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.
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
Among these taxa, autotrophic and heterotrophic picoplankton can play an important role in the functioning of the microbial loop [
For several decades epifluorescence microscopy counts have been the standard method for determining bacterial abundance in plankton samples.
Total microscopic count methods are relatively fast, but limitations of these techniques include time consuming, subjectiveness linked to the operator and the inability to discriminate between living and dead bacteria. To overcome this limit, direct viable counts (DVCs), allowing bacterial growth without division, have been applied in many autoecological studies. These permit the detection of viable cells, but its application to natural samples suffers from methodological limitations ([
Viable But No Culturable Cells (VBNCs) are normally difficult to detect. It is well known that >99 9% of the bacterioplankton community in seawater cannot be cultured on Marine Agar 2216. The study of marine bacteria has strongly advanced with the introduction of molecular techniques [
Due to the low RNA content of marine bacteria, many attempts have been made to enhance the sensitivity of FISH. One example of application is the catalysed reporter deposition (CARD), introduced in 1989 by Bobrow et al. [
On the other hand, flow cytometry (FCM) is a useful tool for counting and identifying microorganisms, offering the prospect of real-time microbial analysis of individual cells, assessing their viability and allowing physical separation of particular bacterial populations and cell sorting for further analysis [
In the present study, data were collected each month for one year with the following objectives to give a preliminary description of the bacterial community composition and diversity in the Central Adriatic Sea using different techniques such as CARD-FISH and FCM and to observe autotrophic and heterotrophic picoplankton seasonal changes.
The Adriatic sea has diverse trophic conditions; the shallow northern basin is a productive system and the deeper central and southern basins are oligotrophic. The northern region, characterized by a high primary productivity, is influenced by the presence of the Po River and other smaller rivers providing nutrients [
A sampling station off the central Adriatic coast (43°49′N; 13°03′EO) was chosen as the area of study for this work (Figure
Location of the study area.
Samples were collected monthly from December 2006 to November 2007; replicates (
The physical-chemical parameters (water temperature, pH, salinity, electrical conductivity) were measured
Flow cytometric analyses were performed by flow cytometry with a FACScalibur (Becton Dickinson, USA) equipped with an Argon Laser set at 15 mV and tuned to an excitation wavelength (488 nm). Multiparametric analysis was performed on both scattering signals (FSC, SSC) and FL1 green fluorescence, 530/30), FL2 (orange fluorescence, 585/42), FL3 (red fluorescence, >670). Fluorochromes with a high affinity for nucleic acids as SYBR Green I (Molecular Probes, Inc., Eugene, Oregon, U.S.A.) and Propidium Iodide (PI, Sigma-Aldrich Corp. St. Louis, MO, USA.) were used to stain bacterial cells for the cytometric analysis; SYBR Green I was used to determine heterotrophic bacteria concentration [
Before processing samples for flow analyses, Cytocount Counting beads (DakoCytomation, Denmark) were added to the stained samples and setup, and calibration procedures were optimized for the absolute counting protocols [
Conversion factors from the literature were used to estimate carbon biomass: 115 fg C cell-1 for picocyanobacteria [
The samples were collected and immediately fixed with paraformaldehyde (2% final concentration), incubated for 24 hours at 4°C, and filtered onto 0.22
Cells on filter sections were hybridized with one domain-specific probe EUB338 (specific for
Hybridization, counterstaining with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Inc., Eugene, USA, 1
Oligonucleotide probes used in the present study.
Probe | 5′-…………………-3′ | % FA (35°C ) | Target | Reference |
---|---|---|---|---|
Eub338 | 5′-GCTGCCTCCCGTAGGAGT-3′ | 55 | Most |
[ |
Eub338 II | 5′-GCAGCCACCCGTAGGTGT-3′ | 55 |
|
[ |
Eub338 III | 5′-GCTGCCACCCGTAGGTGT-3′ | 55 | Verrucmicrobia | [ |
Alf968 | 5′-GGTAAGGTTCTGCGCGTT-3′ | 45 | Alphaproteobacteria | [ |
Gam42a | 5′-GCCTTCCCACATCGTTT-3′ | 55 | Gammaproteobacteria | [ |
Bet42a (comp. for Gam42a) | 5′-GCCTTCCCACTTCGTTT-3′ | 55 | Betaproteobacteria | [ |
CF319a | 5′-TGGTCCGTGTCTCAGTAC-3′ | 55 |
|
[ |
Sar11 | 5′-TACAGTCATTTTCTTCCCCGAC-3′ | 45 |
|
[ |
Non338 | 5′-ACTCCTACGGGAGGCAGC-3′ | 20 |
|
[ |
The slides were examined with an LED fluorescence microscope (Fraen Corporation S.r.l., Milano, Italy), and microscopic counts of hybridized and DAPI-stained cells were produced using 900x magnification (15x ocular and 60x objective lenses). Three parallel sample preparations were counted for each probe, and between 20 and 40 fields were considered on each sample slide. Only free-living bacteria were counted. The counting results were corrected for background fluorescence by subtracting the signals given off by the NON338 probe.
Possible differences in the seasonal variation of heterotrophic and autotrophic picoplankton were evaluated using the analysis of variance (ANOVA) (SPSS v.19 program). Principal component analysis (PCA) was carried out on environmental data in order to visualise the trend of the main abiotic variables. The biological parameters were projected on the factor plane as additional variables, without contributing to the results of the analysis. This can provide an insight into the possible influence of environmental variables on each biological variable (STATISTICA v.8 computer program).
Surface waters during the sample period were characterized by a temperature ranging from 8.9°C to 24.9°C, pH varied from 8.0 to 8.2 and conductivity from 47.9 to 55.7 mS/cm.
The sampling site showed salinities in the range of 30.3 to 37.0‰, with the lowest value in March probably due to intensive rains in that period.
Flow cytometric data provided information on the picoplankton populations in the sampling site (Figure
Examples of dot plot showing bacteria population (HNA and LNA cells) stained with SYBR Green I (a); viable and dead cells stained with SYBR Green I and PI (b).
Total and viable bacterioplankton cell counts (±SD of 3 replicates) measured by FCM from December 2006 to November 2007.
Furthermore, cytometric analyses of fluorescence (FL1) and morphological parameters (SSC) revealed the presence of two distinct bacteria populations: HNA (High Nucleic-Acid) and LNA (Low Nucleic-Acid) cells (Figure
An ANOVA test indicates significant differences of total (
Viable cell values are similar to those obtained by Grégori et al. [
Actively respiring bacteria (CTC+) are reported as a percentage of the total cell concentration.
While the use of this method has increased over the last few years, there have also been a number of studies that are highly critical of CTC as a means of distinguishing metabolically active cells ([
Some fraction of active cells is probably not detected by the CTC method, with the proportion of total cells positive to CTC being low at less than 20% or even much less [
In our samples, CTC-positive cells ranged from 1 to 19% in the sampling period. These results are similar to those obtained by Gasol et al. [
Furthermore, the abundance of actively respiring bacteria was also positively correlated with total, cells, as previously reported in lakes [
Two populations of autotrophic picoplankton were detected by FCM analysis: (1) cyanobacteria are characterised by cells which emit an orange fluorescence (phycoerythrin) and a red fluorescence (chlorophyll); (2) picoeukaryotes only emit a red fluorescence [
An ANOVA test indicates significant differences in picocyanobacteria (
Picocyanobacteria were always more abundant than picoeukaryotes with values ranging from
Picocyanobacteria and picoeukaryotes cell counts (±SD of 3 replicates) measured by FCM from December 2006 to November 2007.
The abundance of picophytoplankton and heterotrophic bacteria measured in this study is comparable to the results of earlier studies, even if under different trophic conditions [
PCA was used to visualize the trends of biological variables in relation to the environmental variables measured at the sampling site (Figures
PCA ordination diagram of sampling based on the selected variables.
Scatter diagram plotting factors 1 and 2 of sampling stations.
The main contribution to total carbon biomass, obtained using the conversion factors, came from heterotrophic bacteria with a mean value of 35 mg C/m3. The contribution of autotrophic bacteria (mean value 9 mg C/m3) is only from picocyanobacteria and picoeukaryotes. This, together with greater heterotrophic pressure, could explain the difference observed between these two functional groups. Furthermore, picoeukaryotes surpass picocyanobacteria in terms of biomass as previously described by Zubkov et al. [
Microscopic DAPI-stained cell counts were in some cases quite difficult to obtain because of the amount of organic and inorganic compounds present in the filters. All samples examined showed bright hybridization signals with a good distinction between probe-specific signals and the background, although cells in some samples showed a bright outline. We thought it could be a problem of endogenous peroxidases [
The microbial communities analysed were dominated by
CARD-FISH counts evidenced the dominance of members of the phyla Alphaproteobacteria, with a strong contribution from SAR11, followed by
Bacterioplankton composition differed each month. In fact, bacterial groups contributed differently depending on sampling time, suggesting possible seasonal patterns.
With relative abundances of
Bacterioplankton community composition expressed as percentages respect to EUB during the sampling period.
The genus
The aim of this work was to preliminarily describe picoplankton community composition in a station localized off the central Adriatic coast.
Seasonal changes of the relative abundance of main bacterial groups were observed during the monitoring period. This could be due to the fact that aquatic bacteria in coastal waters distribute themselves in different ways depending on particular seasons: some bacteria may in fact be motile during particular conditions linked to substrate viability and patchiness [
Viable cell analyses revealed a clear correlation with HNA fraction and actively respiring cells. They represent the really active component, so their study is fundamental to better understanding changes in biogeochemical cycles.
Autotrophs, and in particular picocyanobacteria, are strongly linked to light availability, thus showing their maximum concentration during the summer period.
Even if it is a preliminary study, it is, to our knowledge, the first study in the Central Adriatic Sea using two different approaches, such as FCM and CARD-FISH. Future investigations are needed in order to enhance knowledge of bacterioplankton composition and autotrophic communities. It would also be interesting to evaluate seasonal variations in the relative abundance of bacteria populations at a lower phylogenetic level, using species-specific probes. Such studies are needed considering the importance of picoplankton as a potential sentinel of global climate changes.