Organic matter recycling in a beach environment influenced by sunscreen products and increased inorganic nutrient supply (Sturla, Ligurian Sea, NW Mediterranean).

The beaches are sites where the human influence may be strong and the beach ecosystems have often shown a high sensibility to environmental alterations. These zones may be affected by a large series of anthropogenic-derived pressures, such as unbalanced inorganic nutrient input, that may cause anomalous development of primary production, altering the structure of the trophic webs. Furthermore, the utilisation of cosmetic sunscreen products is reaching unexpected levels, thus assuming a potentially important as well as unknown role in the contamination of marine environments. The present study was planned to test the response of the beach ecosystem to increases in inorganic nutrients (nitrate and phosphate) and to the input of a widely used cosmetic sunscreen product. A short-term laboratory experiment was carried out on microsystems consisting of sediments and seawater from the swash zone of a Ligurian city beach (Sturla). The processes related to organic matter (OM) recycling and some microbial food web components (bacteria and micro-autotrophic organisms) were analysed. The multivariate statistical analysis of the results showed that the increase in inorganic nutrients and sunscreen caused only a transient alteration in the OM recycling processes in the seawater. The sedimentary processes, instead, were different in the different systems, although starting from the same condition. In the sediment, surprisingly, an increase in inorganic nutrients did not lead to an increase in the primary biomass nor to significantly higher bacterial abundance, while the sunscreen caused increased OM recycling, especially devoted to protein and lipid mobilisation, supporting a growing bacterial and autotrophic community by reducing the bottom-up pressure. Additional toxicity tests performed on protozoa highlighted that, while the inorganic nutrients seemed to show no effects, sunscreen decreased the protozoan viability, thus likely favouring microautotrophic and bacterial increases by reducing the top-down pressure.

Action mechanisms of the secondary metabolite euplotin C: signaling and functional role in Euplotes.

Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca(2+) and Na(+), suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.

Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia.

In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays.

Effects of xenobiotic compounds on the cell activities of Euplotes crassus, a single-cell eukaryotic test organism for the study of the pollution of marine sediments.

It is now widely accepted that assays with protists are relevant to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre-chemical screening of estuarine and coastal sediments was evaluated. For this purpose, the effects of exposure to pollutants were tested on the cell viability, fission rate and lysosomal membrane stability of E. crassus. The following toxicants were used: an organophosphate (OP) pesticide, basudin, an organochlorine hydrocarbon, AFD25, both employed especially for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl2) and different mixtures of the above-mentioned compounds, as they might occur in polluted sites. Exposure to these toxicants affected cell viability at concentrations ranging from 96.6 to 966 x 10(3)mg/l for basudin, from 3.3 to 33 x 10(3)mg/l for AFD25 and from 0.1 to 1mg/l for HgCl2. A significant decrease in the mean fission rate (P<0.001) was found after 24- or 48-h exposures to 9.66 mg/l basudin, 3.3 mg/l AFD25 and 7 x 10(-2)mg/l HgCl2. Furthermore, the Neutral Red Retention Assay showed a significant decrease in lysosomal membrane stability after 60- and 120-min exposures to AFD25 (33 mg/l) and HgCl2 (0.33 mg/l). In addition, as it is well-known that the inhibition of acetylcholinesterase activity represents a specific biomarker of exposure to OP and carbamate pesticides in higher organisms, initially the presence of cholinesterase (ChE) activity was detected in E. crassus, using cytochemical, spectrophotometric and electrophoretic methods. Afterwards, this enzyme activity was characterized spectrophotometrically by its sensitivity to specific ChE inhibitors and to variations in pH and temperature. The ChE activity was inhibited significantly by basudin- (9.66 and 96.6 mg/l) or AFD25-exposure (3.3 mg/l). Conversely, exposure to AFD25 (33 mg/l) or HgCl2 (0.1 and 0.3mg/l) caused a significant increase in this enzyme activity. Moreover, exposure to mixtures containing basudin, AFD25 and HgCl2 was found to affect the cell viability, the mean fission rate and the ChE activity differently, in an unpredictable manner. Our results indicate that E. crassus seems to be a suitable test organism to evaluate the toxicity of marine sediments.

Detection of NADPH-diaphorase activity in Paramecium primaurelia.

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.

Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista).

Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions.

Effects of organophosphate compounds on a soil protist, Colpoda inflata (Ciliophora, Colpodidae).

Many investigations on protists indicate that they play an important role in agricultural soils. We have tested the effects of three organophosphate (OP) pesticides, basudin, cidial, and fenix, on the soil ciliate Colpoda inflata, and examined its viability, fission rate, ability to excyst and extrude macronuclear chromatin into cytoplasm. Exposure to these OPs caused a dose-dependent effect on cell viability, and significantly reduced the mean fission rate at a concentration of 1/10(5) v/v. After exposure of resting cysts to 1/10(5) v/v or 1/10(6) v/v concentrations of basudin or cidial, the number of excysted cells was significantly lower than that of the controls. Conversely, exposure to a 1/10(5) v/v fenix concentration did not affect excystment and exposure to 1/10(6) v/v was found to promote excystment. Moreover, exposure to these OPs (1/10(4) v/v or 1/10(5) v/v) interferred with the ability to extrude macronuclear chromatin. The median lethal concentration in 60 min for each OPs tested was at least a hundred times lower than the doses recommended by the manufacturer. Finally, as the inhibition of cholinesterase (ChE) activity is the first target of OPs, the presence of ChE activity was checked in C. inflata. Three ChE activities were found, hydrolyzing the substrates acetyl-beta-methyl thiocholine iodide, propionyl thiocholine iodide and butyryl thiocholine iodide, that appeared to be very low and not inhibited by OP-exposure.

Effects of time-variant extremely low-frequency (ELF) electromagnetic fields (EMF) on cholinesterase activity in Dictyostelium discoideum (Protista).

Recently, we detected propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum using cytochemical, electrophoretic, and spectrophotometric methods. The involvement of this enzyme activity in cell-cell and cell-environment interactions was suggested. In this work, we found that exposure of single-cell amoebae to an extremely low-frequency electromagnetic fields (ELF-EMF) of 300 microT, 50 Hz, from 1 h up to 48 h at 21 +/- 1 degrees C affected PrChE activity.

Detection of molecules related to the GABAergic system in a single-cell eukaryote, Paramecium primaurelia.

Gamma-aminobutyric acid (GABA)-related molecules were identified in Paramecium primaurelia by immunocytochemical methods, and GABA(A) receptors by their histochemical BODIPY-binding sites. Confocal microscope analysis showed different localizations according to the stages of the developmental cycle. A comparison was made with the cholinergic molecules, such as the acetylcholine biosynthetic enzyme (choline acetyltransferase), in double-labelled cells by confocal microscopy. In vivo experiments suggested the involvement of GABA-related molecules in cell-cell interaction.