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Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

Under climate-change conditions, temporary rivers will be the dominant surface-water bodies of the Mediterranean region. In order to manage this kind of water body appropriately, it is necessary to understand the chemical and ecological processes that involve hazardous substances in these environments. The processes during the dry phase have an important influence on the mobility and the availability of hazardous substances. This critical review presents the state of knowledge on the fate and the monitoring of hazardous substances in temporary rivers and highlights future trends in research in this field.

Temporary rivers are characterized by recurrent dry phases, and global warming will stress their hydrology by amplifying extreme events. Microbial degradation and transformation of organic matter (OM) in riverbed sediment are key processes with regard to carbon and nutrient fluxes. In this study, we describe structural and functional changes of benthic microbial communities in a riverine environment subject to hydrological fluctuation. Sampling was carried out in the outlet section of the Mulargia River (Sardinia, Italy) under various water regimes, including one flood event. Overall, sediments were characterized by low bacterial cell abundance (range 0.6–1.8 × 109 cell g−1) as a consequence of their low nutrient and OM concentrations. No major differences were found in the community composition. Alpha-Proteobacteria dominated during the whole year (range 21–30%) followed by Beta-Proteobacteria, Gamma-Proteobacteria, and Cytophaga-Flavobacteria which always contributed

We studied the diversity, community composition and activity of the primary microbial colonizers of the water above freshly re-wetted sediments from a temporary river. Dried sediments, collected from Mulargia River (Sardinia, Italy), were covered with sterile freshwater in triplicate microcosms, and changes of the planktonic microbial assemblage were monitored over a 48 h period. During the first 9 h bacterial abundance was low (1.5 x 10(4) cells ml(-1)); it increased to 3.4 x 10(6) cells ml(-1) after 28 h and did not change thereafter. Approximately 20% of bacteria exhibited DNA de novo synthesis already after 9 h of incubation. Changes of the ratios of (3)H-leucine to (3)H-thymidine incorporation rates indicated a shift of growth patterns during the experiment. Extracellular enzyme activity showed a maximum at 48 h with aminopeptidase activity (430.8 +/- 22.6 nmol MCA l(-1) h(-1)) significantly higher than alkaline phosphatase (98.6 +/- 4.3 nmol MUF l(-1) h(-1)). The primary microbial colonizers of the overlaying water - as determined by 16S rRNA gene sequence analysis - were related to at least six different phylogenetic lineages of Bacilli and to Alphaproteobacteria (Brevundimonas spp. and Caulobacter spp.). Large bacterial cells affiliated to one clade of Bacillus sp. were rare in the dried sediments, but constituted the majority of the planktonic microbial assemblage and of cells with detectable DNA-synthesis until 28 h after re-wetting. Their community contribution decreased in parallel with a rise of flagellated and ciliated protists. Estimates based on cell production rates suggested that the rapidly enriched Bacillus sp. suffered disproportionally high loss rates from selective predation, thus favouring the establishment of a more heterogenic assemblage of microbes (consisting of Proteobacteria, Actinobacteria and Cytophaga-Flavobacteria). Our results suggest that the primary microbial colonizers of the water above dried sediments are passively released into the plankton and that their high growth potential is counteracted by the activity of bacterivorous protists.

In the semiarid Mediterranean regions, water scarcity represents a common physiological stress for microbial communities residing in river sediments. However, the effect of drying has not yet adequately been evaluated when analyzing riverine microbiological processes. The bacterial community structure (abundance, biomass, composition) and functioning (carbon production, live cell percentage) were assessed during experimental desiccation in microcosms with sediments from different Mediterranean temporary rivers (Tagliamento, Krathis, Mulargia, Pardiela). Our results showed that the overall responses to drying of the bacterial community were independent from sediment origin and strictly related to water content. During desiccation, a prompt decline (up to 100%) of the initial bacterial carbon production was followed by a slower decrease in abundance and biomass, with an overall reduction of 74% and 78%, respectively. By the end of the experiment, live cells were still abundant but depressed in their main metabolic functions, thus resulting in a drastic increase in the community turnover time. Only 14% of the initial live cell biomass was available in dry sediments to immediately start the reactivation of the aquatic microbial food web after the arrival of new water. Community composition analysis showed a relative increase in alpha- and beta-Proteobacteria, when passing from wet to dry conditions. Our results suggest that the occurrence of drought events could affect carbon cycling through the freshwater microbial compartment, by temporarily limiting microbial mineralization and altering bacterial community structure.

The results obtained in the four seasonal cruises planned in the PRISMA II project are reported. These concern dissolved and colloidal organic carbon, free amino acids and total dissolved carbohydrates and heterotrophic activity. Main factors controlling organic matter degradation, resulting from laboratory tests not planned in the above project, are also discussed. Dissolved organic matter shows seasonal accumulation, which may be markedly different from year to year, and large contributions by colloidal and saccharide components. Heterotrophic activities play an important role in the carbon cycle, although laboratory runs highlight limitations caused by aging of organic matter and phosphorus deficiency.

The variability of dissolved (DOC) and colloidal (COC) organic matter and of organic biochemical components and their possible relationships with heterotrophic activity have been investigated in the northern Adriatic Sea during four surveys (June 1996, February 1997, June 1997 and February 1998). Samples were collected in two frontal regions, one in the north under the direct influence of the Po River and the other in the south close to the border-line between the northern and the middle Adriatic. The overall set of data ranged from 53 to 281 μM for DOC, 44 to 98 μM for COC, 6 to 72 μM for total dissolved carbohydrates (TDCHO-C), 0·1 to 2·4 μM for free amino acids (DFAA-C), 1·2 to 9·4 μM for total dissolved amino acids (TDAA-C) and 0·01 to 7·1 μg C l−1 h−1for bacterial carbon production (BCP). COC and TDCHO data showed tight relationships with DOC values, which were in turn negatively dependent on salinity. Organic carbon showed increases in surface waters with respect to the background DOC value of 76±10 μM averaged from bottom data in February surveys. Seasonal changes were more or less marked depending on the riverine discharge curve and related trophic dynamics in the investigated year. The most marked increases were observed in June 1996 and, based on average values, amounted to 104 and 62 μM DOC in the north and south frontal regions, respectively; in the successive June 1997, the corresponding increases were 47 and 20 μM. COC concentrations gave a high contribution to DOC and showed a large fraction of the high molecular weight class. Contrary to the increases in DOC and COC concentrations which were stronger in 1996 than 1997, mucous macroaggregates showed a much stronger occurrence in June 1997 compared to the June 1996 survey. Considering that the aggregation of colloids is one of the main processes leading to the formation of macroaggregates, the different behaviour of DOC seasonal changes and mucilage occurrences in the two years may suggest that the qualitative characteristics of the DOC and COC pools are probably more important than their quantitative concentrations in controlling the partitioning between dissolved and aggregate matter. Bacterial production rates showed a large variability during the study period which reflected concentration gradients of inorganic and organic nutrients, differences in the lability of the organic substrate and nutrient limitations. Bioassays carried out on samples collected in different seasons and at various salinities suggested an apparent strong limitation of the bacterial activity by phosphorous.

Nutrients and standing crop over a 2-year period and primary production rates over a 1-year period were measured at three stations located 1·5, 6 and 15 nautical miles (NM) from the coast, 150 km south of the Po river mouth. Both nutrients and standing crop (the latter measured as chlorophyll a concentration and phytoplankton cell density) showed a concentration gradient from west to east with marked changes of average values from 6 to 15 NM off the coast. Nutrients and standing crop appeared to be positively correlated and both showed an inverse dependence on salinity which was mainly influenced by freshwater inputs from the major Italian river, the Po. Primary production rates showed values of about 260 and 210 g C m−2 year−1 at the 1·5 and 6 NM stations respectively, indicating the presence of a narrow coastal belt with productivity levels significantly higher than those previously reported for other Adriatic areas affected by the Po. The offshore station at 15 NM gave an average annual production of about 120 g C m−2 year−1 which lies in the range reported for lagoons and coastal waters in the Po delta area. The photosynthetic efficiency was similar at the 1·5 and 6 NM stations (respectively 4·0 ± 2·3 and 3·5 ± 2·7) but significantly lower at the 15 NM station (1·9 ± 1·7). This difference reflects either different nutrient levels in inshore and offshore stations or different structures in phytoplankton community: diatoms and phytoflagellates prevailed at 1·5 and 15 NM, respectively. Photosynthetic efficiency increased with increasing concentration of nutrients. The behaviour was found to be linear as a function of total dissolved phosphorus, and hyperbolic as a function of total dissolved nitrogen. This different dependence suggests a surplus of nitrogen with respect to phosphorus for photosynthetic processes in the study area, which is consistent with the assumption of phosphorus as limiting element often made for the northern Adriatic.

We studied the diversity, community composition and activity of the primary microbial colonizers of the water above freshly re-wetted sediments from a temporary river. Dried sediments, collected from Mulargia River (Sardinia, Italy), were covered with sterile freshwater in triplicate microcosms, and changes of the planktonic microbial assemblage were monitored over a 48 h period. During the first 9 h bacterial abundance was low (1.5 x 10(4) cells ml(-1)); it increased to 3.4 x 10(6) cells ml(-1) after 28 h and did not change thereafter. Approximately 20% of bacteria exhibited DNA de novo synthesis already after 9 h of incubation. Changes of the ratios of (3)H-leucine to (3)H-thymidine incorporation rates indicated a shift of growth patterns during the experiment. Extracellular enzyme activity showed a maximum at 48 h with aminopeptidase activity (430.8 +/- 22.6 nmol MCA l(-1) h(-1)) significantly higher than alkaline phosphatase (98.6 +/- 4.3 nmol MUF l(-1) h(-1)). The primary microbial colonizers of the overlaying water - as determined by 16S rRNA gene sequence analysis - were related to at least six different phylogenetic lineages of Bacilli and to Alphaproteobacteria (Brevundimonas spp. and Caulobacter spp.). Large bacterial cells affiliated to one clade of Bacillus sp. were rare in the dried sediments, but constituted the majority of the planktonic microbial assemblage and of cells with detectable DNA-synthesis until 28 h after re-wetting. Their community contribution decreased in parallel with a rise of flagellated and ciliated protists. Estimates based on cell production rates suggested that the rapidly enriched Bacillus sp. suffered disproportionally high loss rates from selective predation, thus favouring the establishment of a more heterogenic assemblage of microbes (consisting of Proteobacteria, Actinobacteria and Cytophaga-Flavobacteria). Our results suggest that the primary microbial colonizers of the water above dried sediments are passively released into the plankton and that their high growth potential is counteracted by the activity of bacterivorous protists.

Bacterial degradation of mucilaginous aggregates (creamy layers, stringers and macroflocs) collected during two summer events (2001-2002) was tested. The objective was to describe the temporal trend of the bacterial activity, abundance and composition in the aggregated and dissolved organic matter under different trophic conditions. In the native aggregates proteins and organic phosphorous were actively hydrolyzed as aminopeptidase and alkaline phosphatase activities represented up to 87% and 25% of total activity, respectively; polysaccharides were less hydrolyzed and the highest activities were observed for beta-glucosidase (5% of the total). This hydrolysation pattern tends to a progressive accumulation of long persistent polysaccharides. During short term incubations nutrient addition (P, N and Glucose) differently stimulated bacterial growth in the seawater: P played the main role in stimulating bacterial production from 3 to 6 folds higher than in the control, whereas a secondary C-limitation was observed only for bacteria growing on seawater from macroflocs. This scarce dissolved organic carbon (DOC) bioavailability was confirmed by the lower DOC removal (13% macroflocs, 36% stringers). The total amount of carbon incorporated by bacteria living on aggregates was similar (0.58 mg C L(-1)) both in the control and under P enrichments showing a more balanced condition with respect to the seawater. Hence the well-known P limitation in the Northern Adriatic Sea affects only dissolved organic carbon uptake without influencing the uptake of aggregated organic matter. Organic matter limitation was observed only on stringers--total C incorporated raised to 0.96 mg C L(-1) after PNG addition. Macroflocs release of refractory compounds leads to DOC accumulation (73 microM DOC) contributing to inflate the pool of refractory DOC in the surrounding waters. Several evidences, including different monosaccharide composition of stringers and macroflocs (glucose 15% and 56% on stringers and macroflocs, respectively), bring to the conclusion that stringers are in an older stage in comparison with macroflocs. Community composition described by fluorescence in situ hybridization did not show significant differences between free-living and attached bacteria but it was modified by the different enrichment conditions: Cytophaga-Flavobacteria increased after inorganic nutrients enrichments while organics advantaged gamma-Proteobacteria.

Summary Bacterial communities associated with a variety of benthic detritus types were studied in three streams in the context of the chemical characteristics of the sediment material and the stream water. A cell purification assay was developed for a quantitative microscopic evaluation of bacterial community structure in detritus samples by fluorescence in situ hybridization (FISH). The efficiency of FISH with fluorescently monolabelled probes was compared with FISH with signal amplification by catalysed reporter deposition (CARD-FISH). In detritus types poor in organic carbon and nitrogen, the numbers of prokaryotes were related to the chemical characteristics of the stream water column, whereas no such relationship was found for detritus types rich in organic carbon and nitrogen. These results might help to provide criteria for the selection of detritus types for river ecosystem assessment and monitoring. The percentage of bacteria detected by FISH with monolabelled probes was correlated with the detritus total organic matter (OM). This is likely attributed to a higher ribosome content of microbial cells on substrates rich in OM. Cell detection by CARD-FISH did not show any correlation with OM content, indicating that this technique renders the results more independent from the activity state of cells. Fluorescence in situ hybridization with four group-specific probes suggested a relationship between substrate quality and the composition of the microbial assemblages on the various types of detritus. The improved protocol for cell purification and CARD-FISH may facilitate future investigations on the relationship between the riverine benthic detritus quality and microbial community composition.

The growth and the structure of a coastal bacterioplankton community were monitored in short-term bottle experiments in order to investigate the bacterial uptake of extracellular organic carbon released by the diatom Cylindrotheca closterium grown under P-balanced and P-depleted conditions. Bacterial specific growth rates and carbon demand were significantly lower in the exudates from P-depleted algae (24% and 30% reduction, respectively). The origin of the extracellular carbon appeared also to affect the taxonomic composition of the bacterioplankton assemblage, mainly reducing the development of ?-Proteobacteria. This pattern of bacterial carbon uptake could contribute to a longer persistence of the exudates released in P-depleted conditions affecting the dynamics of the carbon cycle in marine environments. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Dissolved organic carbon (DOC) concentrations in northern Adriatic coastal waters show seasonal variations with increasing values from winter (75-95 ?M in February) to spring-summer periods (120-190 in June 1996 and 85-123 ?M in June 1997). Riverine inputs of refractory organic matter and abiotic transformations of in situ produced biogenic matter contribute to the accumulation of DOC. Phosphorus limitation of bacteria, whose productivity varied widely with time and space (0.01 to 7 ?g Cl-1 h-1), probably concurs to this seasonal DOC behaviour making microbial community unable to contrast the enrichment in DOC concentrations. Possible involvement of DOC accumulation in the gel production in the Adriatic sea are discussed.

Descrizione del progetto PRISMA1

In this study we have carried out a mutational screening of exons 62-79 of the dystrophin gene by SSCP in 38 Italian patients with DMD/BMD and found two novel mutations at exon 70, in 2 mentally retarded DMD patients.

The estuarine processes which can influence the release of nutrients of the River Tiber into the Tyrrhenian Sea have been studied. A conservative behaviour has been found for silicate in different hydrographic conditions. For nitrogen, positive increments in respect of the dilution values were found in summer, due to regeneration processes; on the contrary a conservative behaviour was observed when seawater did not intrude into the river. Dissolved phosphorus showed, in all the hydrographic conditions, a positive deviation in respect of the theoretical dilution values. This deviation has been attributed to regeneration processes which take place during the summer period. The chemical release of phosphate from particulate matter is predominant when the saline wedge is absent.

The need to evaluate the impact of the Tiber River on the receiving marine ecosystem, and the extent of pollution abatement required to safeguard the various coastal water uses, promoted an interdiscipl i nary experimental investigation. First stage results of investigation carried out off the river mouth are presented. The extension of the area influenced by the river has been estimated at 400 km2. Due to the river's high nutrient input rates, eutrophic processes occur with an intensity depending on sample distances from the mouth. Some data concerning the limiting factor have also been derived. Toxicity studies have revealed an incipient threat from Cd and Hg concentration. HCB was only occasionally found in the zone; PCB's were found in surprisingly high levels further away from the river mouth. Bacteriological data led to conclude that the river's impact on beach bathing conditions is only limited to an area immediately adjacent the two outfalls. With the exception of minor changes due to the eurhyaline nature of the zone, no major variation in receiving area ecology was detected (for benthos and nekton).

The processes affecting the in-sea diffusion of nutrients at the Tiber River mouth are strongly dependent on seasonal conditions. In summer, during the low-flow period, a two-layer structure is established and it is then possible to detect the occurrence of biological processes in the lower layer, at the sediment level. During these conditions, the mean concentrations of dissolved organic carbon, inorganic phosphorus and nitrogen increase in the lower saline layer above the theoretical dilution values and, in some cases, the concentrations are double those in the upper layer.

The sources, characteristics and fate of the dissolved organic matter (DOM) in marine ecosystems are reviewed, with particular emphasis on processes relevant to the northern Adriatic Sea. This semi-enclosed basin receives large riverine discharges that strongly affect its trophic dynamics. The high organic load, which results from both autochtonous and allochtonous sources, is responsible for anoxia and massive aggregate formation. The general characteristics of the microbial food web and its role in the cycling of carbon are critically evaluated. Factors affecting DOM uptake by bacteria, including DOM diagenesis and molecular size, nutrient availability, the effect of temperature and the abiotic alterations of DOM, are discussed. Possible causes for DOM seasonality are suggested. A restricted bacterial uptake due to scarce inorganic phosphorus availability, along with strong allochtonous inputs of refractory compounds and strong water stratification in summer, appear to be the most important factors. Finally, the distribution of organic carbon between dissolved and colloidal components and the role of colloids in the aggregation phenomena in marine systems are briefly outlined, and hypotheses for the formation of macroaggregates in the northern Adriatic are reviewed.

The acute toxicity of thallium and chromium to marine phytoplankton was determined in enrichment culture bioassays (1). The results indicated minimal effective concentrations of 0.7 10− 1.1 10−6 M thallium on Phaeodactylum tricornutum growth respectively for the monovalent and the trivalent forms. Separate studies on Dunaliella tertiolecta confirmed the higher toxicity of the monovalent form with respect to the trivalent one (0.4 10−6 M and 0.9 10−6 M, respectively). In experiments with Cr, the minimal effective concentration of Cr (VI) was 1 10−4 M for both algal species in marine waters. Cr (VI) toxicity was found to be strongly dependent on mean salinity due to the competitive role of sulphate. Figure 1 Cr (III) concentrations lower than 4 10−4 M did not significantly reduce the growth rate but produced a longer phase lag, while at concentrations of 4 10−4 M Cr (III) no growth was observed. The toxicity of both Tl (III) and Cr (III) is shown to be reduced by complexation with EDTA.