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. Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147 129
main bacterivorous grazers Fenchel, 1982b; Fuhrman and McManus, 1984; Rassoul- ¨
zadegan and Sheldon, 1986; Wikner and Hagstrom, 1988. The relevance of predation control of bacteria in oligotrophic systems remains, however, controversial and the
coupling between bacteria and bacterivores is not fully understood yet. This is partly due to the difficulty in obtaining precise measurements of bacterivory and biomass of
heterotrophic nanoplankton as these rates and standing stocks are extremely low. Some comparative analysis from different aquatic systems suggest that top-down regulation of
bacteria is more important in eutrophic and bottom-up control in oligotrophic environ- ments, although grazing and bacterial production are generally balanced Sanders et al.,
1992.
After the development of the ‘Microbial Loop’ concept, the prevailing picture was that a highly active and efficient protist grazer community controls bacterial abundance
and consumes new bacterial production, which eventually results in the rather constant and homogenous low bacterial abundance observed in the open ocean e.g. Ducklow,
1983. Efficient grazing control was also supported by experimental microcosm studies which revealed a close coupling between bacteria and bacterivores Wikner and
¨ Hagstrom, 1988; Weisse, 1989 and by substrate addition experiments in which bacterial
abundance remained relatively constant despite strong increases in bacterial production Kirchman, 1990; Kirchman and Rich, 1997. Even less is known about the importance
of grazing as a shaping factor for the phenotypic and genotypic bacterial community composition in oligotrophic systems. Studies in more productive coastal or freshwater
environments have demonstrated that bacterial grazing has an important impact in this
¨ ¨
respect Jurgens and Gude, 1994. The purpose of the present study was to examine the bacteria–protozoan coupling in
warm oligotrophic to ultra-oligotrophic ocean sites in microcosm experiments. Ex- perimental manipulations with substrate additions and size-fractionations were aimed at
stimulating bacterial production and increasing predation pressure by small bacterivores. We were specially interested in 1 revealing the response time of bacterivores to
increases in bacterial production and bacterial biomass, and 2 analysing the impact of grazers on bacterial size distribution and the appearance of grazing-resistant mor-
photypes in response to increased predation.
2. Material and methods
Bottle incubation experiments were carried out during the cruise Latitud II across the Central Atlantic Ocean from the Canary Islands Spain to Mar del Plata Argentina,
´ October–November 1995 aboard the Spanish R V BIO-Hesperides. The positions of
the four stations at which microcosm experiments were performed are shown in Fig. 1. All stations were in oceanic waters over depths . 2000 m. Two stations were located in
the Northern subtropical zone, the station of the first experiment exp. 1 in the vicinity of the Canary Islands Canary Current at 198W, 248N, the second experiment exp. 2
close to the Northwest African upwelling area 208W, 118N. Dominant surface water fluxes in the area are North–South following the coast, and leaving it at around 8–98N
see Dadou et al., 1996. There exists another upwelling in the area created by the
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K . Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147
Fig. 1. Latitude II transect across the Central Atlantic with the sampling stations at which microcosm experiments 1–4 were performed arrows.
Guinea dome, which is centered at 208W, 108N Stramma and Schott, 1999. The Guinea dome appears south of Cape Verde, and creates a cyclonic structure and an upward
displacement of the isolines Siedler et al., 1992. The other two experimental stations were in the southern subtropical zone of the
western South Atlantic 318W, 158S and 328W, 188S. All stations, including those close to the upwelling and Canary Current, were oligotrophic open ocean sites with low
productivity and low chlorophyll a concentrations. Water samples were collected with 12-l Niskin bottles attached to the CTD by a Rosette sampler system. Water for the
experiments was taken from the surface 5 m depth and for three of the stations also from the depth of the fluorescence maximum deep chlorophyll maximum, DCM. DCM
was determined before sampling with a MARK III CTD fitted with a fluorometer. Taking into account the two different depths, 5 m and DCM, altogether seven different
microcosm experiments were performed.
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. Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147 131
All experimental incubations were conducted in duplicate acid-washed 1.5-l poly- carbonate bottles. Experiments started within 2 h after sampling and lasted for 96–160 h.
The bottles with surface water 5 m were incubated in water tanks in order to simulate in situ conditions with approximate ambient light and temperature values. Water
temperature in the tanks varied at the most 0.58 from in situ temperature. Light was reduced with a Nylon net covering the tanks. Light measurements during the cruise
showed that we could simulate the light at roughly 5-m depth. The samples from the DCM were incubated in a climate chamber, at 17–198C, with light of roughly 1 of
surface noon light.
The experiments 1, 2, and 4 consisted of differentially filtered treatments in order to enhance or eliminate predation on bacteria by bacterivores. One fraction, filtered through
a , 5 mm filter 42 mm polycarbonate membrane, Millipore, was designed at eliminating micro- and mesozooplankton, thereby removing predators on HNF and
increasing predation pressure on bacteria. One fraction filtered through a , 0.8 mm filter should contain only bacteria and no eukaryotic bacterivores. In experiment 2 we had an
additional fraction, for 5 m and DCM, filtered through a 50-mm mesh net in order to remove mesozooplankton. We always had two replicate treatments without nutrients and
two replicates which received substrate additions to stimulate bacterial growth. We used yeast extract as a complex substrate source because in previous experiments this resulted
in an immediate growth of bacteria data not shown. The concentration of yeast extract
21
which was added, between 1.5 and 4.5 mg l , was sufficient to increase bacterial
abundance by approximately one order of magnitude. The chemical analysis of the yeast extract we used Difco revealed a molar C:N:P ratio of 93:21:1, resulting in an addition
of 50–150 mM organic C in the experiments. In experiment 3 only unfiltered water incubations from 5 m depth were used to which different substrate sources were added:
21
yeast extract 1.5 mg l , glucose 7.4 mM, mixture of amino acids 5 mM, or
inorganic nitrogen 2 mM NH NO .
4 3
Subsamples for bacteria and HNF were taken daily, fixed with cold glutaraldehyde 1 final concentration and enumerated using epifluorescence microscopy and DAPI
staining Porter and Feig, 1980. Between 1 and 10 ml depending on enrichment and time of incubation were filtered on black 0.2 mm-polycarbonate filter Millipore for
counting bacteria. The filters were frozen at 2 208C until processing of the samples in an epifluorescence microscope Zeiss Axiophot. For measuring bacterial cell size
distribution and calculation of mean cell volume we used an automated image analysis
¨ system SIS GmbH, Munster, Germany. Briefly, the procedure consisted of recording
images of DAPI-stained cells 500–1500 cells per size analysis with a CCD camera and measuring the cell dimensions pixel area, perimeter after edge detection with a second
derivative filter, manual thresholding and binarization e.g. Massana et al., 1997.
3
As an estimation of bacterial activity and production the incorporation of H-leucine was measured Kirchman et al., 1985, following the protocol of Smith and Azam
1992 with centrifugation of samples in Eppendorf vials. 20–40 nM leucine 10 radioactive, 90 cold were added to the samples and TCA-killed controls and
incubated for 90–200 min in the dark at in situ temperature. We used four replicates and two controls and, sometimes, three replicates and one control. For calculation of
bacterial production and bacterial doubling times we used the conversion factor 0.62 kg
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K . Jurgens et al. J. Exp. Mar. Biol. Ecol. 245 2000 127 –147
21
C mol leucine . This factor is an average conversion factor determined in several
experiments during the cruise from parallel measurements of biomass increase and
3
H-leucine incorporation in 0.8-mm filtered bottle incubations Gasol et al. submitted. For some of the substrate-enriched fractions we quantified bacterial abundance in
aggregates at the end of the experiments. Aggregated bacteria were directly counted in normal DAPI preparations when samples contained only smaller aggregates. When large
aggregates with more than several hundred bacteria appeared, these could not be properly quantified and samples were sonicated Branson Sonifier 250, ten bursts of 3 s
to disperse aggregated bacteria. From a first DAPI preparation only the number of freely dispersed single cells was obtained. The total number of bacteria was counted after a
second DAPI preparation of the sonicated sample. The difference of the two counts gives an estimate of the bacterial abundance in aggregates. In addition we counted and
sized maximal dimension bacterial aggregates at the end of the experiments from the DAPI preparations. To visualize whether bacterial aggregates were embedded in a
matrix of extracellular polysaccharides, selected samples were stained with alcian blue according to the procedure described by Logan et al. 1994.
Statistical analysis including t-test and analysis of variance ANOVA were performed using the software package
STATISTICA
.
3. Results
