Renata Zaccone

Enzymatic Activities and Prokaryotic Abundance in Relation to Organic Matter along a West–East Mediterranean Transect (TRANSMED Cruise)

The distribution of extracellular enzymatic activities (EEA) [leucine aminopeptidase (LAP), ß-glucosidase (GLU), alkaline phosphatase (AP)], as well as that of prokaryotic abundance (PA) and biomass (PB), dissolved organic carbon (DOC), particulate organic carbon and particulate total nitrogen (POC, PTN), was determined in the epi-, meso-, and bathypelagic waters of the Mediterranean Sea along a West–East transect and at one Atlantic station located outside the Strait of Gibraltar. This study represents a synoptical evaluation of the microbial metabolism during early summer. Decreasing trends with depth were observed for most of the parameters (PA, PB, AP, DOC, POC, PTN). Significant differences between the western and eastern basins of the Mediterranean Sea were found, displaying higher rates of LAP and GLU and lower C/N ratios more in the eastern than in the western areas. Conversely, in the epipelagic layer, PA and PB were found to be higher in the western than in the eastern basins. PB was significantly related to DOC concentration (all data, n = 145, r = 0.53, P < 0.01), while significant correlations of EEA with POC and PTN were found in the epipelagic layer, indicating an active response of microbial metabolism to organic substrates. Specific enzyme activities normalized to cell abundance pointed out high values of LAP and GLU in the bathypelagic layer, especially in the eastern basin, while cell-specific AP was high in the epi- and bathypelagic zone of the eastern basin indicating a rapid regeneration of inorganic P for both prokaryotes and phytoplankton needs. Low activity and abundance characterized the Atlantic station, while opposite trends of these parameters were observed along the Mediterranean transect, showing the uncoupling between abundance and activity data. In the east Mediterranean Sea, decomposition processes increased probably in response to mesoscale structures which lead to organic matter downwelling.

Dynamics of extracellular enzymatic activities in a shallow Mediterranean ecosystem (Tindari ponds, Sicily)

Three microbial extracellular enzymes, leucine aminopeptidase (LAP), β-glucosidase (β-glu) and alkaline phosphatase (AP), were studied in six small Mediterranean littoral ponds, to evaluate the diversity of microbial activities relative to prevailing environmental conditions. The marked diversification of the trophic states, ranging from oligotrophy to eutrophy, in the ponds was reflected in a range of enzyme patterns at different spatial and temporal scales. There were higher levels and greater variability of microbial activity in the oldest and most ‘confined’ ponds (ranges: 0.55–4360.00 nm h−1, 0.15–76.44 nm h−1, 1.29–1600.00 nm h−1 for LAP, β-glu and AP respectively) compared with the youngest and most seaward ponds (ranges: 22.64–612.0 nm h−1, 0.06–48.89 nm h−1, 0.32–744.0 nm h−1 for LAP, β-glu and AP respectively). The close relationship of the degradative potential with chlorophyll-a and particulate organic carbon could be a consequence of the stimulating effect of phytoplankton-released polymeric compounds (organic matter) and/or a response of the microbial community to warm temperatures, which were recorded from July to September. Within an area less than 1 km2, different aquatic ecosystems coexist and maintain their distinctive properties in terms of microbial biogeochemical processes.

Microbial assemblages for environmental quality assessment: Knowledge, gaps and usefulness in the European Marine Strategy Framework Directive

Abstract The EU Marine Strategy Framework Directive 2008/56/EC (MSFD) defines a framework for Community actions in the field of marine environmental policy in order to achieve and/or maintain the Good Environmental Status (GES) of the European seas by 2020. Microbial assemblages (from viruses to microbial-sized metazoa) provide a major contribution to global biodiversity and play a crucial role in the functioning of marine ecosystems, but are largely ignored by the MSFD. Prokaryotes are only seen as “microbial pathogens,” without defining their role in GES indicators. However, structural or functional prokaryotic variables (abundance, biodiversity and metabolism) can be easily incorporated into several MSFD descriptors (i.e. D1. biodiversity, D4. food webs, D5. eutrophication, D8. contaminants and D9. contaminants in seafood) with beneficial effects. This review provides a critical analysis of the current MSFD descriptors and illustrates the reliability and advantages of the potential incorporation of some prokaryotic variables within the set of indicators of marine environmental quality. Following a cost/benefit analysis against scientific and economic criteria, we conclude that marine microbial components, and particularly prokaryotes, are highly effective for detecting the effects of anthropogenic pressures on marine environments and for assessing changes in the environmental health status. Thus, we recommend the inclusion of these components in future implementations of the MSFD.

MICROBIAL RESPIRATORY AND ECTOENZYMATIC ACTIVITIES IN THE NORTHERN ADRIATIC SEA (MEDITERRANEAN SEA)

The carbon transfer through the microbial community in two areas of the Northern Adriatic Sea was estimated by proteolytic and respiratory activities during four oceanographic surveys carried out in June, 1996, 1997 and February, 1997, 1998. In front of the Po Delta (area A), the mean rates of proteolytic activity range from 4.9 to 9.9 r µg r C r h r l; near Ancona (area B), they range from 3.1 to 7.6 r µg r C r h r l. Respiratory rates vary between 0.19 and 2.29 and between 0.24 and 1.40 r µg r C r h r l in areas A and B, respectively. In general, high rates occur in the surface layers, within the first 10 r m of depth. In area A, proteolytic and respiratory rates undergo seasonal course, with high activity in warm periods. In area B, respiration and bacterioplankton abundance increase from the first to the second year, whilst proteolytic activity decreases. The sequence of metabolic steps in the carbon transfer within the bacteria, from the biotic vs . the abiotic compartment, was drawn in order to define the actual role of bacterial biomass in the biogeochemical fluxes in an ecosystem which often suffers distrophic crises. Respiratory turnover rates, in the upper 10 r m depth, reach low values in cold periods and high values in June, 1997. The carbon transfer versus mineralization flows better in the summer period, in particular in June, 1997. However, the bacterial growth efficiency ranges from 17 to 38% in area A and from 13 to 44% in area B with highest values in February, 1997, when bacteria contribute in a relevant way to the overall respiration.

Microbial hydrolysis of polysaccharides and organic phosphates in the Northern Adriatic Sea

g -glucosidase and alkaline phosphatase, two ectoenzymes involved in the microbial decomposition of polysaccharides and organic phosphates, were evaluated in water samples collected from two areas of the Northern Adriatic Sea during the multidisciplinary Prisma II research project. The distribution of g -glucosidase and alkaline phosphatase is reported together with that of leucine aminopeptidase, an enzyme involved in the degradation of protein compounds. The data obtained showed the prevalence of g -glucosidase in summer months in the northern area, located in front of the Po delta, while in winter it is higher in the southern area (in front of Ancona). Phosphatase activity during February, 1998 had a maximum of 107 r nml m 1 h m 1 at a coastal station in the northern area; this enzyme appeared to be mainly associated with phytoplankton because the increase in alkaline phosphatase was associated with the increase of phytoplankton biomass, measured by chlorophyll a content. The relations between the microbial activities and environmental parameters are discussed.

Deep-chlorophyll maximum time series in the Augusta Gulf (Ionian Sea): Microbial community structures and functions

An integrated study was carried out to follow the temporal evolution of microbiological parameters during a 48 h period, in relation to the deep chlorophyll maximum (DCM) at a coastal station. The micro-organisms showed an active role in the environment and a different distribution, without a clear diel cycle. The phytoplankton community, responsible for the DCM, consisted mainly of diatoms. Their distribution in relation to pycnocline showed an opposite trend with respect to picophytoplankton. Total bacterioplankton contributed to enzymatic degradation of particulated organic carbon (by producing β-glucosidase and aminopeptidase), with peaks related to changes in the main water current. We estimated that about 25% of particulate organic carbon per day may be hydrolysed by bacteria. The living bacterioplankton represented 20% of the total. The picophytoplankton fraction contributed significantly to the high values of alkaline phosphatase, suggesting a fast P regeneration. Respiration showed significant correlations with the physical and chemical parameters as well as with the different planktonic fractions.

Development of a multiplex PCR assay for Photobacterium damselae subsp. piscicida identification in fish samples.

A multiplex polymerase chain reaction protocol for the detection of Photobacterium damselae and subspecies piscicida and damselae discrimination, with internal amplification control, was developed. Assay specificity was assessed by testing 19 target and 25 non-target pure cultures. The detection limit was 500 fg, corresponding to 100 genome equivalents. The optimized protocol was also prevalidated with spleen, kidney and blood samples from infected and uninfected sea bass, without any culture step, and it can be proposed as a valid alternative to culture standard methods for the rapid and specific diagnosis of photobacteriosis in fish.

Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.

Determination of living and active bacterioplankton: a comparison of methods

The purpose of this study was the quantification, through the comparison of different methods, of viable and metabolically active bacteria in marine environments. To quantify the living and active bacterioplankton fractions, we compared the total cell count (TC using DAPI staining), plate count on marine agar (CFU), and three viability-staining methods: nucleoid-containing cell count (NuCC), Live/Dead staining procedure (L/D) and direct viable count (DVC). With respect to TC (mean value 3.0 ± 2.3 × 105 cell ml−1) CFU represented less than 0.1% and DVC cells 1%, both showing significant differences. NuCC and L/D cells were 18.0% and 15.9% of TC, respectively, showing no significant differences and higher percentages in the Ionian Sea than the Adriatic Sea. Moreover, NuCC and L/D were two orders of magnitude greater than the culturable fraction, while active cells (DVC) exceeded CFU by one order of magnitude. The comparison of different staining methods allowed us to confirm the simultaneous presence of different physiological states within the bacterial population in natural marine environments. The NuCC and L/D methods gave comparable values to those of other authors, while the DVC procedure gave lower values than previously reported. This research provides information on the fraction of living and/or metabolically active bacteria in aquatic ecosystems. Since each method has its own detection limits, the study highlights the need to simultaneously compare the different methods to validate their results.

Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.