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Identification and characterisation of microbial populations using flow cytometry in the Adriatic Se

Synechococcus, Prochlorococcus and picoeukaryotes have an important role in primary production and also represent significant food resource for protists and small invertebrates (Callieri and Stockner, 2002), and so participating in the role of prey in the energy flow at higher trophic levels (Liu et al., 2002). With mentioned primary producers, heterotrophic bacteria are important components of marine plankton communities (Azam and Hodson, 1977). On the one hand they are consumers of dissolved organic matter (DOM), and as such they are links in the chain of matter and energy flow through an ecosystem (Cole et al., 1988). On the other hand, they decompose organic matter and transform inorganic compounds in forms suitable for primary producers (Ducklow et al., 1986). Noted principal components of the marine picoplankton community, respectively Prochlorococcus, Synechococcus, picoeukaryotes and heterotrophic bacteria, can be identified and quantified by flow cytometry (Olson et al., 1988 ; Marie et al., 1997). Discovery of Prochlorococcus (Chisholm et al., 1988) and several bacterioplankton groups according to different content of deoxyribonucleic acid (DNA) (Li et al., 1995 ; Marie et al., 1997), using of flow cytometry in marine microbiology resulted with the discovery of several bacterial groups based on different content of DNA: high nucleic acid content group (HNA) and group with low nucleic acid (LNA) content (Gasol and Moràn, 1999 ; Gasol et al., 1999). Therefore, using of flow cytometry in oceanography is helping to explore the heterogeneity of microbial communities in the sea. The objective of this study was to compare two direct counting methods for bacterioplankton for the field samples from different oceanographic regions: the Adriatic Sea and the English Channel. The accuracy of epifluorescence microscopy (EM) was assessed against direct counts made by flow cytometry (FCM). Next objective of the this study was to simulate process of the the seasonal cycles of autotrophic and heterotrophic prokaryotes in the central and southern part of Adriatic Sea and to identify the relevant physico-chemical and biological factors responsible for the observed seasonal and spatial distributions. Samples were collected in two geographically different areas: Adriatic Sea, part of the Mediterranean Sea (Figure 1) and English Channel, part of the Atlantic Ocean ( 50°15’ N, 4° 15’ W, off shore station 6 km off Plymouth, and four shore station from Plymouth Sound UK). From the Adriatic Sea a total of 919 samples spanning offshore and shore areas were collected from 29 sites on monthly basis during 2005. Seawater samples from the Adriatic Sea sites and offshore site in the English Channel were collected by Niskin bottles through vertical profile. At four shore sites from the English Channel samples were collected manually from the surface. Samples were fixed with formaldehyde (2% final concentration), kept in the dark at 4 0C and analyzed within two weeks. For epifluorescence microscopy (EM) preserved samples were stained with 4'-6-diamidino-2-phenylindole (DAPI) (1 µg mL-1 final concentration) for 5 minutes and were filtered through 0.2 µm pore diameter black polycarbonate filters (Millipore, Ireland). Filter were then mounted on microscope slides and stored at 4 0C where they were kept until observation with an Olympus microscope under UV light (PORTER & FEIG, 1980) at magnification of 1000. From 100 to 400 bacteria were counted per sample, depending on concentration. For flow cytometry analysis (FCM) of heterotrophic bacteria, fixed samples were stained with SYBR GREEN I (Molecular probes Inc.) (MARIE at al., 1997 ; LEBARON et al., 1998). Samples from the Adriatic Sea were analyzed on a Beckman Coulter EPICS XL-MCL with a high flow rate from 1 to 1.2 µL/sec. Fluorescent beads were added (Level-II Epics DIVISION of Coulter Corporation Hialeah, Florida) for calibration of fluorescence intensity. Samples from the English Channel were analyzed on a flow cytometer FACSort. Beckman Coulter flow set beads at known concentration were used to calibrate the flow rate. Bacterial abundance was determined in scatter plots of particle side scatter versus Sybr Green I fluorescence related to cellular nucleic acid content to discriminate bacteria from other particles. Two groups of heterotrophic bacteria were distinguished according to their relative green fluorescence as proxy for nucleic content, referred to as high nucleic acid (HNA) and low nucleic acid bacteria (LNA). Abundances of Synechococcus, Prochlorococcus and picoeukaryotes were determined using flow cytometry (Marie et al., 1997 ; Jiao et al., 2002). Samples for autotrophic cells were preserved in 0.5% gluteraldehyde, frozen at -80˚C and stored until analysis, while samples for heterotrophic cells were preserved in 2% formaldehyde and stored at 4˚C until analysis. Samples were analysed on a Beckman Coulter EPICS XL-MCL with a high flow rate from 1 to 1.2 μL sec-1. Fluorescence beads were added to calibrate the cells’ fluorescence intensity (Level-II Epics Division of Coulter Corporation Hialeah, Florida). Autotrophic cells were separated into two groups of cyanobacteria (Synechococcus and Prochlorococcus) and two groups of eukaryotes (picoeukaryotes and Cryptophytes) and were distinguished according to light diffraction, red emission of cellular chlorophyll content and orange emission of phycoerythrin-rich cells.

heterotrophic bacteria, HNA, LNA, cyanobacteria, picoeukaryotes, Adriatic Sea

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