Flow cytometry data was collected in January 2009, in waters off South Australia.
The general purpose of the study is to be able to establish background knowledge on the ecosystem on the continental shelf of South Australia and the impact of upwelling/saline outflow events on microbial communities to ultimately develop a biogeochemical model of the region. Sampling was carried out during cruises conducted on board the RV Ngerin as part of the Southern Australian Integrated Marine System (SAIMOS). During each cruise, the physical, chemical and biological properties of the chlorophyll fluorescence maximum (FM) layer were investigated. Flow cytometry data has been collected for picophytoplankton, bacteria and viruses.
Six main stations have been sampled over the course of the study, five are located on the 100 m isobath, i.e. RS (35.508S, 136.278E), B2 (35.418S, 136.148E), B3 (35.258S, 136.048E), B4 (35.168S, 135.418E) and B5 (35.008S, 135.198E), and one from an offshore station (B1; 36.188S, 136.178E) located southwest of Kangaroo Island. Note that combining the distances between stations (14–25 nautical miles), the average component of the current velocity at middepth along the shelf (0.01 m s21) and the average speed of the vessel (i.e. 9 knots) indicate that different water masses were sampled at each station. Additional samples have on occasion been collected from the National Reference Station (NRS) at Kangaroo Island (35.832S, 136.447E) and the SA Spencer Gulf Mouth Mooring (SAM8SG, 35.25S, 136.690E), where the saline outflow occurs.
At each station, in vivo fluorescence profiles were used to identify the depth of the FM from where we collected seawater with Niskin bottles. These were subsequently homogenized prior to subsampling for nutrients, Chl a concentration, picophytoplankton, bacteria and virus analysis. If no FM could be identify, seawater sampling was done in the surface mixed layer at a depth of 15 m that we previously identified to avoid the effects of photoinhibition.
Seawater samples of 50 mL were filtered through
bonnet syringe filters (0.45 um porosity, Micro Analytix Pty Ltd) and stored at –20oC for nutrient analysis. Nutrient concentrations are not always available for all stations on all cruises. All other
samples were analysed according to the Lachat
Quickchem methods for phosphate (PO3-,4, detection limit; 0.03 uM), nitrate + nitrite (NO-,x, detection limit; 0.07 uM) and ammonium (NH+,4, detection limit; 0.07 uM) on a QuickChem QC8500 Automated Ion Analyser. Chl a concentrations were determined using triplicate 300 mL seawater samples filtered through fibre glass filters (Whatman GF/C, 1.7 um porosity). Filters were stored at –20oC until analysis. Chl a was extracted by placing each filter in 5 mL of methanol for 24 h in the dark at 4oC (Welschmeyer, 1994). Chl a concentrations in the extracts were determined using a Turner 450 fluorometer previously calibrated with Chl a extracted from Anacystis nidulans (Sigma Chemicals, St Louis, MO, USA).
Triplicate 1 mL seawater samples were fixed with
paraformaldehyde (2% final concentration), frozen in liquid nitrogen and stored at –80oC. Samples were processed by flow cytometry (FacsCanto Becton Dickinson) within a month following each cruise. Prior to analysis, 1 um fluorescent marker beads (Molecular Probes, Eugene, OR, USA) were added to each sample (Marie et al., 1999) and each sample was run for 5 min. For each sample, natural orange fluorescence from phycoerythrin and red fluorescence from chlorophyll, together with forward light scatter and side light scatter (SSC), were recorded. All cytograms were then analysed using the software flowJo (TreeStar) following the method described in Marie et al. (Marie et al., 1999). The three known major picophytoplankton
groups, i.e. Synechococcus, Prochlorococcus and picoeukaryotes, could be easily discriminated by their distinct autofluorescence and light scatter properties relative to the beads. Gates or regions around each observed group were drawn such that no adjustment was needed and to maximize cell counts.
After collection samples for heterotrophic
bacteria and viruses were transferred into 2 ml cryovials, fixed with glutaraldehyde (0.5% final concentration)and incubated for 15 min at 4oC (Brussaard, 2004). All samples were then quick frozen in liquid nitrogen, stored at -80oC once returned to the laboratory and analysed within a month of collection.
Heterotrophic bacteria and virus populations were identified and enumerated by flow cytometry (FCM) using a FACScanto flow cytometer (Becton Dickinson). Prior to FCM analysis, triplicate viral
and bacterial samples were quick thawed and diluted 1:10 with 0.2umm filtered TE buffer (10 mM Tris, 1 mM EDTA), stained with SYBR-I Green solution (1:500 dilution; Molecular Probes) and finally incubated in the dark at 80oC for 10 min (Brussaard, 2004). Fluorescent beads with a diameter of 1 umm (Molecular Probes) were added to each sample as an internal size and concentration standard at a final concentration of approximately 10^5 beads per ml (Gasol and del Giorgio, 2000). Phosphate-buffered saline (PBS) solution was added as a sheath fluid, while forward-angle light scatter (FSC), side-angle light scatter (SSC) and green (SYBR-1) fluorescence were recorded for each sample. Data for each sample were collected in list-mode files and analysed using Win MDI 2.8 software ((c) Joseph Trotter).
Viral and heterotrophic bacterial populations were discriminated based on their differences in cell side scatter, a proxy of cell size, and SYBR Green fluorescence, which indicates the amount of nucleic acid present in each cell (Marie et al., 1997, 1999; Brussaard, 2004). Bacterial populations were then split into high DNA (HDNA) and low DNA (LDNA) groups using the differences in green fluorescence
(Li et al., 1995; Gasol et al., 1999, Fig. 2). Viral populations were split into four virus-like particle populations (VLP1, VLP2, VLP3 and VLP4) based on their differences in green fluorescence and SSC. More specifically, VLP1 and VLP2 corresponded to
populations widely observed in seawater samples and identified as bacteriophages (e.g. Marie et al., 1999; Brussaard et al., 2005; Seymour et al., 2006). In contrast, populations VLP3 and VLP4 exhibited similar levels of green fluorescence than VLP2 but a higher SSC. This suggests that they represent groups of phytoplankton viruses that are generally characterised by higher side scatter and/or green fluorescence signatures (Brussaard et al., 1999,
2005, 2008; Baudoux and Brussaard, 2005; Larsen et al., 2008).
Integrated Marine Observing System (IMOS). IMOS is a national collaborative research infrastructure, supported by Australian Government.
South Australian Research and Development Institute (SARDI)