
carried out.

sections). Four layers of the water column were studied:

epipelagic (, 200 m), which includes the euphotic zone,
transition (200, 500 m), mesopelagic (500800 m) and
bathypelagic (. 8002000 m). Average water temperature
ranged from 24.8uC at the surface to 3.9uC at 2000 m
(Fig. 3), and salinity varied from 37.3 to 35.1 between 5 m
and 2000 m (Table 1). Nitrites + nitrates varied between
0.04 mmol L21 at 5 m and 11.77 mmol L21 at 500 m (Fig. 3),
increasing with depth. Phosphate concentration was below
the detection level from the surface to ca. 120 m at far-field
and anticyclonic eddy stations, being detectable, however,
at cyclonic eddy stations (Fig. 3). There, concentration of
increased with depth, reaching a maximum of
PO {3
4
1.23 mmol L21 at 500 m. Ammonium levels varied between
2.19 mmol L21 in the upper 60 m, and below the detection
level at 500 m (Fig. 3). The nitrite + nitrate concentrations
were significantly higher in the epipelagic layer of cyclonic

Bacterial mortality in ocean eddies

889

Fig. 3. Temperature (Temp.), fluorescence (Fluor.), and nutrient values along a depth profile at far-field (FF), cyclonic eddy (CE),
and anticyclonic eddy (AE) stations in the northeast Atlantic during the RODA1 cruise.

eddy stations than at far-field and anticyclonic eddy

stations (F1,13 5 5.4, p , 0.05), and ammonium levels
were significantly higher in the epipelagic layer of
anticyclonic eddy stations than at cyclonic eddy and farfield stations (F1,13 5 74.9, p , 0.01). Fluorescence values,
as indicators of chlorophyll a concentration, ranged from
0.03 to 1.0 relative units (Fig. 3). The DFM changed its
depth with the occurrence of eddies. The DFM in cyclonic
eddies was found at 56 and 68 m (CE2 and CE1,

respectively), whereas at the far-field stations it was located

much deeper, at 120 m (Fig. 3). The DFM in anticyclonic
eddies was observed at 59 and 75 m (AE2 and AE1,
respectively; Fig. 3). No significant differences in fluorescence were found among the epipelagic layer of the three
types of stations.
Distribution, abundance, and production of microorganismsVA ranged between 1.1 3 105 viruses mL21 at

890

Boras et al.

Table 1. Average values of selected physicochemical and biological parameters determined during the RODA1 cruise in the cyclonic
eddy (CE), anticyclonic eddy (AE), and far-field (FF) stations. Depth layers: epi., epipelagic, 5, 200 m; trans., transition, 200, 500 m;
meso., mesopelagic, 500800 m; bathy., bathypelagic, . 8002000 m. Temperature (Temp., uC); fluorescence (Fluor., arbitrary
fluorescence units); viral abundance (VA, 106 viruses mL21); bacterial abundance (BA, 105 cells mL21); heterotrophic nanoflagellates
(HNF, 102 cells mL21); ciliates (102 cells L21); lytic viral production (VPL, 106 viruses mL21 d21); bacterial production (BP;
105 cells mL21 d21); rate of lysed cells (RLC, 104 cells mL21 d21); grazing rate (G, 104 cells mL21 d21). nd, not detected.
Temp.

Salinity

Fluor.

VA

BA

HNF

Ciliates

VPL

BP

RLC

FF1
epi.
trans.
meso.
bathy.

21.0
15.6
11.0
7.0

37.0
36.2
35.6
35.3

0.27
0.05
0.04
0.04

2.5
0.4
0.2
0.2

1.9
0.4
0.3
0.2

2.3
0.9
0.2
0.1

16.2
3.4
0.6
0.2

1.4
0.4
0.9
1.2

2.9
1.0
0.2
0.6

15.8
4.8
2.0
5.7

12.8
2.9
0.4
0.6

FF2
epi.
trans.
meso.
bathy.

20.3
15.7
11.1
7.2

36.9
36.3
35.6
35.3

0.25
0.04
0.04
0.04

3.3
0.6
0.7
0.2

2.6
0.7
0.7
0.3

1.9
0.4
0.9
0.2

8.6
2.8
0.3
0.1

1.7
0.4
0.5
0.1

1.5
0.5
0.2
0.2

9.8
3.2
0.8
1.2

nd
0.7
0.2
0.3

FF average

13.4

36.0

0.09

1.0

0.9

1.0

4.8

0.8

0.9

5.4

2.2

CE1
epi.
trans.
meso.
bathy.

19.2
13.7
9.5
7.1

36.6
35.9
35.4
35.2

0.27
0.04
0.04
0.04

4.3
0.9
0.5
0.4

3.6
1.0
0.6
0.4

3.4
0.7
0.2
0.3

1.1
0.04
0.2
0.2

1.8
0.3
2.0
0.7

4.3
1.3
17.2
5.9

3.3
nd
2.9
,0.1

CE2
epi.
trans.
meso.
bathy.

20.0
14.4
10.5
6.7

36.8
36.0
35.5
35.2

0.39
0.03
0.04
0.04

4.1
0.6
0.3
0.2

2.2
1.2
0.6
0.2

16.5
0.6
0.4
0.1

16.8
3.8
0.6
0.3

0.3
0.7
1.0
1.4

4.3
0.4
0.2
0.2

15.9
2.6
1.6
0.6

20.1
0.6
0.4
nd

CE average

13.6

35.9

0.13

1.8

1.5

3.6

6.5

0.6

1.2

6.2

3.4

AE1
epi.
trans.
meso.
bathy.

22.8
15.7
10.6
8.0

37.0
36.3
35.5
35.3

0.32
0.05
0.04
0.04

4.4
0.7
0.3
0.3

5.4
1.3
0.8
0.5

4.9
1.1
0.4
0.2

11.0
2.3
0.5
0.1

1.0
0.8
0.7
1.0

5.8
0.6
0.4
0.1

52.8
1.2
2.3
0.5

4.7
4.8
1.9
0.4

AE2
epi.
trans.
meso.
bathy.

20.5
14.7
10.3
6.6

36.6
36.1
35.5
35.2

0.42
0.04
0.04
0.04

4.3
0.7
0.3
0.2

2.9
0.9
0.6
0.4

5.7
1.4
0.4
0.3

17.5
2.4
0.7
0.2

0.5
0.1
0.2
2.8

2.0
1.4
0.7
0.9

16.9
8.4
2.6
5.7

3.2
5.8
2.8
1.6

AE average

13.6

35.9

0.12

1.4

1.6

2.0

5.4

0.9

1.5

11.3

3.2

Station and layer

2000 m and 8.1 3 106 viruses mL21 at 50 m, and BA ranged

between 1.5 3 104 cells mL21 at 2000 m and 7.8 3
105 cells mL21 at 5 m (Fig. 4). At all stations, the highest
VAs and BAs were found in the epipelagic layer, above or
in the DFM, and decreased significantly with depth
(Table 2). No differences in VA were found among the
three types of stations, whereas BA was significantly higher
in cyclonic and anticyclonic eddies than at far-field
stations, yet only for the transition layer (F1,15 5 11.9, p
, 0.01). Both VA and BA showed strong positive
correlation with fluorescence (Table 2). Virus to bacterium
ratio (VBR) varied between 1.8 and 26.0, decreasing
significantly with depth (Table 2). The lowest VBR overall
was found between 800 m and 2000 m. HNF abundance
decreased with depth (Table 2; Fig. 5), reaching the lowest
values in the bathypelagic layer (0.1 3 102 cells mL21 at

1000 m), and highest in the epipelagic layer (1.8 3

103 cells mL21 at 5 m). Significant differences in HNF
abundance between the three types of stations were
detected only for the epipelagic layer, with highest
abundances at cyclonic eddy stations and lowest at farfield stations (F2,8 5 8.0, p , 0.02). Slightly, but not
statistically significantly, higher HNF abundances were
detected at anticyclonic eddy stations compared to far-field
or cyclonic eddy stations in transition and mesopelagic
layers (Table 1). Ciliate abundance declined with depth,
from a maximum of 3.0 3 103 cells L21 in the DFM to a
minimum of 5 cells L21 at 1000 m (Fig. 5). At all stations,
except for AE1, a peak of abundance was detected in the
DFM. No significant differences between the three types of
stations were found. The ciliate community was composed
mainly of the autotrophic Mesodinium sp. (range 23.3

Bacterial mortality in ocean eddies

891

Table 2. Significant correlation coefficients among variables.

Viral abundance (VA); fluorescence (Fluor.); bacterial abundance
(BA); virus to bacterium ratio (VBR); heterotrophic
nanoflagellate abundance (HNF); bacterial production (BP);
percentage of lysogenic viral production in total viral
production (%VPLG); virus-mediated mortality of bacteria as a
percentage of bacterial standing stock (VMMBSS); protistmediated mortality of bacteria as a percentage of bacterial
standing stock (PMMBSS); virus-mediated mortality of bacteria
as a percentage of bacterial production (VMMBP).

Fig. 4. Viral and bacterial abundances along a depth profile

at far-field (FF), cyclonic eddy (CE), and anticyclonic eddy (AE)
stations in northeast Atlantic during the RODA1 cruise.

55.6%), followed by Strombidium sp. (range 20.140.8%) in

the euphotic zone, and by Strobilidium sp. (range 19.5
100%) and tintinnids (37.473.9%) in the dark ocean zone.
Bacterivorous ciliates, as scuticociliates, were very rare in

Variables

VADepth
VAFluor.
VABA
BADepth
BAFluor.
VBRDepth
HNFDepth
HNFBA
CiliatesBA
CiliatesHNF
BPFluor.
BPDepth
BPVA
BPBA
BPHNF
%VPLGBP
%VPLGFluor.
%VPLGVA
%VPLGHNF
VMMBSSBP
PMMBSSBP
VMMBPPMMBP

80
80
75
75
75
75
37
37
28
30
24
24
24
22
24
24
24
24
24
24
24
24

20.741
0.753
0.897
20.776
0.625
20.238
20.818
0.919
0.805
0.846
0.759
20.659
0.750
0.631
0.661
0.460
0.462
0.407
0.455
0.490
0.510
20.474

,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.01
,0.05
,0.05
,0.05
,0.05
,0.02
,0.02
,0.02

the collected samples (from 1.7% to 16.4%). Positive

correlations between the abundance of all components of
the microbial food webVA, BA, HNF, and ciliates
were found (Table 2).
BP decreased with depth (Fig. 6A), and increased with
fluorescence and microorganism abundance (VA, BA,
HNF; Table 2). No differences in BP between stations
were detected (Table 1).
VPL varied between 4.3 3 104 viruses mL21 d21 at 200 m
and 2.8 3 106 viruses mL21 d21 at 1000 m (Table 1;
Fig. 6B). At four out of six stations, the highest VPL was
detected in the DFM. High VPL values were also found at
1000 m at four stations; for instance, at Sta. AE2, VPL was
ca. fourfold higher at 1000 m than in the DFM. No
differences in VPL between far-field, cyclonic eddy, and
anticyclonic eddy stations were found in any of the layers.
In 46% of VP measurements, prophage induction was
detected, and the percentage of VPLG (%VPLG) with
respect to total VP varied from 13.4% at 700 m to 84.6%
in the DFM (Fig. 6B). In one of the anticyclonic eddies
(AE2), lysogeny was detected at all analyzed depths. The
%VPLG increased with increasing fluorescence, VA, HNF
abundance, and BP (Table 2).
Bacterial mortalityVMMBP varied between 20.6% d21
at 200 m and 92.0% d21 in the DFM (Fig. 7), and did not
differ significantly among depths (Table 1). Stations in

892

Boras et al.
anticyclonic eddies were characterized by significantly higher
VMMBP in the epipelagic layer (F2,3 5 31.9, p , 0.01) than
cyclonic eddies or far-field stations (Fig. 7). VMMBSS
increased with increasing BP (Table 2), and did not show
any differences among the three types of stations. At far-field
stations, VMMBSS showed a decrease with depth down to a
minimum of 3.8% d21 at 700 m, and then an increase up to
55.8% d21 at 1000 m (data not shown). BP grazed by protists
(PMMBP) ranged from 0.8% d21 at 1000 m to 79.4% d21 at
200 m (Fig. 6). On three occasions, grazing was not
detectable (Table 1). PMMBP did not show a clear pattern
at far-field stations, whereas at cyclonic stations it reached
the lowest (0.8% d21) values at 1000 m (Fig. 7). Significant
differences in PMMBP between the three types of stations
were detected only for the mesopelagic layer (F2,3 5 92.1, p
, 0.01), with the highest values at anticyclonic stations.
Losses of BSS due to protists (PMMBSS) followed the same
pattern as PMMBP (data not shown), and increased
significantly with BP (Table 2). Anticyclonic eddies supported the highest and cyclonic eddies the lowest PMMBSS
values (F2,3 5 10.7, p , 0.05).
Plotted values of rates of lysed against grazed bacteria
(cells mL21 d21) showed that in anticyclonic eddies losses
of bacteria due to viral lysis were higher than losses due to
grazing for the epipelagic layer (the DFM), and close to the
line 1 : 1 for all the other depths (Fig. 8). In cyclonic eddies,
losses due to phages were higher than losses due to protists
at all depths, except for the epipelagic layer (the DFM). At
far-field stations, viral lysis dominated in the majority of
samples for all depths, except for two samples: from the
epipelagic (DFM) and transition (200 m) layers, where both
types of mortality were similar (Fig. 8).
The CCA showed that data were clustered mostly within
the sampling depths (data not shown), indicating that this
was the main factor shaping the biological variability
among samples. However, samples from the same types of
stations also clustered together within each depth. The
permutation test showed that the biotic data had a linear
relationship with abiotic data (p , 0.001)

Discussion

Fig. 5. Heterotrophic nanoflagellate (HNF) and ciliate

abundances along a depth profile at far-field (FF), cyclonic eddy
(CE), and anticyclonic eddy (AE) stations. No ciliate sample was
taken at CE1.

Methodological considerationsThe methods to estimate

complex ecosystem variables, such as BP and losses of
bacteria, have their suite of assumptions and uncertainties.
In most cases these methods give high reproducibility of
results among duplicates, even dealing with natural
communities. However, in some experiments they resulted
in a relatively high range of values, as we observed, e.g., for
PMMBP (FF1, 700 m).
The FLB disappearance technique (Sherr et al. 1987) was
used to measure the grazing rates on bacteria by the whole
community of protists. Taking into account that only a small
part of the ciliates counted was strictly bacterivorous, such
as scuticociliates, the main grazers in our system should be
small HNF (# 5 mm; Sherr and Sherr 2002). The use of FLB
as bacterial surrogates has known limitations, such as
disturbance of the sample and underestimation of bacterial
losses due to prey selection (Christoffersen et al. 1997). This
technique has been widely discussed in Vaque et al. (1994).

Bacterial mortality in ocean eddies

893

Fig. 6. (A) Bacterial production (BP), and (B) lytic (VPL) and lysogenic (VPLG) viral production, detected at far-field (FF), cyclonic
eddy (CE), and anticyclonic eddy (AE) stations. Bars indicate the maximum and minimum values of experimental duplicates. The VPLG
percentages of total VP are shown. At stations FF2 and CE2, the BP and VP values were determined at a depth of 800 m instead of 700 m.

BP was estimated as the sum of the net increase of BA

(BPN) and losses of bacteria due to protists (G) and viruses
(RLCGR). This method was employed in previous studies,
and results of BP were comparable, although slightly
higher, with those obtained with in situ measurements of
BP by the 3H-leucine incorporation method (Boras et al.
2009).
Estimations of bacterial mortality due to viruses are
subjected to the calculation of BS. We estimated BS taking
into account the increase of VA in VP experiments and the
decrease of BA in the same period of time (4 h). We
assumed that the only cause of BA decrease over short time
periods was cell lysis, and we did not take into account the
viral decay and BP during this time interval. BS values
reported in this study, between 5 and 299, are within the
range obtained from different aquatic environments
(Guixa-Boixereu et al. 1996; Parada et al. 2006), and are
lower than values found in the anoxic layer of an eutrophic
lake (, 500; Weinbauer and Hofle 1998). Also, bacterial
losses caused by lysis measured in our study are potential
losses, as we did not consider grazing on infected cells by

protists in our calculations. This process could reduce the

percentage of bacteria that burst because of viral activity in
natural communities.
The VRA used to assess VP and VMM is based on the
assumption that all VP observed during the experiment is a
result of infections prior to incubation. It is also assumed
that no new infections occur, and that both filtration and
incubation do not induce lysogenic bacteria. This method
allows direct observation of changes in VA over time, and
the distinction between production of virulent and temperate phages. In addition, it is relatively easy and inexpensive
to perform (Winget et al. 2005). Detection of lysogeny is
based on lysis induction by mitomycin C. It is known that
in some circumstances (i.e., nutrient availability, pH) this
agent is not sufficient to induce the lytic cycle in all
prophages (Weinbauer and Suttle 1999). However, this
method is widely used, and mitomycin C is better suited
than other inducing agents; hence, the obtained results are
comparable to other studies. Finally, the main drawbacks
of this technique are the considerable levels of sample
manipulation and the loss of a portion of bacterial

894

Boras et al.

Fig. 7. Virus-mediated mortality (VMM) and protist-mediated mortality (PMM) of bacteria

as a percentage of bacterial production (BP) detected at far-field (FF), cyclonic eddy (CE), and
anticyclonic eddy (AE) stations. Bars indicate the maximum and minimum values of duplicates.
At stations FF2 and CE2, the VMM and PMM values were determined at a depth of 800 m
instead of 700 m. nd, not detectable.

community during filtration. Despite these disadvantages,

VRA is considered one of the best-suited incubation-based
methods for VP and VMM estimations (Winget et al.
2005).
Distribution, abundance, and production of microorganismsEddies studied during the RODA1 cruise are
common mesoscale features in the region of the Canary

Islands, northeast Atlantic, and are generated by the

interaction of the islands with the southward-flowing
Canary Current and the intrusion of coastal upwelling
waters into the Canary region. Previous studies have shown
that in addition to the nutrients rising from the lower water
layers, rich upwelling waters are incorporated into cyclonic
eddies in this region (Arstegui et al. 1997), thereby
enriching the otherwise oligotrophic waters around the

Bacterial mortality in ocean eddies

Fig. 8. Plot of the rate of lysed cells (RLC) against grazing

(G) detected at the three types of stations: far-field (FF; open
symbols), cyclonic eddies (CE; black symbols), and anticyclonic
eddies (AE; grey symbols). Different depth layers (epipelagic,
transition, mesopelagic, and bathypelagic) are marked with
diamonds, triangles, circles, and squares; dashed line is the 1 : 1
line. Values above the line represent RLC . G; values below the
line represent RLC , G. Grazing rates 5 0 on the plot correspond
to not detectable.

Canarian Archipelago. Our results show a clear contrasting

pattern of nutrient concentrations in the epipelagic layer of
cyclonic and anticyclonic eddies and far-field stations.
Higher concentrations of nitrates + nitrites and phosphates
in cyclonic eddy than in anticyclonic eddy stations and farfield stations were observed. Moreover, higher ammonium
concentrations at anticyclonic stations suggest intense
recycling processes therein.
In open-ocean waters, VA and BA have been reported to
be highest in the epipelagic layer (down to 200 m), and to
decline reaching relatively constant, low concentrations in
deep waters (Hara et al. 1996). A similar pattern for both
VA and BA was observed in this study, with the highest
abundances between 5 and 120 m. We found peaks of VA
and BA above and in the DFM, consistent with previous
reports from the north Pacific (Hara et al. 1996), and from
the eastern tropical Atlantic Ocean (Winter et al. 2008).
Strong positive correlation between VA and BA throughout the water column, consistent with other reports (Hara
et al. 1996; Magagnini et al. 2007), suggests that
bacteriophages may be an important fraction of the viral
community in that region. Also, the positive correlation of
both VA and BA with fluorescence suggests that viruses of
photosynthetic organisms may also be a significant fraction
of virioplankton in the epipelagic layer. The virusbacterium ratios reported here are similar to the values
reported for other marine waters (Wommack and Colwell

895

2000), but show a decreasing trend with depth, in contrast

with data reported by other studies (Magagnini et al. 2007).
Protist (HNF and ciliate) abundance also decreased with
depth, reaching very low (# 10 cells mL21) values in
transition, mesopelagic, and bathypelagic layers, similarly
to abundances found by Gasol et al. (2009). BP declined
with depth, as reported in the past (Reinthaler et al. 2006),
with a difference of an order of magnitude between the
values in the DFM and at 1000 m.
Stations influenced by eddies, either cyclonic or anticyclonic, showed higher VA and BA than stations away from
the eddy field in most of the layers. This could be due to the
input of nutrients from deeper to shallower waters in
cyclonic eddies, or due to an increase of ammonium
concentration originating from the remineralization of the
organic matter accumulated in the centre of anticyclonic
eddies (Nelson et al. 1995), as observed at the AE2 station.
Similarly to Lochte and Pfannkuche (1987), we have found
no differences in BP between eddies and far-field stations.
However, we detected slightly higher BA and HNF
abundance in the transition, mesopelagic, and bathypelagic
layers of anticyclonic eddy stations than at cyclonic eddy
and far-field stations, suggesting that the deepening of the
surface water masses by anticyclonic eddies could alter the
vertical distribution of microorganisms.
The highest VPL was detected in the DFM, but it was
also high at 1000 m, occasionally exceeding the VPL in the
epipelagic layer (AE2, CE2). It is not a surprising result,
considering the relatively high VAs and VBR found in
deeper waters, which would also suggest a high VP. Parada
et al. (2007) reported similar VAs in bathypelagic waters
(. 1000 m) for the subtropical North Atlantic, and proposed a contribution of allochthonous viral input or VPLG.
In our study, however, the measured VPL would be
sufficient to maintain the VA found in dark waters.
Detected values of VPL are comparable with VP observed
in euphotic (Rowe et al. 2008) and deeper (Parada et al.
2007) waters in the same area. Rowe et al. (2008) reported
VP that ranged from ca. 0.5 3 106 to 7.2 3 106 viruses
mL21 d21 in the upper ocean layer (, 200 m), and in our
study we observed VPL from 0.1 3 106 to 1.7 3 106 viruses
mL21 d21 at that depth (Table 1). Parada et al. (2007)
reported VP values at the level of ca. 0.1 3 106 viruses
mL21 d21 in deeper waters (9001100 m), which are similar
to our observations for stations FF2 and CE1 at 1000 m.
However, at the other stations we detected higher VPL,
which exceeded those numbers by an order of magnitude.
In oligotrophic systems, as well as in deep-ocean waters,
lysogeny should predominate over lytic infections as a
survival strategy of phages at low host densities (Stewart
and Levin 1984). This was confirmed by some studies, as a
higher percentage of lysogens was found in offshore
compared to coastal environments (Weinbauer and Suttle
1999), and in deep compared to surface marine waters
(Weinbauer et al. 2003). Our results show a lower
percentage of lysogeny in samples from deeper waters than
from the upper ocean layers, with the highest values
detected in the DFM at stations influenced by eddies,
although no statistically significant differences between
depths were detected. Moreover, contrary to the results

896

Boras et al.

Table 3. Average carbon and organic nutrients fluxes shunted from bacteria to higher trophic levels by protistan grazing (PMM) and
to dissolved organic matter pool by viral lysis (VMM). Averaged values for all water column (6 SD, n 5 8), for epipelagic + transition
layers (epi., trans.; 6 SD, n 5 4), and for mesopelagic + bathypelagic layers (meso., bathy.; 6 SD, n 5 4).
Carbon (mg L21 d21)
Layer and station
All water column
FF
CE
AE
epi., trans.
FF
CE
AE
meso., bathy.
FFS*
CE
AE

Nitrogen (mg L21 d21)

Phosphorus (mg L21 d21)

PMM

VMM

PMM

VMM

PMM

VMM

0.2760.33
0.4160.31
0.3760.04

0.6560.27
0.7560.17
1.3860.49

0.1360.15
0.1960.14
0.1760.02

0.3160.12
0.3560.08
0.6460.23

0.0160.01
0.0260.01
0.0260.00

0.0360.01
0.0360.01
0.0660.02

0.5060.65
0.7360.73
0.5360.04

1.0260.33
0.7460.56
2.4061.23

0.2360.30
0.3460.34
0.2560.02

0.4860.15
0.3460.26
1.1260.57

0.0260.03
0.0360.03
0.0260.00

0.0460.01
0.0360.02
0.1060.05

0.0560.01
0.1060.11
0.2160.12

0.2960.20
0.7660.90
0.3560.25

0.0260.00
0.0560.05
0.1060.06

0.1360.09
0.3660.42
0.1660.12

0.0060.00
0.0060.00
0.0160.01

0.0160.01
0.0360.04
0.0160.01

* Values of C, N, and P shunted by PMM are significantly lower than by VMM (p , 0.01).

reported in Weinbauer et al. (2003), the percentage of

lysogeny increased with increasing BP, and no relationship
with BA was observed, suggesting that factors other than
host density could also affect the prevalence of lysogeny.
Bacterial mortalityLosses of BP due to viral infection
prevailed over losses due to protists in 92% of the examined
cases, with only two communities in which mortality
caused by protists exceeded that caused by viruses. In
25% of cases, phages caused more than 80% of losses of
BP, mostly in meso- and bathypelagic layers, but also in the
DFM at stations influenced by anticyclonic eddies. These
values are among the highest virus-induced bacterial
mortality rates reported for oligotrophic waters (Wommack and Colwell 2000). In the eastern tropical Atlantic
Ocean, the frequency of infected bacterial cells (which
correspond to VMMBSS in this study) found by Winter et
al. (2008) ranged between 2.4% in deeper waters (75
1000 m) and 8.3% in the mixed surface (1525 m) and
DFM (50 m) layers. Virus-induced mortality of bacterioplankton measured by Weinbauer et al. (2003) in
Mediterranean waters varied between 2.6% in deep waters
(8002000 m) and 14.8% in surface waters (down to 100 m).
On the other hand, Brum (2005) found that an important
part of BSS was lysed because of viral infections at the
oligotrophic station ALOHA, North Pacific Subtropical
Gyre, with a loss of 3.2% of BSS h21 (which corresponds to
63% d21) at 5-m depth, and about 5 times more at 75 m.
These examples show a great variability in rates of bacterial
mortality due to viruses, which may be partly an effect of
the variability of the methods used.
Protist-mediated mortality rates of bacteria obtained
here were comparable to results from mesopelagic waters
(down to 500 m) of the East Sea (Cho et al. 2000), ranging
from 0.1 to 1.1 3 103 cells mL21 h21. Cho et al. (2000)
concluded, however, that HNF were responsible for losses
of most (up to 100%) of BP in this region, but they did not
evaluate experimentally mortality due to viruses. Indeed, a
later study of Hwang and Cho (2002) reported that phages
and protists had a comparable effect on bacterioplankton

in that system. We observed a high grazing activity in

transition, mesopelagic, and bathypelagic layers at stations
under the influence of anticyclonic eddies, possibly related
to the higher abundance of HNF and BP therein.
Finally, we estimated the importance of protistan grazing
vs. viral lysis of bacteria for the nutrient fluxes (C, P, N)
within the microbial web, making a rough estimation of the
POM shunted from bacterial cells to higher trophic levels by
HNF, and to DOM by phages. Values obtained for the farfield stations were comparable to the range of fluxes of N
and C shunted by viruses reported for the oligotrophic
offshore waters of the Gulf of Mexico with relatively steadystate conditions (Wilhelm et al. 1998). In that system, viruses
released 0.120.55 mg C L21 d21, compared to 0.65 6 0.27 mg
C L21 d21 estimated in our system (average for the whole
water column, n 5 8; Table 3). In contrast, fluxes of DOM
assessed for the anticyclonic and cyclonic stations were
similar to or lower than those reported for the Strait of
Georgia, British Columbia (Wilhelm and Suttle 2000),
characterized by turbulently mixed waters. In those conditions, the range of the C flux was 1.031.51 mg C L21 d21 in
stratified waters and 2.008.25 mg C L21 d21 in tidally mixed
waters, whereas our estimations resulted in 0.75 6
0.17 mg C L21 d21 at CE stations and 1.38 6 0.49 mg C
L21 d21 at AE stations (averages from the epipelagic and
transition layers, n 5 4; Table 3). The release of DOM from
bacterioplankton by cell lysis was slightly higher than the
POM ingestion by HNF at far-field and anticyclonic stations
in epipelagic and transition layers, although this trend was
not statistically significant (Table 3). However, in meso- and
bathypelagic layers of far-field stations, nutrient fluxes
shunted by viruses were significantly higher than shunted
by protists (F1,6 5 15.5, p , 0.01; Table 3). This relationship
was not found for other stations. Finally, POM and DOM
fluxes showed slightly higher values (although no significant)
in eddy than at far-field stations for all layers, indicating that
these biological processes could be influenced by mesoscale
features.
In summary, as expected, in anticyclonic eddies HNF
supported the highest pressure on bacteria, and in cyclonic

Bacterial mortality in ocean eddies

eddies the lowest one. Furthermore, in anticyclonic eddies
lysogeny was detected at all depths; however, it was not
always detected in deep waters at the other types of
stations. Contrary to our expectations, loss of BP due to
viruses did not differ significantly among depths, except for
the stations situated in anticyclonic eddies, where it was
significantly higher in the epipelagic layer. In addition, low
values of bacterial losses due to protists were detected at
stations outside the influence of eddies. Our results indicate
that viruses are responsible for losses of an important
fraction of BP, as well as for the release of significant
amounts of C and nutrients to the DOM pool in the studied
system. Furthermore, the importance of viruses in bacterial
C cycling was higher in eddies, both cyclonic and
anticyclonic, and in the deeper layers of the mesopelagic
zone. Regardless of the low BAs in deeper waters (1000 m),
at these depths high VP and a higher percentage of
bacterial losses due to viruses than due to grazers were
found. Our findings indicate that viral lysis could be a
dominant pathway for the flow of bacterial C in the
northeast subtropical Atlantic, particularly significant in
areas dominated by eddy activity.
Acknowledgments
We thank E. Martinez, E. Cabanillas, and A. Gil for helping in
nanoflagellate and ciliate counts, and R. Martnez and J. C. Alonso
for their help with nutrient analyses. We also thank P. Sangra for
providing hydrographic data for Figure 2. We are very grateful to
the staff of Unidad de Tecnologa Marina (Consejo Superior de
Investigaciones Cientficas) and the crew of the Buque de Investigacion OceanograficaHesperides for their help during the cruise. This
study was supported by the following projects: Remolinos
Oceanicos y Deposicion Atmosferica en la Corriente de Canarias
(RODA), Protozoos y Virus: Control de la Biomasa y la Diversidad
de Procariotas y su Repercusion en los Ciclos Biogeoqumicos en
una Zona Costera del Mediterraneo Nor-Occidental (PROCAVIR), and Aislamiento, Identificacion y Especificidad de Virus que
Infectan a Microorganismos Marinos (MICROVIS) (CTM200406842 /MAR; CTM2004-04404-CO2-00/MAR; CTM2007-62140),
funded by the Spanish Ministerio de Ciencia e Innovacion. J.A.B.s
work was supported by a Ph.D. fellowship from the Spanish
Ministerio de Ciencia e Innovacion (Formacion de Profesorado
Universitario grant), and M.M.S. by a postdoctoral I3P-CSIC
contract funded by the Fondo Social Europeo. This is a
contribution to the European Network of Excellence EurOceans.

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Associate editor: Peter G. Verity

Received: 03 April 2009
Accepted: 15 October 2009
Amended: 15 December 2009