Data concerning picoplanktonic community composition and abundance in the Central Adriatic Sea are presented in an effort to improve the knowledge of bacterioplankton and autotrophic picoplankton and their seasonal changes. Flow cytometry analyses revealed the presence of two distinct bacteria populations: HNA and LNA cells. HNA cells showed an explicit correlation with viable and actively respiring cells. The study of viability and activity may increase our knowledge of the part that contributes really to the remineralization and bacterial biomass production. Authotrophic picoplankton abundance, especially picocyanobacteria, was strongly influenced by seasonality, indicating that light availability and water temperature are very important regulating factors. In terms of total carbon biomass, the main contribution came from heterotrophic bacteria with a lower contribution from autotrophic picoplankton. CARD-FISH evidenced, within the Eubacteria domain, the dominance of members of the phyla Alphaproteobacteria, with a strong contribution from SAR11clade, followed by Cytophaga-Flavobacterium and Gammaproteobacteria. The bacterial groups detected contributed differently depending when the sample was taken, suggesting possible seasonal patterns. This study documents for the first time picoplankton community composition in the Central Adriatic Sea using two different approaches, FCM and CARD-FISH, and could provide preliminary data for future studies. 1. Introduction Knowledge of seawater microbial diversity is important in order to understand their community structure and pattern of distribution. In the ocean water column, organisms <200？μm include a variety of taxa: free viruses, autotrophic bacteria (cyanobacteria, which include the group formerly known as prochlorophytes), heterotrophic bacteria, protozoa (flagellates and ciliates), and small metazoans [1] with different morphological, ecological, and physiological characteristics. Autotrophs and heterotrophs constitute two fundamental functional units in ecosystems: the first generally dominate eutrophic systems while heterotrophs generally dominate oligotrophic systems [2, 3]. Among these taxa, autotrophic and heterotrophic picoplankton can play an important role in the functioning of the microbial loop [4], with the former regarded as important templates of assimilable carbon biomass at the base of the microbial food web [5], while the latter acts as both remineralizers of organic carbon and trophic intermediaries within aquatic ecosystems [3, 6]. These represent an important source of food for a
References
[1]
L. Legendre, C. Courties, and M. Troussellier, “Flow cytometry in oceanography 1989–1999: environmental challenges and research trends,” Cytometry, vol. 44, no. 3, pp. 164–172, 2001.
[2]
Q. Dortch and T. T. Packard, “Differences in biomass structure between oligotrophic and eutrophic marine ecosystems,” Deep Sea Research A, vol. 36, no. 2, pp. 223–240, 1989.
[3]
J. M. Gasol, P. A. del Giorgio, and C. M. Duarte, “Biomass distribution in marine planktonic communities,” Limnology and Oceanography, vol. 42, no. 6, pp. 1353–1363, 1997.
[4]
J. Porter, D. Deere, M. Hardman, C. Edwards, and R. Pickup, “Go with the flow—use of flow cytometry in environmental microbiology,” FEMS Microbiology Ecology, vol. 24, no. 2, pp. 93–101, 1997.
[5]
C. Callieri and J. G. Stockner, “Freshwater autotrophic picoplankton: a review,” Journal of Limnology, vol. 61, no. 1, pp. 1–14, 2002.
[6]
T. Fenchel, “Marine plankton food chains,” Annual Review of Ecology and Systematics, vol. 19, pp. 19–38, 1988.
[7]
S. Das, P. S. Lyla, and S. A. Khan, “Marine microbial diversity and ecology: importance and future perspectives,” Current Science, vol. 90, no. 10, pp. 1325–1335, 2006.
[8]
F. Joux and P. Lebaron, “Ecological implications of an improved direct viable count method for aquatic bacteria,” Applied and Environmental Microbiology, vol. 63, no. 9, pp. 3643–3647, 1997.
[9]
C. Pedròs-Aliò, “Diversity of bacterioplankton,” Trends in Ecology & Evolution, vol. 8, no. 3, pp. 86–90, 1993.
[10]
R. I. Amann, W. Ludwig, and K. H. Schleifer, “Phylogenetic identification and in situ detection of individual microbial cells without cultivation,” Microbiological Reviews, vol. 59, no. 1, pp. 143–169, 1995.
[11]
T. D. Mullins, T. B. Britschgi, R. L. Krest, and S. J. Giovannoni, “Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities,” Limnology and Oceanography, vol. 40, no. 1, pp. 148–158, 1995.
[12]
M. Schauer, V. Balagué, C. Pedròs-Aliò, and R. Massana, “Seasonal changes in the taxonomic composition of bacterioplankton in a coastal oligotrophic system,” Aquatic Microbial Ecology, vol. 31, no. 2, pp. 163–174, 2003.
[13]
R. I. Amann, L. Krumholz, and D. A. Stahl, “Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology,” Journal of Bacteriology, vol. 172, no. 2, pp. 762–770, 1990.
[14]
R. Amann and W. Ludwig, “Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology,” FEMS Microbiology Reviews, vol. 24, no. 5, pp. 555–565, 2000.
[15]
F. O. Gl？ckner, B. M. Fuchs, and R. Amann, “Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization,” Applied and Environmental Microbiology, vol. 65, no. 8, pp. 3721–3726, 1999.
[16]
M. N. Bobrow, T. D. Harris, K. J. Shaughnessy, and G. J. Litt, “Catalyzed reporter deposition, a novel method of signal amplification—application to immunoassays,” Journal of Immunological Methods, vol. 125, no. 1-2, pp. 279–285, 1989.
[17]
H. M. J. Kerstens, P. J. Poddighe, and A. G. J. M. Hanselaar, “A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine,” Journal of Histochemistry and Cytochemistry, vol. 43, no. 4, pp. 347–352, 1995.
[18]
A. Pernthaler, J. Pernthaler, and R. Amann, “Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria,” Applied and Environmental Microbiology, vol. 68, no. 6, pp. 3094–3101, 2002.
[19]
E. L. Joachimsthal, V. Ivanov, J. H. Tay, and S. T. L. Tay, “Flow cytometry and conventional enumeration of microorganisms in ships' ballast water and marine samples,” Marine Pollution Bulletin, vol. 46, no. 3, pp. 308–313, 2003.
[20]
J. Vives-Rego, P. Lebaron, and G. Nebe-von Caron, “Current and future applications of flow cytometry in aquatic microbiology,” FEMS Microbiology Reviews, vol. 24, no. 4, pp. 429–448, 2000.
[21]
C. S. Yentsch and C. M. Yentsch, “Single cell analysis in biological oceanography and its evolutionary implications,” Journal of Plankton Research, vol. 30, no. 2, pp. 107–117, 2008.
[22]
A. Manti, S. Papa, and P. Boi, “What flow cytometry can tell us about marine micro-organisms—current status and future applications,” in Flow Cytometry—Recent Perspectives, I. Schmid, Ed., pp. 1–28, InTech, 2012.
[23]
M. Zavatarelli, J. W. Baretta, J. G. Baretta-Bekker, and N. Pinardi, “The dynamics of the Adriatic Sea ecosystem. An idealized model study,” Deep-Sea Research I, vol. 47, no. 5, pp. 937–970, 2000.
[24]
A. Artegiani, D. Bregant, E. Paschini, N. Pinardi, F. Raicich, and A. Russo, “The Adriatic Sea general circulation. Part II: baroclinic circulation structure,” Journal of Physical Oceanography, vol. 27, no. 8, pp. 1515–1532, 1997.
[25]
D. Marie, F. Partensky, S. Jacquet, and D. Vaulot, “Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I,” Applied and Environmental Microbiology, vol. 63, no. 1, pp. 186–193, 1997.
[26]
S. Barbesti, S. Citterio, M. Labra, M. D. Baroni, M. G. Neri, and S. Sgorbati, “Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria,” Cytometry, vol. 40, no. 3, pp. 214–218, 2000.
[27]
G. Grégori, S. Citterio, A. Ghiani et al., “Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining,” Applied and Environmental Microbiology, vol. 67, no. 10, pp. 4662–4670, 2001.
[28]
J. M. Gasol, P. A. del Giorgio, R. Massana, and C. M. Duarte, “Active versus inactive bacteria: size-dependence in a coastal marine plankton community,” Marine Ecology Progress Series, vol. 128, no. 1–3, pp. 91–97, 1995.
[29]
A. Manti, P. Boi, T. Falcioni et al., “Bacterial cell monitoring in wastewater treatment plants by flow cytometry,” Water Environment Research, vol. 80, no. 4, pp. 346–354, 2008.
[30]
W. K. W. Li and W. G. Harrison, “Chlorophyll, bacteria and picophytoplankton in ecological provinces of the North Atlantic,” Deep-Sea Research II, vol. 48, no. 10, pp. 2271–2293, 2001.
[31]
M. V. Zubkov, M. A. Sleigh, G. A. Tarran, P. H. Burkill, and R. J. G. Leakey, “Picoplanktonic community structure on an Atlantic transect from 50°N to 50°S,” Deep-Sea Research I, vol. 45, no. 8, pp. 1339–1355, 1998.
[32]
S. Lee and J. A. Fuhrman, “Relationships between biovolume and biomass of naturally derived marine bacterioplankton,” Applied and Environmental Microbiology, vol. 53, no. 6, pp. 1298–1303, 1987.
[33]
L. A. Pan, J. Zhang, and L. H. Zhang, “Picophytoplankton, nanophytoplankton, heterotrohpic bacteria and viruses in the Changjiang Estuary and adjacent coastal waters,” Journal of Plankton Research, vol. 29, no. 2, pp. 187–197, 2007.
[34]
R. Sekar, A. Pernthaler, J. Pernthaler, F. Warnecke, T. Posch, and R. Amann, “An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization,” Applied and Environmental Microbiology, vol. 69, no. 5, pp. 2928–2935, 2003.
[35]
A. Pernthaler, J. Pernthaler, and R. Amann, “Sensitive multi-color fluorescence in situ hybridization for the identification of environmental microorganisms,” in Molecular Microbial Ecology Manual, 3, pp. 711–726, 2nd edition, 2004.
[36]
R. I. Amann, B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl, “Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations,” Applied and Environmental Microbiology, vol. 56, no. 6, pp. 1919–1925, 1990.
[37]
H. Daims, A. Brühl, R. Amann, K. H. Schleifer, and M. Wagner, “The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set,” Systematic and Applied Microbiology, vol. 22, no. 3, pp. 434–444, 1999.
[38]
A. Neef, Anwendung der in situ Einzelzell-Identifizierung von Bakterien zur populationsanalyse in komplexen mikrobiellen bioz？nosen [Ph.D. thesis], Technische Universit？t München, 1997.
[39]
W. Manz, R. Amann, W. Ludwig, M. Wagner, and K. H. Schleifer, “Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions,” Systematic and Applied Microbiology, vol. 15, no. 4, pp. 593–600, 1992.
[40]
R. M. Morris, M. S. Rappé, S. A. Connon et al., “SAR11 clade dominates ocean surface bacterioplankton communities,” Nature, vol. 420, no. 6917, pp. 806–810, 2002.
[41]
T. Bouvier, P. A. del Giorgio, and J. M. Gasol, “A comparative study of the cytometric characteristics of High and Low nucleic-acid bacterioplankton cells from different aquatic ecosystems,” Environmental Microbiology, vol. 9, no. 8, pp. 2050–2066, 2007.
[42]
E. M. Smith and P. A. del Giorgio, “Low fractions of active bacteria in natural aquatic communities?” Aquatic Microbial Ecology, vol. 31, no. 2, pp. 203–208, 2003.
[43]
P. A. del Giorgio, Y. T. Prairie, and D. F. Bird, “Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry,” Microbial Ecology, vol. 34, no. 2, pp. 144–154, 1997.
[44]
L. Campbell and D. Vaulot, “Photosynthetic picoplankton community structure in the subtropical North Pacific Ocean near Hawaii (station ALOHA),” Deep-Sea Research Part I, vol. 40, no. 10, pp. 2043–2060, 1993.
[45]
P. F. Moreira-Turcq and J. M. Martin, “Characterisation of fine particles by flow cytometry in estuarine and coastal Arctic waters,” Journal of Sea Research, vol. 39, no. 3-4, pp. 217–226, 1998.
[46]
C. Totti, M. Cangini, C. Ferrari et al., “Phytoplankton size-distribution and community structure in relation to mucilage occurrence in the Northern Adriatic Sea,” Science of the Total Environment, vol. 353, no. 1–3, pp. 204–217, 2005.
[47]
F. Cerino, F. Bernardi Aubry, J. Coppola et al., “Spatial and temporal variability of pico-, nano- and microphytoplankton in the offshore waters of the Southern Adriatic Sea (Mediterranean Sea),” Continental Shelf Research. In press.
[48]
D. Marie, C. P. D. Brussaard, R. Thyrhaug, G. Bratbak, and D. Vaulot, “Enumeration of marine viruses in culture and natural samples by flow cytometry,” Applied and Environmental Microbiology, vol. 65, no. 1, pp. 45–52, 1999.
[49]
M. J. W. Veldhuis and G. W. Kraay, “Application of flow cytometry in marine phytoplankton research: current applications and future perspectives,” Scientia Marina, vol. 64, no. 2, pp. 121–134, 2000.
[50]
L. A. Pan, L. H. Zhang, J. Zhang, J. M. Gasol, and M. Chao, “On-board flow cytometric observation of picoplankton community structure in the East China Sea during the fall of different years,” FEMS Microbiology Ecology, vol. 52, no. 2, pp. 243–253, 2005.
[51]
P. A. del Giorgio, Y. T. Prairie, and D. F. Bird, “Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry,” Microbial Ecology, vol. 34, no. 2, pp. 144–154, 1997.
[52]
E. M. Smith, “Coherence of microbial respiration rate and cell-specific bacterial activity in a coastal planktonic community,” Aquatic Microbial Ecology, vol. 16, no. 1, pp. 27–35, 1998.
[53]
H. Eilers, J. Pernthaler, F. O. Gl？ckner, and R. Amann, “Culturability and in situ abundance of pelagic bacteria from the North Sea,” Applied and Environmental Microbiology, vol. 66, no. 7, pp. 3044–3051, 2000.
[54]
H. Eilers, J. Pernthaler, and R. Amann, “Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts,” Applied and Environmental Microbiology, vol. 66, no. 11, pp. 4634–4640, 2000.
[55]
L. Alonso-Sáez, V. Balagué, E. L. Sà et al., “Seasonality in bacterial diversity in North-West Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH,” FEMS Microbiology Ecology, vol. 60, no. 1, pp. 98–112, 2007.
[56]
R. Amann and B. M. Fuchs, “Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques,” Nature Reviews Microbiology, vol. 6, no. 5, pp. 339–348, 2008.
[57]
F. Simonato, P. R. Gómez-Pereira, B. M. Fuchs, and R. Amann, “Bacterioplankton diversity and community composition in the Southern Lagoon of Venice,” Systematic and Applied Microbiology, vol. 33, no. 3, pp. 128–138, 2010.
