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Due to the increasing evidences on dinoflagellate contribution to mucilage phenomena in the Mediterranean Sea, this study was aimed at identifying biochemical and/or genetic markers that could help in relating Gonyaulax fragilis growth with mucilage formation when microscope identification was difficult. The biochemical characterization considered the fatty acid and sterol composition of cultured strains and mucilage samples. The cultured algae displayed the typical fatty acid pattern of dinoflagellates as the 18:0 was the prevalent saturated fatty acid (SFA), 18:1n-7 and 18:1n-9 were the major monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were dominated by 18:5n-3 and 22:6n-3. Two groups of sterols were identified in algal cells: desmethylsterols, dominated by cholesterol and β-sitosterol, and 4α-methylsterols, having dinosterol as the prevailing sterol. The mucilage lipid pattern was characterized by low amounts of PUFAs and appeared deeply influenced by environment dynamics; however, the 20:5n-3/22:6n-3 ratio

Effects of benzo(a)pyrene [B(a)P] (at a nominal concentration of 0.5 mg/L) on immune responses of the clam Chamelea gallina were investigated after 1, 7, and 12 days exposure. Total hemocyte count (THC), hemocyte volume, phagocytic activity, lysozyme-like activity in both hemocyte lysate (HL) and cell-free hemolymph (CFH) were measured. As unexpected alterations in hemocyte adhesion capability were observed in short-term hemocyte cultures for phagocytosis assays after a 1-day exposure, an adhesion test (not included in the original experimental setup) was performed after 7 and 12 days of exposure only. The survival-in-air test was carried out to evaluate general stress conditions in B(a)P-exposed clams. No alterations in THC was observed, whereas exposure for 7 and 12 days to B(a)P significantly decreased phagocytic activity and adhesion capability when compared with controls. Significant decreases in lysozyme activity were observed in CFH and HL, with respect to controls. B(a)P was also shown to alter the resistance to air exposure of clams. The LT(50) values fell from 9 days in control clams to 7 days in 1-day-exposed animals, and from 6 days in control clams to 5 days in 7-day-exposed bivalves. No significant variations in LT(50) values were recorded after 12 days of exposure. Results highlight a relationship between B(a)P exposure and alterations in hemocyte functionality and suggest that the contaminant induced irreversible immunosuppression in C. gallina, by altering phagocytic activity, adhesion capability, and enzymatic activity. Conversely, reduction in resistance to air exposure was reversible, suggesting that impairment of important physiological functions of clams occurred in the first phases of exposure only.

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.

Chloramphenicol (CA) is a largely used antibiotic and it is an inhibitor of protein synthesis that also induces ROS production. In this work there were investigated activities and expressions in the Adriatic bivalve Chamelea gallina of some antioxidant and detoxification proteins like superoxide dismutase (Mn–SOD, Cu/Zn–SOD), catalase (CAT) and Cytochrome P450 (CYP1A). Clams exposed to 5 mg l −1 of chloramphenicol were sampled 2, 4 and 8 days after treatment (CA2, CA4 and CA8). SODs, CAT, and CYP1A activity and/or expression were detected in pooled digestive glands by Western blotting and by spectrophotometrical analysis. Enzymes activities increase during the entire antibiotic exposure. With respect to the control Cu/Zn–SOD expression increases, while Mn–SOD expression decreases significantly after 4 days. Two CYP1A immunopositive-proteins (57.7 and 59.8 kDa) were detected. The lower band significantly decreases in CA8, the upper one also in CA4 condition. High levels of Mn–SOD, CAT activity and Cu/Zn–SOD expression, indicate intense ROS production while Mn–SOD expression inhibition might be ascribable to mitochondrial alterations due to CA and indirectly to ROS. CYP1A1 action determines H 2 O 2 production that would contribute to a CYP1A1 gene promoter down regulation, a response to oxidative stress with the antioxidant enzymes activation as a final result. This study highlights the close association, in C. gallina , in presence of chloramphenicol, between SOD/CAT and CYP system, and it appear particularly interesting to the lack of similar researches on mollusc species.

The effects of different salinity levels (28, 34 and 40‰) on functional responses of Chamelea gallina haemocytes were evaluated in a two part study dealing with modulations of immune parameters. This part (Part II) of the study was focused on the superoxide dismutase (SODs) activity and expression in haemocyte lysate and cell-free haemolymph. Results of this study established that the exposure of C. gallina specimens at 40‰ salinity provoked a decrease in Mn-SOD and Cu/Zn-SOD activities in haemocyte lysate suggesting a declining superoxide anion generation at the highest salinity tested. Expression of MnSOD was coherent with activity values, while Cu/ZnSOD showed two immunoreactive bands. The former corresponds to the cytosolic Cu/Zn-SOD (16 kDa) was not coherent with the enzyme activity and the second (28–30 kDa) probably attributed to EC-SOD. In cell-free haemolymph, Mn-SOD activity decrease and Cu/Zn-SOD activity increase at 40‰ were observed, likely due to EC-SOD contribution, strongly induced at the same salinity. After EC-SOD detection with two different antibody, we postulate that the EC-SOD like-protein band (29 kDa) may be constitute partly by EC-SOD and probably by Cu/Zn-SOD dimeric form not completely dissociated under reducing condition or a Cu/Zn-SOD degenerated but still recognized by antibody. The cell-free haemolymph increase of EC-SOD at high salinity values plays an important role in immune defence of C. gallina. According to the conclusion of Part I of this study, our data confirmed the destabilizing effect of 40‰ salinity on haemocyte functionality, while to 28‰ exposure, data don’t confirm its stressful action as instead stated by results of Part I. Further studies are necessary to clear up this discrepancy.

An analytical method based on high-performance liquid chromatography (HPLC) combined with fluorescence detection (FL) has been developed for the simultaneous determination of fumonisin B 1 (FB 1 ) and its totally hydrolized metabolite aminopentol-1 (AP 1 ) in pig liver. The sample preparation is based on a single solid phase extraction (SPE). o -Phthalaldehyde (OPA) was used for pre-column derivatization before the programmed reversed-phase analysis on phenylhexyl column. The developed method shows good repeatibility for inter- and intra-day precision as well as adequate linearity of calibration curves ( r 2 was 0.9855 for FB 1 and 0.9831 for AP 1 ). Average recoveries from the matrix were 93.6% for FB 1 and 95.3% for AP 1 . The limit of quantification (LOQ) in swine liver was 75 μg/kg for FB 1 and 42 μg/kg for AP 1 .