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GOALS
To determine how
metazooplankton grazing
impacts the trophic
structure of the
microbial assemblage and
the protistan species
composition in the San
Pedro Channel. The
number of metazoan
predators (eg, naupliar,
copepodite and adult
stages of copepods,
other metazoa) is
altered in
exclusion/addition
experiments (see details
below) and the effect of
this manipulation on
abundance and community
composition of natural
prey populations
(phototrophic and
heterotrophic micro-,
nano-, and picoplankton)
examined.
Protistan community
structural changes due
to zooplankton predation
are investigated using
both traditional (microscopical,
flow cytometry)
methodologies with
molecular techniques
(sequencing of small
subunit ribosomal RNA
genes, quantitative PCR).
This new and powerful
approach provides
essential information on
energy and material flux
through the phototrophic
and heterotrophic
components of the
plankton and yields
significant new details
of prey-predator
dynamics in the San
Pedro Channel.
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EXPERIMENTAL APPROACH
Seawater for
predation addition/exclusion
experiments is obtained
by opening the spigot
of 50 L carboys and
immersing them under water.
Zooplankton for
addition treatments are
collected by towing a 200 μm
net through the upper water
column (5-10 m).
Treatments:
Whole seawater is
prefiltered through a mesh
to exclude metazoan
predators and filled into
4 L incubation
bottles.
The mesh size is
chosen depending on the
predator assemblage that is
target of the investigation.
In this treatment changes
in the microbial assemblage
in the absence of the
grazers is monitored (“no
copepods”, see diagram
above).
Next bottles are filled with
whole seawater to examine
biomass and species
composition changes within
the microbial community in
the presence of a natural
predator assemblage (“whole
seawater”). In a third
treatment the number of
metazooplankton is increased
to study the impact of
amplified grazing pressure
on the microbial community.
Zooplankton are
picked from the plankton
tows, rinsed in
0.2 µm filtered
seawater and added to
bottles filled with whole
seawater (“+ copepods”).
Healthy specimens are added
to the addition treatment
bottles to reach abundances
that are about 10 times
natural abundances.
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SAMPLING
TO: Immediately after this addition to the appropriate bottles the contents of 3 bottles that contain whole seawater are prescreened using the Nitex mesh to remove any metazoa, and the water is then sampled to determine abundances of micro-, nano-, and picoplankton and chlorophyll a concentrations at the onset of the experiments (details below). In addition aliquots of water are concentrated onto GF/F filters to collect genomic DNA (Refer to DNA sequencing protocols).
T1 and T final: The remaining bottles are incubated under light and temperature conditions close to in situ (eg, outdoor seawater tanks) and at each sampling point the contents of three additional bottles for each treatment are sampled following the same exact protocol from T0. The metazoa are rinsed off the mesh using filtered seawater and preserved in Formalin (5% final concentration) to be counted, sized and identified later.
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Plankton Enumeration and Biomass Calculations
Microplankton: (Also see sampling protocol for microplankton) Protists in the 2.5 - 80 µm size fraction are enumerated by light microscopy (Leica DM IRBE) using standard settling techniques after sample preservation (100 mL) with both Lugols solution (10% final concentration) and formalin (1% final concentration) - the latter allowing to distinguish heterotrophic and autotrophic cells based on presence/absence of cell pigment. The protists are assigned to broad taxonomic groups based on morphological examinations (diatoms, ciliates, dinoflagellates and other flagellates). Geometric shapes are employed that best approximate cell shape for ~50 individual organisms for each protistan group to calculate biovolume. Average biovolumes (BV) are then converted into carbon biomass by applying published conversion factors for ciliates (0.19 pgC µm-3, Putt and Stoecker 1989), diatoms (pgC µm-3 = 0.288BV.811, Menden-Deuer and Lessard 2000), dinoflagellates (pgC µm-3 = 0.76BV0.819, Menden-Deuer and Lessard 2000) and other flagellates (pgC µm-3 = 0.433BV0.863, Verity et al. 1992).
Nanoplankton: (Also see sampling protocol for nanaoplankton) Heterotrophic and phototrophic protistan plankton <10 µm in size are enumerated by flow cytometry (FACSCalibur/Becton Dickinson) from samples preserved with formalin (1% final concentration) and stained using DAPI (25 µg ml-1 final concentration, (Sherr et al. 1993). Nanoprotistan biomass is calculated as described for microplankton using published biovolume-carbon conversion factors (Verity et al. 1992).
Picoplankton: (Also see flowcytometry protocol) Enumeration of heterotrophic (bacteria + archaea) and phototrophic prokaryotes (Synechococcus spp. + Prochlorococcus spp.) is performed using flow cytometry (Olson et al. 1990a; Olson et al. 1990b; Rose et al. 2004). Carbon biomass for Synechococcus spp. and Prochlorococcus spp. is calculated using mean cell carbon values of 0.203 and 0.031 pg C cell-1, respectively (Heldal et al. 2003). For heterotrophic picoplankton (bacteria + archaea) a cell carbon value of 0.015 pg C cell-1 is applied (Caron et al. 1995).
Chlorophyll a: Pigment samples are collected onto GF/F Whatman filters and stored at -20˚C until analyses. Chlorophyll a fluorescence is measured before and after acidification with 5% HCl using a Turner TD 700 fluorometer and concentrations are calculated using equations from Parsons et al. (Parsons et al. 1984).
Zooplankton ingestion rates: Zooplankton ingestion rates (µg C ind. -1 d-1) on protozoa (ciliates and heterotrophic flagellates) are calculated from differences in prey biomass between the bottles from which metazoa are removed and the bottles that contain natural and elevated abundances of predators (Frost 1972). Zooplankton dry weights are measured for individuals (groups of individuals) picked from the tow and stored frozen. These samples are thawed, dried at 60˚C for 24 h and reweighed. Dry weights are converted into carbon weight by using published conversion factors.
Creation of 18s DNA Clone Libraries: (Refer to DNA sequencing protocols).
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REFERENCES
CARON, D. A. and others 1995. The contirbution of microorganisms to particulate carbon and nitrogen in surface waters of the Sargasso Sea near Bermuda. Deep-Sea Research 42: 943-972.
FROST, B. W. 1972. Effects of size and concentration of food particles on the feeding behavior of the marine planktonic copepod Calanus pacificus. Limnology and Oceanography 17: 805-815.
HELDAL, M., D. J. SCANLAN, S. NORLAND, F. THINGSTAD, and N. H. MANN. 2003. Elemental composition of single cells of various strains of marine Prochlorococcus and Synechococcus using X-ray microanalysis. Limnology & Oceanography 48: 1732-1743.
MENDEN-DEUER, S., and E. J. LESSARD. 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnology & Oceanography 45: 569-579.
OLSON, R. J., S. W. CHISHOLM, E. R. ZETTLER, M. A. ALTABET, and J. A. DUSENBERRY. 1990a. Spatial and temporal distribution of prochlorophyte picoplankton in the North Atlantic Ocean. Deep-Sea Research 37: 1033-1051.
OLSON, R. J., S. W. CHISHOLM, E. R. ZETTLER, and E. V. ARMBRUST. 1990b. Pigments, size and distribution of Synechococcus in the North Atlantic and Pacific Oceans. Limnology and Oceanography 35: 45-58.
PARSONS, T. R., Y. MAITA, and C. M. LALLI. 1984. A manual of chemical and biological methods for seawater analysis. PERGAMON PRESS, OXFORD (UK): 187.
PUTT, M., and D. K. STOECKER. 1989. An experimentally determined carbon:volume ratio for marine oligotrichous ciliates from estuarine and coastal waters. Limnology & Oceanography 34: 1097-1103.
ROSE, J. M., D. A. CARON, M. E. SIERACKI, and N. POULTON. 2004. Counting heterotrophic nanoplankton protists in cultures and in aquatic communities by flow cytometry. Aquatic Microbial Ecology 34: 263-277.
SHERR, E. B., D. A. CARON, and B. F. SHERR. 1993. Staining of heterotrophic protists for visualization via epifluoresecence microscopy, p. 223-235. In B. F. Sherr and E. B. Sherr [eds.], Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers.
VERITY, P. G., C. Y. ROBERTSON, C. R. TRONZO, M. G. ANDREWS, J. R. NELSON, and M. E. SIERACKI. 1992. Relationships between cell colume and the carbon and nitrogen content of marine photosynthetic nanoplankton. Limnology & Oceanography 37: 1434-1446.
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